M Y C
Journal Name
1 1 5 7
Manuscript No.
Abstract
A0.2
ECMM Drouhet lecture. The history of
antifungal combination therapy. To make a
virtue out of necessity
A. Polak
Aesch, Switzerland
Antifungal combination therapy has a more than
30-year history. In 1971 Medoff and coworkers
observed for the first time a synergistic effect of
flucytosine (5FC) with amphotericin B (Amph B)
in vitro. At the same time the monotherapy with 5FC
caused in clinical trials a significant increase of
resistant mutants. These two events are the roots
of an exciting scientific development; from the finding
of additive effects in vitro, over sophisticated animal
models to clinical trials. Combination therapy of 5FC
plus Amph B became the gold standard for the acute
phase of cryptococcal meningitis and was also used
for other opportunistic fungal diseases in severely
immunosuppressed patients. Over the years new
antifungals with different mode of actions and better
clinical efficacy appeared; however, the combination
therapy has still its value. In modern time the term
combination therapy has widened: not only antifungals are combined, but new combinations exist, f.e.
an antifungal with an immunostimulant drug or an
antifungal with a virulence inhibitor. Additionally
systemic and topical therapy schedules can be
combined showing a significant benefit. Generally,
combination therapy broadens spectrum, increases
efficacy, reduces appearance of resistant mutants and
may even show a better cost/benefit value.
A1.1
State of the art: antifungal therapy in cancer
patients
G. Maschmeyer
Klinikum Ernst von Bergmann, Potsdam, Germany
Invasive fungal infections have become a major cause
of morbidity and mortality in patients with high-grade
hematological malignancies and allogeneic stem cell
transplant recipients. Patients at high-risk, e.g. those
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
B
Dispatch: 12.8.05
Author Received:
Journal: MYC CE: Kavi
No. of pages: 173 PE: Vijay
with prolonged profound neutropenia or those with
severe graft-vs.-host disease and intensive immunosuppressive treatment, have been subject to studies on
primary antifungal prophylaxis using oral, parenteral
or nebulized agents. A significant reduction of fungal
infection-related mortality has been demonstrated in
alloSCT patients given prophylactic fluconazole in the
early 1990s. Itraconazole reduces the number of
invasive aspergillosis more effectively than fluconazole
in alloSCT recipients who tolerate the drug in a
sufficient dose over a long period of time. Prophylactic
inhalation of amphotericin B has shown promising
results in single reports, but tolerance has been poor,
particularly when conventional AmB is used. Longterm antifungal prophylaxis with voriconazole in
alloSCT patients is attractive, but still under investigation. Fatality rates in patients with documented
invasive aspergillosis have been significantly reduced
by primary use of voriconazole as compared with
amphotericin B. In patients not tolerating or refractory
to voriconazole or an amphotericin B formulation,
complete or partial response can be achieved by a
second-line therapy with caspofungin. Pre-clinical
data suggest promising activity of primary combination antifungal treatment, however, results from
prospective clinical trials should be awaited before
this approach is used outside controlled studies. Early
detection of invasive aspergillosis and prompt start of
an effective antifungal therapy are the most essential
factors for treatment outcome in these patients.
Thorough clinical examination, early high-resolution
thoracic CT scans and non-culture based diagnostic
techniques using galactomannan or PCR assays may
help to identify patients in early stages of invasive
pulmonary aspergillosis. Invasive and oropharyngeal
or esophageal candidiasis in cancer patients may be
due to species other than Candida albicans, particularly
C. glabrata, which may be resistant to fluconazole.
However, in patients not treated with fluconazole for
prophylaxis, C. albicans is still predominant, and
therefore, fluconazole may be effective in the majority
of Candida infections in cancer patients. For initial
antifungal intervention in neutropenic patients with
candidemia, amphotericin B or caspofungin are
recommended, before species identification and in vitro
susceptibility are known. It should be kept in mind that
clinical outcome in patients with candidemia depends
not solely on the choice of antifungals, but rather on
the severity of the underlying disease.
With the advent of new broad-spectrum antifungals
with favourable safety profiles, our armamentarium to
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combat life-threatening fungal infections has broadened significantly, but prudent use of these agents
based upon rationale definition of indications is
mandatory.
C2
Interactive case session 2: infections in the ICU
K. Vandewoude and S. Blot
Ghent University Hospital, Ghent, Belgium
Fungal infection is a prevalent problem in critically ill
patients [1]. Candida spp. cause the majority of these
infections. In critically ill patients, invasive candidiasis is associated with a poor prognosis but early
initiation of adequate antifungal therapy might limit
the attributable mortality [2]. Adequate management, however, is hampered by a problematic
diagnosis as the clinical picture of invasive candidiasis often is non-specific and blood cultures have a
low sensitivity. Moreover, it is difficult to differentiate
Candida colonization from infection and many critically ill patients are heavily colonized with Candida
spp., especially when receiving broad-spectrum antibacterials. A range of therapeutic strategies for the
management of invasive candidiasis has been developed: antifungal prophylaxis, pre-emptive therapy,
empiric and definite antifungal therapy [3]. Each of
these strategies has a specific target population, as
defined by specific underlying conditions and/or
individual risk factors. Antifungal prophylaxis – in
order to prevent candidal infection – is based on type
of underlying diseases with a high risk for invasive
candidiasis. Individual risk factors are not taken into
account. Potential indications are bone marrow
transplantation, liver transplantation, recurrent gastro-intestinal perforations or leakages, and surgery
for acute necrotizing pancreatitis. Pre-emptive therapy is also a preventive strategy. It can be recommended on basis of an individual risk profile including
overt candidal colonization. Empiric therapy is started
in patients with a risk profile for invasive candidiasis.
It is recommended in the presence of clinical signs of
infection, clinical deteriorating parameters, or a
clinical picture of infection not responding to antibiotics, but in the absence of a clear causative
pathogen. Definitive antifungal therapy is defined as
therapy in patients with documented invasive infection. Invasive aspergillosis is increasingly recognized
as an emerging disease in critically ill patients [4]. In
a single center study in a medical ICU, an incidence of
2
6.7% was found using strictly the EORTC/MSG
criteria. Current diagnosticcriteria are based on
clinical, radiological, microbiological indices and host
risk factors for the acquisition of invasive aspergillosis
[5]. The latter are mainly neutropenia and severe
immunosuppression. The diagnostic algorithm used
for hemato-oncologic patients is difficult to apply in
apparently immunocompetent ICU patients, since
clinical signs and radiological data are atypical,
hampering a timely diagnosis and institution of early
appropriate antifungal therapy. The presence of
Aspergillus spp. in respiratory tract isolates should
not be discarded in the absence of typical host risk
factors, but should urge for prompt CT-scan, broncho-alveolar lavage, and lung biopsy if appropriate.
Solid diagnostic criteria for invasive aspergillosis in
critically patients must be developed. This should
allow early antifungal therapy which, hopefully, can
result in better outcomes, as in ICU patients invasive
aspergillosis carries a high fatality rate as well as a
significant attributable mortality [6].
References
1. Eggimann P, Garbino J, Pittet D. Epidemiology of Candida species
infections in critically ill non-immunosuppressed patients.
Lancet Infect Dis 2003; 3: 685–702.
2. Blot SI, Vandewoude KH, Hoste EA, Colardyn FA. Effects of
nosocomial candidemia on outcomes of critically ill patients.
Am J Med 2002; 113: 480–5.
3. Blot S, Vandewoude K. Management of invasive candidiasis in
critically ill patients. Drugs 2004; 64: 2159–75.
4. Vandewoude K, Blot S, Benoit D, Depuydt P, Vogelaers D,
Colardyn F. Invasive aspergillosis in critically ill patients:
analysis of risk factors for acquisition and mortality. Acta Clin
Belg 2004; 59: 251–7.
5. Meersseman W, Vandecasteele SJ, Wilmer A, Verbeken E,
Peetermans WE, Van Wijngaerden E. Invasive aspergillosis in
critically ill patients without malignancy. Am J Respir Crit Care
Med 2004; 170: 621–5.
6. Vandewoude KH, Blot SI, Benoit D, Colardyn F, Vogelaers D.
Invasive aspergillosis in critically ill patients: attributable
mortality and excesses in length of ICU stay and ventilator
dependence. J Hosp Infect 2004; 56: 269–76.
W0.1
Molecular biology and new species in the
Pseudallescheria boydii complex
J. Cano, F. Gilgado, J. Gené and J. Guarro
Unitat de Microbiologia, Universitat Rovira i Virgili,
Reus, Tarragona, Spain
Pseudallescheria boydii (anamorph Scedosporium apiospermum) is the species responsible for human
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scedosporiosis, a fungal infection with a high mortality rate and which is difficult to treat. Recently, it
has been demonstrated that high genetic variation
exists within this species. We have performed a
morphological and molecular study involving
numerous strains of clinical or environmental origins
and from different countries. The analysis of partial
sequences of the b-tubulin (two loci) and calmodulin
genes and the ITS region of the ribosomal RNA gene
have demonstrated that P. boydii is a species
complex. The combined analysis of the four loci
sequences of 60 strains has showed the presence of
44 haplotypes in the in-group. Three species morphologically related to P. boydii sensu stricto, i.e.
P. angusta, P. ellipsoidea and P. fusoidea, which had
previously been considered synonyms, could be
differentiated genetically from P. boydii in our study.
The new species Pseudallescheria minutispora and
Scedosporium aurantiacum are clearly phylogenetically
and morphologically separated from the other species
of the genus. Further studies are needed to demonstrate if all the species included in the P. boydii
complex have different clinical spectra and antifungal susceptibility.
W0.2
A case of a child with cystic fibrosis and
infection with Aspergillus fumigatus and
Pseudallescheria boydii: clinical parameters
and serology
B. L. Rottier,1 S. van der Heide,2 H. Hovenga2 and
H. F. Kauffman2
1
Beatrix Children’s Hospital and 2Laboratory for
Allergology and Pulmonology, University Medical
Centre Groningen, University of Groningen, The
Netherlands
Background: A 5-year-old girl diagnosed with
cystic fibrosis (CF) at that age developed the clinical
picture of Allergic Bronchopulmonary Aspergillosis
(ABPA) at the age of 7 years. The diagnosis was
based on clinical manifestations (shortness of breath,
increased productive cough, decreased exercise tolerance, wheeze on auscultation) and laboratory
examinations (decreased pulmonary function tests
[FEV1 59%], a rise of total IgE from 71 to
1254 kU l–1,
elevated
total
eosinophils
[4.54 109 l–1], a sputum culture positive for
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
Aspergillus fumigatus (Af) [first 4 months before
ABPA-diagnosis] and positive IgE and IgG against
A. fumigatus). The patient is (with a temporary
interruption) still being treated with itraconazole.
Further, initial therapy consisted of prednisolon for a
period of 3 months daily and 4 months later a period
of alternate day prednisolon. Five months after the
ABPA was diagnosed Pseudallescheria boydii (Pb) was
cultured in sputum. The aim of this paper is to
discuss the clinical characteristics of this patient in
time together with IgG ELISA for both Af and Pb as
well as the development of a specific test for Pb.
Material and methods: Clinical parameters and
outcome of sputum cultures were compared with
serology on multiple occasions. A culture medium
was prepared through filtration and dry-freezing
using the Pb sputum strain according to a routine
Aspergillus protocol. An ELISA assay and doubleimmuno-diffusion (DID) test to Pb were developed
using the culture medium extract. We tested sera
from our case, aspergillus positive sera and
aspergillus negative controls by means of ELISA
and DID.
Results: After treatment the patient’s symptoms
improved and FEV1 returned to normal. The IgE
level drastically lowered under prednisolone therapy,
but has risen again since discontinuation. Initially,
titres for both Af and Pb were negative. At first, only
Af was cultured from the sputum and serum
demonstrated specific IgG and IgE to Af at the time
of diagnosis for ABPA. The IgG titre for Pb was
already positive before the sputum cultures became
positive for Pb and remained positive for both Af and
Pb during the period that both fungi were present in
the sputum cultures. Later Af disappeared from the
sputum cultures, while Pb remained. During the
latter period titres for both Af and Pb remained
strongly positive. ELISA IgG cross-inhibition experiments and DID suggest that there is no cross
reactivity between Af and Pb. Positive ELISA titres
against both micro-organisms suggest parallel sensitisation. Sera of patients with CF and patients after
lungtransplantation (negative or positive serology to
Af) showed different titres against Pb. DID showed
strong specificity for both fungi and could be negative
(no precipitation lines with Pb) in some cases with
positive ELISA against Pb.
Conclusion and discussion: This is the first report
describing infection with Af and Pb together with IgG
ELISA titres and DID and corresponding clinical
parameters in a child with CF. Pb gradually replaces
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Af in the sputum, but serology for both remains
strongly positive. We have developed a specific ELISA
assay to Pb, without cross reactivity with Af. We
think that continuing exposure results in positive
titres against both fungi whereas a positive DID test
may be more specific to identify a disease state with a
fungus.
environmental isolates revealed a huge genotype
diversity, but a common genotype was identified for
one patient between environmental isolates and
clinical isolates from sputum sample. Factors allowing the development of the fungus in potted plants
remain to be defined.
W0.4
W0.3
Recent advances in epidemiology and physiopathology of Scedosporium apiospermum
infections in patients with cystic fibrosis
J.-P. Bouchara, B. Cimon, O. Lima and G. Larcher
GEIHP Groupe d’Etude des Interactions
Hôte-Parasite, Angers, France
Despite a relatively high occurrence of Scedosporium
apiospermum in patients with cystic fibrosis (CF)
contrasting with its paucity in the environment,
little information are available about virulence
factors of the fungus. In this context, our attention
has been focused on anti-oxidant systems. By means
of enzyme assays and of negative staining after nondenaturing electrophoresis, a superoxide dismutase
(SOD) has been detected in a cytoplasmic extract of
the fungus cultivated in iron-restricted culture
medium. The enzyme which was purified by chromatography on Amberlite XAD16, presents a
molecular mass of 16.5 kDa under reducing conditions, and its properties, including its susceptibility to
commercially available inhibitors, were investigated.
More, the gene encoding this enzyme has been
sequenced, showing a high homology with other
fungal cytoplasmic Cu/Zn SOD, and its expression
has been studied according to culture conditions or
to the origin of the strains. Moreover, in order to
identify the reservoirs of the fungus which might be
responsible for the contamination of the patients, the
indoor environment of six CF patients followed in our
University Hospital has been investigated. For each
patient, air and surface samples have been collected
in the patient’s bedroom, in the livingroom and in his
bathroom. Likewise, water (from shower) and soil
(from the garden and from all potted plants) were
sampled. All air and water samples were negative,
and a single colony of the fungus was recovered from
one surface sample. In contrast, the fungus was
isolated, sometimes in large amount, from soil of
almost all potted plants. Moreover, RAPD typing of
4
Comparison of broth microdilution, Etest and
disk diffusion methods for susceptibility testing
of Scedosporium against licensed antifungal
agents and posaconazole
A. Velegraki,1 E. Alexopoulos1 and G. S. de Hoog2
1
Mycology Reference Laboratory, Medical School,
National University of Athens, Athens, Greece and
2
Centraalbureau voor Schimmelcultures, Utrecht,
The Netherlands
Scedosporium species, which are in part anamorphs of
Pseudallescheria, have been described as agents of
mycosis already in 1889 by Siebenmann. Histopathology findings are identical to that of aspergillosis
and consequently it is frequently identified and
treated as such. Scedosporium is notoriously refractive
to therapy and therefore detailed studies regarding
in vitro susceptibility are necessary. In this study 12
S. prolificans and 37 S. apiospermum (including 2
S. aurantiacum and 1 P. minutispor; Gilgado et al.
2005) clinical and environmental strains obtained
from Centraalbureau voor Schimmelcultures,
Utrecht, The Netherlands, were tested against posaconazole (POS; Schering Plough Research Institute,
New Jersey, USA), amphotericin B (AMB; Sigma, St
Louis, Mo., USA), Caspofungin (CAS; AS MedCare,
Lexington, HY, USA), itraconazole (ITZ; Janssen,
Beerse, Belgium) and voriconazole (VOR; Pfizer,
Sandwich, Kent, U.K) using broth microdilution
(BMD), YeastOne (YO; Trek Diagnostic Systems,
West Sussex, UK) and Etest (AB Biodisk, Solna,
Sweden). POS was also tested by disk diffusion (DD)
using commercially prepared disks at final concentration 5 lg disk–1 (OXOID, Basingstoke, UK). BMD
was performed by the NCCLS M38-A method. CAS
was not tested by YO, as the drug was not included in
the panel. For DD the adjusted inoculum was
swabbed onto the Miller Hinton 90 mm plates and
the POS disk was placed in the centre of the plate.
Plates were incubated at 35<H>0<H> C and read at
24, 48 and 72 h. The following QC isolates were run:
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Aspergillus fumigatus ATCC 2004 304, A. fumigatus
ATCC 2004 305 and Candida parapsilosis ATCC
22019. MIC range (lg ml–1) for AMB against
S. apiospermum and S. prolificans was 2->16 and 8>32 respectively, CAS 8->32 for both species, VOR
0. 06–1 (S. apiospermum) and 0.12–2 (S. prolificans),
ITZ 0.25–4 (S. apiospermum) and 1->64 (S. prolificans), POS 0.12–1 (S. apiospermum) and 0.25–4
(S. prolificans).MICs of the new species did not differ
from those of S. apiospermum. No intrazonal growth
was observed in any of the DD method trials using
POS and the inhibition zone diameters ranged from
10–30 mm. Interclass correlation coefficients (ICCs)
and 95% confidence intervals for comparing the
methods were calculated using log2 transformed
data. ICC for BMD vs. Etest, vs. YO were 0.91
(P < 10–4) for AMB, 0.62 (P = 0.036) for CAS BMD
and Etest, 0.94 (P < 10–4) for VOR BMD, Etest and
YO, 0.93 (P < 10–4) for ITZ BMD, Etest and YO,
and 0.94 (P < 10–4) for POS BMD and Etest. The ICC
for POS BMD, Etest and disk diffusion was 0.86
(P < 10–4). Disk diffusion offers an attractive alternative for rapidly testing Scedosporium isolates,
provided it is further evaluated. Standardized susceptibility testing can aid the selection of the most
relevant antifungal therapy for the management of
Scedosporium infections.
W0.5
Population dynamics in Pseudallescheria boydii:
factors affecting distribution and frequency
J. Rainer, A. Zacke, E. Lackner and J. Kaltseis
Institute of Microbiology, Leopold Franzens
University Innsbruck, Austria
Pseudallescheria boydii is an opportunistic fungus
causing subcutaneous but also cerebal infections in
humans. Among the supposed routes of infection
traumatic inoculation, inhalation, and aspiration of
contaminated water are the most prominent. Existing data on the abundance of P. boydii in the
environment, which may help to understand epidemiological questions, are scarce. Available ecological
data on strains from culture collections and publications suggest that polluted environments are important habitats. The aim of the presented experimental
work was to determine factors that could influence
the occurrence and distribution of P. boydii in the
human-impacted environment. We also tried to find
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parameters that could enhance the preponderance of
certain strains within populations. Data from experiments and systematic sampling should help to define
environments which hide higher risks for infection
with P. boydii. For the experiments strains of P. boydii
representing molecular subgroups, Scedosporium prolificans, Aureobasidium pullulans and Exophiala dermatitidis, which were originally isolated from human
infections and soil, were used. They were exposed to
different concentrations of various N- and P-sources
for 2 weeks. Competitive abilities of strains were
tested by confronting strains with each other on solid
complex media (MEA), on solid media with KNO3
and in liquid media with KNO3 and H2PO4 as
sole N- and P-sources. Quantitative sampling of
P. boydii was carried out in nature protection areas
in the Austrian Alps and in the Dutch Wadden
estuarium, as well as in urban parks, city ponds,
industrial estates and agricultural soil. The main
results are:
1. P. boydii and S. prolificans were generally more
tolerant to high KNO3 concentrations than the
other tested species.
2. Tolerance to KNO3, KH2PO4 and (NH4)2SO4
within P. boydii was strain dependent and could
not be related to the origin of the strain.
3. The extent of suppression is stronger under the
influence of high KNO3 or H2PO4 concentrations compared to non-stress conditions.
4. Species and strains strongly influence each
other if grown together, with the effect of
strong production of sexual reproduction stages
in P. boydii or barrage zones.
5. We also showed, that a strain which is tolerant
to higher levels of N or P, is not necessarily the
one which dominates a population. Factors
such as aeration also play a role for strain
selection.
6. The frequency of P. boydii in urban and
industrial areas and agricultural soil is much
higher than in habitats with low human
influence.
We conclude that high concentrations of N- and
P-compounds, which are used in agricultural fertilisation, have an impact on populations of P. boydii but
it cannot be foreseen if the selection tends towards
strains belonging to a certain molecular subgroup.
Enrichment of P. boydii in urban areas is more
probable and therefore the general risk of infection in
human-impacted environments is higher than elsewhere.
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W0.6
W1.1
The Pseudallescheria database and culture
collection
Immunological responses against yeast cell
antigen in different animal species
F. Symoens,1 D. Garcia Hermoso,2 V. Robert,3
E. Dannaoui,2 G. S. de Hoog3 and R. Horré4
1
BCCM/IHEM Culture Collection, Scientific
Institute of Public Health, Brussels, Belgium,
2
Molecular Mycology Pasteur Institute,
Paris, France, 3Centraalbureau voor
Schimmelcultures, Utrecht, The Netherlands and
4
Federal Institute for Drugs and Medical Devices,
Bonn, Germany
W. Schroedl
Institute of Bacteriology and Mycology, University of
Leipzig, Leipzig, Germany
Pseudallescheria and Scedosporium are among the very
few fungi that are truly emerging as opportunistic
agents of human disease. An analysis of 528
published cases of P. boydii infections in humans
revealed marked changes in its clinical spectrum.
Initially the fungus was observed mainly as a cause
of mycetoma after traumatic inoculation. Since the
seventies of the previous century, other disease
entities have become preponderant. Two disease
entities are particularly notable: colonization of
lungs of patients with cystic fibrosis, and the unique
fungal syndrome of delayed cerebral infection after
near-drowning. The ECMM Network on Pseudallescheria/Scedosporium Infections (PSI) unites partners
from 15 European countries and has a multidisciplinary approach of clinicians, medical microbiologists
and fundamental scientists. An infrastructure has
been implemented to collect and preserve large
numbers of clinical and environmental strains. These
are made available by IHEM (Brussels) and CBS
(Utrecht). In addition, on the basis of the BioloMICS
software, a web-accessible database is being built up
(www.Scedosporium-ECMM.com), with a number of
interesting features such as on-line identification
tools, an interactive phylogenetic tree option, a
picture gallery, and full literature data. Ample
possibilities will be provided for regular updating.
Strain-specific clinical information combined with
susceptibility and microbiological data will enable
retrospective monitoring of disease processes for
precise understanding of virulence and therapeutic
success.
6
Yeasts are a component of the microflora in the
gastrointestinal tract (GIT) and mucosa, but in
different yeast cell counts. Different factors (food,
feeding, antibiotics, stress state etc.) influence the
yeast counts in the GIT and can have an affect on the
specific humoral response against yeast components.
The antibody level (IgG, IgM, IgA) against extract
antigen of Candida albicans, C. glabrata, C. krusei,
C. tropicalis and Saccharomyces cerevisiae were determined in different animal species (horse, cattle, pig,
dog and chicken). In all investigated animal samples
antibody (Ab) levels against the different Candidaantigens could be measured. In blood samples from
horses the highest relative IgG-concentrations
against yeast antigen were measured in comparison
to the other investigated animal species, particularly
against the antigen from C. albicans, C. glabrata and
C. tropicalis. The situation was quite different with
IgM-anti-Candida where the pig showed the highest
Ab-level. Regarding IgA-against-yeast antigen there
were not obvious differences between the examined
animal species. By means of immunoblot analysis it
could be shown that between and within the animal
species similar but also different yeast components
were bound by the antibodies (based on the molecular weight). Presuming that yeast cells stimulate
continuously the humoral response on GIT-mucosa,
the aim was to check the influence of special clinical
situations like displacements or distortions in the GIT
on a stronger immunological, specific humoral
response to Candida-antigen. In cows suffering from
dislocatio abomasi it could be observed that animals
with strong increased concentration of haptoglobin
in the blood also significantly showed more Candidaspecific antibodies. Dogs with gastric torsion showed
an alteration of the Ab-level against Candida and also
a characteristic alteration of the CRP-concentrations
(a typical acute phase protein in dogs). The statistical
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
evaluation of the measured values showed a positive
correlation trend between the tumor necrosis factor
(TNF)-activity and the relative concentration of IgMagainst-Candida in dogs with stomach dislocation.
Also a positive correlation trend could be evaluated
between CRP and IgM-against Candida-extract antigen. These results are an indication for the variability
of the specific humoral immune response against
yeast as a microflora member on the GIT-mucosa.
The results speak for a continuous interaction
between the mucosal immune system and the yeast
microflora in all investigated animal species. In some
clinical situations, especially in the GIT, the measurement of antibodies against yeast components
could be important. Two or better three bloodsamples with a time shift of two to four days should
be investigated. The measurement of typical acute
phase proteins of the animal species in the same
blood samples is important for the interpretation of
the diagnostic results.
W1.2
Genetic diversity of the neurotropic black yeast
Exophiala dermatitidis
M. Sudhadham,1 G. S. de Hoog,1 A. H. G. Gerrits van
den Ende,1 M. Th. Smith,1 G. Haase2 and F. C. Odds3
1
Centraalbureau voor Schimmelcultures, Utrecht,
The Netherlands, 2Institute for Medical Microbiology,
University Hospital RWTH Aachen, Aachen, Germany
and 3Institute of Medical Sciences, University of
Aberdeen, Aberdeen, UK
Objective: The black yeast Exophiala dermatitidis is
an uncommon etiologic agent of fatal infections of
the central nervous system in otherwise healthy,
mainly adolescent patients in East Asia. The route of
infection is still a mystery. The steam bath apparently
provides a novel environmental opportunity for this
fungus, but its natural niche is still unknown. Two
preponderant ITS rDNA genotypes are known, which
might be used as markers in population dynamic
processes. It is our aim to reveal the natural niche
and to establish whether the transition to the
human-dominated environment may be accompanied by natural selection and/or evolutionary adaptation to the new habitat.
Methods: Strains were isolated by preincubation in
Raulin’s solution, and subsequently on Erythritol-
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
Chloramphenicol Agar (ECA) at 40 C. Strains were
purified with Tween 0.1%. The rDNA ITS region was
sequenced for most strains, and elongation factor 1a
for a selection of strains. Genotype-specific assays
were developed using Single-Strand Confirmation
Polymorphism (SSCP), by restriction analysis (RFLP)
and by applying selective primers. dDNA homology
was performed spectrophotometrically. Animal
experiments were performed by intravenous injection
into BALB/c mice.
Results: The species was recovered in small but
significant amounts in the faeces of fruit-eating
tropical animals, and on tropical fruits. The
human-dominated niche is known to be the public
steam bath. Genotype detection was enhanced by the
use of specific primers and SSCP. The distribution of
genotypes in environmental niches is very different
from that of intestinal and cerebral strains in
humans. Virulence of strains tested in the animal
model proved to be strain-dependent.
Conclusion: The preponderance of one ITS genotype cannot be explained by differences in invasive
potential, as virulence proved to be strain-dependent
in the animal model. The existence of two separate species rather than one was excluded by
sequencing of elongation factor 1a and by nDNA
hybridization. The phenomenon therefore must be
explained by population dynamics, such as founder
effects.
W1.3
Genetic variability among animal and human
strains of Microsporum canis
R. Sharma and Y. Gräser
Institut für Mikrobiologie und Hygiene, Berlin,
Germany
The zoophilic dermatophyte species Microsporum
canis is the causative agent of Tinea capitis et
corporis in prepubescent children. Microsporum canis
is distributed worldwide and transmitted by contact
with a range of mammals, e.g. cats, dogs and horses
where it occurs asymptomatically or causes lesions
on the furred skin. Phylogenetically closely related species are the anthropophilic dermatophytes
M. audouinii and M. ferrugineum. Our aim was the
development of molecular markers to analyse the
epidemiology and population structure of M. canis. In
7
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a recent study, 13 DNA loci have been used which
were able to separate the three species but were little
polymorphic within. This time we have used the
‘enrichment technique’ to develop microsatellite loci
which are known for their high mutation rate. A
collection of about 100 isolates from human and
animal origin have been analysed which were from
geographically distant locations (Austria, Germany,
Mexico, Turkey, Korea, Netherlands, Dominican
Republic and USA). The preliminary analysis of the
multilocus genotype of two markers containing GTrepeats [Mc(GT)17 and Mc(GT)9GC(GT)3] detected 11
alleles among the M. canis strains and a total of three
among M. audouinii and M. ferrugineum. Using the
software STRUCTURE (V2.0) genetic distances were
calculated based on allele frequencies under the noadmixture model revealing three major clusters in
which the animal isolates were equally distributed.
Interestingly, 80% of the isolates from horses
grouped together and independent of their geographic origins most of the human isolates formed
another cluster. The latter result suggests that only
single M. canis genotypes primarily from cats have
the potential to infect humans which are distributed
worldwide.
W1.4
The chytrid fungus Batrachochytrium
dendrobatidis – a threat to global amphibian
populations
T. Ohst,1 Y. Gräser,2 F. Mutschmann3 and J. Plötner1
1
Museum für Naturkunde, Institut für Systematische
Zoologie, Berlin and 2Institut für Mikrobiologie und
Hygiene, Berlin, Germany and 3Exomed, Berlin,
Germany
Batrachochytrium dendrobatidis is a fungus in the class
Chytridiomycetes. In contrast to most Chytridiomycetes, B. dendrobatidis does not occur as free-living
saprophyte but as a parasite in the skin of amphibians, invading the keratinised epidermis. In advanced
stages of chytridiomycosis typical clinical symptoms
appear including of lethargy, discoloration and
sloughing of the skin. The zoosporangia are mostly
observed in the thickened epithelium layer (stratum
corneum) just below the surface. The mortality rate
8
of infected amphibians is very high, the mechanisms
causing death are not known yet. Batrachochytrium
dendrobatidis has been implicated as an important
factor of global decline in amphibian populations.
The fungus has not only been shown to be highly
virulent in the laboratory, but was also found in
areas where catastrophic declines of wild populations
of several frog species were reported. The pathogen is
supposed to be the prime cause of the extreme
mortalities observed during declines, although alternative explanations should not be ruled out since
studies to detect B. dendrobatidis have not been at
random. Because B. dendrobatidis occurs under a
wide range of climatic conditions the species might
be composed of genetically isolated populations
adapted to more restrictive conditions. Sequences
from the ITS region of B. dendrobatidis revealed
around 10% sequence divergence comparing strains
from Australia and the Americas. The genetic
identity of some fungal isolates from different continents suggests that recent transcontinental transport
of the pathogen may occur. In Europe only one case
of mass mortality of wild toads was reported
presumably caused by chytridiomycosis. Molecular
identification of the causative agent was not applied.
The present study was undertaken to evaluate the
presence of B. dendrobatidis in European water-frogs
using molecular tools. We collected more than 650
tissue samples from water frogs (Rana sp.) captured
at 10 localities in Greece and in western Turkey,
together with specimens caught along the river
Rhine. Species-specific PCR-based tests amplifying
the ITS region were applied to detect B. dendrobatidis,
and microsatellite markers were developed for subtyping of strains. So far 357 samples have been
analysed. The chytrid was found in nine (2.5 %)
specimens from three sites. At one site in southern
Greece the fungal infection was found in 9.4% of all
examined water frogs (64 samples) without any sign
off mass die off in the frog population. Some infected
captive frogs showed no clinical signs or mortality for
a period of at least 10 months. These results suggest
that water frogs are less susceptible to the fungus
then other amphibians that were the subject of
recently publications. Subtyping of strains in this
fungal species is problematic because sequence
analysis of variable DNA markers suggests a polyploid genome structure or multi-nucleate cells of
B. dendrobatidis.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
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W3.2
M-CHiPS – a data warehouse for statistical
analysis of a microarray database’s entire
content, including e.g. experimental or clinical
annotations and GO terms
N. C. Hauser,1 C. Busold,2 S. Winter,3 J. Dippon,3
K. Fellenberg,2 J. D. Hoheisel2 and S. Rupp1
1
Genomics – Proteomics – Screening, Fraunhofer
IGB, Stuttgart, 2Funktionelle Genomanalyse,
Deutsches Krebsforschungszentrum, Heidelberg and
3
Institut für Stochastik und Anwendungen,
Universität Stuttgart
Current technologies for genome, transcriptome or
proteome analysis often result in large amount of
data. The analysis and functional interpretation of
such datasets still represents a challenging task. The
Multi-Conditional Hybridisation Intensity Processing
System (M-CHiPS) is a data warehouse, which
provides a structure suitable for statistical analysis
of a microarray database’s entire content, including
components such as the experimental and clinical
annotations, for example. The storage concept is
flexible and accounts for future developments. Currently there are 11 species represented in the system,
including human biopsies and the fungal pathogen
Candida albicans. Although these databases may
contain different ontologies of experimental and
other annotations, they share the same structure
and therefore can be accessed by the very same
statistical algorithms. For overall data analyses as
well as the identification of associations between
transcriptional variations and annotated factors,
including clinical information and GO-terms, correspondence analysis is used extensively. It is an
explorative computational method for the study of
associations between variables and proved its usefulness for identifying factors, which are associated to
certain phenotypes, for example. One major advantage of the process is its ability to present different
parameters of a multi-dimensional data matrix (e.g.
genes and experimental conditions) in a single plot.
Localisation of genes and individual experiments is
an indicator for an association between them.
Moreover, additional information, such as GO-term
or clinical annotations, can be displayed also,
permitting an immediate identification of regulated
functional groups or pathways. In addition, algorithms have been established to identify from the
annotated data the factors, which are likely to be
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
causative for the establishment of certain sub-groups
(clusters) of factors (e.g., genes or patient groups) by
a statistical data evaluation. We use the M-CHiPSystem for transcript profiling analysis for various
aspects on virulence and resistance with one focus on
host-pathogen interaction. For such analysis we
have established a genome-wide microarray containing virtually all genes of C. albicans. Within the DFG
Priority Programme ‘Colonisation and infection by
human-pathogenic fungi’ the M-CHiPS platform has
been made accessible to the network for analysis on
both transcriptional and proteome data.
S01.3
Pharmacodynamics of antifungal combinations
J. Meletiadis
National Cancer Institute, Bethesda, USA
Combination antifungal therapy is often employed to
manage difficult-to-treat fungal infections with the
premise to increase the antifungal activity, decrease
toxicity, broaden antifungal spectrum, and decrease
emergence of resistant strains. However, antifungal
combinations may be disadvantageous particularly
when a negative pharmacodynamic interaction
occurs. In order to assess in vitro pharmacodynamics
of antifungal combinations, different methodologies
have been employed such as the checkerboard broth
dilution, agar diffusion and time-kill assays where
drug effects on growth, metabolism and fungal killing
are assessed turbidimertrically, colorimetrically or
with viability assays. In vivo pharmacodynamics can
be assessed in animal models based on tissue fungal
burden, survival and other biomarkers, taking into
account pharmacokinetic, toxicological and immunological factors. A central role in the analysis of the
results obtained from in vitro and in vivo combination
studies has the zero interaction theory, based on
which a drug combination will be classified synergistic or antagonistic when its effect is greater or less,
respectively, than the expected non-interactive effect
determined from a zero interaction theory such as the
Loewe additivity (LA) or Bliss independence (BI). The
LA-based fractional inhibitory concentration index is
the most commonly used model to assess antifungal
combinations. Various other models have been
developed in order to assess statistically departures
from LA and BI such as the isobolographic analysis
of LA and the three-dimensional response-surface
9
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analysis of BI. Response-surface modeling is an
attractive approach because a mathematical equation
is fitted to the entire data and interactions can be
assessed statistically based on model parameters.
Although different methodologies and analytical tools
have been employed to assess antifungal combinations against Candida spp., Cryptococcus neoformans,
Aspergillus spp. and other filamentous fungi, additive/
independent to synergistic interactions were found for
the combination of amphotericin B plus 5-fluctytosine
or echinocandins and azoles plus echinocandins,
terbinafine or 5-flucytosine whereas a more complicate pattern of interaction may exist for amphotericin
B plus azoles with antagonism being the most potent
interaction. However, the nature and magnitude of
pharmacodynamic interactions may be strain
dependent and different at different concentrations
of antifungal drugs. Finally, pharmacodynamic synergy does not always correspond to therapeutic
synergy and pharmacodynamic antagonism is not
necessarily correlated with therapeutic failure. Therefore, pharmacodynamics of antifungal combinations
should be properly analyzed and carefully translated
in clinical settings.
S01.4
Cytochrome P450 and antifungal agents
R. Stahlmann
Institute for Clinical Pharmacology and Toxicology,
Charité – Universitätsmedizin Berlin, Campus
Benjamin Franklin, Berlin, Germany
Systemically acting antifungal agents can cause
pharmacokinetic or pharmacodynamic drug–drug
interactions by a variety of mechanisms. The systemic
azoles, such as ketoconazole, itraconazole, fluconazole
and voriconazole, differ with respect to their lipophilicity and metabolism, but all of them are inhibitors of
cytochrome P450 (CYP) isoenzymes. They exert their
fungistatic effect by inhibiting the specific fungal
CYP51A1-dependent C-14 a-demethylase (lanosterol
demethylase), the enzyme necessary for the conversion of lanosterol to ergosterol. Despite significant
differences in their affinity to fungal and human
cytochromes, the systemic azoles are also substrates
and inhibitors of human CYP isoforms, such as
CYP3A4, CYP2C9 and CYP2C19, to varying degrees.
In addition, some are substrates of the MDR-1 gene
product, P-glycoprotein (P-gp). These features are the
basis for most of the interactions occurring during
10
azole therapy, e.g. in severely ill patients in the
hospital who are treated with multiple drugs. It must
be considered that a dose adjustment of concomittantly given drugs is not only necessary when an azole
is added to the medication list of a patient, but also
when therapy with the azole is stopped. In addition it
seems noteworthy to mention that antifungal substances like fluconazole and itraconazol are used
increasingly often for the treatment of genital mycoses
and mycoses of the skin in outpatients. This has
increased the likelihood for drug interactions also in
an ambulatory setting. For evaluation of the clinical
importance of drug interactions, a specific combination of drugs can either be (a) absolutely contraindicated, (b) acceptable when accompanied by drug level
monitoring or dose adjustment or (c) have a sufficiently low drug interaction potential allowing concomitant administration without precautions.
S02.1
Gene regulation during morphogenesis in
Candida albicans
A. J. P. Brown
Aberdeen Fungal Group, School of Medical Sciences,
University of Aberdeen, Institute of Medical Sciences,
Foresterhill, Aberdeen
In response to environmental signals, Candida albicans undergoes reversible morphogenetic transitions
between yeast-like (budding), pseudohyphal and
hyphal growth forms. The regulation of morphogenesis has been the focus of attention for many groups
because morphogenesis is thought to contribute to
the virulence of this major fungal pathogen of
humans. This is probably true, and there is a
considerable body of evidence that supports this
presumption. However, I know of no experiment that
has confirmed this unambiguously. The combined
efforts of many laboratories, and an elegant combination of approaches including cell biology, molecular genetics and genomics, have helped to unravel a
complex set of signal transduction pathways that
regulate morphogenesis in C. albicans. These include
cAMP-protein kinase A, MAP kinase, and Tup1
signalling pathways. However, the specific molecular
signals that activate these pathways, and relationships between these pathways remain to be clarified.
One of the outputs of morphogenetic signalling
pathways is morphogenetic gene regulation. A small
number of hypha-specific genes have been identified,
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
most of which encode (or are thought to encode) cell
wall adhesins (such as ALS3, HWP1, HYR1). None of
these are essential for hyphal development. More
recently, Zheng et al. [2440, EMBO J. 23, 1845–56]
identified a cyclin-like gene, HGC1, which is activated specifically during hyphal development. The
significance of their work is that HGC1 is the first
hypha-specific gene that is essential for hyphal
development, and the attenuated virulence of hgc1
cells provides some of the best genetic evidence in
support of the idea that morphogenesis does contribute to the virulence of C. albicans. An interesting
observation that has arisen from relatively unbiased
genomics approaches is that morphogenetic regulators (such as Efg1, Tup1, Nrg1) do more than
regulate morphogenesis. These regulators also influence the expression of metabolic and stress genes.
Hence these regulators appear to be coordinating the
expression of virulence factors (morphogenesis and
adhesins) with fitness attributes (metabolism, stress
responses). Presumably both virulence and fitness
attributes contribute to the pathogenicity of this
fungus during disease establishment and progression.
the immune system. Fundamental insights into the
pathogenesis of fungal surface infections and the
strategy of fungal invasion of a host can be achieved by
identifying the genes of C. albicans which are expressed
during infection. We are using in vitro and ex vivo
infection models, samples from infected animals and
patient samples and genome-wide transcriptional
profiling to elucidate how the fungus responds to local
environmental changes during infection. Transcriptional regulation may not only help the fungus to
adapt to the host environment but also to actively
change the local microenvironment. By comparing
in vivo transcriptional profiling data from different
infection models we aim to elucidate (i) whether
C. albicans expresses genes unique for certain types of
infection and by comparing the transcriptional pattern from infected tissue with the pattern from in vitro
cultures determine (ii) whether C. albicans provides
genes which are expressed only during infection.
S02.2
M. A. Ghannoum
Case Western Reserve University and University
Hospitals of Cleveland, Cleveland, USA
In vivo transcriptional profiling of Candida
albicans
B. Hube,1 K. Zakikhany,1 C. Fradin,1 S. Thewes,1
M. Schaller,2 J. Naglik,3 A. Schmidt-Westhausen,4
R. Almeida1 and A. Mavor1
1
Robert Koch-Institut, Berlin, Germany, 2Department
of Dermatology, University of Tübingen, Germany,
3
Department of Dermatology and Allergology, Guy’s
Hospital, London, UK and 4Charité, Campus
Benjamin Franklin, Berlin, Germany
An intact immune system and a normal microbial
flora are generally sufficient to protect an individual
from infections with Candida albicans. However, certain critical events such as extensive antibacterial
treatment or dysfunction of the immune system may
enable this fungus to overgrow the microbial flora on
mucosal surfaces, to cause surface infections and, in
severe cases, life-threatening systemic infections.
Therefore, C. albicans is able to survive and proliferate
in radically changing environments with drastic
changes in oxygen and carbon hydrate, pH, osmolarity, availability of nutrients, or temperature. In
addition, it has to resist the constant surveillance of
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
S02.3
Biofilm formation and Candida non-albicans
yeasts
While Candida albicans is the most commonly isolated
species of Candida, other species are being isolated with
increasing frequency. Although research on the
C. albicans biofilm has been forthcoming, little is
known about fungal biofilm formed by the nonalbicans Candida species. Recent efforts compared the
ability of various Candida species to form biofilm. These
studies have shown that C. albicans isolates produce
more biofilm than Candida parapsilosis, Candida glabrata, and Candida tropicalis isolates, as determined by
dry weight assay and confocal scanning laser microscopy. Moreover, confocal laser scanning microscopic examination compared biofilms formed by
various Candida species, and showed that C. albicans
form biofilm that are more complex in architecture.
Interestingly, although Candida seems to produce
quantitatively more biofilm than non-albicans strains,
the percentage of the latter isolates to form biofilm is
much higher than those produced by C. albicans (79%
of non-albicans Candida species produced biofilm
compared with 8% of C. albicans isolates). Similar to
studies showing that significant differences exist
11
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between invasive and non-invasive Candida albicans
isolates in their ability to form biofilm, invasive
C. parapsilosis, obtained from the bloodstream are
more likely than isolates from other sites to produce
biofilm (86% vs. 47%). Of the non-albicans species,
C. parapsilosis has received the most attention with
respect to biofilm characterization. This is not surprising, since this organism is known to be associated
with the use of central line and parenteral nutrition.
Our group recently undertook genotypic and phenotypic characterization of a set of C. parapsilosis isolates
obtained from patients in an outbreak setting. In our
study, we showed that the outbreak clone produced
more biofilm than all other strains. Interesting, no
clear relationships were apparent for other putative
virulent factors (adherence, phospholipase, and proteinase production), suggesting that they are not
critical to outbreak behavior. Importantly, this study
indicated that biofilm production plays a role in C.
parapsilosis outbreak. Studies directed at understanding the biology of biofilm found by non-albicans
Candida is warranted.
Using this approach, we have observed that diverse C.
albicans biofilms have homogeneous transcript profiles that differ significantly from those of planktonic
cultures (Garcia-Sanchez et al. Euk. Cell 2004; 3:
536). Furthermore, we have identified a set of genes
that are significantly over-expressed in mature biofilms. Inactivation of several of these genes results in
alterations in the formation of biofilms by C. albicans.
The recent sequencing of the genome of the distantly
related, haploid and pathogenic species Candida
glabrata provides alternative ways to investigate
biofilm formation at a genomic scale. In particular,
we have performed a screen for insertional mutants
that have alterations in their ability to form biofilms
and shown that a Ser/Thr protein kinase controls
biofilm formation through the regulation of telomeric
silencing and expression of specific adhesions (Iraqui
et al. Mol. Microbiol. 2005; 55: 1259). Interestingly,
the orthologous kinase in C. albicans also appears as a
critical regulator of biofilm formation and hyphal
differentiation.
S03.1
S02.4
Postgenomic approaches to the study of biofilm
formation by pathogenic Candida
C. d’Enfert,1 I. Iraqui,2 S. Goyard,1 M. Chauvel,1
S. Garcia-Sanchez,1 J-M Ghigo3 and G. Janbon2
1
Unité Postulante Biologie et Pathogénicité
Fongiques, 2Unité Postulante de Mycologie
Moléculaire and 3Groupe de Génétique des Biofilms,
Institut Pasteur, Paris, France
Biofilms are three-dimensional communities of microorganisms that develop attached to a surface. They
contain an exopolymeric matrix and exhibit distinctive phenotypic properties. Candida albicans biofilms
are found in association with catheters and prosthetic
devices and are associated to mucosal candidiasis and
intestinal colonization, constituting a risk factor for
invasive candidiasis. One of the distinctive features of
C. albicans biofilms is their reduced susceptibility to an
array of antifungals. Consequently, biofilms represent
an important cause of relapses after therapy. In this
context, our aim is to decipher the molecular mechanisms that underlie the development of C. albicans
biofilms and their unique physiology. We have taken
advantage of several models of biofilms formation to
investigate the transcriptome of C. albicans biofilms.
12
What is the optimal catheter management in
patients with fungemia?
M. Nucci
University Federal, Rio de Janeiro, Brazil
The reasons for removing central venous catheters
(CVC) in patients with candidemia are: (a) the
catheter is frequently the source of candidemia;
(b) catheter removal reduces the frequency of complications; (c) catheter removal is associated with
decreased mortality. Although these arguments are
strong, the evidences supporting these assumptions
are not. Evidences for a cutaneous origin of candidemia are weak. Catheter retention is associated with
prolonged candidemia, but the clinical significance of
persistent candidemia is not well defined: in one study
in 96 children, persistent candidemia was associated
with higher rates of focal complications and renal
candidiasis, but the death rates were not statistically
different; in another study in 153 children, disseminated candidiasis was more likely to occur in patients
with persistent candidemia. Finally, the association
between catheter retention and death has been widely
reported, but most studies lacked appropriate methodology. We reviewed the literature between 1966
and 2004, and found 23 studies evaluating catheter
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
removal and survival. However, because death is
strongly associated with other important variables,
such as disseminated disease, persistence of immunosuppression and severity of illness score, we selected
only studies that performed multivariate analysis,
expressed the results in odds ratios and 95% confidence intervals, and included a validated severity of
illness score. Only eight studies fulfilled these criteria.
A strong association between catheter removal and
lower mortality was evident in only 1 study. Despite
these limitations, it seems reasonable to change CVC
in patients with candidemia, especially non-neutropenic patients.
S03.2
What does a positive Candida culture in
tracheal secretions mean?
E. Azoulay
Service de Réanimation Médicale, Hôpital
Saint-Louis, Paris, France
Candida is an opportunistic yeast normally found in
the oral cavity and gastrointestinal tract. Candida
colonization in ICU patients reflects acquired immunodepression with alterations in both neutrophil
function (killing of bacteria, production of oxidants)
and alveolar macrophage function. Studies in ICU and
surgery patients have confirmed that a continuum
exists from colonisation to infection with Candida:
colonisation is an independent risk factor for systemic
candidiasis. Candidal ‘pneumonia’ seems to exist as
two variants. One is secondary to haematogenous
dissemination with selective tropism for the blood
vessels. This is probably true invasive pulmonary
candidiasis: an exceedingly rare entity. In the other
variant, Candida colonizing the oropharynx and gastrointestinal tract spreads along the respiratory tract,
ultimately filling the alveoli, so that endobronchial
specimens are positive but no clinical or pathological
evidence of pneumonia is detectable. Many ICU
patients have pulmonary specimens containing Candida counts above the significant ‘thresholds’ validated
for distinguishing colonization from nosocomial pneumonia. Two clinical ICU studies investigated the
clinical relevance of ‘positive’ tracheal or protected
distal specimens, bronchoalveolar lavage fluid, or
bronchial or transbronchial biopsies. Both studies
included ICU patients who received mechanical ventilation for longer than 3 days and had no evidence of
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
systemic candidiasis. In most patients, lung biopsies or
lung autopsy specimens found tracheobronchial colonisation without evidence of invasive candidiasis
despite positive respiratory specimens. Thus, the usual
diagnostic criteria for nosocomial pneumonia do not
seem valid for pulmonary candidiasis. Nevertheless,
endobronchial specimens yielding Candida remain
clinically relevant: they indicate ‘relative’ immunodepression, particularly in ICU patients or after surgery
(postaggression immunodepression). In this situation,
curative (pre-emptive) antifungal treatment should be
discussed. Because a respiratory specimen yielding
Candida indicates bronchial colonization, a search for
colonisation at other sites is in order to allow
estimation of the colonization index, which is known
to correlate well with secondary emergence of systemic candidiasis. The beneficial effects of prophylactic
treatment has been suggested by several ICU studies.
One recent study has suggested that pre-emptive
antifungal treatment in patients with Candida colonization might be protective.
S03.3
What is the value of non-culture fungal
diagnosis in intensive care patients?
L. Klingspor
Mycology Unit, Division of Clinical Bacteriology,
Karolinska Institutet, Karolinska University Hospital
Huddinge, Stockholm, Sweden
Invasive fungal infections (IFI) are important causes of
morbidity and mortality in intensive care patients,
such as patients undergoing abdominal surgery,
transplant recipients, patients with hematological
malignancies, burn patients, and very low birth
weight neonates. Candida blood stream infections
(BSI) carry an attributable mortality of approximately
30–50%. Candida albicans accounts for the majority of
all such infections, but there has been a shift towards
more non-albicans spp. in BSI causing IFI. Invasive
aspergillosis (IA) in allogeneic stem cell transplant
recipients have an incidence of 4–10%, and a mortality rate of 80–90%. There is also the emergence of
other moulds, such as Scedosporium, Fusarium and
Zygomycetes spp. Although conventional diagnostic
tests, such as microscopy and culture, remain the
corner-stone of mycology diagnosis, their sensitivity
varies in different patient groups and cultures are timeconsuming. There has been some progress in the early
13
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diagnosis of IA, in recent years. This has mainly been
due to use of high-resolution CT-scanning and other
imaging procedures. The value of non-culture based
diagnostics, especially molecular methods, such as the
polymerase chain reaction (PCR), is that it offers great
promise for the rapid diagnosis of fungal infections.
Different protocols for sensitive detection of fungal
DNA by amplification with PCR (conventional PCR,
nested PCR, real-time PCR) has been established using
universal fungal PCR primers and species-specific
probes for a broad variety of fungal pathogens.
Furthermore, it can diagnose fungi that does not grow
in blood cultures such as Aspergillus spp. New methods
including real-time PCR, also allows for the online
quantification of the DNA-load. In our clinical mycology laboratory we have established an assay, using a
robot for automated extraction of fungal DNA, in a
various of clinical specimens including biopsies,
achieving high purity and speed in combination with
real-time PCR for Candida and Aspergillus spp. The
whole assay takes approximately 5–6 h to perform.
Prospective studies evaluating the potential benefits of
early therapy based on real-time PCR in patients at
high risk for invasive Candida and Aspergillus infections
are ongoing at the, Karolinska University Hospital
Huddinge. Different genetic analyses methods for long
length sequencing is helpful to rapidly identify fungi
that are slow growing and/or difficult to identify with
conventional methods. The Pyrosequencing technology is based on the principle of sequencing by
synthesis. It is a rapid and easy method for short to
medium length sequences. This method has been
developed in our laboratory for rapid typing (6 h) of
Candida, Cryptococcus, Saccharomyces and Aspergillus to
species level. Nucleic acid sequence-based amplification (NASBA) is an isothermal amplification technology which specifically amplifies RNA sequences in a
DNA background by using T7 RNA polymerase.
NSBA-based assays have been described for both
Candida and Aspergillus spp. The technique may
become a valuable tool for sensitive, specific, fast and
reliable detection of both Candida and Aspergillus RNA,
with potential for routine diagnosis, including the
possibility to test the viability of cells. PCR fingerprinting, such as arbitrarily (AP)-PCR mediated genotyping, and pulsed-field gel electrophoresis are methods
used as an epidemiological tool to investigate for
example transmission of Candida strains between
individuals in the ICU. In conclusion: the potential
value of molecular-based diagnostics in critically ill
patients in the ICU are many: (i) Rapid detection of
14
fungi compared to cultures, (ii) More sensitive; may
detect fungi that does not grow in cultures or grow
very slowly, (iii) Rapid identification to species level
compared to conventional tests. Especially for invasive
fungal infections, rapid and reliable methods to
establish a definitive diagnosis are not available to
date, although survival of the patients depends on
prompt diagnosis followed by initiation of antifungal
therapy. Fungal disease is a significant threat mainly
to immunocompromised patients seen in the transplant and cancer chemotherapy settings. In these
settings, mortality rates of up to 90% can be observed
which are mainly due to invasive fungal infections
with the yeast Candida and the mould Aspergillus.
Culture as the most important diagnostic tool lacks
sensitivity (culture is positive in <50% for invasive
Candida infections and negative in almost all cases of
invasive Aspergillus infections) and is time-consuming
(diagnosis takes up to 3 weeks). Hence, the inadequate
status quo of conventional diagnosis demands for a
reliable alternative of diagnosis in the early stage of
some mycotic and bacterial infections. The cultureindependent detection of fungal or bacterial DNA by
molecular biological techniques such as polymerase
chain reaction (PCR) promises to become an important
tool in the early diagnosis of life-threatening infections
and might raise the chance of survival of invasive
infections. In order to update the epidemiological and
mycological profile of candidaemia in Europe, the
European Confederation of Medical Mycology conducted a prospective, sequential, hospital populationbased study from September 1997 to December 1999.
A total of 2089 cases were documented by 106
institutions in seven European countries. Rates of
candidaemia ranging from 0.20 to 0.38 per 1000
admissions were reported. Candida albicans was identified in 56% of cases. Non-albicans Candida species
were most frequently isolated from patients with
haematological malignancies (65%). With increasing
age, an increasing incidence of Candida glabrata was
seen. The 30-day mortality rate was 37.9%. The
survey results underline the burden of candidaemia in
a wide range of patient populations, confirm the
importance of non-albicans species, and provide baseline data for future surveillance studies at a European
level. The DNA sequence is compared to avaible
sequences in the GenBank.
Molecular methods for epidemiologic typing of
fungal pathogens
DNA-based methods Restriction
fragment length poymorphism (RFLP)
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xxx
Puls -field gel electrophoresis
PCR fingerprinting.
Here we present an assay using the MagNa Pure LC
Instrument for fully automated extraction of Candida
DNA, achieving high purity and speed in combination
with the Light Cycler System. The whole assay takes
approximately 5–6 h to perform. This assay has a
sensitivity of 10 fg of fungal DNA, corresponding to
1–10 fungal cells per ml of blood (7). Furthermore to
asses its clinical applicability, a large number of blood
samples, and samples from other normally sterile
body fluids, from patients with suspected IFI were
analysed with real time PCR and compared to culture
assay, and /or CT-scan results. Treating these infections at an early stage is often essential for a
favourable outcome. ability of improved tests for early
diagnosis of IFI would reduce the number of patients
who are treated unnecessarily with anti-fungal drugs
and their side effects. Although conventional diagnostic tests, such as microscopy and culture, remain
the cornerstone of mycology diagnosis, their sensitivity in immuno-compromised patients are low. IA is
common in allogeneic SCT recipients, with an incidence of 4–10%. The majority of these infections are
diagnosed several months after SCT and they are
frequently associated with GVHD. The diagnosis is
difficult and often delayed. Established IA is difficult to
treat with a death rate of 80–90%. There has been
some progress in the early diagnosis of IA, in recent
years. This has mainly been due to use of highresolution CT-scanning and other imaging proce.
S04.1
Hypotheses on the natural occurrence of black
yeasts
G. S. de Hoog,1 G. Fernandez-Zeppenfeldt,2
F. Prenafeta-Boldú,3 M. Sudhadham,1 A. Nishikaku,4
C. Ruibal1 and B. Gerrits van den Ende1
1
Centraalbureau voor Schimmelcultures, Utrecht,
The Netherlands, 2National Experimental University
‘‘Francisco de Miranda’’ (UNEFM), Coro, Venezuela,
3
Laboratory of Environmental Engineering, Centre
UdL-IRTA, University of Lleida, Lleida, Spain and
4
Laboratory of Immunopatology of Mycosis,
Department of Immunology, São Paulo University,
São Paulo-SP, Brazil
Objective: Black yeasts are supposed to be opportunistic fungi. This supposition is based on the fact
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
that most species lack a pathogenic phase, transmission to new hosts is unknown, clinical pictures are
frequently dependent on the condition of the host,
and are associated with disorders of innate host
immunity or with metabolic diseases. Nevertheless
the order of the black yeasts (Chaetothyriales) has
a remarkable pathogenic potential, containing
specific agents of disease in cold-blooded vertebrates, as well as neurotropic agents in apparently
healthy humans. Our research aims to find a
common factor in the ecology of different members
of Chaetothyriales.
Methods: Strains were identified and attributed to
the order by sequence data of Small SubUnit (SSU)
and Internal Transcribed Spacer (ITS) rDNA. Goodness of 18S trees is compared by calculating the
number of trees generated in Paup when different
constraints are used. Physiological abilities focused
on assimilation of monoaromatic compounds.
Results: In addition to pathogenicity to humans
and animals, members of the Chaetothyriales were
frequently found to assimilate xenobiotic monoaromatic compounds, which may be of plant as well as
of animal origin. Extremotolerance is encountered as
a condition ancestral to the order. Constraints did
not decrease the number of possible trees found in
Paup.
Conclusion: The SSU trees were optimal when
repetitive host shifts from environmental sources
to humans and animals were allowed. This excludes
the likelihood that vertebrate pathogenicity, or
extremotolerance through the production of meristematic forms is a mainstay in the evolution of
the order. The supposition of a dual potential –
environmental vs. invasive – is potentially present in
each species. The consistent assimilation of monoaromatic compounds, which are found as precursors in
humans as well as in plants, might be a clue to
explanation.
S04.2
Zygomycosis: reemergence of an old pathogen?
C. A. Kauffman
University of Michigan and Veterans Affairs Ann
Arbor Healthcare System, Ann Arbor, MI, USA
The zygomycetes, most commonly the order Mucorales and the genera Rhizopus, Rhizomucor, and
Mucor, cause devastating infections that continue to
15
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cause high mortality. The underlying risk factors
include hematologic malignancies, neutropenia,
diabetes mellitus, solid organ (SOT) or hematopoietic
stem cell transplantation (HSCT), corticosteroids,
burn or trauma wounds, and deferoxamine chelation therapy. Rhinocerebral, pulmonary, and disseminated infection predominate; cutaneous and GI
infections occur less often. Rhinocerebral infection is
seen most often in diabetics and pulmonary infection
in those with hematologic malignancies and posttransplantation. An apparent increase in infections
with these fungi has been noted since the introduction of voriconazole, an expanded spectrum triazole
that lacks activity against the zygomycetes. Reports
from at least nine cancer centers in Europe and the
US have noted increased rates of infection with the
zygomycetes among patients with leukemia and
HSCT. Most patients were markedly immunosuppressed and many had GVHD. Voriconazole was
used for prophylaxis in the majority of cases and for
empiric or definitive treatment in the others. The
CDC TRANSNET surveillance system in 25 US
transplant centers noted an overall increase in
zygomycoses among HSCT and SOT from 2001 to
2004. A nested case–control study using cases of
invasive aspergillosis as controls showed that voriconazole exposure was highly associated with development of zygomycosis in both HSCT and SOT
patients. The zygomycetes are ubiquitous in the
environment, but point source outbreaks in hospitals
are uncommon. None of the reports noted above
have noted isolation of a single outbreak strain.
Although the association with voriconazole is
strong, the epidemiological factors that have led to
this increase in zygomycete infections have yet to be
fully elucidated. Treatment of infection with the
zygomycetes is problematic. Amphotericin B remains
the agent of choice. Lipid amphotericin B formulations are almost always used, and the dosage is
increased to 10 mg kg–1 day–1 for most patients.
Posaconazole, an expanded spectrum oral triazole
that has activity against the zygomycetes, has been
used in a salvage therapy protocol for patients
refractory to or intolerant of standard therapy with
success rates od approximately 70%. It is possible
that posaconazole will become a less toxic alternative to amphotericin B, but the efficacy of primary
therapy of zygomycosis with posaconazole is not
known at this time. Surgical debridement remains a
crucially important aspect of treatment of zygomycete infections.
16
S04.3
Pneumocystis jiroveci
E. J. Calderón (Coordinator of Eurocarinii Project)
Department of Internal Medicine, Virgen del Rocı´o
University Hospital, Seville, Spain
Pneumocystis jiroveci, previously known as Pneumocystis carinii f. sp. hominis, remains a major opportunistic infection among persons with AIDS and in
HIV-negative immunocompromised patients. The
incidence of P. jiroveci pneumonia (PcP) has
decreased in AIDS patients in developed countries
with the use of specific chemoprophylaxis and, above
all, with highly active antiretroviral therapy. In spite
of this, PcP is still an important cause of morbidity
and mortality in the western world and is a problem
that is being identified more and more in developing
countries. Nowadays, interest in P. jiroveci infection
is not only confined to AIDS patients; it also
represents a common and serious opportunistic
infection in other immunocompromised groups, such
as organ transplant recipients, patients with autoimmune diseases who receive immunosuppressive therapy, or patients with neoplasias, especially in those
with lymphoproliferative diseases. Despite the advances made in understanding human Pneumocystis
infection, many aspects about its epidemiology and
natural history remain unclear. Eurocarinii project,
an extensive epidemiological study carried out in
seven European countries, has demonstrated that in
Europe up to 40% of the patients with chronic
pulmonary diseases present P. jiroveci colonisation,
and some of these subjects have P. jiroveci strains
with DHPS gene mutations linked to sulfa-drugs
resistance. The potential role of P. jiroveci colonization in the natural course of chronic pulmonary
diseases is not currently known. The complex host–
Pneumocystis relationship is still incompletely understood. It has been shown that proliferation of
P. jiroveci is accompanied with anatomical and
physiological changes. Animal models have been
used to demonstrate changes in alveolar-capillary
permeability followed by degenerative changes in
type I pneumocytes, regenerative hypertrophy of type
II pneumocytes, and diffuse alveolar damage leading
to pulmonary fibrosis. Studies performed on individuals with PcP and AIDS have also shown changes in
the permeability of the alveolar-capillary membrane,
in the pulmonary perfusion capacity, and in total
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xxx
pulmonary and vital capacity. These changes depend
on the ability of Pneumocystis, as demonstrated in
animal models, to induce at very early stages in the
infection activation of alveolar macrophages, an
increase in proinflammatory interleukins, and changes in pulmonary surfactant even with very low
numbers of microorganisms. This ability has also
been demonstrated in humans by showing the ability
of the major surface glycoprotein (MSG) antigen of
P. jiroveci to induce production of IL-8 and monocyte
chemoattractant protein-1 (MCP-1) by human alveolar epithelial cells. Taking this into account, it is
possible that pulmonary carriage of P. jiroveci in
patients with chronic pulmonary diseases could
favour the progression of these process by means of
the mechanisms described above, which are currently considered to be involved in the pathophysiology of Chronic Obstructive Pulmonary Disease and
Pulmonary Fibrosis. The understanding of Pneumocystis infection mechanisms is a crucial clinical and
epidemiological challenge, since source of the infection in humans and reservoir have not yet been
established. The infection can be transmitted by
airborne route and cross infection experiments have
shown that Pneumocystis from a given mammal
cannot infect other host species. Pneumocystis jiroveci
could behave in a similar manner, and humans could
constitute therefore the reservoir for this species.
Several studies from European countries had suggested transmission person-to-person. Besides, an
experimental animal model carried out into Eurocarinii project has showed that Pneumocystis organisms
are able for replicating in the lung alveolus of the
immunocompetent host, keeping intact their infectious power, which suggests that patients with
chronic pulmonary diseases colonized by this pathogen could play a role of reservoir and source of
infection in human populations. In this sense,
patients with chronic pulmonary diseases who are
colonized by P. jiroveci could represent a reservoir
and, through habitual expectoration, a source of
infection for immunocompromised individuals who
are susceptible to pneumonias caused by this pathogen. If proven, this would be of particular importance
in hospitals, where it is relatively common to find
subjects with chronic pulmonary diseases and
immunocompromised patients hospitalized in the
same areas. Moreover, if mutations in the DHPS
gene exist in the strains carried by subjects with
chronic pulmonary diseases, this may lead to a worse
prognosis for patients who become infected with
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
these strains. In conclusion, there is a high prevalence of patients with chronic pulmonary diseases
who are P. jiroveci carriers who could potentially act
as sources of infection for susceptible individuals.
This finding could represent an important public
health problem and merits the development of
further research to assess the true scope of the
problem and to design rational preventative strategies if necessary. Moreover, it is also necessary to
elucidate the role of P. jiroveci infection in the natural
history of chronic pulmonary diseases in order to
improve the clinical management of these diseases.
S05.1
Tryptophan-dependent metabolites of
Malassezia furfur: From chemistry to skin
disease
P. Mayser
Center of dermatology, Justus Liebig University,
Giessen, Germany
Recently we have shown that Malassezia furfur can
produce pigments and fluorochromes if tryptophan is
offered as the main nitrogen source. This newly
discovered metabolic pathway may explain clinical
phenomena of pityriasis versicolor (PV), a common
Malassezia-related skin disease. To date, 18 compounds have been isolated, of which 14 are new in
terms of chemical structure. They have interesting
pharmacological properties, such as UV protection
(pityriacitrin, pityrialacton), stimulus-specific inhibition of the oxidative burst of granulocytes and
IL12p70 release of dendritic cells (pityriarubins),
induction of apoptosis in human melanocytes and
activation of the AHR arylhydrocarbon receptor as
well as induction of cytochrome P450 in keratinocytes
(malassezin), properties that are closely associated
with clinical signs of PV. As it has not been impossible
so far to demonstrate these compounds in skin
scrapings from patients with pityriasis versicolor
because of scant material with high degree of contamination the subtraction technology according to
Diatchenko was used for detection of pigment-associated genes, which does not need knowledge of
complete sequences. Sixty-eight of 600 screened
sequences showed differential expression and some of
these genes are coding enzymes which are responsible
for steps in the above mentioned pigment synthesis.
17
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Interestingly some smut fungi phylogenetically related
to M. furfur are also able to share the same indol
pathway, which was detected by us in M. furfur. In
Ustilago maydis until now we have detected malassezia
carbazole A, malassezia indole A, pityriacitrin, pityria
anhydride and pityriarubin B. As the genome of
U. maydis is nearly totally sequenced and partly
annotated we have a second promising way to find
the genes of pigment synthesis. The identification of
their transcripts in lesional skin of pityriasis versicolor
will help us to get further insights into the pathogenesis of this common skin disease. Furthermore we were
able to detect this new pathway in ascomycetic yeasts
for example C. glabrata. At the moment about 50
fungal species of different genera are able to produce
these indolic pigments with some variations. Once we
have identified pigment-synthesis-associated genes we
can look for the evolution of these genes and on the
other hand, by knock out mutants, we can determine
whether these are factors of pathogenicity in human,
animal or plant disease.
S05.2
Tinea faciei – an often misdiagnosed facial
eruption
J. C. Szepietowski
Department of Dermatology, Wroclaw Medical
University, Wroclaw and Department of
Dermatology, Venereology and Allergology,
University of Medicine, Wroclaw, Poland
Tinea faciei is a relatively uncommon and rarely
discussed superficial dermatophyte infection. It
occurs on the non-bearded regions of the face. Tinea
faciei accounts for 3–4% of the cases of tinea
corporis, however in pediatric population it has been
recently shown to constitute slightly <19% of all
superficial fungal infections. Tinea faciei may appear
in persons of any age, but has two peaks. One peak
involves children, often due to direct contact with
pets. The other peak occurs in those aged 20–40
years. This may be due the heightened physical
activity common in this age group. Recent study
indicates the female : male ratio as 1.06 : 1, however some authors consider tinea faciei more common in women. The causative agent of tinea faciei
varies by geographic region and the potential
reservoirs located in that environment. In the US,
Trichophytan tonsurans, T. rubrum, and Microsporum
18
canis are the most common causative species.
Trichophytan mentagrophytes and T. rubrum were the
most common in some European countries. It is often
confused with other dermatoses since fungal infections occur more frequently on other parts of the
body. Atypical features are more common in tinea
faciei than other forms of ringworm, possibly due to
the complex anatomy of the face. Therefore, tinea
faciei has been known to mimic several dermatologic
disorders, including contact dermatitis, rosacea,
seborrheic dermatosis, bacterial infections and others. As many as 70% of patients with tinea faciei are
initially misdiagnosed as having other dermatoses.
For this reason, diagnosis and treatment of tinea
faciei are often delayed.
S05.3
Differential drug treatment for tinea capitis:
griseofulvin or not?
G. Ginter-Hanselmayer
Department of Dermatology, Medical School Graz,
Austria
Tinea capitis, is primarily a disease of preadolescent
children with increasing incidence in the US as well as
in European countries, mainly in urban areas. The
causative agent varies according to the geographical
location with Trichophyton tonsurans accounting for
the majority of infections in North America and
Microsporum canis in Europe. As a dermatophyte
infection involving the hair shaft on the scalp systemic
antifungal treatment is required to achieve cure. Since
more than four decades griseofulvin is considered to be
the drug of choice for tinea capitis. It is FDA approved
for this indication, highly efficacious with an excellent
long-term safety record, and is not expensive. Accompanying the overall increase in the incidence and the
changing epidemiology of tinea capitis is the suggestion of a decrease in the sensitivity of scalp infections to
griseofulvin. This development has been followed by
recommendations for increase of both dosing and
duration with this drug in the last decade. Currently
most experts use 20 mg kg–1 daily (microsized) for
8 weeks for the treatment of tinea capitis in children.
Microsporum species appear to be more sensitive to
griseofulvin than are Trichophyton species. The newer
antifungals like terbinafine, itraconazole and fluconazole are with a few exception not yet FDA approved
although several studies and meanwhile a more than
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xxx
10 years experience proof them to be safe and
efficacious. Like in adults with these drugs – owing
to their longer half-lives and pharmacokinetic property to persist in the hair follicle for several weeks after
discontinuation of therapy – cure can be achieved by
shorter and more convenient dosing regimens. Terbinafine dosing is usually based on weight (children
10–20 kg require 62.5 mg day–1; 20–40 kg –
125 mg day–1 and >40 kg – 250 mg/day–1), with
the duration of therapy between 2 and 4 weeks. When
the causative organism is M. canis, terbinafine may
need to be administered for a longer time period or in a
higher dosage. Itraconazole and Fluconazole are
available as capsules and in suspension form and
may although be used with short courses of therapy
including continuing-, pulse- and intermittent dosing
regimens. The most common daily dosing regimen of
itraconazole in the literature is 5 mg kg–1 or
100 mg day–1, with duration varying between 2 and
6 weeks, depending from the etiologic pathogen and
the clinical course. In tinea capitis caused by Trichophyton-spp. treatment duration of 2 weeks is sometimes sufficient. With 6 weeks itraconazole treatment
long-term results for children with tinea capitis caused
by Microsporum canis were nearly identical compared
to 6 weeks of griseofulvin. Problems with administration of itraconazole to small children may be the
capsule formulation as well as the content of cyclodextrine in the liquid formulation, which may cause
diarrhea in children. For fluconazole currently there is
no standard dosing regimen. Compared to endothrix
tinea capitis for M. canis scalp infection fluconazole
treatment has to be given for a longer duration. When
fluconazole is used as an oral suspension, the reconstituted solution should be used within 2 weeks in
order to ensure optimal stability.
Conclusion:
Griseofulvin has been the gold-standard treatment of
tinea capitis for the past decades; however, patient
compliance poses a challenge for a number of
reasons like the long treatment duration, the development of drug-resistant dermatophyte strains and
adverse events with this drug. Short term terbinafine,
itraconazole and fluconazole therapy have been
shown to be comparable in efficacy and safety with
griseofulvin, but are more expensive and with a few
exceptions not FDA-approved in this indication. To
our opinion they may serve as alternative medications for cases in which griseofulvin has failed or as
primary therapy. However, the varying susceptibility
with regard to the causative dermatophyte and the
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
clinical presentation have to be taken into account
regarding the selection and treatment modalities
with these new oral antimycotics. As griseofulvin
alternatives they should be licensed for children.
S06.1
Epidemiology and clinical importance of in vitro
resistance in fungi
J. L. Rodriguez Tudela
Servicio de Micologı´a, Centro Nacional de
Microbiologı´a, Instituto de Salud Carlos III,
Majadahonda, Espagna
Since 1997, there are available standardized
methodologies for detecting antifungal resistance of
human pathogenic fungi. CLSI was the first organization devoted to create antifungal susceptibility
standards. Thus, two standards, for yeasts and for
filamentous fungi can be used for establishing the
susceptibility or resistance to antifungal drugs. Later,
but with great enthusiasm, Antifungal Susceptibility
Testing Subcommittee of European Committee on
Antibiotic Susceptibility Testing has produced their
own European Standards. One of them that dedicated
to detect the resistance to antifungals in yeasts able
to ferment glucose, is already available (Edis 7.1). In
addition, the standard devoted to detect the resistance to antifungals in Aspergillus is in the final phase.
With those tools, many epidemiological studies have
been performed in order to ascertain the rate of
resistance for antifungals of clinical use in human
fungal pathogens. The most important and valuable
studies are those based in population active surveillance. However, for low incidence fungal infections,
active surveillance is complex because many centers
need to be involved together with a long period of
study. In those cases, data obtained from passive
surveillance are, frequently, the only available. The
data obtained from these studies identify those
strains, species or genera that have high MICs to
antifungal drugs. The comparison, in time, of
surveillance studies indicate if species are developing
secondary resistance or maintain the primary susceptibility pattern. In a broad sense, the data
obtained from the studies is very important because
identify species with intrinsic resistance to antifungals and also the species that have a tendency to
develop secondary resistance. In addition, if the study
19
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is population based, the data reflects risk factors and
the real rate of resistance and therefore public health
interventions can be delineated and promoted.
Lastly, the isolates showing high MICs can be
analysed in order to identify mechanism of resistance
in comparison with those strains with low MICs. The
last phase, and very important, is to identify the
clinical value of resistant strains. In other words, if
the results obtained with antifungal susceptibility
testing could predict the outcome of fungal infections. The answer is not easy; MIC value is a variable
that need to be in context and the circumstances in
fungal infections are very complicated to have a
simple solution. However, there are many data in the
literature showing that microorganisms with high
MICs are more related with clinical failures than
those with low MICs (the outstanding 90–60 rule).
S06.4
Biofilms and antifungal drug resistance
L. J. Douglas
Division of Infection and Immunity, University of
Glasgow, Glasgow, UK
Biofilms are structured microbial communities that
are attached to a surface and encased within a matrix
of extracellular polymers. Individual biofilm organisms characteristically display a phenotype markedly
different from that of planktonic (free-floating) cells;
most importantly, they are significantly less susceptible to antimicrobial agents. Recent estimates suggest
that a substantial proportion of human infections
involve biofilms. Many of these are implant-related
infections in which adherent microbial populations
are found on the surfaces of devices such as catheters,
prosthetic heart valves and joint replacements.
Because of the drug resistance of biofilm organisms,
and the ability of biofilms to withstand host immune
defences, these infections are usually extremely
difficult to treat. Most fungal biofilm research to date
has concentrated on Candida albicans and related
Candida species. Resistance of Candida biofilms in vitro
to clinically important antifungal agents such as
amphotericin B, fluconazole, flucytosine and itraconazole was first demonstrated in 1995 and has been
subsequently confirmed by several research groups
using a variety of biofilm model systems. Newer azoles
(voriconazole and ravuconazole) are also ineffective
against biofilms. The molecular basis of this pheno-
20
typic drug resistance is not understood and is likely to
be multifaceted. Possible mechanisms include (i)
restricted penetration of drugs through the biofilm
matrix; (ii) phenotypic changes resulting from a
decreased growth rate or nutrient limitation; and
(iii) expression of resistance genes induced by contact
with a surface. The biofilm matrix does not appear to
constitute a major barrier to drug penetration since
C. albicans biofilms with little or no matrix material
are just as resistant as those with an extensive matrix.
Moreover, high levels of drug diffusion through
biofilms fail to produce complete killing of biofilm
cells. Similarly, drug resistance is not simply attributable to a low growth rate because biofilm organisms
grown in a perfused biofilm fermenter are resistant
over a range of growth rates. Upregulation of genes
coding for multidrug efflux pumps would result in a
multidrug-resistant phenotype. To date, evidence for
this as a resistance mechanism is equivocal.
Although genes encoding efflux pumps appear to be
upregulated during biofilm formation, mutants carrying deletions in these genes are susceptible to
antifungal agents when growing planktonically but
retain their resistant phenotype during biofilm
growth. There is, however, evidence that mature
biofilm cells contain decreased levels of ergosterol, a
factor which might contribute to their drug resistance. Some newer antifungal agents, such as the
echinocandins (caspofungin and micafungin) and
liposomal formulations of amphotericin B, show
activity against Candida biofilms. Aspirin and other
non-steroidal anti-inflammatory drugs, which inhibit
fungal prostaglandin synthesis, also display antibiofilm activity in vitro. Finally, inhibitors of quorum
sensing, a cell–cell signalling mechanism, may be
effective against biofilms as has already been demonstrated for bacteria. In C. albicans two quorum sensing
signal molecules have been identified: farnesol, which
acts as a negative signal and inhibits the formation of
hyphae, and tyrosol which acts as a positive signal
and promotes hyphal formation. Both of these
molecules are likely to be involved in biofilm development and maturation; compounds which antagonize their action could therefore represent novel
antifungal/antibiofilm agents.
References
Alem MAS, Douglas LJ. Effects of aspirin and other nonsteroidal
anti-inflammatory drugs on biofilms and planktonic cells of
Candida albicans. Antimicrob Agents Chemother 2004; 48: 41–7.
Al-Fattani MA, Douglas LJ. Penetration of Candida biofilms by
antifungal agents. Antimicrob Agents Chemother 2004; 48: 3291–7.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
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Baillie GS, Douglas LJ. Candida biofilms and their susceptibility to
antifungal agents. Methods Enzymol 1999; 310: 644–56.
Douglas LJ. Candida biofilms and their role in infection. Trends
Microbiol 2003; 11: 30–6.
Mukherjee PK, Chandra J, Kuhn DM, Ghannoum MA. Mechanism of fluconazole resistance in Candida albicans biofilms: phasespecific role of efflux pumps and membrane sterols. Infect Immun
2003; 71: 4333–40.
Ramage G, Bachmann S, Patterson TF, Wickes BL, Lopez-Ribot JL.
Investigation of multidrug efflux pumps in relation to fluconazole
resistance in Candida albicans biofilms. J Antimicrob Chemother
2002; 49: 973–80.
S07.3
Recognition of fungal pathogens by Toll-like
receptors
M. G. Netea, J. W. M. Van der Meer and
B.-J. Kullberg
Department of Medicine, Radboud University
Medical Center, Nijmegen, The Netherlands, and
Nijmegen University Center for Infectious Diseases,
The Netherlands
Toll-like receptors (TLRs) have been identified as a
major class of pattern-recognition receptors. Recognition of pathogen-associated molecular patterns
(PAMPs) by TLRs, either alone or in heterodimerization with other TLR or non-TLR receptors, induces
signals responsible for the activation of innate
immune response. Recent studies have demonstrated
a crucial involvement of TLRs in the recognition of
fungal pathogens such as Candida albicans, Aspergillus
fumigatus and Cryptococcus neoformans. By studying
fungal infection in knock-out mice deficient in either
TLRs or TLR-associated adaptor molecules, it
appeared that specific TLRs such as TLR2 and
TLR4 play differential roles in the activation of the
various arms of the innate immune response. In
addition, interaction between TLRs and non-TLR
receptors such as the lectin-like receptors dectin-1,
mannose receptor, and DC-SIGN, are crucial for the
effective activation of the normal host defense.
Recent data also suggest that TLRs offer escape
mechanisms to certain pathogenic microorganisms,
especially through TLR2-driven induction of antiinflamatory cytokines. These new data have substantially increased our knowledge of the recognition of
fungal pathogens, and this remains one of the most
active area of research in the field of fungal
infections.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
S07.4
Role of complement in invasive fungal
infections
R. Würzner
Department of Hygiene, Microbiology and Social
Medicine, Innsbruck Medical University, Innsbruck,
Austria
Fungi, as most other pathogens invading the human
body, are attacked by the host immune system
directly following entry and usually also during
consecutive stages of disease, especially when they
are in contact with blood. Complement represents a
central part of innate immunity, facilitating chemotaxis, opsonisation and lysis, and is also a fine tuner
of adaptive immunity in the pathogenesis of invasive
fungal infections, such as aspergillosis, candidiasis,
cryptococcosis, paracoccidioidomycosis, blastomycosis and histoplasmosis. In order to escape detection
and evade destruction by complement, fungi have
developed an effective battery of specific strategies,
like other micro-organisms. As the innate immune
system forms the most important part of the immune
defence against fungi – evident via the high proportion of fatal fungal infections in neutropenic patients
– evading the interplay between complement and the
phagocytic system plays a major role for fungi in
order to survive the hostile environment within the
host. The measures to avoid recognition by complement and eradication (phagocytosis rather than
lysis) involves several fungal molecules which can
be considered as virulence factors and which are
secreted into the near vicinity or expressed on the
fungal cell surface. Of all these highly sophisticated
mechanisms employment of complement or its
imitation (molecular mimicry) appear to be the most
evolutionary elaborated.
S08.1
How can we eradicate fungi from foods?
J. P. Gangneux
Laboratoire de Parasitologie-Mycologie, Faculté de
Médecine de Rennes- Centre Hospitalier Universitaire
Pontchaillou, Rennes, France
Fungal growth in foods is generally responsible for
food spoilage and sometimes for foodborne illness.
Foodborne fungal hazards are known to cause
21
xxx
considerable morbidity and mortality, but are much
rarely investigated or even mentioned compared to
foodborne diseases due to bacteria, parasites and
viruses1. Fungal proliferation in the digestive tract
can lead to systemic infections like invasive aspergillosis, particularly in severely immunosuppressed
patients. Fungemia has been reported after ingestion
of probiotics such as Saccharomyces boulardii, even in
immunocompetent patients. Ingestion of moldy foods
can lead to poisoning by mycotoxins such as
aflatoxin, ochratoxin and fumonisin. It is thus
surprising that very few foodborne disease surveillance programs cover these fungal risks and there is a
need to improve our knowledge of fungal involvement in food-related illness and death, and to develop
epidemiologic studies and preventive measures2, 3. In
sterile hospital units, it is of prime importance to
prevent nosocomial infections acquired by ingestion
and/or inhalation of microorganisms. Sterile diets or
diets with low microbial content have been recommended for immunocompromised patients for several
years. One safe recommendation is to avoid consumption of contaminated foods that cannot be
sterilized. Remington and Schimpff recommended in
1981 to eliminate vegetable salads from the diets of
granulocytopenic patients because previous studies
recovered considerable counts of Pseudomonas aeruginosa and Enterobacteriaceae in numerous samples4.
Filamentous fungi and yeasts are also frequently
present on food5–7. Massive Aspergillus contamination was first reported in pepper, leading to ban
black-pepper sachets from food-trays served in
Hematology Units. Nuts, beans, smoked meat,
cheese, regular and herbal tea, fruits and even
freeze-dried foods can be a potential source of fungal
exposure. However, food restriction may affect the
patient’s well-being. One alternative is to sterilize
foods and dishes for at risk patients, but no consensual protocol has been adopted yet. Food wellcooking is generally recognized as an efficient
method8. Besides, little is known on the susceptibility
of filamentous fungi to biocides. Using an experimental model of contamination of fluid and surfaces,
we examined the efficacy of several physical and
chemical procedures that can be applied on foods and
wrappings for the eradication of A. fumigatus contamination. These procedures were then assessed on
naturally or artificially contaminated foods. Results
showed that exposure to a temperature of at least
100 C using oven heating or microwave irradiation
is necessary to eradicate A. fumigatus spores from
22
foods. For foods that cannot be exposed to high
temperature or microwave, 70% ethanol only partially reduces the level of surface contamination9.
More anecdotal physical and chemical procedures
affecting fungal growth have been reported in vitro:
gamma irradiation, spice essential oils, oils extracted
from some medicinal plants, extracts of garlic, leek,
chive, or onion or schallot bulbs for example10–11.
The use of such methods for the prevention of
foodborne illness in susceptible individuals has still to
be evaluated. Nevertheless, whatever the process
applied to sterilize foods at the end of the cooked
dishes chain, toxins already present may not be
inactivated. Then the risk of invasive fungal infection
in a limited population of cancer patients due to
fungal growth shifts to the deterioration of liver and
kidney function by mycotoxins in the whole population.
References
1. Adak GK et al. Emerg Infect Dis. 2005; 11: 365–72.
2. Dalton CB et al. Commun Dis Intell 2004; 28: 211–24.
3. Majkowski J. Emerg Infect Dis 1997; 3: 551–4.
4. Remington JS, Schimpff SC. N Engl J Med 1981; 304: 433–5.
5. Vargas et al. Lancet 1990; 336: 881.
6. Nolard N. Path Biol 1994; 42:706–10.
7. Bouakline et al. J Clin Microbiol 2000; 38: 4272–3.
8. Vermorel-Faure et al. Presse Med 1993; 22: 157–60.
9. Gangneux et al. Blood. 2004; 104: 2000–2.
10. Tsao SM, Yin MC. J Med Microbiol 2001; 50: 646–9.
11. Soliman KM, Badeaa RI. Food Chem Toxicol 2002; 40: 1669–75.
S08.2
Presence of mycotoxins in food
B. Andersen
Center for Microbial Biotechnology, BioCentrumDTU, Technical University of Denmark, Lyngby,
Denmark
Mycotoxins are defined as ‘natural products from
moulds which initiate a toxic response in vertebrates, when introduced in small concentrations by
a natural orifice, i.e. the mouth, the respiratory
system or the skin’ (Gravesen et al. Microfungi,
1994). Certain food products, such as cereals, fruit
and vegetables, are more susceptible to fungal
spoilage than bacterial spoilage. This is because of
their lower water activity and lower pH compared
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
to food products of animal origin. Since cereals,
fruit and vegetables have specific properties, each
product type is spoiled by specific fungi, which, in
turn, produce specific mycotoxins. Therefore, different mouldy products contain different mycotoxins.
As an example, apples are often spoiled by the
patulin-producing fungus Penicillium expansum. Patulin is one of the few mycotoxins which are
regulated by legislation and products like apple
juice and apple sauce are analysed regularly for
patulin. However, P. expansum also produces a
variety of other metabolites in apples (citrinin and
roquefortine C) that may be ingested, even if
patulin levels are below the legal limit or not
produced at all. Tomatoes, on the other hand, are
spoiled by P. tularense, which produces other
metabolites (janthitrems, paxillins, roquefortine C)
not covered by legislation. Tomato juice or paste
might therefore get a ‘clean bill of health’ and still
contain mycotoxins. Other fungal species, such as
Alternaria tenuissima and Stemphylium eturmiunum,
may also participate in the spoilage of apples and
tomatoes, but biologically active metabolites (macrosporin, stemphol, tentoxin) from these fungi are
not even registered by the authorities. A part of
our intake of cereals, fruit and vegetables consists
of fresh produce, where mouldy items are easily
discovered and discarded by the consumer. Another
larger part consists of processed produce such as
beer, bread, breakfast cereals, coffee, ketchup, jams,
juices, purees, wine etc. Mouldy raw materials or
faulty production hygiene can result in mycotoxin
contamination of the finished product, because
mycotoxins can withstand most of the processes
in the food industry. Barley grain, grapes and
coffee beans are often spoiled by P. verrucosum,
Aspergillus carbonarius and A. ochraceus, respectively,
resulting in the formation of ochratoxin A. It has
been shown that this mycotoxin survives processing and can be found in beer, wine and coffee,
which are enjoyed in the large quantities in the
northern hemisphere. With the exception of aflatoxins (acute liver damage, carcinogenic), ochratoxin A (nephrotoxic, teratogenic and reproductive
effects) and patulin (gastrointestinal hyperaemia,
distension haemorrhage and ulceration), little is
known about the toxicity of other mycotoxins in
mammals. Animal experiments show that mycotoxins can have a negative biological activity and
that additive and synergistic effects may occur
when two or more metabolites are ingested at the
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
same time. In vitro experiments have shown that
the effect of patulin and roquefortine C is additive
and the effect of ochratoxin A plus either citrinin
or fumonisin B is synergistic. Hundreds of biologically active metabolites are known from fungi,
but little is known on their toxicity and even less
on their presence in food.
S08.4
Structural and clinical aspects of fungal
allergens
R. Crameri
Swiss Institute of Allergy and Asthma Research
(SIAF), Davos, Switzerland
For many years fungi have been recognised as
potential causes of allergy. Moulds, in particular
Aspergillus fumigatus, Alternaria alternata and Cladosporium herbarum, basidiomycetes, and yeasts, particularly of the species Malassezia, have been shown
to be involved in the pathogenesis of respiratory
allergies and atopic eczema, respectively. However,
since the prevalence of fungal allergy is not known
indirect methods have to be used to estimate the size
of the problem. Most cases of fungal allergy are
associated with severe asthma or in the case of
sensitisation to yeasts to atopic dermatitis. In the
USA around 16 millions of self-reported cases of
asthma have been recorded in 2002. Depending on
the definition about 10–20% of these patients might
be classified as subjects suffering from severe asthma
and in this group 30–70% can be expected to be
sensitised to at least one fungal species. Extrapolating this figure to the industrialised countries we
have to assume that several millions of asthmatic
patients are affected by fungal allergy. However,
with exception of special cases like workplace
exposure or ABPA which are well documented,
the contribution of fungal sensitisation to the
severity of asthma remains to be investigated.
A. fumigatus is the only mould which, according to
the literature, is involved in a well documented
broad variety of pulmonary complications ranging
from benign colonisation of the lung to life-threatening disease like invasive aspergillosis or ABPA.
However, also in the case of A. fumigatus large
variations in the incidence of A. fumigatus sensitisation and ABPA have been reported and are mainly
due to the lack of reliable extracts. Since most of the
23
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clinical criteria used for the diagnosis of ABPA are
not specific for the disease, serologic findings
showing sensitisation to A. fumigatus are essential
to confirm or exclude an ABPA suspected from
clinical signs. ABPA is a disease with a high risk for
the development of irreversible end stage fibrosis.
Rapid cloning of allergen-encoding cDNAs by phage
surface display and high-density arrays allowed
elucidating and comparing the repertoire of allergens expressed by different fungal species1. All
fungal allergen repertoires investigated to date are
complex and each one contains more than 25
different IgE-binding proteins. Interestingly the
allergens can be classified as species specific allergen
representing secreted proteins like the ribotoxin Asp
f 1 or the major allergen of A. alternata Alt a 1, and
in cross-reactive allergens. Cross-reactive structures
represent mainly phylogenetically highly conserved
intracellular proteins like manganese-dependant
superoxide dismutase (MnSOD), cyclophilin and
thioredoxin, among others. The three examples
cited share also a high degree of sequence identity
and homology with the corresponding human
MnSOD, cyclophilin and thioredoxin enzymes. Interestingly serum IgE to the human proteins as well as
T-cell reactivity can be detected in vitro, indicating a
possible implication of B- and T-cell mediated
autoreactivity in severe atopic diseases. The biological relevance of these reactions has been demonstrated in vivo by the ability of the human proteins
to elicit Type I skin reactions upon challenge in
sensitized patients, as well as by the ability of
human MnSOD to elicit eczematous reactions in
Atopy Patch Tests in patients suffering from severe
atopic eczema2. We have now solved the crystal
structures of A. fumigatus MnSOD and cyclophilin,
as well as the structures of M. sympodialis cyclophilin and thioredoxin. These structures, together with
the already solved structures of the homologous
human proteins allow exact comparison of conserved residues between pairs of self/non-self antigen to figure out surface regions potentially forming
cross-reactive B cell epitopes. Obviously only homologous, surface exposed amino acids can account for
cross-reactivity at humoral level. This work allowed
us to understand how cross-reactivity between
environmental allergens and homologous self antigens, which would be expected to induce self
tolerance, can occur. However, mutational analysis
of the common residues will be required to prove
that the structure-derived prediction of B cell
24
epitopes really correspond to functionally active B
cell epitopes.
Acknowledgements: This work was supported by
the Swiss National Science Foundation and by the
OPO-Stiftung, Zürich.
References
1. Crameri R, Kodzius R, Konthur Z, Lehrach H, Blaser K, Walter
G. Tapping allergen repertoires by advanced cloning technologies. Int Arch Allergy Immunol 2001; 1124: 43–7.
2. Schmid-Grendelmeier P, Flückiger S, Disch R, Trautmann A,
Wüthrich B, Blaser K, Scheynius A, Crameri R. IgE-mediated
and T cell-mediated autoimmuinity against manganese superoxide dismutase in atopic dermatitis. J Allergy Clin Immunol
2005; 115: 1068–75.
S09.2
CNS pharmacokinetics of systemic antifungal
agents
M. Cavling Arendrup
Unit of Mycology and Parasitology, Statens Serum
Institut, Copenhagen, Denmark
Fungal infections in the central nervous system
(CNS) are on the rise due to an increasing number of
patients with predisposing diseases, and yeasts,
moulds as well as dimorphic fungi may be involved.
Although the knowledge of susceptibility of these
fungi to the various antifungal compounds is now
available and helpful in guiding the clinician choosing among the different antifungal compounds, the
unique CNS pharmacokinetics influence the availability of the drug at this site of infection. The
penetration of antifungal agents into the CSF and
CNS is dependent on molecular size and ionisation,
protein binding, lipid solubility, capillary and choroid
plexus efflux pumps, and the degree of inflammation.
The systemic antifungals, which include conventional amphotericin B deoxycholate and various lipid
associated formulations hereof, azoles including
fluconazole, itraconazole and voriconazole, caspofungin and flucytosine, exhibit major differences with
respect to these parameters. A summary of pharmacokinetic parameters of antifungal compounds is
shown in the table below.
Conventional amphotericin B (AmB) and the three
lipid associated formulations vary strikingly in
pharmacokinetic parameters. Thus, the peak
serum concentration (Cmax), which is the pharmacodynamic parameter most strongly associated with
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
effect, ranges from 1.7 lg ml–1 for ABLC over 2.9
and 3.6 lg ml–1 for ABCD and AmB to 29 lg ml–1
for L-AmB, respectively. These differences compare
with similar differences in volume of distribution,
and agree with the findings in animal models that
brain tissue concentrations were significantly higher
in animals receiving L-AmB and resulted in a more
efficient fungal clearance (Groll et al. JID 2000).
Similarly, a comparative study of L-AmB and AmB in
patients with cryptococcal meningitis demonstrated
significantly earlier clearance of the CSF in patients
receiving L-AmB (Leenders et al. AIDS 1997).
Flucytosine shows excellent penetration into the
CSF. It exhibits a short T½ and efficacy is T/MIC
dependent, why flucytosine is dosed four times daily.
It is given only as part of combination therapy as
resistance emerges if given as monotherapy. Among
the azoles flu- and voriconazole penetrates well into
the CSF while the amount of itraconazole, although
lipophilic that reaches the CSF is minimal. This is in
part due to its 99% protein binding and 0.5%
erythrocyte binding, but probably also in part due to
P-glycoprotein efflux-transporter-protein activity of
the endothelial capillaries and parenchyma (Imbert
et al. Drug Metabolism and Disposition 2003). In
animal models caspofungin has been shown to
penetrate very poorly into the CSF, probably as a
consequence of the high protein binding and the
poor diffusion of large molecules through the blood
brain barrier. In an animal model, caspo- and
micafungin failed to reduce the CNS fungal burden
significantly, in contrast to various formulations of
amphotericin B (Clemons et al. Abstract ECCMID
2004). However, case reports have been published
reporting successful outcome of cerebral aspergillosis
and candida meningitis and further studies are
needed to clarify this.
Drug
MW T½
CSF/serum
Protein
binding PD parameter# ratio
Amphotericin B 924 15 days
>90%
Fluconazole
306 25–30 h
12%
Itraconazole
705 22 h
99%
Voriconazole
349 6 h
58%
Caspofungin
1093 9–11 h (+) 97%
Flucytosine
129 3–6 h
2–4%
Cmax/MIC
AUC/MIC
AUC/MIC
AUC/MIC
Cmax/MIC
T/MIC
fl*
75%
fl
46%
fl
74%
*Low or immeasurable concentrations in the CSF.
#
The pharmacodynamic parameter most important for effect.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
S09.3
Cryptococcal meningitis
A. E. Brouwer, T. S. Harrison and A. A. Siddiqui
St George’s University of London, London, UK
Cryptococcal meningitis is a common life-threatening
infection in patients with advanced HIV disease. It is a
major cause of death from AIDS in Asia and
accounted for 13, 17 and 44% of all deaths in three
cohorts of HIV-infected patients in Africa1–4. As a
consequence of the increase in HIV-associated cryptococcosis, there has been a shift in the epidemiology
of meningitis: cryptococcal meningitis is now the
leading cause of community-acquired meningitis,
ahead of tuberculous and bacterial meningitis,
accounting for 20–45% of laboratory-confirmed cases
of meningitis in Southern Africa5, 6. The expansion of
antiretroviral programmes now raises the prospect of
transforming the long-term prognosis of these
patients provided that they survive the acute phase
of the illness. Factors contributing to the high
mortality are the inadequacy of current drug regimes
to sterilize the CSF, and the high intracranial pressure
in over a half of the patients7, 8. In animal models of
cryptococcal infection, protection is associated with
an active granulomatous inflammatory response and
depends on intact cell-mediated immunity and, in
particular, a Th1 pattern of cytokine release9. In a
study of combination antifungal treatment involving
64 HIV positive patients with cryptococcal
meningitis, we assessed the mycological response of
individual patients accurately by means of serial
quantitative CSF fungal cultures. In order to explore
the correlates of human immunity to cryptococcal
infection in vivo, we analysed the immune response at
the site of infection over time in the same patients.
Survival was associated with increased concentrations of the pro-inflammatory cytokines IFN-c, TNF-a
and IL-6 in the CSF. Furthermore IFN-c concentrations were independently associated with rate of
clearance of infection10. The results increase the
scientific basis for adjunctive immunotherapy with
IFN-c for cryptococcal meningitis. One phase 2 study
with adjuvant recombinant IFN-c 1b showed a trend
towards improved mycological success and was well
tolerated11. The fungicidal activity of new treatment
strategies can be assessed accurately with small
numbers of patients using serial quantitative cultures
to determine the rate of clearance of infection7.
25
xxx
References
1. Chariyalertsak S, Sirisanthana T, Saengwonloey O, Nelson KE.
Clinical presentation and risk behaviors of patients with
acquired immunodeficiency syndrome in Thailand, 1994–
1998: regional variation and temporal trends. Clin Infect Dis
2001; 32(6): 955–62.
2. Corbett EL, Churchyard GJ, Charalambos S, Samb B, Moloi V,
Clayton TC et al. Morbidity and mortality in South African
gold miners: impact of untreated disease due to human
immunodeficiency virus. Clin Infect Dis 2002; 34(9): 1251–8.
3. French N, Nakiyingi J, Carpenter LM, Lugada E, Watera C, Moi
K et al. 23-Valent pneumococcal polysaccharide vaccine in
HIV-1-infected Ugandan adults: double-blind, randomised and
placebo controlled trial. Lancet 2000; 355(9221): 2106–11.
4. Okongo M, Morgan D, Mayanja B, Ross A, Whitworth J.
Causes of death in a rural, population-based human immunodeficiency virus type 1 (HIV-1) natural history cohort in
Uganda. Int J Epidemiol 1998; 27(4): 698–702.
5. Gordon SB, Walsh AL, Chaponda M, Gordon MA, Soko D,
Mbwvinji M et al. Bacterial meningitis in Malawian adults:
pneumococcal disease is common, severe, and seasonal. Clin
Infect Dis 2000; 31(1): 53–7.
6. Hakim JG, Gangaidzo IT, Heyderman RS, Mielke J, Mushangi E,
Taziwa A et al. Impact of HIV infection on meningitis in Harare,
Zimbabwe: a prospective study of 406 predominantly adult
patients. AIDS 2000; 14(10): 1401–7.
7. Brouwer AE, Rajanuwong A, Chierakul W, Griffin GE, Larsen
RA, White NJ et al. Combination antifungal therapies for HIVassociated cryptococcal meningitis: a randomised trial. Lancet
2004; 363(9423): 1764–7.
8. Graybill JR, Sobel J, Saag M, van Der HC, Powderly W, Cloud G
et al. Diagnosis and management of increased intracranial
pressure in patients with AIDS and cryptococcal meningitis.
The NIAID Mycoses Study Group and AIDS Cooperative
Treatment Groups. Clin Infect Dis 2000; 30(1): 47–54.
9. Casadevall A.PJ. Cryptococcus neoformans. Washington DC:
American Society for Microbiology; 1998.
10. Siddiqui AA, Brouwer AE, Wuthiekanun V, Jaffar S, Shattock
R, Irving D et al. IFN-gamma at the site of infection determines
rate of clearance of infection in cryptococcal meningitis.
J Immunol 2005; 174(3): 1746–50.
11. Pappas PG, Bustamante B, Ticona E, Hamill RJ, Johnson PC,
Reboli A et al. Recombinant interferon- gamma 1b as
adjunctive therapy for AIDS-related acute cryptococcal meningitis. J Infect Dis 2004; 189(12): 2185–91.
S09.4
Intracerebral infections due to filamentous
fungi
S. Schwartz
Medizinische Klinik III, Charité Campus Benjamin
Franklin, Berlin, Germany
Invasive fungal infections are increasingly recognised
as a major cause of morbidity and mortality in
26
immunocompromised patients. Among various types
of filamentous fungi Aspergillus has been most
frequently recognised as a cause of invasive fungal
infections, but infections caused by Zygomycetes,
Fusarium or Scedosporium species have been
increasingly reported in recent years. Infections
caused by filamentous fungi are usually air-borne
diseases resulting in pneumonia, sinusitis or less
frequently in skin infections and are notoriously
difficult to treat. From the initial sites of infection
central nervous system (CNS) involvement might
arise from extension of sinus/ear infections or
hematogenous spread. Autopsy studies indicate that
the CNS is the second most frequent organ affected in
invasive aspergillosis. Typically, CNS-infections
caused by filamentous fungi present as brain abscess,
infarction or hemorrhage and less frequently as
meningitis, mycotic aneurysm or granuloma formation. In immunocompromised patients, CNS-infections caused by filamentous fungi have a devastating
prognosis and a fatality rate of almost 100% has
been repeatedly reported in patient with neuroaspergillosis. The main contributory factor for this
extremely poor prognosis is the limited penetration
of most available antifungal drugs across the blood–
brain barrier. Treatment with standard doses of
amphotericin B (deoxycholate or liposomal preparations), itraconazole and caspofungin result in negligible levels in the cerebrospinal fluid and brain tissue.
However, high-dose liposomal amphotericin B
(10 mg kg–1) has been successfully used in few
patients. Voriconazole, which is derived from fluconazole, has been repeatedly found in fungicidal
concentrations in cerebrospinal fluid. Furthermore,
high brain tissue concentrations of voriconazole have
also been reported. No treatment guidelines for the
management of CNS-infections caused by filamentous fungi are available, but a successful neurosurgical approach in addition to medical treatment has
been repeatedly reported. In a recent retrospective
analysis of 81 patients with proven or probable
neuroaspergillosis, treatment with voriconazole
resulted in an improved response rate of 35% and
31% of patients had a prolonged survival for a
median observation time of 310 days. Interestingly,
neurosurgery was significantly associated with an
improved survival in this series of patients. In
conclusion, CNS-infections caused by filamentous
fungi are threatening conditions urgently requiring
improved therapies. Antifungal drugs with improved
penetration into the CNS should be preferentially
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
used to treat these infections. Furthermore, a neurosurgical management should be considered
whenever feasible, which might reduce the fungal
burden and prevent complications (e.g. cerebral
hemorrhage). Further studies using higher doses of
medication or combination therapies and evaluating neurosurgical treatment approaches are clearly
needed.
S10.1
Cell wall dynamics in Saccharomyces cerevisiae
and Candida albicans
F. M. Klis, G. Sosinska, P. De Groot, S. Brul,
K. Hellingwerf and C. de Koster
Swammerdam Institute for Life Sciences, University
of Amsterdam, Amsterdam, The Netherlands
The cell walls of S. cerevisiae and C. albicans are
similarly organized. In both organisms we find (i) an
internal skeletal layer consisting of the stress-bearing
polysaccharides 1,3-b-glucan and chitin, and (ii) an
external layer of mannoproteins, which are covalently linked to the underlying skeletal polysaccharides4, 5. There are two main classes of covalently
linked cell wall proteins (CWPs). GPI-CWPs are
linked through a remnant of their original glycosylphosphatidylinositol (GPI) anchor to 1,6-b-glucan,
which in turn is linked to 1,3-b-glucan or chitin.
GPI-CWPs are the predominant form of CWPs. The
second class is formed by ASL-CWPs, which are
directly linked to 1,3-b-glucan through an alkalisensitive linkage (ASL) of unknown nature3, 6. Some
GPI-CWPs may also be linked through an alkalisensitive linkage to 1,3-b-glucan. Five different CWPpolysaccharide complexes can be distinguished in
this way4, 5. This cell wall model has strong
predictive power for other ascomycetous fungi including mycelial species3. Proteolytic digestion of isolated walls followed by mass spectrometric analysis
(LC-MS/MS) of the released peptides has identified
about 15–20 different CWPs in each organism.
CWPs may be involved in adhesion and virulence
and in regulating cell surface hydrophobicity; they
may have a cell wall-reinforcing function, and they
may be involved in iron uptake3. Interestingly, a
considerable number of CWPs are putative polysac-
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
charide-processing enzymes2, 6. Although CWPs
show only limited turnover after having been
incorporated into the wall, the cells may use different
CWPs for incorporation into the cell wall, depending
on both internal and external cues such as (a) the
phase of the cell cycle, (b) the presence of pheromones, (c) growth form (yeast vs. filamentous
growth), (d) oxygen availability, (e) nutrient availability, (f) pH of the medium. As a result, the protein
composition of the cell wall may vary widely.
Recently, we have begun to investigate the cell wall
proteome of Candida albicans cells grown under
conditions mimicking the microenvironment of the
vagina. To this end, we have developed a vaginasimulative (VS) medium buffered at pH 4.2. In VS
medium the cells mainly grow in the yeast and
pseudohyphal form. Initial results show that the cell
wall proteome of cells growing exponentially in VS
medium is largely comparable to that of exponentially growing cells cultured in rich medium, except for
the appearance of Phr2p, a known low pH-inducible
protein, and of Als3p, which is known as a putative
adhesion protein specific for the hyphal growth form,
and which is also expressed in human clinical
vaginal specimens1 (Grazyna Sosinska, unpublished
data).
References
1. Cheng G, Wozniak K, Wallig MA, Fidel PL Jr, Trupin SR, Hoyer
LL. Comparison between Candida albicans agglutinin-like
sequence gene expression patterns in human clinical specimens
and models of vaginal candidiasis. Infect Immun 2005; 73:
1656–63.
2. De Groot PW, de Boer AD, Cunningham J, Dekker HL, de Jong
L, Hellingwerf KJ, de Koster C, Klis FM. Proteomic analysis of
Candida albicans cell walls reveals covalently bound carbohydrate-active enzymes and adhesins. Eukaryot Cell 2004; 3:
955–65.
3. De Groot PW, Ram , Klis FM. Features and functions of
covalently linked proteins in fungal cell walls. Fungal Genet Biol
2005; May 17 [Epub ahead of print].
4. Klis FM, Mol P, Hellingwerf K, Brul S. Dynamics of cell wall
structure in Saccharomyces cerevisiae. FEMS Microbiol Rev 2002;
26: 239–56.
5. Klis FM, de Groot P, Hellingwerf K. Molecular organization of
the cell wall of Candida albicans. Med Mycol 2001; 39(Suppl 1):
1–8.
6. Yin QY, de Groot PW, Dekker HL, de Jong L, Klis FM, de Koster
CG. Comprehensive proteomic analysis of Saccharomyces cerevisiae cell walls: Identification of proteins covalently attached
via GPI remnants or mild-alkali sensitive linkages. J Biol Chem
2005; Mar 21 [Epub ahead of print].
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S10.2
The role of cell surface mannans in Candida
albicans
N. A. R. Gow, J. M. Bain, S. Bates, G. Bertram,
R. P. Hobson, H. B. Hughes, C. A. Munro,
D. MacCallum, F. C. Odds and A. J. P. Brown
Aberdeen Fungal Group, Institute of Medical
Sciences, University of Aberdeen, Aberdeen, UK
The outer layer of the cell wall of Candida albicans is
heavily enriched in glycosylated proteins that play
critical roles in cell adherence, and act as major
antigens and in the immunoregulation of the host. A
null mutant in the Golgi manganese transporter gene
PMR1 was viable in vivo, had greatly reduced N- and
phosphomannan and was attenuated in virulence.
Therefore protein mannosylation is required for
pathogenesis. The C. albicans O-linked mannan
consists of a tetrasaccharide in which Mnt1p and
Mnt2p participate as partially functionally redundant
enzymes in the assembly of the terminal two alpha1,2-mannose residues. Deletion of either MNT1,
MNT2 or both MNT1 and MNT2 resulted in strains
with reduced adherence to epithelia and attenuation
of virulence. This suggests that O-mannan functions
as a ligand in interactions with host surfaces.
Mutants with deletions in the MNN4 gene are almost
devoid in phosphomannan, which has been implicated in recognition of C. albicans by macrophages ,but
were phagocytosed normally. Deletion of the OCH1
resulted in elimination of the outer N-mannan
chains, induction of the cell wall salvage pathway
and loss of virulence. Analysis of glycosylation
mutants demonstrates that the carbohydrate
epitopes of mannoproteins play key roles in pathogenesis of C. albicans.
S10.3
The cell wall of Candida glabrata
M. Weig
Institute of Medical Microbiology and German
Reference Centre for Systemic Mycoses, University of
Goettingen, Germany
Candida glabrata is an important cause of human
mucosal and bloodstream infections. In comparison
to the polymorphic C. albicans, C. glabrata does not
28
form true hyphae in vivo and shows a high intrinsic
resistance to antifungal agents, such as fluconazole.
In fungi, the cell wall provides physical strength,
limits permeability and protects the cell from hostile
degrading enzymes. However, the cell wall is not
static but a highly dynamic structure that facilitates
morphological plasticity and adaptation to environmental conditions. For pathogenic fungi, the cell wall
is the first point of interaction with host cells. For
these reasons, the cell wall has become recognized as
an attractive target for antifungal drug development,
especially since the successful clinical use of the
echinocandins. In order to explore the cell wall of
C. glabrata, we have analysed its composition and
architecture. It consists of a carbohydrate backbone
of beta-1,3-glucan, beta-1,6-glucan and chitin to
which proteins are covalently attached. These cell
wall proteins (CWPs) possibly mediate adhesion,
foster biofilm formation and modulate the immune
response. We have performed a systematic in silico
analysis of the putative glycosylphosphatidylinositolmodified (GPI) proteins, a large part of which are
covalently bound to the cell wall glucan network.
Among the 106 GPI proteins that we have predicted,
are adhesive proteins (51), potentially implicated in
fungus-host interactions, glycoside hydrolases (12),
involved in cell wall expansion and remodelling or in
biofilm formation, phospholipases (3), aspartic proteases (9), homologues of Ecm33p (4) and Kre1p (1)
of Saccharomyces cerevisiae, and structural CWPs
(14). These predictions were confirmed by extracting
GPI-proteins from cell walls with HF-pyridine followed by their identification using Electrospray
Ionization Tandem Mass Spectrometry. The most
abundant GPI-dependent CWP in C. glabrata has
homology to Cwp1 of S. cerevisiae. Functional
analysis revealed that Cgcwp1 limits permeability
and protects the carbohydrate backbone of the cell
wall from degrading enzymes. Interestingly, Cwp1homologues are absent in C. albicans. A second class
of CWPs can be extracted with NaOH (ASL-CWPs).
Within this group, in silico methods revealed five
putative Pir-proteins (Pir1–5p), and five members of
the Bgl2 glycoside hydrolase family 17. Using
immunological methods and MS/MS-analysis we
have unequivocally demonstrated the incorporation
of CgPir1-4p and two members of the Bgl2 family in
the cell wall of C. glabrata. Together, our experimental data and our in silico predictions represent the first
systematic analysis of the C. glabrata cell wall and its
proteome.
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S10.4
Galactomannan has multiple sources in
Aspergillus fumigatus
J. P. Latgé,1 T. Fontaine,1 W. Morelle,1
12 J. P. Debeaupuis1 and M. Tabouret2
1
Aspergillus Unit, Institut Pasteur and Bio-Rad,
Steenworde, France2
1 Galactomannan has been recognized for years as a
key antigen in Aspergillus fumigatus. Previous studies
have shown that galactomannan has a comb like
structure with an a 1,2, 1,6 mannan central chain
and b1,5 galactofuran side chains. It has been shown
that this molecule is present both in the alkaliinsoluble and alkali-soluble fractions of the cell wall.
Carbohydrate analysis as well as Western blot
experiments using an anti-galactofuran monoclonal
antibody have shown that polysaccharides and
glycoproteins bearing the galactofuran epitope are
actively secreted in culture filtrates. Further studies
have recently characterized two new galactofuranose-containing molecules: (i) a glycosylphosphatidyl
inositol linked galactomannan and (ii) glycoproteins
with a N-glycan containing galactofuranose linked to
the terminal non-reducing end of the N-glycan. The
diverse origin of molecules reacting with the anti-GM
monoclonal antibody and the versatility of their
secretion under different environmental growth conditions may explain the efficiency of this anti-GM Mab
in the diagnosis of A. fumigatus infections.
S11.1
Invasive candidiasis in preterm neonates
E. Roilides
3rd Department of Paediatrics, Hippokration
Hospital, Aristotle University of Thessaloniki,
Thessaloniki, Greece
Invasive candidiasis is a serious infection in the
preterm neonates. In population studies, neonatal
age has been shown to be the most susceptible to
candidiasis. Its frequency increases from <1% in
neonates to >5% in very-low-birth-weight neonates
(VLBW, <1500 g) to even ~10% in extremely-lowbirth-weight infants (ELBW, <1000 g). Colonization
of the neonate from the genital tract of the mother
(vertical transmission) or during the stay in NICU
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
(horizontal transmission) usually precedes the invasive infection. Neonatal candidiasis most frequently
manifests as bloodsteam infection (almost 100%).
Candidemia can be followed by secondary localization
in the CNS (usually meningitis, 5%), sceletoarticular
infection (3%), endocarditis (5%), positive urine
culture (60%). Risk factors for Candida colonization
and/or invasive candidiasis have been found intravascular catheters, parenteral nutrition, use of
broad-spectrum antimicrobial agents, especially third
generation cephalosporins and carbapenems, necrotic
enterocolitis and abdominal surgery. In general,
Candida albicans remains the single most frequent
causative Candida spp. However, during the last
decade non-C. albicans species cause candidiasis with
increasing frequency. Among non-C. albicans species,
Candida parapsilosis is the most frequent species in
neonates due to invasive catheters and parenteral
nutrition usually employed in the very young infants.
Candida tropicalis and other Candida spp. are much less
frequent in neonatal age. Candida albicans isolates
derived from neonates are uniformly susceptible to
amphotericin B and almost always to fluconazole.
However, among non-C. albicans species, Candida
glabrata and Candida krusei may be fluconazole nonsusceptible. Nevertheless, these are infrequent isolates
in neonatal candidiasis. Positive cultures of blood or
other sterile body fluids are the cornerstone of
diagnosis of neonatal candidiasis. Persistence of
positive blood cultures may follow despite the initiation of active antifungal therapy. Polymerase chain
reaction and serum glucan concentration are promising tools for the early diagnosis of candidemia or
when cultures are negative. Non-specific laboratory
indices, such as elevated CRP and decrease of platelet
count, may suggest generalized infection. The antifungal agents that are useful in the management of
neonatal candidiasis are deoxycholate (conventional)
amphotericin B, which is considered the drug of choice
for neonatal candidiasis. Fluconazole is a second-line
therapeutic agent with good activity for susceptible
isolates and very favorable pharmacokinetic properties (good penetration to CNS, peritoneal cavity and
urinary tract). Flucytosine is still used mainly in
Candida meningitis in combination with amphotericin
B. The great majority of neonatologists and pediatric
infectious disease specialists in USA and Greece prefer
deoxycholate amphotericin B as the first line therapeutic agent in neonates due to lack of toxicity seen in
older patients. In addition, lipid formulations of
amphotericin B such as liposomal amphotericin B
29
xxx
and amphotericin B lipid complex have been studied in
neonates with good results. Caspofungin has been
used in few neonates with refractory and resistant
neonatal candidiasis. The management strategies of
neonatal candidiasis include antifungal prophylaxis,
empiric therapy and therapy of culture-proven infection. While antifungal prophylaxis has been advocated in centers with extremely high colonization and
infection rate due to Candida spp., most centers do not
practice it since they do not see neonatal candidiasis so
frequently. One exception would be the ELBW infants
(<1000 g) or even <750 g in whom the rate of
candidiasis is the highest. Although not studied
extensively, empiric or preemptive initiation of
antifungal therapy before the result of the cultures
may be a useful method of early management of
neonatal candidiasis. Outcome of this infection is
dependent on early initiation of therapy. Without
early therapy, mortality and permanent sequelae are
high. By comparison, early initiation of antifungal
therapy decreases mortality of this infection.
S11.2
Fungal infections in immunocompetent children
F.-M. Müller
Pediatric Pulmonology & Infectious Diseases,
Department of Pediatrics III, University Heidelberg,
Germany
Fungal infections have not only emerged in immunocompromised patients, but also in immunocompetent adults and children. Certain children are at
higher risk to develop fungal infections: Patients
undergoing complicated abdominal surgery, longterm use of broad-spectrum antibiotics, systemic or
inhaled steroids, patients with implanted catheters
(oncology and cystic fibrosis patients), female adolescents suffering from recurrent Candida vaginitis and
preterm infants below 1.500 g birth weight. Even a
fullterm newborn is not considered to be immunocompetent and due to a high Candida colonization rate
of the mother during pregnancy, it may develop
oropharyngeal candidiasis and diaper dermatitis or
congenital cutaneous candidiasis. Candidaemia and
disseminated candidiasis is inversely correlated with
the gestational age and birth weight: Extremely low
birth weight infants (ELBW) have a risk of up to 20%
for developing neonatal candidiasis during their stay
in the neonatal intensive care unit (NICU). From a
30
recent epidemiological study conducted in Germany,
50% of ELBW infants are colonized with fungi, and
from those that develop disseminated infection the
mortality rate is 30%. C. albicans is the most common
isolated pathogen (70%), but 23% were non-albicans
Candida spp. with C. parapsilosis the leading one.
Patients with allergic asthma and cystic fibrosis
patients may suffer sensitisation to various mould
species or allergic bronchopulmonary aspergillosis
(ABPA) and the diagnosis is often cumbersome. The
respiratory tract of cystic fibrosis patients is often
colonised with Candida spp. and long-term persistence
of isogenic strains may occur. If Candida spp. have an
impact on chronic infection and inflammation in the
CF-lung is still controversial and currently revisited. It
is a future task to assign more attention and focus in
research to fungal infections in children with special
regard to preterm infants and immunocompetent
(cystic fibrosis, asthmatics) children at high risk for
developing fungal infection or mould allergy. The
new antifungals need to be investigated in this
population including in preterm infants and new
concepts of antifungal pre-emptive therapy and
prophylaxis need to be carefully evaluated in children
to reduce morbidity and mortality caused by fungi.
S12.1
Invasive aspergillosis: update on conventional
diagnosis
J. Bille
Institute of Microbiology, University Hospital,
Lausanne, Switzerland
The clinical presentation of invasive aspergillosis is
rather non-specific, and its diagnosis still relies on
conventional microbiology, histopathology and radiology in an appropriate host setting. Diagnosis of
proven aspergillosis requires the isolation by culture
of Aspergillus spp. from a normally sterile site ( which
is clinically or radiologically abnormal – by an
invasive procedure infrequently attempted. Probable
aspergillosis relies on the combination of host,
clinical (nodules, wedge-shaped infiltrate, air crescent sign or cavity on lung CT), and microbiological
criteria (sputum, BAL or sinus positive culture).
Sensitivity of direct examination and/or culture of
lower respiratory tract specimen is widely variable
for the diagnosis of IA (0(89%, overall 43%), and
PPV of a positive culture varies according to the type
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
of patient from <20% (in an unselected hospital
population) to 60% (in high risk patients such as
bone marrow transplants), and can be graded
according to different scores. Multiple positive
samples and/or higher scores increase the PPV.
Practically, direct examination of clinical specimens
should include special stains such as Calcofluor to
better detect fungal filaments, often rare or truncated. Culture should be done on non-inhibitory
(Sabouraud, BHI ± blood) and inhibitory agar media
(BHI + antibiotics), and kept 2 weeks at 30 C.
Cytology or histopathology (samples for typical
hyphae positive) offer only probable diagnosis, unless
immuno-histochemical staining specific for Aspergillus is positive. Special stains (PAS, MSS) increase the
sensitivity compared to the classic HE stain. CT scan
of lung has become a very useful investigation for the
early diagnosis of IA, and several more or less specific
images have been described according to the stage of
the disease and the type of patients. For example, the
halo sign is positive in <60% of IA occurring in
neutropenic patients, but only at the early stage and
for 5–7 days, whereas crescent shape nodular lesion
is a later finding. IA is occurring today in a wider
range of patients (ICU, COPD, cortico-therapy,
mechanically ventilated pts.) than the classic neutropenic host, underlying the pivotal role of a high
index of suspicion. In summary, IA is still difficult to
diagnose early on, and the conventional diagnostic
tools are still the corner stone; antigen and molecular
tools are very useful complementary tests.
number of commercially available methods to detect
fungal glucan there has been little experience outside
Japan where the various test formats were developed.
A new ELISA based chromogenic test to detect fungal
glucan has been recently commercialized, formerly
known as Glucatell, renamed as Fungitell, Associates
of Cape Cod, USA. A number of retrospective studies
have described the characteristics and performance of
this test in various clinical settings as a surrogate
marker of invasive fungal disease. In patients with
acute myeloid leukemia or myelodysplastic syndrome
at least one serum sample was positive for fungal
glucan at a median of 10 days before the clinical
diagnosis in 100% of subjects with a proven or
probable invasive fungal infection. Absence of a
positive glucan finding has a 100% negative predictive value, and the specificity of the test was 90% for a
single positive test result and ‡96% for ‡2 sequential
positive results. In a second study the performance of
the glucan test was compared with the galactomannan assay for invasive aspergillosis. A similar experience with the glucan test was reported. However, the
glucan test became positive earlier than the galactomannan test. False/positive reactions occurred at a
rate of 10.3% in both tests, but the patients showing
false/positive results were different in each test. A
combination of the two tests improved the specificity
to 100%. More recent case reports have demonstrated
the potential usefulness of the glucan assay in other
clinical settings, and as a biomarker of fungal
exposure in water damaged buildings.
S12.3
S13.2
Update on glucan detection
More echinocandins – better therapies?
M. Richardson
Department of Bacteriology and Immunology,
University of Helsinki, Finland
B. de Pauw
University Medical Center St Radboud, Nijmegen,
The Netherlands
The innate recognition of fungal pathogens in
humans occurs through both opsonic and nonopsonic mechanisms of host effector cells. A number
of pattern recognition receptors have been implicated,
including those that recognize the major carbohydrate constituents of fungal cell walls. It has been
known for some considerable time that soluble
glucans are also released into plasma and other body
fluids during fungal infections, including infection
with the most common pathogens such as Aspergillus
and Candida. Although there have been available a
During the past two decades, Candida has evolved into
an key pathogen in the category of nosocomial
infections. Neutropenic patients and recipients of
organ transplants are at high risk followed by patients
admitted to intensive care units and neonates.
Multiple logistic regression analysis learned that
repeated bowel surgery, long-term use of central
venous catheters, Candida colonization, use of broadspectrum antibiotics, and hemodialysis constitute
independent predisposing factors. In patients with
hematologic malignancies the use of antifungal
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
31
xxx
prophylaxis, particularly with absorbable drugs, was
linked to a higher incidence of non-albicans candidemia. This change in the pattern has been attributed to
the prophylactic use of fluconazole. The attributable
mortality from candidemia ranges from about 40% in
patients with uncomplicated candidemia to over 90%
for those who suffer from an acute disseminated
disease. In patients with malignancies aspergillosis
has even superseded candidiasis as the most prominent invasive fungal infection. Reports on the overall
incidence of acute aspergillosis are confusing. The
figures vary between 0% and 25% depending on the
study population. Amphotericin B has been the drug
of choice for all invasive fungal infections in severely
ill patients for more than 30 years. Clinical failure due
to development of resistance is rare but the drug has a
very narrow therapeutic index. Lipid formulations
offer a much safer alternative but there are limits such
as a compromised kidney function and increased
cyclosporine levels in transplant recipients. The use of
azoles is limited by their interactions with cytochrome
P450 which prohibits their uncomplicated exploit in
patients who require other drugs that depend on the
same metabolic pathway. Echinocandins, of which
caspofungin is the best studied representative, constitute a totally new class of antifungal agents. Whilst
polyenes and azoles interact with ergosterol in the cell
membrane, echinocandins inhibit non-competitively
the synthesis of 1,3-b-D-glucan. This 1,3-b-D-glucan
is an essential component of chitin in the cell wall, a
structure that human cells do not share. Interference
with glucan synthesis ultimately leads to lysis of the
fungal cell. This mechanism of action makes that
cross-resistance with azoles and polyenes is not
expected and holds promise for their use against
resistant fungi both as single agents and in combinations. Candins exhibit fungicidal activity against
Candida species, including fluconazole-resistant species, and showed activity against Aspergillus species
with a notable lack of activity against Cryptococcus
neoformans and Zygomycetes. The safety profile of
caspofungin proved excellent. There is no evidence
of histamine release related to the administration of
caspofungin. The incidence of caspofungin-related
clinical and laboratory adverse event was similar to
fluconazole. Mild elevations in transaminases have
been noted when caspofungin had been given concurrently with cyclosporin A. It appears feasible to
co-administer caspofungin with drugs like mofetil,
tacrolimus, itraconazole and amphotericin B without
added toxicity.
32
S13.3
When will amphotericin B die?
F. C. Odds
Aberdeen Fungal Group, School of Medical Sciences,
Institute of Medical Sciences, University of Aberdeen,
Scotland, UK
Amphotericin B has been the mainstay of broad
spectrum treatment for systemic mycoses for more
than 40 years. With new antifungal agents now
introduced to the clinic it is reasonable to examine
the place of amphotericin B in current antifungal
therapy. The presentation will discuss amphotericin
B, its history and its many formulations in the
context of today’s therapeutic needs. Will amphotericin B die? If so, when?
S13.4
Who should be treated with high dose
liposomal amphotericin B?
R. Herbrecht
Department of Hematology and Oncology, Hôpital
de Hautepierre, Strasbourg, France
Liposomal amphotericin B has been on the market for
many years but we still do not know the optimal daily
dose for the treatment of invasive fungal infections.
Most initial clinical trials were performed with a 1–
3 mg kg–1 day–1 dose and did not show superior
efficacy over conventional amphotericin B. To date,
the only study demonstrating a higher efficacy rate of
liposomal amphotericin B over conventional amphotericin B in invasive aspergillosis was conducted with
a 5 mg kg–1 day–1 dose (Leenders et al., Br J Haematol
1998). An attempt to investigate the dose–efficacy
relationship failed to demonstrate the superiority of a
4 mg kg–1 day–1 dose over a 1 mg kg–1 day–1 dose in
the first line therapy of invasive aspergillosis (Ellis
et al. Clin Infect Dis 1998). However only a limited
number of the patients enrolled in this study had a
documented infection making the conclusion of
absence of difference less relevant. This study never
significantly influenced clinical practice with physicians being reluctant to lower the dose to 1 mg kg–
1
day–1. A dose escalating clinical study demonstrated that daily dosages ranging from 7.5 to 15 mg/kg
were well tolerated in patients with a filamentous
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
fungal infection (Walsh et al. Antimicrob Agents
Chemother 2001). Mean maximal plasma drug concentrations of the drug have been as high as
120 lg ml–1 in patients treated with a daily dose of
10 mg kg–1. Interestingly, 52% of the patients treated
for at least 7 days responded favourably to the
treatment. These data created a rational to explore
higher doses of liposomal amphotericin B in clinical
practice although subsequent animal studies showed
no improvement in survival between 1, 3 and 5
mg kg–1 day–1 in a murine model of invasive aspergillosis (Martin et al. J Antimicrob Chemother 2003).
Many physicians have treated patients with doses
higher than the approved 3–5 mg kg–1 day–1 regimen but with few published evaluation of this
approach. An open labelled non-comparative study
was conducted in Spain in 10 patients with probable
or proven invasive aspergillosis treated in first line
with 10 mg kg–1 day–1 of liposomal amphotericin B
(Ruiz et al., ICAAC, 2003). Nine patients responded
favourably suggesting excellent efficacy of this regimen. A similar approach is currently being explored
in United Kingdom in filamentous fungi infections
(Kinsey et al., Meeting of the British Society of
Haematology, 2004). An interim analysis showed
that 12 of 20 patients responded favourably with a
12-week survival of 60%. A large double blind trial
comparing 3 vs. 10 mg kg–1 day–1 for 14 days
followed by 3 mg kg–1 day–1 in patients with invasive
filamentous fungi infections has been recently completed and the results should be presented soon. A
large number of the patients included had documented invasive aspergillosis. These results should definitively answer the question of a beneficial effect of
high dose liposomal amphotericin B. Regarding the
extremely high cost of liposomal amphotericin B,
the only suggestion we can make is to stay with the
standard dose of 3–5 mg kg–1 day–1 for invasive
aspergillosis till more information are available and
to only discuss higher doses for the most severe
invasive fungal infections such as zygomycosis.
L1
kingdom of fungi also offers a great variety of
influences to human life as there are mind-altering
psychedelic mushrooms, deadly poisonous toadstools, incredibly expensive delicasies or just beautiful-looking creatures. These attributes have inspired
numerous artists to immortalize fungi in artwork.
From the beginning of our history we find rockcarvings (petroglyphs) of mushrooms in the African
desert, in the high north of Europe and all over the
Americas. Beautiful paintings sustained centuries
since the time of the ancient Greeks and have even
survived volcanic eruptions in the Roman Empire.
There are numberless Italian still lives depicting the
imperial mushroom Amanita caesarea, others, mainly
from Dutch masters show arrangements with bolets
(Boletus edulis). In Central America, the ritual use of
hallucinogenic mushrooms bore many artwork
influenced by Teonanacatl, the flesh of the gods.
But we do not find fungi in paintings and sculptures
only. There are numerous books, from the great
Russian writers like Tolstoy and Pushkin to modern
American thrillers, from culinary French tales to
Japanese haikus. Even in movies mushrooms sometimes play a crucial role. In some they dance to the
music of Tchaikovsky, in others again they – when
being eaten up – convert people into mushrooms
themselves. Stravinsky wrote a song depicting the
war between mushrooms and beetles, and the 1960s
Rock Legends Jefferson Airplane set the Alice-inWonderland-story to music, where a mushroom (the
one with the hookah-smoking caterpillar sitting on
it) can change the size of the consumer. There are
even paintings of human mycoses. Above all the
works of the 17th Century Spanish painter Bartolomé Esteban Murillo. He greatly depicted the heads
of children suffering from a favus.
O1.1
Temporal trends of post-mortem invasive
fungal infections among AIDS patients at a
University hospital in Italy
W. Buzina
Institute of Hygiene, Medical University Graz, Graz,
Austria
S. Antinori,1 M. Nebuloni,2 C. Magni,1 M. Fasan,1
S. Corvasce,1 C. Parravicini2 and L. Vago2
1
Infectious Diseases and Tropical Medicine, Clinical
Science ‘L. Sacco’, Milan, Italy and 2Patology, Clinical
Science ‘L. Sacco’, Milan, Italy
The important role of fungi as pathogens is well
known among mycologists and physicians. Yet the
Objectives: Analyze temporal trends of invasive
fungal infections (IFI) observed at autopsy performed
Fungi and art
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33
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between 1984 and 2002 among AIDS patients;
verify the concordance between clinical and postmortem diagnosis; verify anatomical sites involved.
Methods and design: Retrospective study from the
electronic archive records of the Institute of Pathology; all patients with a post-mortem diagnosis of IFI
were included. Fungal findings on the mucosal
surface of the upper GI and respiratory tracts up to
tracheal bifurcation were not considered. Autopsy
data of the identified patients were cross-checked
with the chart records. The histological preparations
were stained with H–E, period acid–Schiff and
Grocott–Gomori’s. The study period was subdivided
into four time according to the antiretroviral therapy
used: 1984–88 (no therapy/monotherapy); 1989–
93 (monotherapy); 1994–97 (double therapy/triple
therapy); 1998–2002 (triple therapy). Trends of
proportions of invasive fungal infections was evaluated over time using a Chi square test (P = 0.05).
Results: During the study period 2101 AIDS patient
died in-hospital and 1630 underwent autopsy
(77.6%); the autopsy rate fell from 87.8% (84–88)
and 88.1 (89–93) to 77.5 (94–97) and 33.4 in 1998–
2002 (P < 0.001). A total of 297 cases (18%) met the
criteria for IFI. The etiological assignment was as
follows: 131 pneumocystosis (44.1%), 83 aspergillosis
(27.9%), 62 cryptococcosis (20.8%), 15 candidiasis
(5.1%), four histoplasmosis (1.4%) and two zygomicosis (0.7%). The overall concordance between clinical
and post-mortem diagnosis was 46.1% (137/297),
with wide variations between the single mycoses:
95.2% for cryptococcosis; 50.3% for pneumocystosis;
25% for histoplasmosis; 11% for aspergillosis and
1.3% for candidiasis. A disseminated disease (two or
more non-contiguous organs involved) was observed
in all cases of histoplasmosis, 61.3% of cryptococcosis,
22.9% of aspergillosis, 40% of candidiasis, 11.4% of
pneumocystosis. The lung was the organ more
frequently involved by Pneumocystis jiroveci (98.5%)
and Aspergillus spp. 96.4 % whereas Cryptococcus
neoformans was identified in the SNC in 83.9% of cases.
The trend analysis of IFI overtime revealed a temporal
increase of aspergillosis; on the contrary, infections
caused by P. jiroveci and C. neoformans showed a
temporal drop although not statistically significative,
particularly in the period 97–02. Analysis of candidiasis revealed a significant increase of frequencies of
such infection (P = 0.035), when comparing the
periods 1984–1997 vs. 1998–2002 (P < 0.01).
Conclusion: Although decreasing, IFI are still
important among patients dying with AIDS;
34
cryptococcosis and pneumocystosis showed a trend
in reduction in the last period compared with the
previous periods examined whereas aspergillosis and
candidiasis showed an opposite trend. A high rate of
discrepancy between clinical and post-mortem diagnosis was observed for histoplasmosis, aspergillosis
and candidiasis.
O1.2
A prospective study of the ocular
manifestations of candidemia – results from the
Voriconazole Global Comparative Candidemia
Study
A. M. L. Oude Lashof,1 J. D. Sobel,2 M. Ruhnke,3
P. G. Pappas,4 C. Viscoli,5 H. T. Schlamm,6
I. T. Oborska7 and B. J. Kullberg1
1
Radboud University Nijmegen Medical Centre,
Nijmegen University Centre for Infectious Diseases,
Nijmegen, The Netherlands, 2Wayne State University
School of Medicine, Detroit, USA, 3Charité,
Humboldt University, Berlin, Germany, 4University of
Alabama, Birmingham, USA, 5University of Genova
and National Institute for Cancer Research, Genova,
Italy, 6Pfizer Global Research and Development, New
York, USA and 7Pfizer Global Research and
Development, Sandwich, UK
Background: Retinal lesions are among the most
frequent complications of candidemia. The incidence,
risk factors, and outcome of eye involvement during
candidemia are largely unknown. We prospectively
studied the ocular manifestations of candidemia in a
large, worldwide, randomized multicentre trial comparing voriconazole with amphotericin B followed by
fluconazole for the treatment of candidemia.
Methods: Non-neutropenic patients with at least
one positive blood culture for Candida species were
randomized for treatment with voriconazole or
amphotericin B/fluconazole in a 2 : 1 ratio. Dilated
fundoscopy was performed in each patient at baseline
and on day 7, at 2 and 6 weeks after end of treatment
(EOT) and, if clinically indicated, at 12 weeks after
EOT. Definite Candida chorioretinitis was defined as a
retinal lesion with positive histology for yeasts.
Definite Candida endophthalmitis was defined as a
positive vitreal aspirate culture or positive biopsy plus
white fluffy balls in the vitreous body. In patients
without diabetes mellitus, bacteraemia, or other
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
known risk factors for retinal lesions, probable
chorioretinitis was defined as focal white infiltrates,
haemorrhages, Roth spots, or infarctions. Additionally, for probable endophthalmitis, extension into the
vitreous body was required. In patients with diabetes,
bacteraemia or other risk factors for retinal lesions,
abnormalities seen at fundoscopy as described above
were classified as possible Candida infection.
Results: Of 370 patients, 51 patients had abnormal
fundoscopy at baseline, and an additional 10 patients
developed abnormalities during treatment (total 61
patients (16%)). Probable Candida eye infection was
diagnosed in 41 patients (35 chorioretinitis, six
endophthalmitis), possible Candida eye infection in
20. None of the ocular infections were culture or
biopsy proven. Therapy with either voriconazole (45
cases) or amphotericin B/fluconazole (16 cases) was
successful in 62%; outcome was not evaluable in
38%, and unfavourable in none of the cases.
Conclusion: Ocular involvement is frequent (16%)
in patients with candidemia. Probable Candida chorioretinitis occurred in 9.5%. Full-blown endophthalmitis is rare (1.6%). Treatment with either voriconazole
or amphotericin B/fluconazole was successful in all
cases in which follow-up was available.
difference in the incidence of proven or probable IFI
(primary endpoint). P-values were two-sided.
Results: Pt. characteristics: Eligible pts 219; arm A:
110; arm B: 109. Reasons for exclusion were:
absence of N (eight), infection prior neutropenia
(three) and patient’s decision (one). Baseline characteristics were balanced for age (mean 53.8 years),
underlying disease (119 AML, 27 ALL, 64 NHL, nine
other), duration of N (mean 14.8 D) and treatment
modality (primary 149, secondary 42, transplant
28). Primary endpoint: The incidence of IFI was five
of 110 pts (4.6%) in arm A and 22 of 109 pts
(20.2%) in arm B (P = 0.001, RR = 2.9, CI 1.3–
6.5). Key secondary endpoints: pneumonia of
unknown origin occurred in six pts (5.5%) vs. 28
pts (25.7%) (P < 0.001), the incidence of possible,
probable and proven IFI was 11 pts (10.2%) vs. 42
pts (39.6%) (P < 0.001), Toxicity: no grade 3 or 4
toxicity was noted. Laboratory abnormalities, including creatinine and liver function tests, were not
different between the treatment groups.
Conclusion: The significant lower incidence of IFI
in pts treated with L-AmB prophylaxis supports its
use in prolonged N.
O1.4
O1.3
Low dose liposomal amphotericin B (L-AmB) as
prophylaxis of invasive fungal infections (IFI) in
neutropenic patients (pts): a phase-III trial
O. Penack, M. Reinwald, M. Schmidt-Hieber,
P. Martus, S. Schwartz, E. Thiel and I. W. Blau
Charite Campus Benjamin Franklin, Hematology,
Oncology and Transfusion Medicine, Berlin, Germany
Background: This trial was designed to evaluate
the efficacy of L-AmB prophylaxis in high-risk
neutropenic patients.
Methods: 231 pts with hematological malignancies
and expected neutropenia (N) of more than 10 days
(D) following intensive chemotherapy or autologous
stem cell transplantation were enrolled, 219 pts
became neutropenic and were randomized to receive
either 50 mg L-AmB i.v. every second D (arm A) or no
systemic antifungal prophylaxis (arm B). Treatment
with L-AmB started 1–3 D before onset of N and was
continued until neutrophil recovery, breakthrough
IFI, intolerable toxicity or death. With 82 eligible pts
in each arm the study had 0.80 power to detect 50%
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
A multicenter trial of oral posaconazole vs.
fluconazole for the prophylaxis of invasive
fungal infections in recipients of allogeneic
hematopoietic stem cell transplantation with
graft-vs.-host disease
A. J. Ullmann,1 J. H. Lipton,2 D. H. Vesole,3
P. Chandrasekar,4 A. Langston,5 S. Tarantolo,6
H. Greinix,7 C. Hardalo,8 H. Patino8 and S. Durrant9
1
Johannes Gutenberg University, Mainz, Germany,
2
Princess Margaret Hospital, Toronto, ON, Canada,
3
Medical College of Wisconsin, Milwaukee, WI, USA,
4
Harper Hospital, Detroit, MI, USA, 5Emory
University, Atlanta, GA, USA, 6University of Nebraska
Medical Center, Omaha, NE, USA, 7University Clinic
of Vienna, Vienna, Austria, 8Schering-Plough
Research Institute, Kenilworth, NJ, USA and 9Royal
Brisbane Hospital, Brisbane, Australia
Background: Allogeneic hematopoietic stem cell
transplant (HSCT) recipients are at risk for lifethreatening invasive fungal infection (IFI). In patients
with graft-vs.-host disease (GVHD), IFIs are mainly due
to moulds, limiting the utility of prophylactic
35
xxx
fluconazole (FLU). We compared posaconazole (POS)
with FLU in preventing IFI in HSCT recipients with
GVHD on intensive immunosuppressives.
Methods: Patients in this double-blind study received oral POS (200 mg tid) or FLU (400 mg qd)
for up to 16 weeks. Incidence of IFI was determined
at 16 weeks and up to 7 days after last dose by EORTC/
MSG criteria adjudicated by a blinded expert panel.
Results: A total of 600 patients were enrolled (301
POS; 299 FLU). Incidence of proven/probable IFIs is
shown below. Mortality rate due to IFI was 1% in the
POS group vs. 4% in the FLU group; overall mortality
rate was 25% vs. 28%. Safety and tolerability were
comparable for both POS and FLU. Discontinuations
due to treatment failure were lower for POS vs. FLU
(3% vs. 8%), while those due to adverse events were
similar (33% each).
Proven/probable
IFIs
POS, n
(%)
FLU, n
(%)
Odds ratio
(95% CI)
P-value
At any time
Study period
(16 weeks)
Total
Aspergillus
Breakthrough
infections
Total
Aspergillus
20 (7)
42 (14)
0.43 (0.25–0.75)
0.003
16 (5)
7 (2)
27 (9)
21 (7)
0.56 (0.30–1.1)
0.31 (0.13–0.75)
0.07
0.006
7 (2)
3 (1)
22 (8)
17 (6)
0.30 (0.12–0.71)
0.17 (0.05–0.57)
0.004
0.001
Conclusions: POS was superior to FLU in preventing aspergillosis and as effective in preventing other
breakthrough IFIs, in HSCT patients with GVHD in
this study. Both POS and FLU were well tolerated.
O1.5
Safety, tolerance and outcome empirical
antifungal therapy with liposomal amphotericin
B in pediatric cancer/HSCT patients: a
postmarketing analysis
H. Kolve, J. Ritter, H. Juergens and A. H. Groll
University Children’s Hospital, Pediatric Hematology/
Oncology, Muenster, Germany
Objectives: Liposomal amphotericin B (AmBisome;
LAMB) is approved for empirical antifungal therapy in
patients (pts) with fever and granulocytopenia. However, little published information exists on its use in the
36
postmarketing setting. We therefore conducted a
single-center prospective survey on safety, tolerance,
and outcome in a large unselected cohort of pediatric
cancer/hematopoietic stem cell transplant (HSCT) pts
treated empirically with LAMB.
Methods: The survey identified 62 consecutive
children and adolescents (mean (±SEM) age:
9.48 ± 0.65 years; 26 f, 36 m) with hematological
malignancies (37), solid tumors (15), bone marrow
failure syndromes (nine) and non-neoplastic hematologic disorder (one) who received 75 courses of
LAMB started as empirical antifungal therapy
between Sept. 00 and Sept. 03. LAMB was administered until intolerance or maximum efficacy.
Timing of initiation and dosages of empirical therapy
were individually determined by the responsible
physician. Institutional guidelines recommended a
1–3 mg kg)1 starting dose to be administered within
48 h of persistent/recurrent fever and dosage adjustment as clinically indicated.
Results: Twenty-seven courses were postHSCT
(36%; 17 allogeneic, 10 autologous in pts with
solid tumors), and all courses were started during
granulocytopenia
(mean
duration:
15.3 ± 1.16 days; range 2–45). LAMB was administered for a mean duration of 15.8 ± 1.7 days
(range 1–109). The mean starting dose was
1.99 ± 0.09 mg kg)1 (95% CI: 1.80–2.19; range
0.94–3.94), and the mean maximum dose was
2.45 ± 0.10 mg kg)1 (95% CI: 2.24–2.66; range,
0.98–5.10). The dose was escalated (19) and/or
de-escalated (25; mostly to an every-other-day
regimen) in 38 of the 75 courses (50.6%). Mild
to moderate adverse events were noted during 54
courses (72%; hepatic, 56%; electrolyte wasting,
44%; renal, 17.3%; infusion related reactions,
15.6%); adverse events necessitating discontinuation of LAMB occurred in four courses (5.3%:
renal, 1; anaphylaxis, 2; hepatic, 1). While mean
GOT, GPT, AP and BUN values were slightly higher
at end of treatment (EOT), mean bilirubin and
creatinine values were not different from baseline
(BL). Sixty-four of the 75 courses (85.3%) of
empirical therapy were completed as success, as
defined by the absence of possible/probable/proven
breakthrough infection, no discontinuation due to
intolerance, and survival through EOT. Apart from
the four AE-related discontinuations and one
death with bacterial sepsis, failures included six
(8%) proven (one), probable (two) or possible
(three) breakthrough infections with ultimately
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
fatal outcome in two cases (2.7%). Overall survival
at EOT was 96% (72/75) and 92% (69/75) at
3 months postEOT.
Conclusion: In this postmarketing prospective survey in high-risk granulocytopenic pediatric cancer/
HSCT patients, empirical antifungal therapy with
LAMB was safe and provided an effective preventive
intervention against life-threatening invasive fungal
infections.
O1.6
Monitoring of voriconazole blood levels: 1-year
experience at a University Hospital
A. Pascual, S. Bolay and O. Marchetti
CHUV, Infectious Diseases, Lausanne, Switzerland
Background: Voriconazole (VRC) is first choice
therapy for aspergillosis and a new Rx option for
candidiasis. Inter- and intra-individual variations of
VRC blood levels related to non-linear pharmacokinetics, polymorphism of cytochrome CYP2C19, drug
interactions and hepatic dysfunction have been
reported and may affect efficacy or tolerance. The
aim of the study was to evaluate the clinical impact
of monitoring of VRC blood levels.
Methods: Retrospective analysis of 35 VRC Rx
courses in 33 adult patients (Pts) during 2004.
Standard definitions for diagnosis of mycosis, response
to therapy, and serious adverse events (SAE) (NCI
criteria). VRC blood levels were measured by HPLC.
Results: Indications for VRC therapy (median dose:
4 mg kg)1 bid, duration: 50 d) were aspergillosis
(58%), candidiasis (26%), and other mycosis (15%).
VRC blood levels were measured in 19 of 35 (54%) Rx
courses. Median # and intervals between VRC blood
levels measurements: 4.5 (1–9) and 7 d (1–62).
Among eight Pts with VRC levels >5.5 mg L)1, four
presented with neurological SAE (hallucinations and
encephalopathy, two each), which resolved after VRC
dose reduction (one) or discontinuation (three).
Median VRC troughs levels were not different in Pts
with or without SAE (6.3 vs. 7.0 mg L)1). However,
adjustment of VRC dosing limited median exposure to
VRC levels >5.5 mg L)1 in Pts without compared to
Pts with neurological SAE (5 vs. 12.5 d) (P=0.06).
Cholestasis was observed in 26% of Rx courses: no
correlation with VRC levels was found. No SAE
occurred in the 16 Rx courses during which VRC
blood levels were not measured.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
<0.2 (mg L)1) 0.2–5.5 (mg L)1) >5.5 (mg L)1) (c)
Success
1/2 (a,b)
SAE
- Neurological None
- Hepatic
None
7/9
8/8
None
2/9
4/8
1/9
(a) 1 Pt with rifampin interaction: persistent fever resolved.
(b) 1 Pt received VRC 400 mg d)1 orally: relapse of aspergillosis
responded to VRC dose increase (600 mg d)1).
(c) Median VRC dose 4 mg kg)1 bid (3.5–4.5). Tacrolimus interaction in one pt.
Conclusions: One-year experience of monitoring of
VRC blood levels suggests that prolonged overdosing
may be associated with neurological SAE. Monitoring
may help to improve management in this subset of
patients.
VRC trough levels (mg L)1) and clinical course:
O1.7
When an inexpensive initial therapy for
invasive aspergillosis with amphotericine B
becomes more expensive than voriconazole
J. Garbino,1 G. Schnetzler,2 C. Roberts3 and D. Lew1
1
University Hospital, Infectious Disease Division,
Geneva, Switzerland, 2Pfizer Schweiz AG, Medical
Department, Zürich, Switzerland and 3Pfizer Inc,
Outcomes Research, New York, USA
Introduction: New antifungal drugs for the treatment of invasive aspergillosis, e.g. voriconazole (VRZ)
show superior clinical outcome and tolerability
compared to conventional amphotericine B (CAB)
but the later is often used as initial treatment due to
lower drug acquisition costs. Therefore a cost-effectiveness analysis for VRZ as initial therapy has been
performed taking into account not only drug acquisition but also hospitalization and AE-related costs.
Material and methods: The cost-effectiveness model compares a regimen of VRZ followed by CAB to a
regimen of CAB followed by VRZ. Switch therapy
occurs based on the likelihood of toxicity or nonresponse. Costs are based on local drug acquisition
costs, local cost estimates for hospitalization and
locally observed frequencies for acute renal failure
(30%). Overall length of hospitalization and length of
stay on the ICU as well as changes in the initial therapy
due to treatment failure (13% for VRZ and 16% for
CAB) are taken from the Global Clinical Aspergillosis
study. Adjusted (without hospital and ICU compo-
37
xxx
nents) additional costs of CAB-induced acute renal
failure are calculated according to Bates et al.
Results: Based on this model initial therapy of IA
with VRZ is cost saving when compared to initial
therapy with CAB (CHF 37 894/patient vs. CHF
49 735/patient, respectively). Results are sensitive to
percent acute renal failure, but cost savings is
sustained for VRZ over a wide range of assumed
values. Additionally adjusted for the superior survival
rate for VRZ initial therapy, this results in a cost
benefit of CHF 32 378 per successfully treated patient.
Conclusion: Considering that initial therapy with
VRZ is both cost saving and results in better clinical
outcomes, VRZ is the dominant cost-effective option
for initial therapy of IA, despite very low drug
acquisition costs of CAB.
O2.1
Surveillance of waterborne Aspergillus in a
Portuguese University Hospital
R. Araujo,1 C. Pina-Vaz1 and A. G. Rodrigues2
1
Faculty of Medicine, Microbiology, Porto, Portugal
and 2Faculty of Medicine and Hospital S. Joao,
Anesthesiology, Porto, Portugal
Aspergillus species cause serious infections among
immunocompromised and transplant patients. Water
has been described as a possible route of infections
caused by Aspergillus. Our objective was to quantify
Aspergillus in water supplied to hospital wards, intensive care units and operating theatres of a University
Hospital during the summer (four consecutive weeks
from 23 June to 22 July of 2004) and the autumn (four
consecutive weeks from 22 September to 15 October of
2004). Cold and hot water supplied to 16 rooms was
evaluated in this study, corresponding to two operating theatres, four intensive care units, two hematological units and 10 common wards. From operating
theatres, samples of sterile (filtered) water used for
washing hands of medical personal were also collected. Water quality was studied by analysis of 500 ml of
water filtered through cellulose membranes of
0.22 lm. Membranes were incubated in DG18 culture
medium during 10 days, at 37 C. Water samples were
not commonly contaminated with Aspergillus species.
A. fumigatus, A. flavus and A. niger were rarely isolated
from water samples and were never detected in water
from the same room in consecutive weeks. No other
Aspergillus species were detected. No significant differ-
38
ence was found comparing between cold and hot
water. Filtered water from operating theatres never
presented any contamination with Aspergillus species.
It is possible to detect occasionally in hospital water
some Aspergillus strains that may cause, in last
instance, infections in high-risk patients. However, it
is difficult to consider water an important route for
infections in our Hospital. The use of filters may limit
this route, preventing further infectious.
Acknowledgment: This study was supported by
grant no. 60901 from Fundaçao Calouste Gulbenkian.
O2.2
Rare mycotic infections in tsunami survivors
C. Garzoni,1 M. Djordjevic,1 I. Uçkay,1
K. Bouchuigui-Waf,2 L. Legout,3 B. Rilliet,4
L. Bernard3 and J. Garbino1
1
Infectious Diseases, University Hospitals of Geneva,
Geneva, Switzerland, 2Bacteriology, University
Hospitals of Geneva, Geneva, Switzerland,
3
Orthopaedic Surgery, University Hospitals of
Geneva, Geneva, Switzerland and 4Neurosurgery,
University Hospitals of Geneva, Geneva, Switzerland
Hundred thousands people died or were severely injured
during tsunami on December 26. First aid was performed in local hospitals in very difficult situations. Two
Swiss tourists were locally treated and referred to our
institution. Both developed severe infections caused by
multi-resistant bacteria and unusual fungal infections.
Case 1: A previous healthy 59-year-old man was
initially treated due to an aspiration pneumonia and
septic shock caused by Acinetobacter baumanii multiresistant and Escherichia coli. Three weeks later he was
repatriated. Several residual pulmonary abscesses and
an empyema were drained and finally a lobectomy was
performed. After 6 weeks of treatment with piperacillin-tazobactam he was discharged. One month later,
he was readmitted to drain a paravertebral collection
with spodylodiscytis (T8–T9) caused by Scedosporium
apiospermum. The spine was immobilized with a corset
and voriconazole 200 mg p.o. bid was started. No
neurology complications were evidenced.
Case 2: A healthy 51-year-old woman survived for
more than 24 h in the mud with severe wounds of the
legs, pelvis fractures and bladder rupture. She was
stabilized and repatriated 5 days later. At admission,
wounds were infected with multi-resistant bacteria
and different Candida species. Pelvic fractures were in
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xxx
direct contact with urine due to a bladder rupture. A
panresistant Acinetobacter baumanii only sensible to
colistin was cultured in the urine. Piperacillinetazobactam with concomitant debridements and local
colistin were started. A Nocardia africana abscess of the
tight was treated with surgical drainage, meropenem
and trimethoprim/sulfamethoxazole. Persistent fever
and conscious state alteration motivated a cerebral CT
scan on week 8, which showed an intracerebral
abscess with hydrocephalus. The abscess was drained
and a ventriculo-peritoneal drainage was posed. The
abscess cultured Scedosporium apiospermum. Voriconazole 4 mg kg)1 IV bid was started.
These two patients represent a paradigm of unusual
and difficult-to-treat pathogens, which could be
encountered in tsunami survivors. The tragedy constitutes an exceptional event and several factors put
wounded at risk in the short and the long-term followup. Patients inhaled tepid water and lied several hours
in warm stagnant slush, where bacteria and fungi
found ideal conditions to colonize open bone wounds
and disseminate in immunocompetent patients. Taking advantage from the large inoculum, extended soft
tissue and internal injuries, hospitalization in emergency conditions and the selection pressure with
longstanding broad-spectrum antibiotic-therapy, emergence of multi-resistant bacteria and unusual fungal
infections should be suspected. Every patient should be
considered at very high risk for severe infections which
constitute a difficult challenge for diagnosis and
treatment even for infectious disease specialists.
O2.3
the incidence of opportunistic infections, particularly those caused by fungi. Among them, infections caused by Scedosporium spp. have emerged as
an important entity. In response to an increase in
Scedosporium infections in Australia a national
observational study, the Australian Scedosporium
(AUSCEDO), was initiated. Epidemiological, clinical
and outcome data on all infections reported from
January 2003 for 3 years were collected prospectively. Scedosporium isolates were forwarded to a
central laboratory for molecular analysis. One
hundred and fifty six Scedosporium isolates (most
of the AUSCEDO study isolates plus some previously collected isolates; 69 S. apiospermum and 87
S. prolificans) were analysed for genetic relatedness
by PCR-fingerprinting. PCR-fingerprinting analysis,
using the core sequence of phage M13, clearly
separated S. apiospermum and S. prolificans from
each other and showed varying degrees of intraspecies variation (more than five subtypes per
species). There was no correlation between subtypes or types and their geographical origin, the
body site of isolation, and the patient’s Scedosporium status (colonisation or infection). The S.
prolificans population caused a higher percentage
of invasive infections than S. apiospermum and is
being studied further. The amplified fragment
length polymorphism (AFLP) method has been
developed to analyse the genetic diversity of
epidemiologically related S. prolificans isolates (isolates from two nosocomial hospital outbreaks).
These isolates will be compared with a panel of
unrelated Australian isolates representative of our
S. prolificans population.
Scedosporiosis in Australia: epidemiology of infection and molecular typing of Scedosporium species
L. Delhaes,1 S. Chen,2 C. Heath,3 Q. Nguyen,4
M. Slavin,5 C. Halliday,2 T. Sorrell2 and W. Meyer2
1
Molecular Mycology Laboratory, Westmead
Hospital & University of Sydney, Sydney, Australia,
2
Westmead Hospital, Centre for Infectious Diseases
and Microbiology, Sydney, Australia, 3Department of
Microbiology and Infectious Diseases, Royal Perth
Hospital, Perth, Australia, 4Department of
Microbiology, St Vincent’s Hospital, Sydney, Australia
and 5Department of Infectious Diseases and
Microbiology, Alfred Hospital, Melbourne, Australia
The ever increasing numbers of immunosuppressed
individuals has led to a significant increase in
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
O2.4
Isolation of Cryptococcus adeliensis from
clinical samples and the environment in
Germany
K. Tintelnot, E. Antweiler and H. Losert
Robert Koch-Institut, Abt.1, FG 14 Mykologie,
Berlin, Germany
1
A first case of meningitis caused by Cryptococcus
adeliensis in a patient with acute myeloid leukemia
has been published recently (1). C. adeliensis has
been described as a separate species within the C.
albidus clade (2) and can be misidentified as C.
39
xxx
albidus. C. adeliensis is phenotypical variable: creamcolored and smooth to dry on Sabouraud-agar, a
touch of brown-color-effect on Guizotia abyssinicaagar after 2 weeks of incubation, no tolerance of
35 C in vitro. In contrast to the type strain from
the Antarctic the European isolates do not tolerate
10% NaCl and 0.01% cycloheximide. C. adeliensis
has been isolated from clinical samples of the
respiratory tract as well as in bird droppings from
Berlin and Hannover. Reidentification of so-called
C. albidus-isolates of our strain collection revealed,
that C. adeliensis has been isolated from clinical
2 samples and the environment since 19763). We
should pay attention of this rather unknown
Cryptococcus species.
References:
(1) Rimek D, Haase G et al. First report of a case of meningitis
caused by Cryptococcus adeliensis in a patient with acute
myeloid leukemia. J Clin Microbiol 2004; 42: 481–3.
(2) Scorzetti G, Petreswcus I et al. Cryptococcus adeliensis sp. nov., a
xylanase producing basidiomycetous yeast from Antarctica.
Antonie Leeuwenhoek 2000; 77: 153–7.
(3) Tintelnot K, Losert H. Isolation of Cryptococcus adeliensis from
clinical samples and the environment in Germany. J Clin
Microbiol 2005; 43: 1007.
O2.5
Multi-locus sequence typing of pathogenic
fungi
M. D. Jacobsen, A. D. Davidson, J. A. Whyte,
N. A. R. Gow, D. J. Shaw and F. C. Odds
Aberdeen Fungal Group, Institute of Medical
Sciences, Aberdeen, UK
Multi-locus sequence typing (MLST) affords an
unambiguous approach to epidemiological typing
of microbial species based on the variability of
nucleotide sequences in a number of housekeeping
genes. With pathogenic fungi, schemes have already
been published for Candida albicans and Candida
glabrata. Here, MLST schemes for Aspergillus fumigatus, Candida tropicalis and Candida krusei are described. Isolates from a wide range of geographical
and anatomical sources have been analysed to
demonstrate the value of MLST in determining
epidemiological relationships between isolates and
population structures within the species. Data
presented will reflect the current status of these
on-going projects.
40
O2.6
Two days diagnosis of onychomycosis including
epidemiological typing of Trichophyton rubrum
strains
V. Kardjeva,1 T. Kantardjiev,1 R. Summerbell,2
D. Devliotou-Panagiotidou,3,4 T. Koussidou3 and
Y. Graeser5
1
Microbiology, National Centre of Infectious and
Parasitic Diseases, Sofia, Bulgaria, 2Centraalbureau
voor Schimmelcultures (CBS), Utrecht, The
Netherlands, 3Mycological Laboratory, State Hospital,
Thessaloniki, Greece, 4Dermatology, Aristotle
University of Thessaloniki, Thessaloniki, Greece and
5
Department of Parasitology (Charitŭ), Institute of
Microbiology and Hygiene, Berlin, Germany
Onychomycosis is a fungal infection of the nail unit
caused by dermatophytes, yeasts and moulds. Onychomycosis occurs worldwide and its prevalence is
2%–18% of the whole human population or even
higher. The most significant causative agent is
Trichophyton rubrum with an incidence of more than
90% in Europe. Less commonly, Trichophyton interdigitale is involved. Nail invasion by moulds is
considered uncommon. Treatment of onychomycosis
is closely linked to the identity of causative agent. The
most commonly used oral antifungals include itraconazole, terbinafine and fluconazole; these drugs have
superseded the traditional therapies with griseofulvin
and ketoconazole. While terbinafine and itraconazole
demonstrate efficacy against T. rubrum and T. interdigitale, griseofulvin and fluconazole are ineffective
against T. interdigitale and moulds, e.g. S. brevicaulis,
which may cause the infection. Current diagnosis and
treatment procedures for onychomycosis rely on
direct microscopic examination of fungal elements
in nail material using 30% KOH, in combination with
and the identification of the etiological agent based on
the examination of its colony and microscopic
morphology. These methods are low in specificity
and very time consuming, because growth, sporulation, and routine physiological testing of the fungi
involved may take 2–4 weeks. In addition, 15%–50%
of microscopically true-positive samples fail to grow a
culture. We present a strategy for the molecular
identification of fungal agents of onychomycosis
using species-specific primer pairs as well as universal
primers in conjunction with a commercial kit allowing the extraction of DNA directly from the nails. The
microsatellite marker T1 which is based on a (GT)n
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xxx
repeat has been applied for the species-specific identification of T. rubrum. To detect the remaining species
of onychomycosis the internal transcribed spacer
(ITS) region of the rDNA has been used. In addition,
using separation of the T1-PCR product on a polyacrylamide gel subtyping of strains of T. rubrum was
possible. We studied 195 nail specimens and 66
selected etiologic strains from 261 onychomycosis
patients from Bulgaria and Greece. T. rubrum was the
most common organism detected (76%) followed by
T. interdigitale and other moulds. Subtyping revealed
that all but four strains were of the common type B of
T. rubrum which is prevalent in Europe. In comparison
to microscopy and culture the PCR was more sensitive
(77%) than the culture (22%) and more specific
(100%) compared to the non-specific microscopy.
Using the molecular approach the time for diagnosing
the identity of fungi causing onychomycosis could be
reduced to 24 h. The early detection and identification of the infecting species in nails will facilitate
prompt and appropriate treatment and may be an aid
for the development of new antifungal agents.
O2.7
PCR in onychomycosis: direct detection of
dermatophytes in clinical specimens offers new
opportunities for diagnosis
A. Y. Sergeev,1 S. N. Scherbo,2 Y. V. Sergeev,1
P. G. Bogush,3 V. M. Leschenko3 and N. V. Nekhaeva1
1
All-Russian National Academy of Mycology, Moscow,
Russia, 2NPO Genetech, Moscow, Russia and
3
Moscow City Mycological Center, Moscow, Russia
Several new techniques were proposed recently
to improve the accuracy of laboratory diagnosis of
superficial fungal infections. Being expensive or timeconsuming, none of them are suitable for routine
confirmation of clinical diagnosis. Limitations of
conventional methods: KOH direct microscopy and
culture make current laboratory diagnosis of onychomycosis far from being perfect. With sensitivity of
microscopy approximating 80% and culture reaching
50–60% in best mycology units, both methods depend
strongly on quality of nail samples collection and skills
of laboratory staff. PCR provides a new opportunity for
diagnosing cutaneous fungal infections. We report
results of multi-centre study evaluating the clinical
application of PCR for laboratory diagnosis of onychomycosis. Direct PCR probes for detection of dermatophyte DNA in clinical specimens were designed in
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
Russia in late 2003. Using a DNA fragment encoding
fungal topoisomerase II, species-specific probes were
developed for Trichophyton rubrum and T. mentagrophytes var. interdigitale, together contributing to at
least 95% of dermatophyte onychomycosis in Russia.
Preliminary blind testing of new duplex method with
44 samples succeeded in early 2004 (Sergeev et al.,
2004). In a current study, 1358 specimens from
patients with onychomycosis and other nail conditions were collected by eight dermatologists for several
clinical centres of Moscow. Microscopy was performed
in 1006 cases, PCR in 941, culture in 258; combinations of different tests were used in estimations of
sensitivity and specificity. Among valid cases, microscopy was positive in 51.7% of samples taken, culture
in 40.7%, PCR in 55.5%. True positive cases were
assigned as positive results by any method (60.7%) or
conventional only (53%). Sensitivity of PCR was
estimated as 82.5% and 98.9% when excluding nondermatophyte culture cases. Specificity of PCR was
found 100% (no false-positives) or 77% when relying
on either conventional method positive. When both
positive culture and microscopy were used as referent
results, sensitivity of PCR was 92.9% and specificity
68.5%. In contrast to conventional methods, results of
PCR were not dependent (with statistical significance)
on doctor taking sample. PCR may become the new
gold standard for laboratory diagnosis of onychomycosis, since its accuracy surpasses conventional
methods. It may be used as a standard approach to
confirm the diagnosis of onychomycosis together with
KOH microscopy or even alone.
O2.8
Kerion Celsi: the changing face of Microsporum
species infection?
M. Skerlev
Department of Dermatology and Venereology,
Zagreb University Hospital Center and Medical School
of Zagreb University, Zagreb, Croatia
Kerion Celsi is a highly inflammatory, suppurative
fungal infection of the scalp caused by zoophilic
dermatophytes in the majority of (but not in all) cases.
Trichophyton (T.) mentagrophytes, rubrum, and violaceum have been traditionally recognized as typical
pathogens in such cases, however, the clinical
features and the etiologic agents of fungal scalp
infections these days might sometimes be quite
41
xxx
different from the routine we have been used to. Thus,
some atypical and unusual variations of tinea capitis
due to Microsporum (M.) species are presented
regarding the clinical pattern. There has been an
epidemic outbreak of M. canis infection in Croatia in
the last 25 years, from one positive culture in 1978
up to 407 positive isolates in 2004. The scalp was
involved in about 30% of all these infections. In the
majority of cases, the clinical pattern was the
superficial tinea capitis with the small spored ectotrix
type. However, during the last 5 years, 48 cases of
typical kerion Celsi due to Microsporum species
(belonging to the both M. canis and M. gypseum
species) were observed (not mixed infection with
T. mentagrophytes). The clinical features consisted of
painful inflammatory mass on the scalp with loose
hairs, pustular discharge, sinus formation, mycetoma
like grains and thick crusting. In 39 cases, M. canis,
and in nine cases, M. gypseum were isolated by
culture. Some examples of the kerion Celsi due to M.
canis in adults are presented, as well. Staphylococcus
aureus was isolated from the scalp surface overlying
the kerion and from the pus within the kerion in
about 70% of patients. Gram-negative bacteria were
found in the same locations in about 10% of patients,
respectively. These data indicate that bacteria are
frequently cultured from kerions. Kerion Celsi (especially due to M. canis) represents a certain therapeutic
problem because of the impressive clinical features
and children’s age in the majority of cases. In the first
stage of the treatment, the antibacterial agents should
be topically applied in the thick layer. Otherwise,
using only topical antimycotic therapy in kerion Celsi,
the course of disease may be prolonged and the
therapeutic result may be poor. The antimycotics for
oral use should be applied, as well. However, it should
be pointed out that kerion due to M. canis represents a greater therapeutic problem as compared to
T. mentagrophytes. Moreover, the new or previously
very sporadic etiologic agents causing kerion have
been recently observed in Croatia, such as T. tonsurans, mostly in wrestlers, so far. The ‘open questions’
regarding kerion, such as the role of bacteria, and the
appropriate treatment including the use of steroids to
modify the intensity of the inflammatory response
resulting with scarring have not been completely
answered, so far. The most recent advances require
corresponding evolution of diagnostic and treatment
strategies of kerion in order to provide the suitable
laboratory testing and antifungal therapy and to
prevent the unnecessary surgical procedures.
42
O3.1
Expression of epithelial Toll-like receptor (TLR) 4
in a model of oral candidosis supplemented by
polymorphonuclear leukocytes is crucial for
host defence
M. Schaller,1 H. C. Korting2 and G. Weindl1
1
Department of Dermatology, Eberhard Karls
University, Tübingen, Germany and 2Department of
Dermatology, Ludwig-Maximilians-University,
München, Germany
In humans, cells of the innate immune system can
discriminate between pathogens and self by using
signals from a family of ten Toll-like receptors (TLRs).
TLRs recognise conserved motifs called pathogenassociated-molecular-patterns (PAMPs), which represent broad groups of microbial pathogens or
components (bacteria, fungi, RNA, DNA). Despite
extensive studies in this new field, little is known
regarding the role of TLRs in mucosal immunity
against common fungal pathogens like C. albicans. In
addition, the interaction of epithelial cells with
principal cells of the mucosal innate defence system,
such as polymorphonuclear leukocytes (PMNs), has
not been studied in detail. In this study, using an
established model of oral candidosis based on reconstituted human oral epithelium (RHE), we examined
the role of Toll-like receptors (TLRs) in sensing C.
albicans by oral epithelial cells and the affect of PMNs
on this interaction. Stimulation of oral epithelium by
C. albicans induced a strong cytokine response (GMCSF and IL-8), but failed to induce TLR expression by
quantitative RT-PCR, confocal laser and immunoelectron microscopy. However, when the C. albicansinfected oral RHE model was supplemented with
PMNs we detected a strong increase (>100-fold) in
epithelial TLR4 expression. This upregulation was
observed after direct application of PMNs to the
epithelial surface and also when PMNs were placed to
the basal side of the model, which has a microporous
filter that inhibits cell migration and hence direct
interaction of the PMNs with the epithelial cells.
Confocal laser and immunoelectron microscopy confirmed stimulation of epithelial TLR4 expression on
the protein level and demonstrated TLR4 presence
only by epithelial cells directly in contact with
C. albicans. Upregulation of epithelial TLR4 in the
presence of PMNs was also associated with an
attenuated virulence phenotype of C. albicans, which
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
could be reversed by anti-TLR4 neutralising antibodies. The data implicates a pivotal role for ‘immunological cross-talk’ between C. albicans-infected human
epithelium and PMNs, resulting in the TLR-mediated
sensing of C. albicans by human epithelial cells. In
addition, our RHE model provides an ideal tool to
characterise host/pathogen interactions and to study
the mechanisms involved in the TLR-mediated sensing of C. albicans at mucosal surfaces.
O3.2
Innate immunity against Aspergillus fumigatus
M. Dubourdeau,1 R. Athman,2 V. Balloy,3 M. Brock,4
M. Chignard,3 D. Philpott,2 J. P. Latgé1 and
O. Ibrahim-Granet1
1
Aspergillus, 2Immunité Innée et Signalisation,
3
Défense Innée et Inflammation. Institut Pasteur,
Paris, France and 4Institute for Microbiology,
University of Hannover, Germany
Aspergillus fumigatus is a human pathogen, able to
cause invasive aspergillosis in immunosuppressed
patients. In the immunocompetent situation inhaled
conidia are easily cleared by the immune system.
Therefore, one aim of our investigations was the
identification of factors involved in the clearance of
conidia. One of the major cytokines involved in
immune defence is TNF-alpha. A high level of TNFalpha synthesis is directly accompanied with an
increased resistance against an A. fumigatus infection.
TNF-alpha production is triggered by MAP kinases
like ERK and p38. Our investigations revealed that
both MAP kinases of alveolar macrophages are
activated under in vitro conditions, when conidia of
A. fumigatus were applied. Actually, in vivo experiments showed that most likely only ERK is directly
involved, because under these conditions activation
of p38 was negligible. Immunosuppression with
corticosteroids inhibited phosphorylation of ERK and
was directly accompanied with a strongly decreased
level of TNF-alpha and additional cytokines. Therefore, ERK seems to be an essential MAP kinase in the
defence system against A. fumigatus. Another system,
which is able to stimulate the excretion of cytokines
for immune defence is the direct activation of
NFkappaB after stimulation by toll-like receptors.
Due to controversial opinions about the involvement
of TLR2 and TLR4 in signalling and response towards
A. fumigatus we investigated the in vivo response
towards conidia of respective knock-out mice.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
Interestingly, in our study neither one of these
receptor mutants showed a significantly altered
cytokine expression and the immunocompetent mice
did not show a higher susceptibility to obtain an
invasive aspergillosis. Therefore we conclude, that
these receptors are dispensable in the clearance of A.
fumigatus under immunocompetent situations.
O3.3
Multilocus Microsatellite Typing (MLMT) of
Aspergillus fumigatus: problems with sizing
repeats and the development of an alternative
system using longer repeat units
A. C. Pasqualotto,1,2 D. W. Denning1,2 and
M. J. Anderson1
1
School of Medicine, The University of Manchester,
Manchester, UK and 2Wythenshawe Hospital,
Manchester, Germany
Aspergillus fumigatus is the most common mould
pathogen of humans, causing both life-threatening
invasive disease in immunocompromised patients and
allergic disease in patients with atopic immune
systems. Several phenotypic and genotypic techniques have been developed for examining the
variability of A. fumigatus, but only a limited number
of these approaches have been found to be useful for
the intraspecific clustering and typing of isolates.
Multilocus sequence typing (MLST) is a nucleotide
sequence-based approach for the unambiguous characterisation of isolates of bacteria and other organisms and it has become the method of choice for
epidemiological investigations. Although it is useful
for phylogenetic studies, preliminary data from other
laboratories have revealed that MLST does not
provide sufficient discrimination for typing A. fumigatus isolates. Microsatellites are 1–6 bp motifs that are
tandemly repeated. The higher mutation rates at
these loci [10)2 to 10)6 mutations per generation as
compared to 10)9 for point mutations (as detected by
MLST)] allow variation to accumulate at a far higher
rate, leading to greater genetic diversity in a population. A microsatellite scheme has been developed for
A. fumigatus, based on the amplification of four
dinucleotide repeats (1). This MLMT scheme has been
used to type isolates in our laboratory, either by sizing
alleles using an automatic sequencer with fluorescently labelled primers or by direct sequencing.
However, comparison of the sizes between the two
techniques revealed differences of up to 5 bp. These
43
xxx
data will be presented. These differences demonstrate
the problem of using an automatic sequencer for
sizing PCR fragments. Although a precise size was
obtained, it was not always accurate as was seen
when it was compared to the actual size as determined
by sequencing. This can mean that laboratories could
report differing repeat sizes for the same isolate and
means that a microsatellite-based typing scheme is
not readily portable. The aim of this study is to develop
a new microsatellite-based scheme for A. fumigatus
typing. It is our hope that by using 5 and 6 bp repeat
units, it will possible for sizes to be determined
accurately on automatic sequencers as well as
enabling sizing to be done on agarose gels. Therefore
the scheme should provide a rapid and cheap way of
discriminating between isolates of A. fumigatus if
required. The availability of genomic sequence from
two A. fumigatus isolates has enabled us to identify
suitable loci that are present in both isolates and that
differ in the number of repeat units. Preliminary data
will be presented on the polymorphism of a selected
few loci amongst 16 isolates. The appropriate number
of loci will be developed to provide sufficient discrimination to generate a unique type for most strains.
Reference:
1. Bart-Delabesse et al. J Clin Microbiol 2001; 39:
2683–6.
observe selection of certain strains of P. boydii under
the influence of high concentrations of nitrate and
phosphate sources. A further aim of this study was to
detect possible molecular changes under the given
experimental settings.
Methods: For the experiment a selection of strains
was used, representing molecular subgroups of
P. boydii, which were isolated from clinical materials
and from the environment. The strains were confronted with each other pairwise in liquid culture
with high concentrations of KNO3 and KH2PO4 as
sole nitrate and phosphate sources for 2 weeks at
20 ordm;C. After the exposure to KNO3 and KH2PO4
the strains were characterised with molecular methods, namely fingerprinting and RAPD-analyses
(M13-, UBC 701-Primers).
Results and discussion: In each experiment the
environmental strain had the most competitive potential compared to the strains from clinical materials. In
similar experiments on solid media a clinical strain
had the highest competitive potential. Molecular
changes were assumed in one experiment for 12
strains with KH2PO4. We showed that high concentrations of N- and P-sources, in agricultural areas
originating from fertilisation, may have a selective
effect on populations. The route of selection in the
environment is hard to estimate, because other influences like aeration can also be determining factors.
O3.4
O3.5
Selection and molecular changes of
Pseudallescheria boydii under the influence of
high N- and P-concentrations
Cyclic peptides as specific markers for
diagnosing fungal infections
A. Zacke, E. Lackner and J. Rainer
Institute of Microbiology, Leopold-FranzensUniversity, Innsbruck, Austria
Introduction: Pseudallescheria boydii is an opportunistic human pathogen. This fungus can cause
several severe clinical pictures, among them infections of the CNS. P. boydii has been isolated from
eutrophic and anthropogenic influenced habitats like
excessively fertilised areas, city ponds, manure and
ornithogenic soil. P. boydii is a molecular-biologically
markedly variable species, which is reflected in strainspecific virulence and N- and P-tolerance. From that it
is hypothesised that eutrophication could support the
accumulation of specific strains, presumably more
virulent individuals of P. boydii. Do we propagate our
own pathogens? A laboratory model was created to
44
V. Havlicek,1 M. Sulc,1 A. Jegorov,2 M. Zabka,1
M. Hajduch3 and O. Ditrich4
1
Institute of Microbiology, Prague 4, Czech Republic,
2
IVAX-Pharmaceuticals, Ceske Budejovice, Czech
Republic, 3Palacky University, Olomouc, Czech
Republic and 4Institute of Parazitology, Ceske
Budejovice, Czech Republic
Several new cyclic peptides and depsipeptides were
detected by mass spectrometry on fungal spores or in
medium extracts from Pseudallescheria, Beauveria,
Paecilomyces and Trichothecium. These compounds
are synthesized non-ribosomally by multienzyme
systems and hence can serve as extremely specific
fungal markers. Cyclic peptides can easily be recovered
from the whole blood (low picomol range with 2.1 mm
ID HPLC column). Beauverolides and roseotoxins
demonstrated cytotoxic activity in vitro. The most
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
sensitive were hematopoietic cell lines (CEM, K562
and B2.4), but significant cytotoxicity was observed
also in solid tumor representatives (A549, HT29,
MCF7). Unfortunately, the mycotoxins were also
cytotoxic for resting human lymphocytes suggesting
their usefulness as potential anticancer drugs. However, direct toxicity against immune cells (lymphocytes and macrophages) indicates that both compounds may play a role in host–pathogen interaction.
Rosetoxins but not beauverolides were potent inhibitors of LPS mediated induction of macrophage NOsynthase activity. This result indicates a potential antiinflammatory activity of roseotoxins, which again
may be involved in host–pathogen interaction. Both in
the immunocompromised or immunodeficient mice
no gliotoxin, verruculogen or fumagillin were found.
Particularly, the immunosuppressive, but non-specific
gliotoxin, was formerly assumed as a plausible marker
candidate. Despite the mice exhibited neurological
symptoms and histopathology has revealed a positive
fungal invasion, no gliotoxin was found in any case.
Acknowledgments: This work was supported by
the Czech Science Foundation (203/04/0799),
Czech Ministry of Education, Youth and Sports
(LC545 and MSM6198959216), European Commission (MTKD-CT-2004-014407).
Mn and AMn were measured by ELISA (Platelia,
Bio-Rad). Positive tests defined by two consecutive
values of GMn > 0.4 or >0.5 OD Index, of
Mn > 0.5 ng mL)1 and of Amn > 10 AU mL)1.
Results: 11 IA (three proven, eight probable) and
13 IC (one proven, 12 probable) occurred in 125
neutropenic episodes. 16 samples per episode (3–35)
were analyzed over 35 d (17–122).
GMn (IA)
Cut-off
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
>0.3
91
88
53
98
>0.4
82
95
69
97
>0.5
64
96
70
95
Mn/Amn
(IC)
GMn/Mn/AMn
(IA+IC)
>0.5/10
77
95
77
95
>0.4/0.5/10
91
89
77
96
GMn: sensitivity of 0.4 cut-off was higher than that
of 0.5, specificity was similar. Mn/AMn: in 90% of
Pts with IC, positive tests preceded clinical diagnosis
by a median of 10 d. GMn/Mn/AMn: in 89% of Pts
with IFI positivity anticipated conventional diagnosis
by a median of 6 d.
Conclusions: In neutropenic cancer patients, twice
weekly monitoring of GMn/Mn/AMn may facilitate
early non-invasive diagnosis of invasive fungal
infections.
O3.6
Combined galactomannan (GMn), mannan
(Mn), and anti-mannan (AMn) for diagnosis of
invasive fungal infections (IFI) in neutropenic
cancer patients (Pts)
L. Senn,1 J. O. Robinson,1 S. Schmidt,2 M. Knaup,1
B. Duvoisin,2 J. Bille,1 T. Calandra1 and O. Marchetti1
1
CHUV, Infectious Diseases, Lausanne, Switzerland
and 2CHUV, Radiodiagnostic, Lausanne, Switzerland
Background: Invasive aspergillosis (IA) and candidiasis (IC) are life-threatening complications in
neutropenic cancer Pts. Diagnosis remains difficult
and is often delayed when relying on conventional
methods. Detection of fungal antigens and antibodies
may speed up diagnosis and facilitate antifungal Rx.
Aim: To evaluate the utility of a combined use of
GMn, Mn and AMn.
Methods: Prospective evaluation of 125 consecutive
episodes of neutropenia (median duration 23 d) in Pts
with acute leukemia. IFI was diagnosed according to
EORTC-MSG criteria. Blood was collected 2· weekly
before onset of fever and daily thereafter. GMn,
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
O3.7
Caspofungin: antifungal activity in vitro,
pharmacokinetics, and effects on animal
survival and fungal load in neutropenic rats
with invasive pulmonary aspergillosis
W. van Vianen,1 S. de Marie,1,2 R. A. A. Mathot3 and
I. A. J. M. Bakker-Woudenberg1
1
Medical Microbiology & Infectious Diseases,
Erasmus MC, University Medical Center Rotterdam,
Rotterdam, The Netherlands, 2Internal Medicine,
Section of Infectious Diseases, Erasmus MC,
University Medical Center Rotterdam, Rotterdam,
The Netherlands and 3Hospital Pharmacy, Clinical
Pharmacology Unit, Erasmus MC, University Medical
Center Rotterdam, Rotterdam, The Netherlands
Testing of new antifungals for the treatment of
invasive pulmonary aspergillosis (IPA) in vitro and in
animal models, in relation to pharmacokinetics, is
important in order to evaluate their potential and
optimize the design of clinical trials. The antifungal
activity of caspofungin (CAS) vs. amphotericin B
45
xxx
(AMB) was investigated in vitro as well as in an
inhalation model of Aspergillus fumigatus infection in
neutropenic rats, using survival and decrease in
fungal burden as parameters for therapeutic efficacy.
Whereas AMB produces concentration-dependent
reduction in fungal growth with a sharp endpoint in
vitro, CAS shows a gradual decrease in fungal growth.
This makes the visual reading of CAS endpoints using
the NCCLS recommended method challenging. A
quantitative XTT assay measuring fungal metabolism
appears more appropriate for CAS susceptibility testing. Using this assay, CAS was less active than AMB
evidenced by a fourfold difference in the concentration
required for ‡50% inhibition of fungal growth in vitro.
The efficacy of CAS was also measured in a rat model of
pulmonary aspergillosis. Therapy was started 16 h
after fungal inoculation when hyphal growth in the
left lung was established, and continued once-daily for
10 days. From the start of the infection until the end of
dosing, rats were persistently neutropenic, but neutrophil numbers gradually increased after day 11
postinfection. Treatment regimens included CAS
administered intraperitoneally at 1, 2, 3 or
4 mg kg)1 d)1 (CAS 1, 2, 3 or 4), AMB at the
maximum tolerated dose in this animal model
(1 mg kg)1 d)1; AMB1), or a combination of CAS 1
and AMB 1. Treatment with CAS 1 or AMB 1 showed
comparable moderate efficacy (rat survival 60% and
55% respectively), at the end of the treatment period,
and no significant difference after the whole 21-day
observation period (P = 0.24). The combination of
CAS and AMB did not show additive effects. Increased
dosage of CAS up to 2, 3 or 4 mg kg)1 d)1 resulted in a
significant, dose-dependent increase in therapeutic
activity which was, superior to AMB 1. There was
100% survival among rats in the CAS 4 group which
correlated with a significant decrease in fungal
burden. The galactomannan concentration in serum
and lung tissue as well as the amount of A. fumigatus
DNA in lung tissue was used to quantify fungal
burden. The pharmacokinetics of the CAS 4 dosage in
rats shows a 24-h AUC that is similar to the 24-h AUC
achieved by 50 mg CAS dosage in men, which is the
current maintenance dose in the clinical treatment of
IPA. Given the dose-dependent efficacy and the
excellent safety profile of CAS, it is worthwhile to
investigate the therapeutic efficacy of CAS administered at further increased dosage against advanced
stage of severe aspergillosis in rats. Such studies may
give further insight into optimal dosage of CAS in
severe fungal infections in patients.
46
P001
Evolutionary fitness in itraconazole susceptible
and resistant Aspergillus fumigatus clinical
isolates
A.M. Albarrag, M.J. Anderson and D.W. Denning
Department of Medicine, University of Manchester,
Manchester, UK
Introduction: Aspergillus fumigatus is the most
common aetiological agent of aspergillosis. Invasive
aspergillosis is a major cause of death in leukaemic
and organ transplant patients. The increasing rates
of recovery of antimicrobial resistant micro-organisms in hospital and community settings are of
growing concern. The evolutionary changes of drug
resistance and its fitness penalty have been extensively studied in bacteria, viruses and yeasts. In
many organisms, mutations that confer resistance to
drugs confer a biological fitness cost that is expressed
as decreased growth rate, survival or virulence. Loss
of fitness causes resistant organisms to become
disadvantaged relative to susceptible strains in the
absence of drug pressure.
Methods: Selected itraconazole susceptible and
resistant clinical isolates of A. fumigatus were studied.
Various measures of growth rate (colony radial
growth rate and specific growth rate), germination
time and conidial yields have been determined on
various media. Mutations in the 14-alpha lanosterol
demethylase gene, cyp51A, were determined.
Results: There was a correlation between resistance
and specific growth rate (P = 0.03). Generally,
resistant isolates grow more slowly than susceptible
isolates. Three resistance profiles were observed in
these clinical isolates. One isolate was resistant to
itraconazole only, two isolates were resistant to
itraconazole and ravuconazole, and two isolates were
resistant to itraconazole, voriconazole, ravuconazole,
and posaconazole. Sequencing the cyp51A gene of
these isolates revealed three different amino acid
substitutions at three different codons.
Conclusions: It is hoped that this and additional
molecular studies will highlight mutations that are
the potential cause(s) of resistance in the various
isolates and that correlations can be made between
different types of mutation (e.g. single nucleotide
polymorphisms in the demethylase gene vs. overexpression of efflux pumps) and measures of evolutionary fitness.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
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P002
Evaluation of the NCCLS M27-2A, Sensititre
YeastOne, disk diffusion, and Etest methods for
determining susceptibilities of Candida
dubliniensis to Voriconazole, Fluconazole and
Itraconazole
M.P. Arévalo,1 F.J. Salgado,1,2 J. Alcoba,1,2 A. Arias,1
3 M.D. Moragues3 and G. Quindosv
1
Universidad de La Laguna, Facultad de Medicina,
Medicina Preventiva y Salud Pública, La Laguna.
Tenerife, Spain, 2Hospital Universitario de Ntra. Sra.
de Candelaria, Microbiologia, Santa Cruz de Tenerife,
Spain and 3Universidad del Paı´s Vasco. Facultad de
Medicina, Inmunologı´a, Microbiologı´a y
Parasitologı´a, Bilbao, Spain
Objectives: Candida dubliniensis is a recently (1995)
described Candida species closely related to Candida
albicans, which has been primarily associated with
oral candidiasis in HIV-infected patients but has also
been recovered from a wide range of anatomical sites
and clinical samples. C. dubliniensis is prevalent
throughout the world. The great majority of Candida
dubliniensis isolates are susceptible to all of the
commonly used antifungal agents; however, reduced
susceptibility to azole drugs has been reported in
clinical isolates. The purpose of this study was to
compare the in vitro activity of Voriconazole, Fluconazole, and Itraconazole, against 62 isolates of
Candida dubliniensis by the NCCLS M27-2A (BMD),
Sensititre YeastOne, disk diffusion and Etest methods
and to study the effect of the time of reading (24 h vs.
48 h) in the susceptibility testing.
Results: All of isolates were susceptible for the
Voriconazole by the Sensititre, Etest, and disk
diffusion methods, three isolates were considered
resistant by the reference BMD method at 48 h
(agreement 95.1%) while at 24 h only one isolate
was resistant (agreement 98.4%). Concerning Fluconazole, all the isolates were susceptible by the
Sensititre and Etest methods and only one showed
dose-dependent susceptibility by disk diffusion method. By the reference BMD method at 48 h, there were
one isolate S-DD and three resistant (agreement
93.5%). At 24 h none of the isolates was S-DD and
only one was resistant (agreement 98.4%). For
Itraconazole, all the isolates were susceptible by Etest
method, although two isolates showed dose-dependent susceptibility by Sensititre (agreement 96.8%),
ten by reference BMD method at 48 h (agreement
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
82.2%) and three at 24 h (agreement 95.2%). Only
one isolate was resistant at 48 h by the reference
BMD method. The highest rate of strains with
decreased susceptibility was obtained against Itraconazole as it has been previously described by other
authors. The lowest MICs were observed for Voriconazole and these results suggested that the in vitro
potency of Voriconazole is greater than that of the
rest of azoles tested, which is concordant with
the results that have been reported by other investigators.
Conclusions: The MICs results obtained by all the
methods demonstrated that C. dubliniensis is very
susceptible to all the antifungal agents tested.
Voriconazole was the most potent in vitro followed
by Fluconazole, and Itraconazole. The highest rate of
resistance was obtained against Itraconazole with a
high number of isolates defined as susceptible dosedependent. The agreement between the MICs registered at 24 and 48 h by the reference BMD was
better for Voriconazole.
P003
Voriconazole under anaerobic conditions:
implications of in vitro data for the treatment of
Candida infections
H. Bernhardt,1 M. Knoke,1 K. Zimmermann,2
G. Schwesinger,3 J. Bufler,4 K. Ludwig5 and
J. Bernhardt5
1
Faculty of Medicine, University of Greifswald,
Greifswald, Germany, 2Institute of Medical
Microbiology, University of Greifswald, Greifswald,
Germany, 3Institute of Pathology, University of
Greifswald, Greifswald, Germany, 4Pfizer GmbH,
Karlsruhe, Germany and 5Clinical Center Suedstadt,
Clinic of Surgery, Rostock, Germany
Background: Two in vitro culture systems were
used to investigate the effect of voriconazole (VORI)
on Candida colonization and morphology in aerobic
vs. oxygen-depleted environments.
Methods: Growth and morphological alterations of
Candida albicans were studied on cover slips in 24well microtiter plates in the presence or absence of
VORI (16 mg l-1). The Candida test panel included the
wild-type strain SC5314, the mutant strain CSSK215 with a deleted ssk1 response regulator gene, and
several clinical blood-culture and esophagitis isolates.
47
xxx
Growth patterns on glass surfaces were visualized
by vital staining with FUN 1 (Molecular
Probes). Anaerobic conditions were obtained using
Anaerocult A (Merck). In addition, we investigated
the differential effects of VORI under aerobic
vs. anaerobic growth conditions for up to 9 days in
a long-term continuous flow culture (CFC) system
adapted to a 15–20 h generation time. For anaerobic conditions, the system was flushed with a
mixture of nitrogen and carbon dioxide. Aerobic
conditions were obtained by ventilation with pressurized air.
Results: VORI-induced growth inhibition was evident by diminished colonization of surfaces and
reduction of colony-forming units in all strains.
VORI inhibited the growth of both yeast cells and –
especially under anaerobic conditions very strongly –
mycelia, which was apparent by large numbers of
dead cells. The mutant CSSK21-5 formed no mycelia
at all. Clinical isolates from candidemia patients
showed less pronounced and variable inhibition. In
the anaerobic CFC, a fungicidal effect (99.9% growth
inhibition) of VORI was observed comparing the
germ-counts in the control trials without VORI
administration. In contrast, fungistatic activity
(90% inhibition) was apparent under aerobic conditions. In the anaerobic CFC without VORI, fluorescence microscopy of fungal morphology in biofilms
on glass surfaces after 8 days revealed differentiation
into blastospores, germ tubes, pseudomycelia and
mycelia. However, in anaerobic cultures containing
VORI, only few live yeast cells were detected,
sometimes among cell detritus. Surface adhesion
was clearly reduced. Under aerobic conditions, more
blastospores but no differentiated mycelia were
detected.
Discussion: The enhanced antifungal effect of VORI
under anaerobic conditions may confer a therapeutic
advantage. In vivo, VORI is expected to be particularly active in infected tissue with low oxygen
levels, especially in sequestered areas like mycotic
abscesses. We suggest that this enhancement is
caused by the low fungal metabolic rate in environments depleted from oxygen. Residual synthesis of
ergosterol in the presence of VORI may be sufficient
to maintain some fungal growth under aerobic but
not anaerobic conditions.
Summary: Two in vitro culture systems revealed
that growth inhibition of Candida albicans by VORI is
much stronger under anaerobic than under aerobic
conditions.
48
P004
Interaction fluconazole–flucytosine against
Candida spp. with low susceptibility to FZ
determined by checkerboard, Etest and killing
curve methods
E. Cantón,1 J. Pemán,2 M. González de Cárdenas,3
N. Borrel3 and M. Gobernado2
1
Centro de Investigación, Hospital Univ La Fe,
Valencia, Spain, 2Servicio de Microbiologı´a, Hospital
Univ La Fe, Valencia, Spain, 3Servicio de
Microbiologı´a, Hospital Univ Son Dureta, Palma de
Mallorca, Spain, 4Servicio de Microbiologı´a, Hospital
Univ Son Dureta, Palma de Mallorca, Spain and
5
Servicio de Microbiologı´a, Hospital Univ La Fe,
Valencia, Spain
Background: A combination of antifungal agents is
a good alternative to avoid therapeutic failure in
invasive fungal infections but standard methodology
to study the in vitro activity of drugs in combination
is not yet available. Checkerboard and time-killing
methods to determine drug interaction are timeconsuming for use in a clinical laboratory. The aim of
this study was to evaluate the in vitro activity of
fluconazole (FZ) and flucytosine (FC) when given in
combination by three methods in order to find a
method that facilitates the in vitro interaction studies
in clinical laboratories.
Methods: Four strains (two C. krusei and two
C. glabrata), isolated from blood cultures, with
decreased susceptibility to FZ were tested. Drug
interaction was determined by checkerboard (M27A2 guidelines), Etest and time-killing curve methods.
Concentrations tested ranged from 0.016–56 mg l)1
for FZ, and from 0.002–32 mg l)1 for FC. Drug
interaction was evaluated by the fractional inhibitory
concentration index (FICI) (£0.5 synergism, >4
antagonism). Concentrations (mg l)1) tested by killing curves were (FZ + FC): 8 + 8, 8 + 32, 64 + 8
and 64 + 32 for C. krusei and 32 + 0.25, 32 + 1,
64 + 0.25, 64 + 1 for C. glabrata. CFU were determined at 0, 3, 6, 24 and 48 h. E-test was performed
following the methodology described in Rev Esp
Quimioterap 2004, 17: 48–56.
Results: No interaction FZ–FC was detected by
checkerboard, Etest and killing studies. MIC of the
combination was that of the most active drug (FICI
>0.5 and <4) and the decrease in CFU determined by
killing curves was similar to the most active drug
alone.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
Conclusions: Correlation among methods was
good. Etest could be a rapid and suitable method to
determine antifungal interactions in clinical laboratories. Clinical studies are needed to determine the
in vitro–in vivo correlation of combined therapy.
P005
Acquired resistance to echinocandins in
Candida albicans
M.T. Baixench,1 N. Aoun,2 D. Garcia-Hermoso,3 S.
Ramires,2 D. Hoinard,3 C. Piketti2 and E. Dannaoui1,3
1
Unité de Parasitologie-Mycologie, Laboratoire de
Microbiologie, Hôpital Européen Georges Pompidou,
AP-HP, Paris, France, 2Service d´’Immunologie
Clinique, Hôpital Européen Georges Pompidou,
AP-HP, Paris, France and 3Centre National de
Référence Mycologie et Antifongiques, Institut
Pasteur, Paris, France
isolates were recovered from the oral cavity 12 days
before admission (isolate 1), and on days 18, 63 and
131 (isolates 2, 3 and 4). In vitro antifungal susceptibility testing was performed by Etest on RPMI agar
and by EUCAST reference method both in RPMI and
AM3 medium. Strain genotyping was performed by
analysis of three polymorphic microsatellite markers.
Results: Isolates 2, 3 and 4 showed a similar
genotype. All four isolates were resistant to fluconazole (MIC = 32–64 lg ml)1); for voriconazole, MIC
were 0.50 lg ml)1 for isolates 1, 2 and 3 and
2 lg ml)1 for isolate 4. Table shows MICs for
echinocandins. There was a clear MIC increase for
caspofungin and micafungin for isolates 3 and 4
recovered after clinical failure of caspofungin therapy.
MIC (lg ml)1)
Etest
EUCAST
Caspofungin
Caspofungin
Micafungine
RPMI
AM3
RPMI
0.25
0.25
2
2
0.03
0.03
1
1
0.03
0.03
0.5
1
Isolate
Background: Caspofungin and micafungin are two
antifungal drugs inhibiting the synthesis of 1,3-betaD-glucan and exhibiting in vitro fungicidal activity
against Candida species. Caspofungin has been successfully used for treatment of fluconazole-resistant
esophagitis. Few cases of acquired resistance to
caspofungin in Candida species have been reported
to date.
Patient and methods: A 34-year-old male with
AIDS (CD4 count of 6 cells ll)1) was admitted with a
diagnosis of thrush and Candida esophagitis lasting for
3 months. Previous therapy with fluconazole had
failed. On day 1, he was treated with voriconazole
(400 mg day)1) with rapid clinical improvement by
day 4. On day 11, treatment for a Mycobacterium avium
infection was started and Candida infection relapsed on
day 15. From day 24 to day 37 he was treated with
caspofungin (70 mg daily). His esophagitis responded
partially with persistent oral confluent plaques. By the
end of treatment (day 38), thrush and esophagitis
relapsed. Treatment with amphotericin lipid complex
(3 mg kg)1) once weekly was done (day 39 and 45)
and stopped after two injections for systemic intolerance (chills and fever). At the same time (day 39),
antiretroviral treatment was started. From day 55 to
65, a second course of caspofungin was initiated
without clinical efficacy. At day 66, treatment with
high doses of voriconazole (800 mg day)1 followed by
600 mg day)1) was initiated. Thrush and esophagitis
did resolve completely on the third day. C. albicans
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
1
2
3
4
0.047
0.064
>8
8
Conclusion: Few cases of caspofungin resistance in
Candida species have been described to date and all
occurred with a caspofungin dosage of 50 mg day)1.
We report, for the first time in Europe, the acquisition
of caspofungin resistance in Candida albicans. Clinical
resistance occurred despite high caspofungin dosage
of 70 mg day)1 and was associated with an increase
of in vitro MICs for both caspofungin and micafungin.
P006
Non-responsiveness to first line empirical
caspofungin in patients with febrile
neutropenia. A single unit experience
M. Das, C. Barnes, S. Natarajan, M. Dennis, J. Cavet,
4 D.W. Denning, E. Liakopoulou
Introduction: Caspofungin has been licensed for salvage treatment of invasive aspergillosis and recently
for empiric antifungal treatment in patients with
febrile neutropenia. We report a single unit experience
with echinocandins (caspofungin) as first line empirical treatment for persistent neutropenic fever.
49
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Patients and methods: Since September 2004 to
April 2005, Caspofungin (70 mg then 50–70 mg
daily) was administered as first line empirical antifungal treatment 96 h after persistence of neutropenic fever in patients treated for haematological
malignancies (n = 30 acute leukaemia, n = 5 autograft) as per our revised antifungal protocol. Upon
initiation of empirical antifungal treatment, high
resolution CT scan of thorax was requested and
performed within 24–48 h. BAL was performed to
support the diagnosis when clinically recommended.
Failure to respond to first line empirical antifungal
treatment and the need to switch to 2nd line
treatment was defined as worsening of clinical
condition and/or continued temperature for at least
72 h postinitiation of first-line treatment.
Results: Thirty-five patients (M = 22, F = 13) with
total of 40 episodes of neutropenic fever required
initiation of empirical antifungal treatment with
Caspofungin. In 10 of these episodes (25%) a change
to second line antifungal was required as per clinical
criteria. In 8 (20%) episodes this change to liposomal
amphotericin resulted in clinical (n = 8) and radiological (n = 4) resolution of signs.
Conclusion: Echinocandins (Caspofungin) are efficacious and well-tolerated agents against fungal infection. In this group of patients, we report a substantial
proportion of non-responders to first-line empirical
treatment with this agent. We believe that this proportion may vary in each unit but needs to be considered
for requiring early modification of treatment, and in
designing antifungal treatment algorithms.
P007
Influence of the incubation time to MIC’s and
the in vitro-susceptibilities of Voriconazole,
Fluconazole, Itraconazole, and Flucytosine
W. Fegeler,1 K. Becker,1 A. Schmalreck2 and the
DMykG Antifungal Study Group3
1
Institution of Medical Microbiology, University of
Muenster, Muenster, Germany, 2MBS, Munich,
Germany and 3German Speaking Mycological Society
(DMykG), Germany
Background: As already shown, the susceptibility
of yeast isolates to antifungal agents can be influenced by the incubation/reading time. However, data
using different standardized MIC – determination
methods, and taking these phenomena into account
are still rare. Moreover, data addressing the recently
50
established tentatively CLSI-breakpoints for voriconazole (VOR) are missing.
Methods: In order to determine the frequency of
incubation-time-depending MIC-shifts of voriconazole
(VOR), fluconazole (FLC), itraconazole (ITR), and
flucytosine (FCY), the MICs of 420 clinical Candida
isolates including: C. albicans (49.5%), C. glabrata
(14.8%), C. tropicalis (15.0%), C. parapsilosis (5.2%),
C. krusei (I. orientalis) (9.8%), C. lusitaniae (3.6%),
C. guilliermondii (0.5%), C. inconspicua (1.0%), C. kefyr
(0.5%), and C. famata (0.2%) were analyzed by
susceptibility pattern analysis (SPA) and pair-to-pair
analysis. The MIC’s collected in a German multicenter
study were determined by microdilution according to
DIN-58940-84 in YST medium, and NCCLS M27 A2
in RPMI medium. All media contained 2% glucose.
The MIC readings were done visually after an incubation time of 24 and 48 h at 36 C. Each isolate was
categorized to be susceptible (S), intermediate or
resistant (R) by the following assumed breakpoints:
S: FLC £ 8 mg l)1, ITR £ 0.125 mg l)1, VOR £
1 mg l)1,
FCY £ 2 mg l)1;
R:FLC ‡ 64 mg l)1,
)1
)1
ITR ‡ 1 mg l , VOR ‡ 4 mg l , FCY ‡ 32 mg l)1,
(I = intermediate: MIC values between S and R).
Results: The increased number of isolates categorized to be resistant after prolonging the incubation
time form 24 h to 48 h differed depending on the
antifungal agent and the medium as follows: for
YST : FCY 1.2%; FLC 7.4%; VOR 0.7% and ITR
15.2% and for RPMI : FCY 4.1%; FLC 15.9%; VOR
9.8% and ITR 20.0% respectively. The percentage of
isolates with the same susceptibility result after 24 h
and 48 h (24/48 h; S/S, I/I, R/R) ranged between
94.8% (FCY) and 62.9% (ITR in YST) and was
significant influenced by the medium only for FLC
(81.4% in YST and 75.7% in RPMI), but species
dependent variations could be found for all antifungal agents. Considering the results of all four
antifungal agents the number of isolates with a shift
from susceptible to resistant (SR-shift) was three
times higher in RPMI (7.2%; 121/1680) than in YST
(2.4%; 41/1680) and differed significantly for FLC
(YST n = 3 vs. RPMI n = 35) and VOR (YST n = 3
vs. RPMI n = 39), whereas, there was no significant
difference for FCY (YST n = 3 vs. RPMI n = 5) and
ITR (YST n = 32 vs. RPMI n = 42).
Conclusion: Although there was a good correlation
between the susceptibility results read after 24 h and
48 h, respectively, shifts in susceptibility depending
on the antifungal agent, and the appropriate yeast
species will necessitate a second reading of the
susceptibility to confirm the MIC.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
P008
Involvement of lipid rafts in the antifungal
mode of action of miconazole
I.E.J.A. François,1 K. Thevissen,1 J. Vandercappellen,1
G.D. Dispersyn,2 J. Ausma,2 M. Borgers2 and
B.P.A. Cammue1
1
Katholieke Universiteit Leuven, CMPG, Heverlee,
Belgium and 2Barrier Therapeutics, N.V. Geel,
Belgium
The class of synthetic azole antimycotics constitutes
the largest group of antifungal agents currently in
clinical use. The generally accepted mode of action of
azoles is the inhibition of 14a-lanosterol demethylase,
a key enzyme in ergosterol biosynthesis, resulting in
depletion of ergosterol and accumulation of toxic
14a-methylated sterols in membranes of susceptible
yeast species. Recently, we and other research groups
could demonstrate that generation of reactive oxygen
species (ROS) is important for the antifungal activity
of miconazole, pointing to an ancillary mode of action
for this azole. Furthermore, using a genome-wide
approach, we could demonstrate that Saccharomyces
cerevisiae deletion mutants affected in sphingolipid
and ergosterol biosynthesis are resistant to miconazole, suggesting a possible involvement of lipid rafts in
the antifungal mode of action of miconazole. In
contrast, S. cerevisiae deletion mutants affected in
mitochondrial function were found to be hypersensitive to miconazole. Hence, the antifungal mode of
action of miconazole appears to involve more than
mere ergosterol biosynthesis inhibition.
P009
Antifungal activity of BAL4815, a novel azole,
against dermatophytes
M.A. Ghannoum and N. Isham
Center for Medical Mycology, Case Western Reserve
University, Dermatology, Cleveland, OH, USA
Background: There remains the need for welltolerated agents to treat dermatophytoses.
BAL4815 is the active component of the antifungal
agent BAL8557 (the water-soluble prodrug) entering
phase III clinical development as an oral and IV
formulation. BAL4815 has a long half-life suitable
for once daily or even less frequent dosing. We
report the minimum inhibitory and fungicidal
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
concentrations (MICs and MFCs) of BAL4815 against
common dermatophytes.
Methods: Organisms tested included the dermatophytes Trichophyton rubrum (including terbinafineresistant strains), T. mentagrophytes, T. tonsurans,
Epidermophyton floccosum, and Microsporum canis.
Ten strains of each species were tested. MIC testing
was performed according to Clinical and Laboratory
Standards Institute (formerly NCCLS) standard
M38A. MFC was defined as the lowest concentration
that exhibited two or fewer colonies on subculture.
Results: The MIC ranges of BAL4815 and terbinafine against all dermatophyte strains were 0.06–0.25
and 0.002–16 lg ml)1, respectively. The mean MIC
of BAL4815 was 0.09 lg ml)1 against all strains,
including the T. rubrum strains with elevated terbinafine MICs. The mean MIC of terbinafine against all
dermatophyte strains was 0.013 lg ml)1, excluding
the T. rubrum strains with elevated terbinafine MICs,
which had a mean MIC of 5.38 lg ml)1. Neither
BAL4815 nor terbinafine showed cidal activity
against the majority of the strains tested.
Conclusion: The new azole BAL4815 showed
potent activity against all the dermatophytes tested,
including the terbinafine-resistant T. rubrum isolates.
P010
Caspofungin treatment in severely ill,
immunocompromised patients: a case
documentation study of 118 patients
A. Glasmacher,1 O.A. Cornely,2 K. Orlopp,1
S. Reuter,3 S. Blaschke,4 M. Eichel,5 G. Silling,6
G. Egerer,7 M. Siemann8 and G. Just-Nübling5
1
Department of Internal Medicine I, University of
Bonn, Bonn, Germany, 2Department of Internal
Medicine I, University of Cologne, Cologne,
Germany, 3Department of Internal Medicine III,
University of Ulm, Ulm, Germany, 4Department of
Nephrology, University of Goettingen, Goettingen,
Germany, 5Department of Internal Medicine III,
University of Frankfurt, Frankfurt, Germany,
6
Department of Internal Medicine A, University of
Muenster, Muenster, Germany, 7Department of
Haematology, Oncology and Clinical Immunology,
University of Duesseldorf, Duesseldorf, Germany and
8
Staedtisches Krankenhaus, Institute of Pathology
and Bacteriology, Kiel, Germany
Background: Caspofungin has shown efficacy in
empirical antifungal therapy in neutropenic patients,
51
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refractory invasive Aspergillus infections and invasive Candidiasis. Here, we report the currently largest
series of patients treated with caspofungin outside
clinical trials.
Methods: Centres in Germany that were known to
treat patients with invasive fungal infections were
asked to fill out detailed questionnaires for all
patients treated with caspofungin. No effort was
made to influence the decision to use caspofungin.
95% – confidence intervals (95%CI) were calculated
for response rates.
Results: A total of 118 patients were evaluable
(median age 48 years, interquartile range 38–58).
Forty-one (35%) suffered from acute leukaemia, 31
(26%) had allogeneic stem cell transplants, 16
(14%) lymphoma or myeloma, 8 (7%) autologous
stem cell transplants and 22 (19%) other causes
for immunosuppression. One hundred and six
patients were evaluable for efficacy. 68 (64%,
95%CI 54–73) patients achieved a complete or
partial remission. 81/115 (70%, 95%CI 61–79)
patients were alive 30 days after the end of
caspofungin therapy. Response rates were similar
in proven (20/32, 63%) and probable (27/46,
59%) infections, in neutropenic patients (41/55,
75%) and in patients who were (44/70, 63%) or
were not (24/36, 67%) refractory to antifungal
pretreatment. The response rate in mechanically
ventilated patients was 29% (7/24). Caspofungin
was well tolerated, even in 14 patients, who were
concomitantly treated with cyclosporine A, no
drug-related elevations of bilirubin, ALT or creatinine were found.
Conclusions: This open case study of severely ill
patients with invasive fungal infections demonstrates
both excellent efficacy and very low toxicity of
caspofungin.
Further authors: M. Florek (Dresden), R. Schnitzler (Cologne), P. Ebeling (Essen), J. Ritter
(Münster), S. Kathöfer (Heidelberg), H. Reinel
(Nürnberg), C. Tapprich (Düsseldorf), A. Schüttlöffel
(Köln), P. Schütt (Essen), H. Fischer (Tübingen),
T. Kees (Tübingen), W. Torka (Tübingen), C. Hahn
(Bonn)
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P011
Effects on hepatic and renal function of
concomitant cyclosporine A in
immunocompromised pediatric patients
receiving caspofungin salvage therapy
T.H. Lehrnbecher,1 A. Attarbaschi,2 F. Schuster,3
N. Herzog,4 L. Grigull,5 M. Dworzak,2 H.J. Laws6 and
A.H. Groll7
1
Pediatric Hematology/Oncology, University
Children’s Hospital, Frankfurt, Germany, 2Pediatric
Hematology/Oncology, University Children’s
Hospital, Wien, Austria, 3Pediatric Hematology/
Oncology, University Children’s Hospital, Muenchen,
Germany, 4Pediatric Hematology/Oncology,
University Children’s Hospital, Wuerzburg, Germany,
5
Pediatric Hematology/Oncology, University
Children’s Hospital, Hannover, Germany, 6Pediatric
Hematology/Oncology, University Children’s
Hospital, Duesseldorf, Germany and 7Pediatric
Hematology/Oncology, University Children’s
Hospital, Muenster, Germany
Objectives: Reversible and mild increases in hepatic
transaminases have been observed in healthy adult
volunteers enrolled on a pharmacokinetic interaction
study of caspofungin (CAS) and cyclosporine A (CSA).
While a pediatric dosage has not been established,
CAS is occasionally used in pediatric patients (pts).
Here, we report the effects of concomitant CSA on
hepatic and renal function in immunocompromised
pediatric patients considered to require CAS therapy
included in a recent multicenter survey.
Methods: The survey identified 64 pts (mean age:
10.4 years; 25 female, 39 male) with hematological
malignancies (48), marrow failure (nine), solid tumors (three), non-neoplastic hematological disorders
(two) and congenital immunodeficiency (two) who
received CAS for proven (17), probable (14) and
possible (17) invasive infections or empirically (16).
CAS was administered as single antifungal agent (20)
or in combination with other antifungal agents (44)
until intolerance or maximum efficacy at dosages
individually determined by the responsible physician
for refractory infection (38), intolerance of standard
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
agents (10), or as best therapeutic option (16). In
order to assess hepatic and renal adverse effects of
concomitant CSA therapy, we compared laboratory
hepatic and renal function parameters of the 19
patients receiving CSA for posttransplant immunosuppression to the remaining 45 patients receiving no
concomitant CSA therapy.
Results: The 19 pts on concomitant CSA (+CSA)
received CAS for a mean (±SEM) duration of
36 ± 6.8 days (r, 6–131), when compared with
52 ± 7.1 days (r, 2–218) in the 45 pts not receiving
CSA (–CSA) (P = 0.2285). The mean maintenance
dosages were 1.13 ± 0.12 mg kg)1 (r, 0.40–2.92) or
34.35 ± 2.34 mg m)2 (r, 20–57) and 1.25 ±
0.07 mg kg)1 (r, 0.44–2.33) or 34.5 ± 1.5 mg m)2
(r, 16–54) respectively (P = 0.1811 and P =
0.9606). Increases in hepatic and renal function
parameters during therapy were frequent. However,
in none of the patients, CAS or CSA was discontinued
because of laboratory adverse events. While mean
GOT values were slightly higher in both cohorts at end
of treatment (EOT; P = 0.0194 and 0.0070), mean
GPT, bilirubin, alkaline phosphatase and creatinine
values were not different from baseline (BL). No
differences between the two cohorts in the proportion
of patients with elevated (‡1.5 and ‡3.0 · BL) values
of GOT, GPT, bilirubin, alkaline phosphatase, and
creatinine at EOT (P = 0.2.969–P = 1.0000) were
observed. Overall survival at EOT was similar in both
cohorts (68.4 vs. 77.8%, P = 0.5302).
Conclusion: In this retrospective survey, in severely
immunocompromised pediatric patients without
therapeutic alternative, there was no indication of
increased laboratory hepatic or renal toxicity or
increased mortality at EOT in the subset of patients
receiving concomitant CSA therapy.
P012
Invasive pulmonary aspergillosis in a University
Hospital in Taiwan, 1999–2004
P.R. Hsueh,1 C.C. Lai2 and L.N. Lee3
National Taiwan University Hospital, Internal
Medicine, Taipei, Taiwan, 2National Taiwan
University Hospital, Laboratory Medicine and Internal
Medicine, Taipei, Taiwan and 3National Taiwan
University Hospital, Laboratory Medicine and Internal
Medicine, Taipei, Taiwan
1
of patients with invasive pulmonary aspergillosis
(IPA).
Setting: National Taiwan University Hospital, an
1800-bed medical center located in northern
Taiwan.
Patients and methods: All clinical and microbiological records with pulmonary isolation of Aspergillus species at the hospital from January 1999 to
December 2004 were retrospectively analyzed. IPA
was defined based on the consensus of European
Organization for Research and Treatment of Cancer
(EORTC).
Results: A total of 26 patients with proven and
probable IPA were identified. Fourteen (54%) of
these infections were hospital-acquired. The mean
age of these patients was 47.5 years old (range 4–
87 years). Among these patients, 11 (42%) had
hematological malignancy and 2 (7%) had solid
organ cancers. Seventeen (65%) patients had ever
received various immunosuppressive agents and
eight (31%) had received steroid. Five patients
(19%) had undergone bone marrow transplantation. Productive cough (88%) was the most common presentation, followed by fever (85%). Two
consecutive Platelia Aspergillus tests (ELISA) were
performed in only six patients and all of them had
positive results (OD index >1.5 ng ml)1). Among
20 culture-proven infections (77%), the most frequently encountered fungi were Aspergillus fumigatus (46%), followed by A. flavus (23%), and A. terreus
(8%). Twenty-four patients (92%) received antifungal therapy (including amphotericin B, caspofungin,
or voriconazole) and seven (27%) underwent
surgical intervention. Seventeen patients (65%)
had respiratory failure necessitating ICU stay.
The overall mortality rate was 62%. Univariate
logistic regression analysis showed that only disseminated intravascular coagulopathy (DIC) was
significantly associated with death of IPA
(P = 0.0014).
Conclusions: Invasive pulmonary aspergillosis is
associated high mortality rate, particularly for
patients with DIC. Application of novel diagnostic
modality, such as Aspergillus antigen test, to make
early diagnosis and prompt and appropriate antifungal therapy combined with surgical intervention are critical for management of patients with
IPA.
Study objectives: To investigate the clinical
manifestations, predisposing factors, and outcomes
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
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P013
In vitro susceptibility testing of vaginal Candida
albicans isolates against boric acid
fungus be inhibited by the highly concentrated drug
in vivo, resulting in apparent clinical cure.
P014
M. Hui, M.L. Chin, K.C. Chu and C.Y. Chan
The Chinese University of Hong Kong, the Prince of
Wales Hospital, Microbiology, Shatin, Hong Kong
Background: Boric acid therapy for the treatment of
vulvovaginal candidiasis was described >30 years
ago. It is prescribed as a vaginal capsule at 600 mg
once daily for 14 days, and twice weekly for maintenance therapy. In this study, we aimed to evaluate the
susceptibility profile of vaginal Candida albicans isolates
to boric acid by the use of agar dilution method.
Materials and methods: A total of 12 vaginal yeast
isolates of Candida albicans were tested. Type strains
used include Candida albicans ATCC 24433, Candida
glabrata ATCC 90030, Candida krusei ATCC 6258,
Candida tropicalis ATCC 750, Candida parapsilosis ATCC
22019, Candida guilliermondii ATCC6260, Candida
lusitaniae ATCC 42720 and Candida dubliniensis
ATCC MYA)66. Boric acid in different concentrations
was incorporated into Petri plates with RPMI 1640
medium with final concentrations ranging from 0.015
to 32 mg l)1. Each isolate was suspended in 5 ml
(0.85%) of sterile saline and adjusted to a turbidity
equivalent to that of a 0.5 McFarland standard. The
final concentration of the inoculum was 1 · 103 cfu
ml)1 yeasts per spot and confirmed by viable count.
Plates were incubated aerobically at 35 C. The first
visual reading was carried out after 24 h of incubation, and the minimum inhibitory concentration
(MIC) value was recorded as the lowest concentration
that inhibited visible growth; and also again after 48 h
of incubation. Quality control of the system was tested
by using amphotericin B at various concentrations
(0.015–32 mg l)1) against the type strains.
Results: At 24 h, all vaginal isolates and types
strains of Candida demonstrated an MIC of
>32 mg l)1 against boric acid, with the exception
of Candida krusei ATCC 6258 which showed an MIC
of 32 mg l)1. By 48 h, the Candida krusei ATCC 6258
also demonstrated an MIC of >32 mg l)1.
Discussion: Boric acid is generally regarded as a
fungistatic and bacteriostatic agent. Currently, there
is no recommended interpretative cutoff MIC values
for boric acid as an antifungal drug. Despite the high
MIC values recorded, boric acid is administered
topically at a concentration much higher than was
used in our test system. It is thus plausible that the
54
A randomized controlled trial of itraconazole
capsule vs. fluconazole capsule for the
prophylaxis of systemic fungal infections in
patients with haematological malignancies
Y. Ito,1 K. Ohyashiki,1 M. Takeuchi,2 A. Mugitani,3
H. Wakita,4 M. Matsuda,5 M. Hino,6 T. Kiguchi,7
A. Kanamaru5 and T. Masaoka8
1
First Department of Internal Medicine, Tokyo
Medical University, Tokyo, Japan, 2Department of
Internal Medicine, National Hospital Organization
Minami Okayama Medical Center, Tsukubo,
Okayama, Japan, 3Department of Hematology,
Fuchu Hospital, Osaka, Japan, 4Departments of
Internal Medicine, Narita Red Cross Hospital, Narita,
Japan, 5Department of Internal Medicine, Kinki
University School of Medicine, Osakasayama, Japan,
6
Department of Clinical Hematology and Clinical
Diagnostics, Graduate School of Medicine, Osaka
City University, Osaka, Japan, 7Department of
Internal Medicine, Kagawa Rosai Hospital,
Marugame, Japan and 8Osaka Medical Center for
Cancer and Cardiovascular Diseases, Osaka, Japan
Objectives: Although fluconazole (FLCZ) is widely
used for the anti-fungal prophylaxis in patients with
neutropenia, it is known to have low anti-fungal activity
for Aspergillus, Candida glabrata or Candida krusei. To date,
there is insufficient evidence of benefit for the prophylaxis of respiratory fungal infection such as invasive
Aspergillosis. Meta-analysis of studies with itraconazole
(ITCZ) oral solution, which has anti-fungal activity for
Aspergillus spp, has revealed utility of ITCZ in prophylaxis
during neutropenia. In Japan, ITCZ is available only in a
capsule formulation. We report the results of a randomized controlled trial between ITCZ capsules and
FLCZ capsules in patients with haematological myeloid
malignancies treated with chemotherapy.
Subjects and methods: After written informed
consent was obtained, patients with acute myeloid
leukemia (AML) or myelodysplastic syndromes (MDS)
receiving chemotherapy were enrolled and randomized to either the ITCZ capsule (200 mg day)1)
therapy group or to the FLCZ capsule (200 mg day)1)
treatment group. Oral administration of anti-fungal
prophylactic drugs was started along with 300 mg
levofloxacin when they received chemotherapy.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
Prophylactic use of ITCZ or FLCZ was continued until
neutrophil count normalized or when therapeutic use
of anti-fungal drugs was started. The clinical guidelines for systemic fungal infection (SFI) issued by the
Japanese compiling committee were used to diagnose
SFI. Serum b-D glucan, a commonly measured bioparameter for SFI, was measured to assist diagnosis.
Results: A total of 156 courses of remission induction chemotherapy or consolidation therapy in 107
patients managed at 33 nationwide facilities were
enrolled in this study. 76 (ages 16–80) were assigned
to the ITCZ group and 80 (ages 16–78) to the FLCZ
group. Among them, 72 in the ITCZ group and 78 in
the FLCZ group were evaluated. No case of proven SFI
was observed in any of the two groups, but the number
of cases with probable or possible fungal infection was
8 (11.1%) in the ITCZ group and 13 (16.7%) in the
FLCZ group (P = 0.46). The number of cases where
prophylactic therapy had to be switched to therapeutic
anti-fungal therapy was similar between the ITCZ
group (15 cases, 20.8%) and the FLCZ group (15
cases, 19.2%) (P = 0.81). Adverse reactions were
seen in three cases (4.2%) from the ITCZ group and
two cases (2.6%) from the FLCZ group (P = 0.93).
Conclusion: In patients with chemotherapy-treated
AML or MDS, ITCZ capsules were found to be useful
anti-fungal prophylactic agents, comparable to FLCZ
capsule. Both ITCZ and FLCZ were proven to be well
tolerated. At this time, there is no evidence of an
effect on mortality.
P015
In vitro activity of fluconazole against Candida
species isolated from patients in two
hematological centers of Russia
G. Kliasova,1 S. Ptitcin1 and A. Maschan2
1
National Research Center for Hematology,
Laboratory of Clinical Microbiology, Mycology and
Antibiotic Treatment, Moscow, Russia and 2Institute
of Pediatric Hematology, Moscow, Russia
Objectives: Yeasts are frequent pathogens in
immunocompromised patients. Resistance to azoles
has become an emerging problem in Candida spp. The
aim of this study was to evaluate the susceptibility of
Candida isolates by disk diffusion method.
Methods: Candida isolates were tested by the NCCLS
standard M44-A disk diffusion method. Test plates
were read and results recorded with the BIOMIC
Vision System.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
Results: All 888 Candida species (580 C. albicans, 115
C. glabrata, 54 C. kefyr, 46 C. krusei, 28 C. parapsilosis,
21 C. tropicalis, 21 C. norvegensis, 10 C. lusitaniae, eight
C. guilliermondii, two C. dubliniensis, one C. intermedia,
one C. famata, one C. inconspicua) were recovered from
clinical samples isolated between July 2003 and May
2005. Candida isolates were obtained from blood
(n = 13), peritoneal (n = 2) and cerebrospinal fluids
(n = 1), urine (n = 48), biopsies (n = 21), wounds
(n = 5), BAL (n = 43), sputum (n = 9), pleural fluid
(n = 1), genitals (n = 8), throat (n = 476) and gut
(n = 261). The susceptibility data (%) for main species
are summarized in Table:
Species (n)
S
SDD
R
C.
C.
C.
C.
C.
C.
C.
C.
99
67
100
2
96
100
52
90
0
21
0
30
0
0
38
0
1
12
0
68
4
0
10
10
albicans (580)
glabrata (115)
kefyr (54)
krusei (46)
parapsilosis (28)
tropicalis (21)
norvegensis (21)
lusitaniae (10)
Conclusion: Fluconazole was active against main
Candida species. Despite widespread use of azoles in
recent years, only 1% of C. albicans were resistant to
fluconazole. The predominant isolates of C. glabrata
(67%) were susceptible to fluconazole.
P016
Non-comparative study of caspofungin for
treatment of fungal infections
T. Lejko-Zupanc,1 S. Zver,2 A. Špec-Marn,3
J. Tomažič,1 V. Papuga,4 M. Jereb,1 B. Šibanc,5
B. Ožek,6 Z. Novak7 and I. Šeruga8
1
Clinical Centre Ljubljana, Infectious Diseases Clinic,
Ljubljana, Slovenija, 2Haematology Department,
Clinical Centre Ljubljana, Ljubljana, Slovenija,
3
Department for Anesteziology and Intensive Care
Medicine, Clinical Centre Ljubljana, Ljubljana,
Slovenija, 4Department for Intensive Care Medicine,
General Hospital Celje, Celje, Slovenija, 5Infectious
Diseases Department, General Hospital Celje, Celje,
Slovenija, 6Department for Intensive Care Medicine,
General Hospital Novo mesto, Novo mesto, Slovenija,
7
Infectious Diseaes Department, General Hospital
Maribor, Maribor, Slovenija and 8Merck Sharp &
Dohme, Ltd., Ljubljana, Slovenija
Objective: To
evaluate
the
efficacy
and
tolerability of caspofungin for the treatment of
55
xxx
presumed or proven fungal infections in everyday
practice.
Study design: Prospective non-comparative study
conducted in University Medical Centre and in three
general hospitals in Slovenia in 2004. Patients who
received caspofungin were included. Demographic
data, data on concomitant diseases, previous antifungal therapy, fungal pathogen, caspofungin dosage
and treatment outcome were collected.
Results: A total of 50 patients (33 male, 17 female;
mean age 57.3 years; age range 19–84 years) were
included in the study. Underlying diseases were:
haematological malignancy in 14 patients, treatment
in medical/surgical ICU for various serious conditions
in 18 patients, polytrauma in four patients, other
immunocompromised states in seven patients, various serious medical diseases, requiring long-term
antibiotic treatment in seven patients. In majority of
the patients caspofungin was used as 70 mg loading
dose and 50 mg day)1 thereafter. The average duration of treatment was 19.2 days (3–85 days). Antifungal treatment was empirical in 10 patients. Fungal
infection was presumed in 16 patients and proven in
24 patients. Various Candida species (albicans in nine
patients, non-albicans in 19 patients) were isolated in
28 patients (56%) and Aspergillus spp. in 10 (20%)
patients. In two patients, infection as the result of
Pneumocystis carinii was proven. Both of the patients
were cured. Thirty-six (72%) patients have previously
received other antifungal treatment. Thirteen
patients (26%) died, six of them due to fungal
infections. In seven patients, the death occurred in
the first week of treatment. Overall the clinical success
was observed in 32 (64%) patients. Overall success
rate in Candida infections was 71%. More than 50% of
Candida species were resistant or less susceptible to
fluconazole. Clinical success rate in fluconazoleresistant group was 87%. Of the 10 patients with
Aspergillus infection five died, three of them due to
fungal infections (failure in 30%), clinical success was
documented in 3/10 (30%). In the group of patients
with empirical antifungal therapy improvement was
noted in 7/10 patients. The tolerability of caspofungin
was estimated as good or very good in 90% of the
patients. Only in two (4%) patients the treatment was
discontinued due to adverse event.
Conclusion: Our mixed population of patients represents the everyday clinical situation. Overall success rate of caspofungin in this setting was 64% and
in patients who received caspofungin for ‡7 days
was 76%. The most notable clinical success was in
56
the group of patients with C. glabrata and C. krusei
infections. The success rate was lower in patients
with invasive aspergillosis, probably due to late
diagnosis and poor clinical condition of the patients.
Caspofungin was well tolerated in various clinical
settings in patients with serious underlying medical
or surgical conditions.
P017
In vitro evaluation of the susceptibility of fungi
isolated from clinical materials to Voriconazole
A.B. Macura, B. Pawlik and A. Szul
Microbiology, Mycology, Krakow, Poland
The objective of the study was in vitro evaluation of the
susceptibility of 103 fungal strains to Voriconazole.
The fungi were isolated from clinical materials:
sputum, bronchial lavage, stool, urine, vaginal
smears, skin and nails. A slide-dilution method was
applied: the drug tested was incorporated into YNB
medium (Yeast Nitrogen Base) at concentrations 0.1,
1, 10 and 100 mg l)1. Fungal cultures on YNB
without the drug added served as controls. Sixty-three
strains of yeast-like fungi were evaluated after 48 h
incubation at 37 C. Eighteen mould strains were
evaluated after 72 h incubation at 27 C. The outcome was recorded as either total or partial growth
inhibition. Additionally, 22 dermatophyte strains
were evaluated after 7 and 14 day incubation at
27 C. The drug at concentration 0.1 mg l)1 inhibited
17.5% of the yeast-like fungal strains; 1 mg l)1 –
23.8%; 10 mg l)1 – 33.3%. The remaining 25.4% of
the strains were inhibited at 100 mg l)1. Considerably, lower drug concentration was sufficient to
partially inhibit fungal growth: 0.1 mg l)1 – 50.8%;
1 mg l)1 – 44.4%; 10 mg l)1 – 4.8%. Low values of
fungal growth inhibiting concentration were found
with other than C. albicans Candida strains (C. glabrata,
C. krusei, C. kefyr, C. guilliermondii). In the 18 mould
strains, total growth inhibition was achieved at
0.1 mg l)1 in 11.1% of them, at 1 mg l)1 in 66.7%,
at 10 and 100 mg l)1 in 11.1% each. Partial growth
inhibition, when compared with controls, occurred at
0.1 mg l)1 in 72.2% of the strains, at 1 mg l)1 in
22.2%. Only a single strain (5.5%) was inhibited at
10 mg l)1. Very low inhibiting concentrations were
found with Aspergillus fumigatus. Voriconazole turned
out to be particularly effective against dermatophytes.
No matter whether the culture was evaluated after 7
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
or 14 days, the growth of 95.5% of the strains was
totally inhibited at concentration of 1 mg l)1. The
strains of Trichophyton mentagrophytes and Trichophyton rubrum were most susceptible to Voriconazole. It is
noticeable that Voriconazole is especially effective
against A. fumigatus and other than C. albicans yeastlike fungi which may be beneficial in the mycoses
caused by those fungal species.
P018
Comparison of Etest and NCCLS M38-A methods
for testing susceptibilities of filamentous fungi
to posaconazole
E. Lopez Oviedo,1 C. Martin Martı́n de la Escalera,1
A.I. Aller,1 M. Ramirez,1 C. Castro,1 A. Romero,1
J. Peman,2 E. Cantón2 and E. Martı́n Mazuelos1
1
H.U.Valme, Microbiologı´a, Sevilla, Spain and
2
H.U.La Fe, Microbiologı´a, Valencia, Germany
Background: Posaconazole is a new triazole derivate and structural analogue of Itraconazole. This
drug has fungicidal activity against yeast and
filamentous fungi. The purpose of our study was to
compare Etest method (ET) with broth microdilution
NCCLS M38-A (MD) for ‘in vitro’ susceptibility testing
of filamentous fungi isolates against posaconazole.
Methods: Etest method and MD MICs were realized
simultaneously for 79 isolates: 56 Aspergillus spp (19
A. terreus, 17 A. fumigatus, 14 A. flavus, 3 A. glaucus, 2
A. niger and 1 A. nidulans), 10 Zygomicetes (6 Rhizopus
spp., 4 Mucor spp.), 9 Scedosporium spp. (6 S. apioespermum, 3 S. prolificans) and 3 Fusarium spp. Quality
control strains C. parapsilosis 22019, C. krusei 6258, A.
flavus 204304 and A. fumigatus 204305. ET MICs were
determined on solidified RPMI 1640 medium (2%
glucose) after 24 and 48 h of incubation at 35 C,
M38-A MICs were read after 48 h. With both methods,
MICs were determined by the lowest drug dilution that
showed 100% inhibition (MIC48). The agreement
(percentage of MICs pairs within a two dilutions
range) between ET and MD MICs were calculated.
Results: At 24 h ET/48 h MD the MIC50 and MIC90
(mg l)1) were as follows respectively: Aspergillus spp,
ET 0.125/0.25 and MD 0.06/0.25; Zygomicetes, ET
1/2 and MD 0.25/0.5; S. prolificans, ET ‡32/‡32 and
MD 8/8; S. apioespermum, ET NG/NG (NG, nongrowth) and MD 1/1; Fusarium spp., ET ‡32/‡32 &
MD 8/8. At 48 h ET and 48 h MD not difference
were observed except for Zygomicetes which showed
overgrowth at this time of reading and S. apioesper-
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
mum which showed MICs value 48/48 of 2/8 and
0.5/1 The agreement (ET/MD):in the different groups
were Aspergillus spp. 24/48: 93%, 48/48: 83.3%;
Zygomicetes 24/48: 60%, 48/48: 10%; S. apioespermum 48/48: 50%, S. prolificans :24/48 100% 48/
48:100% Fusarium spp. 24/48: 100%, 48/48: 100%.
Conclusions: (i) The best ‘in vitro’ activity was
observed for Fusarium spp. and Aspergillus spp.
followed by Zygomicetes and S. apioespermum. (ii)
The highest agreement was observed for Fusarium
spp. and Aspergillus spp. (iii) Etest method by reading
after 24 h incubation time could easily replace the
time-consuming NCCLS M38A reference method,
except S. apioespermum.
P019
Does preoperative colonisation with Candida
warrant systematic prophylactic antifungal
therapy in patients undergoing bowel resection
for mesenteric ischemia?
F. Meurant,1 J.P. Koch1 and F. Romano2
1
Kirschberg Hospital, Intensive care, Luxembourg
and 2Kirschberg Hospital, public health, Luxembourg
Introduction: Many cases of systemic candidiasis
are thought to originate from the intestine, especially
when partial mucosal ischemia (PMI) occurred. The
purpose of this retrospective study is to confirm the
high-risk of developing fungal infection in patients
who are precontaminated by Candida spp. (Cs.).
Methods: We examined 115 patients who underwent laparotomy for acute suspected bowel ischemia:
the patients were separated into two subgroups
depending on whether PMI was present (G1:
n = 25) or not (G2: n = 90) at the moment of
surgery. Each subgroups received intravenously
preoperative antibiotic prophylaxis with amoxicillinclavulanic acid (2 g every 8 h) plus amikacin
(15 mg kg)1 as a single dose) after bacterial cultures
were done (sputum, urine, nasograstric, as well as
blood-cultures) on day 1 (D1). The candida colonisation index (CCI = colonised sites/expored sites) was
calculated every 4 days (on DA, D8 and D12).
Fluconazol was started (400 mg 12 h) as soon as
CCI showing evidence of fungal infection (CCI > 0.7).
Results: The mean hospitalisation time was
12.6 ± 3.5 days for G1 and 7.86 ± 0.5 for G2
(P < 0.001). At D1 we had a 54% (n = 13) positive
Cs rate in G1 and a 58% (n = 52) positive rate in G2
(CCI < 0.47) (P = 0.6). At D4 we had a 60% (n = 54)
57
xxx
Cs colonisation rate in G2 with one Cs infection rate
(CCI > 0.7).At this time in G1 however we had a 81%
(n = 20) incidence of fungal pneumonia (sputum+)
with a CCI > 0.7 showing evidence of fungal infection
(P < 0.001). At D8 21% (n = 19) of G2 patients also
had a CCI > 0.7 warranting institution of antifungal
therapy. At D12, 55% (n = 50) of patients were
cured in G2 as compared with 89% (n = 22) resolution rate in G1 patients (P = 0.003).
Conclusion: Real intestine ischemia is rare. As the
high rate of fungal translocation, the existence of
only one site colonised by Cs. in patients with PMI
seems to be enough to consider them preinfective
and to warrant systematic prophylactic antifungal
therapy. The significance of the first CCI must be
re-evaluated for these high-risk patients.
revealed a good in vitro activity All C. krusei species
were resistant to flucytosine whereas the other species
were highly susceptible.
Amphotericin
Fluconazole
Itraconazole
Ketoconazole
5-flucytosine
Voriconazole
MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90
C. albicans (38)
0.19
0.25
0.25
0.75 0.05
0.13
0.03
0.03
0.06
0.13
0.03
0.03
C. tropicalis (15)
0.25
0.5
1
2
0.09
0.75
0.03
0.06
0.06
0.25
0.05
0.13
C. parapsilosis (19)
0.5
0.75
1
2
0.03
0.38
0.03
0.06
0.03
0.06
0.03
0.03
C. glabrata (18)
0.25
0.5
16
48
1.5
8
0.25
1.5
0.03
0.06
0.25
0.5
C. krusei (4)
0.5
0.75
48
256
0.5
1
0.5
1
C. lusitaniae (4)
0.5
1.5
0.5
1.5
0.03
0.5
0.03
0.03
0.03
0.03
1
2
0.09
0.13
0.03
0.03
0.03
C. guilliermondii (2) 0.03
32
32
0.25
0.38
0.03
0.03
0.03
0.03
0.03
0.03
P020
Conclusions: Fluconazole seems to be a very good
tool in the treatment of non-krusei and non-glabrata
Candida infections and amphotericin B is still an option
for all non-lusitaniae Candida infections. Voriconazole
appears to be an attractive alternative to antifungal
therapy.
EM > in vitro activity of six antifungal agents
against clinical Candida isolates
P021
V. Monteiro Lopes, J. Manuel Pereira and
J. Manuel Amorim
Hospital Geral de Santo António, Microbiology,
Porto, Portugal
Introduction: The purpose of this study was to
evaluate the in vitro activity of six antifungal agents
against clinical significant Candida species isolated in
our Hospital.
Material and methods: We tested 100 Candida
species isolated specimens clinically significant. The
species were identified with Vitek products. The in
vitro activity was determined by the Etest strips of
amphotericin B, fluconazole, itraconazole, ketoconazole, flucytosine and voriconazole with RPMI agar,
2% glucose, as recommended by the manufacturer.
The quality control was performed with Candida
parapsilosis ATCC 22019, Candida krusei ATCC 6548
and Candida albicans ATCC 90028. We used the MIC
interpretive criteria from NCCLS and those published
by Rex et al CID 1997; 24:235–247.
Results and discussion: The MIC50 and MIC90
lg ml)1obtained are represented in the following
table: Amphotericin B showed a good in vitro activity
against all species with the exception of C. lusitaniae, as
expected. Fluconazole was highly active against
C. albicans, C. tropicalis, C. parapsilosis, C. lusitaniae
and C. guilliermondii. The classical azoles were poorly
or no active against C. glabrata and C. krusei; voriconazole MIC’s were higher, for the latest species, but it
58
In vitro activity of a new triazole BAL4815
against Candida isolates with decreased
fluconazole susceptibility
J.W. Mouton,1 P.E. Verweij,2 P. Warn,3 D. Denning,3
M. Heep,4 N. Isham5 and M. Ghannoum5
1
Canisius Wilhelmina Hospital, Nijmegen, The
Netherlands, 2RUNMC, Nijmegen, The Netherlands,
3
University of Manchester, Stopford Building,
Manchester, UK, 4Basilea Pharmaceutica, Basel,
Switzerland and 5Case Western Reserve University,
Cleveland, OH, USA
Background: BAL4815 is a new triazole, the active
component of BAL8557, a water-soluble prodrug for
oral and IV application. BAL4815 has a long halflife
suitable for once daily or possibly once weekly dosing
and is currently entering phase III clinical development. Little data exist on the activity against clinical
isolates, especially against isolates resistant to other
azoles. We compared the in vitro activity of BAL4815
with that of fluconazole (FLC) and voriconazole
(VRC) against 231 Candida isolates. The panel was
preselected at three centers to include isolates
demonstrating decreased susceptibility to FLC.
Methods: Strains were isolated during clinical routine, FLC non-susceptible clinical strains were added
from culture collections. Microdilution testing was
done in accordance with CLSI M27-A2 guidelines in
RPMI 1640 MOPS broth with L-glutamine without
bicarbonate. Plates were incubated at 35 C for 48 h.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
The MIC was determined visually and/or spectrophotometrically as the lowest concentration of drug
showing ‡50% reduction of growth compared with
that of the growth control.
Results: MIC distributions (lg ml)1) for BAL4815,
VRC, and FLC were as follows. Over all strains,
MIC50s of BAL4815, VRC and FLC were 0.03, 0.06
and 4 lg ml)1 respectively. The MIC50s for C. glabrata
were 0.25, 1, and 16 lg ml)1, BAL4815 being
significantly more active than VRC.
BAL4815
C. albicans FLC-s
(n = 62)
C. albicans FLC-r
(‡32; n = 29)
C. glabrata (n = 45)
C. krusei (n = 22)
C. parapsilosis (n = 21)
C. tropicalis (n = 20)
C. norvegensis (n = 2)
C. guilliermondii
(n = 10)
C. lusitaniae (n = 10)
C. dublinensis (n = 10)
VRC
FLC
Range
MIC50 Range
MIC50 Range
MIC50
0.001–8
0.008 0.004–1
0.008 0.06–16
0.5
0.004–8
0.25
0.25
32–128
64
0.001–8
0.03–2
0.001–16
0.001–8
0.125–0.5
0.002–1
0.25
0.25
0.03
0.015
1
0.25
0.06
0.03
0.125–128
4–128
0.25–64
0.25–128
64–128
2–8
16
32
2
1
0.06–32
0.008–8
0.06–4
0.008–32
0.008–8
1
0.125 0.015–0.25
0.06
4
0.001–0.015 0.001 0.004–0.015 0.008 0.25–1
0.5
0.001–0.008 0.008 0.008
0.008 0.125–0.5 0.25
Conclusions: BAL4815 shows promising antifungal activity against Candida species including azole-R
strains. Overall, the activity of BAL4815 was at least
equal to VRC.
P022
Distribution and antifungal susceptibility of
Candida species causing nosocomial candiduria
with E-test and microdilution methods
B. Ozhak,1 D. Ogunc,1 D. Colak,1 G. Ongut,1 F. Gunseren,2
D. Inan,2 M. Gultekin,1 M. Gonul1 and T. Vural1
1
Akdeniz University School of Medicine, Medical
Microbiology, Antalya, Turkey and 2Akdeniz
University School of Medicine, Infectious Diseases
and Clinical Microbiology, Antalya, Turkey
Introduction: The incidence of nosocomial fungal
infections has increased substantially over the past
several decades and Candida species are the most
common pathogens. The aim of the study is to
determine; (i) the distribution of Candida spp. (ii) antifungal susceptibilities, and (iii) correlation between
Etest and microdilution methods in Candida spp.
causing nosocomial candiduria.
Materials and methods: A total of 100 urine
nosocomial Candida spp. were isolated from urine
specimens between March 2003 and May 2004 at
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
Central Laboratory of Akdeniz University Hospital,
Turkey. Identification of the isolates was performed by
standard microbiologic procedures. Organisms were
identified to species level by germ tube test, evaluation
of morphologic properties on cornmeal tween 80 agar,
carbohydrate assimilation tests with API 32C strips
(bioMèrieux, France). Susceptibility to fluconazole,
voriconazole, caspofungin, and amphotericin B were
determined by broth microdilution method as specified
by National Committee for Clinical Laboratory Standards (NCCLS) document M27-A2. Susceptibility to
fluconazole, voriconazole, itraconazole, and amphotericin B were determined by E-test method according
to manufacturer’s recommendations. E-test MICs were
read at 48 h and were compared with reference to
microdilution MICs read at 48 h.
Results and discussion: Although Candida albicans
was the most common species identified, non-albicans
Candida spp. accounted for 56% of all our Candida
isolates. The most potent azole antifungal was voriconazole, followed by fluconazole and itraconazole.
MIC?L?90?L? values of voriconazole, fluconazole,
caspofungin, amphotericin B by broth microdilution
method were found as 0.125, 16, 1, 2 respectively.
MIC?L?90?L? values of itraconazole were 0.38 by Etest.
The overall agreement between the results of the two
methods (Etest at 48 h within ± two log<L>2?L?
dilutions of standard MICs at 48 h) were 71% for
amphotericin B, 94% for voriconazole, 84% for
fluconazole. The majority of nosocomial fungal
urinary tract infections are caused by C. albicans and
other Candida spp. Identification of Candida to the
species level and antifungal susceptibility patterns are
important clinical and epidemiological markers for the
5 presence of resistant Candida strains.
P023
In vitro activity of fluconazole, Ketoconazole
and amphotericin B against Candida isolated
from Candida vaginitis by disk diffusion method in Shiraz, Iran
Pakshir,1 Rezaie,2 Yazdani3 and Kimiaghalam4
1
Faculty of Medicine, Parasitology & Mycology,
Shiraz, Iran, 2Faculty of Pharmacy, Medicinal
Chemistry, Shiraz, Iran, 3Faculty of Medicine,
Obsterics & Gynecology, Shiraz, Iran and 4Faculty of
Pharmacy, Medicinal Chemistry, Shiraz, Iran
Introduction: Candida vaginitis is the most common
problem in women. Recurrences, relapses and chronic
59
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courses are considered as serious clinical problems in
these patients. Candida albicans is the major cause of
Candida vaginitis and the other species are less common
but an increase of non-albicans species has been
observed, particularly in recurrent cases. Candida albicans
is usually sensitive to Immidazoles but other species are
occasionally resistant to them. In this study, we
investigated the activity of three antifungal drugs against
Candida vaginitis isolates by disk diffusion method.
Patients and methods: A total of 80 Candida
species from 183 patients suspected Candida vaginitis
were isolated and identified by Traditional methods
and Chromagar Candida media. Fluconazole, ketoconazole and amphotericin B powders were dissolved
in their solvents and loaded on free drug disks in
25.15 and 10 lg per Disk concentration respectively.
Yeast inoculum suspension were prepared according
to 0.5 MCFarland density standard and inoculated on
RPMI 1640 agar plates by dipping and streaking a
sterile cotton swab in to the surface of the plates and
incubated at 35 C. Inhibiting zone diameters of disks
were measured after 24 and 48 h incubation.
Results: By using disk diffusion method, Antifungal
susceptibility test on 80 Candida species including:
Candida albicans 63 (87.75%), Candida glabrata 7
(8.75%), Candida krusei 5 (8.75%), Candida tropicalis 2
(2.5%) and non-albicans 3 (3.75%) reveal 8 (10%),
43 (53.75%) and 2 (2.5%) resistance to fluconazole,
ketoconazole and amphotericin B respectively.
Conclusion: In this study, non-albicans species had
no significant differences in resistance to antifungal
drugs, compared with Candida albicans isolates.
Antifungal susceptibility test by disk diffusion method
is a quick and simple method for determination of
resistance in Candida species, specially in routine
laboratories.
P024
Antifungal activity of thyme and oregano oils
against Scopulariopsis brevicaulis
E. Pinto,1 C. Cavaleiro,2 M.J. Gonçalves3 and
L. Salgueiro4
1
Faculty of Pharmacy, Microbiology/CEQOFF, Porto,
Portugal, 2Faculty of Pharmacy, Pharmacognosy/CEF,
Coimbra, Portugal, 3Faculty of Pharmacy,
Pharmacognosy/CEF, Coimbra, Portugal and 4Faculty
of Pharmacy, Pharmacognosy/CEF, Coimbra, Portugal
tions caused by Scopulariopsis species (like S. brevicaulis) have been described, namely onychomycosis,
mycetoma and keratitis. Scopulariopsis spp. appears to
have inherent broad-spectrum resistance to different
classes of antifungal. The increasing resistance to
antifungal compounds and the reduced number of
available effective drugs urged the search for therapeutic alternatives. Previous research of our team
demonstrated the antifungal activity of thyme and
oregano oils against Candida species (1–3) and
dermatophytes (4, 5). The aim of this study is to
evaluate the antifungal activity of the essential oils of
Lippia graveolens and Origanum virens (oregano oils)
and Thymus vulgaris, used in folk medicine, in order
to support their use as antifungal agents in the
treatment of infections by Scopulariopsis brevicaulis.
The composition of the oils was investigated by gaschromatography and gas-chromatography/mass
spectroscopy. The minimal inhibitory concentration
(MIC), determined according to the NCCLS protocol
M38-A and the minimal lethal concentration (MLC)
were used to evaluate the antifungal activity against
Scopulariopsis brevicaulis (clinical isolate). The antifungal activity of their major components (carvacrol,
gama-terpinene, para-cymene and thymol) was also
evaluated. The essential oils showed similar antifungal activity. MIC and MLC values are similar, ranging
from 0.16 to 0.32 ll ml)1. This is probably due to
their chemical composition, mainly composed by
carvacrol (70.3% T. vulgaris; 68.1% O. virens and
44.8% L. graveolens). Thymus vulgaris and Origanum
virens, which have the highest content of carvacrol,
were the most active. The MIC value for both samples
and carvacrol was the same. This study showed the
antifungal activity of these essential oils on Scopulariopsis brevicaulis, which may contribute to the
scientific basis for supporting future therapeutical
trials on onychomycosis due to Scopulariopsis species
and dermatophytes.
Acknowledgements: FCT, POCTI and FEDER
(POCTI/40167/ESP/2001) for financial support.
References
1. Salgueiro LR et al. Planta Medica 2003; 69:871–874.
2. Pina-Vaz C et al. Europ Acad Dermatol Venereol 2004; 18:73–
78.
3. Salgueiro L et al. Revista de Fitoterapia 2002; 2 suppl 1:204
(A244).
4. Pinto E et al. Clin Microbiol Infect 2003; 9(suppl 1):222 (P966).
5. Pinto E et al. 33rd International Symposium on Essential Oils,
2002; p. 120.
Scopulariopsis species are common soil saprobes with
a wide geographical distribution. A variety of infec-
60
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
P025
Reduced toxicity and improved compliance with
chemotherapy courses in patients with AML
receiving caspofungin therapy for systemic
fungal infection in the AML15 trial
C.H. Poynton,1 H. Gover,2 S. Laretta3 and
A.K. Burnett1
1
University Hospital of Wales, Haematology, Cardiff,
UK, 2University Hospital of Wales, Pharmacy, Cardiff,
UK and 3University Hospital of Wales, Cancer Trials
Unit, Cardiff, UK
The purpose of this retrospective study was to
establish whether the lack of toxicity of Caspofungin
(particularly nephrotoxicity) translated into improved
patient outcome. In a single centre, the outcome of
128 intensive courses of chemotherapy in 44 patients
entered into the UK AML15 trial was studied from
July 2002 until March 2005. Patients received
systemic antifungal therapy at some time during
their treatment either with amphotericin B (colloidal),
AmBisome or caspofungin. Each neutropenic cycle
was evaluated according to whether the patient
received caspofungin (group 1) or other antifungal
treatment not including caspofungin (group 2).
Cycles where combinations of caspofungin with other
antifungal were used were excluded from the analysis. There was a significant difference in time cohorts
since more AmBisome was used in years 2002–2003
and more caspofungin from 2004–2005 but no other
demographic differences. There were 23 males and
19 females with a median age of 60 years (19–63)
with no difference in FAB subtypes between the two
groups. Duration of severe neutropenia ranged from
6 days (FAB M3 on ATRA) to 96 days (FAB M1 after
FLAG-Ida). To assess the toxicity of antifungal
therapy, renal and liver function chemistry were
compared at baseline, at the start of a course of
antifungal therapy and after 7 days of therapy. For
each course of chemotherapy the time to neutrophil
recovery (1 · 109 l)1), the gap between cycles of
therapy, the number of days of systemic antibiotics
and hospitalisation days were all compared between
the cycles where caspofungin was used (empiric or
proven infection) compared with cycles where other
antifungals were used. A cumulative number of days
delay was compiled based on the time from neutrophil
recovery to the next cycle. It was felt this best reflected
when the physician felt the patient was fit enough to
continue to the next cycle.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
Results: There was no significant difference in
neutrophil recovery time or hepatic toxicity between
the two groups. However there was a major difference in renal toxicity amounting to a reduction in
creatinine from baseline (median 15 lmol) in group
1 compared with a rise in creatinine (median
35 lmol) in Group 2. Moreover, there was a
significantly shorter time delay in group 1 cycles
between courses compared to group 2 patients
(P = 0.02) and a cumulative time delay over four
cycles that amounted to 39 days difference. Group 1
cycles of chemotherapy also had a trend to shorter
overall hospital stay and less use of systemic
antibiotics. In conclusion, caspofungin appears to
be less nephrotoxic than amphotericin preparations
and this seems to translate into a reduced delay in
receiving further chemotherapy.
P026
Voriconazole but not caspofungin exhibits
additive antifungal effects with human
neutrophils against Aspergillus fumigatus
M.S. Simitsopoulou,1 M.D. Dalakiouridou,2
T.K. Konstantinou,2 P.S. Siourda,3 J.I. Ioannidis,3
K.K. Kanellou,3 T.J.W. Walsh4 and E.R. Roilides2
1
Aristotle University of Thessaloniki and
Technological Institute of Thessaloniki, Medical
Laboratories, Thessaloniki, Greece, 23rd Department
of Pediatrics, Aristotle University of Thessaloniki,
Thessaloniki, Greece, 3Technological Institute of
Thessaloniki, Medical Laboratories, Thessaloniki,
Greece and 4National Cancer Institute,
Immunocompromised host Section, MD, USA
Background: Voriconazole (VRC), a broad-spectrum ergosterol synthesis inhibitor, and caspofungin
(CAS), a fungal cell-wall glucan synthesis inhibitor,
constitute new potent therapies for invasive aspergillosis with different mechanisms of activity. Little is
known about the combined effects of VRC or CAS
with neutrophils (PMN), a critical component of
innate immunity, in damaging hyphae of Aspergillus
fumigatus.
Methods: PMN isolated from healthy blood donors
and purified by dextran sedimentation/Ficoll centrifugation were incubated alone or combined with
0.1 mg l)1 VRC or 0.01 mg l)1 CAS and 5 · 103
A. fumigatus hyphae at 37 C and 5% CO2 for 6 and
20 h at a PMN : hyphae ratio of 10 : 1 or 20 : 1.
61
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The PMN-induced % hyphal damage (HD) against
A. fumigatus was then assessed by XTT colorimetric assay assessing metabolic activity of the hyphae
that remained alive. Statistical analysis was performed by ANOVA with Dunnett multiple comparison test.
Results: After incubation for 20 h (seven experiments), PMN and VRC showed an additive antifungal
effect against A. fumigatus hyphae at both 10 : 1 (HD
of PMN + VRC was 41.1 ± 7.7% vs. 16.0 ± 1.2% of
PMN, P < 0.01, or 29.1 ± 6.0% of VRC) and 20 : 1
ratios (HD of PMN + VRC was 45.9 ± 8.4% vs.
20.4 ± 1.3% of PMN, P < 0.01, or 29.1 ± 6.0% of
VRC, P < 0.05). By comparison, the combination of
PMN and CAS at either 10 : 1 or 20 : 1 PMN :
hyphae ratio did not show significantly different HD
from PMN or CAS alone against A. fumigatus (HD of
PMN, CAS and PMN+CAS ranged within 16.0–
22.9% for 10 : 1 or 18.9–20.3% for 20 : 1). After
incubation for 6 h, neither VRC nor CAS showed any
combinational effect with PMN against A. fumigatus.
Conclusions: VRC but not CAS exhibits additive
activity with human PMN in damaging A. fumigatus
hyphae. These discordant combinational results may
be due to the different mechanisms of antifungal
activities of the two drugs and may have important
implications in the overall management and potential improved outcome of invasive aspergillosis.
P027
Efficacy and safety of higher doses of
caspofungin (CAS)
N. Kartsonis, H. Teppler, A. Ngai, M. Bourque,
M. White, A. Williams-Diaz, A. Taylor, R. Lupinacci
and C. Sable
Merck Res Labs, W Point, PA, USA
Objectives: CAS is approved for use at 50 mg day)1
(with a 70-mg load on Day 1 for invasive fungal
infections). In Phase I studies, single CAS doses up to
210 mg and multiple CAS doses up to 100 mg day)1
have been well tolerated. Herein, we review the CAS
efficacy and safety at doses >50 mg day)1 in patients
(pts) from phase II–III experience.
Methods: Ninety-six patients received CAS 70 or
100 mg day)1 as initial therapy in four clinical
trials, including three esophageal candidiasis (EC)
trials (Protocol 003 [P003], P004, P007) and an
invasive aspergillosis (IA) study (P019).
62
Efficacy: EC: three CAS doses (35, 50 and
70 mg day)1) were studied in EC. All pts had
symptomatic, endoscopic and microbiological evidence of EC. A favorable clinical response required
complete symptom resolution and substantial (‡2
grade) reduction in endoscopic lesions. IA: three CAS
doses (50, 70, and 100 mg day)1) were sequentially
evaluated in P019. All pts had proven/probable IA,
refractory or intolerant to standard antifungal therapy (Rx). A favorable response (complete or partial)
required significant clinical and radiographic
improvement. In both EC and IA, efficacy was
assessed using a modified-intention-to-treat (MITT)
approach (‡1 CAS dose and documented fungal
diagnosis).
Safety: Adverse events (AE) were collected from all
pts with EC or IA. Investigators identified the
seriousness, causality, and action on study Rx for
all clinical and lab AE during Rx and for 14 days
postRx. CAS safety data at higher doses (70 and
100 mg day)1) was compared with safety at lower
doses (35 mg and 50 mg day)1) across all four
studies.
Results:
CAS 35 mg CAS 50 mg CAS 70 mg CAS 100 mg
day)1
day)1
day)1
day)1
(n = 34; %) (n = 274; %) (n = 90; %) (n = 6; %)
Clinical drug-related (DR) AE
Fever
Phlebitis/infused-vein
complications
Diarrhea
Nausea
Headache
Rash
Lab DRAE
› ALT
› AST
› Alkaline phosphatase
fl Potassium
› Creatinine
fl Hematocrit
Any DR serious AE (SAE)
Discontinuations due to DRAE
50
21
12
33
8
12
48
20
16
0
0
0
2
9
12
9
49
24
27
24
3
0
9
0
6
3
3
6
1
30
7
8
7
3
1
7
1
1
3
3
6
3
35
11
11
9
9
1
1
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
Efficacy: In EC, the pt characteristics were similar
across the three CAS doses. Higher success rates were
noted at 50 and 70 mg day)1 (79 and 82%) vs.
35 mg day)1 (67%). In IA, the pt characteristics
were also similar across the three doses. No efficacy
differences were noted across the 50 and 70 mg
groups (48 and 44% respectively). Data at the
highest dose (100 mg day)1) were limited (2/6,
33%).
Safety: Mean duration of CAS Rx in pts receiving
70 mg day)1 (n = 90) and 100 mg day)1 (n = 6)
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
was 15.4 days (range 2–90) and 34.8 days (range
7–80) respectively. The AE incidence and safety
profile was relatively similar among pts receiving either higher (>70 mg day)1) or lower
(<50 mg day)1) CAS doses.
Conclusion: CAS at doses up to 100 mg day)1 has
been generally well tolerated. Additional safety and
efficacy data at multiple doses up to 150 mg daily is
currently being collected.
the use of high-resolution qTof-MS and MS/MS
allowed the structural elucidation of the metabolites.
Conclusion: With the formation of the monooxidized BAL4815 in animals, similarly to man, all
species were qualified as suitable toxicological species.
P029
P028
Metabolic profile of BAL8557 and BAL4815 in
liver microsomes rat, human, mouse, rabbit,
dog, cynomolgus and rat hepatocytes
A. Schmitt-Hoffmann, J. Spickerman and M. Wind
Basilea Pharmaceutica Ltd., Basel, Switzerland
Background: BAL8557 is a water soluble pro-drug,
rapidly converted in plasma to the active azole
BAL4815 (BAL). In vitro, BAL showed broad-spectrum antifungal activity against all major opportunistic fungi and true pathogenic fungi, including
fluconazole-resistant strains. The objective of the
present study was to compare the metabolite profiles
of all toxicological species with that in man.
Methods: Mixtures (1 : 1, w : w) of BAL8557 and
4D-BAL8557 (tetra-deuterated) or BAL and 4D-BAL
were incubated at final concentrations of 1, 10 and
100 lg ml)1 with mouse, rat, rabbit, dog, cynomolgus monkey and human liver microsomes for 60 min
and with rat hepatocytes for 24 h. Incubations were
analyzed by capillary-LC-qTof-MS and MS/MS.
Results: In human and cynomolgus monkey microsomes almost no metabolic biotransformation
occurred. However, qualitatively the same metabolite
pattern of BAL was observed in all species, including
human, with the formation of mono-oxidized BAL,
most probably epoxyde or phenol. In rabbit microsomes, an additional di-oxidized metabolite of BAL
was obtained, resulting from the hydrolysis of the
epoxyde. In rat hepatocytes, the glutathione-,
cystein- and N-acetylcystein-phase 2 conjugates of
the mono-oxidized BAL could be identified. No
specific metabolite for BAL8557 could be observed.
Despite the low amounts of metabolite formed, the
unique isotopic pattern of the mixture combined with
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
The in vitro activity of highly lipophilic triazoles
against Candida albicans – underestimated?
M. Seibold,1 G. Just-Nübling,2 A. Kruggel3 and
K. Tintelnot1
1
Robert Koch-Institut, FG 14/Mykologie, Berlin,
Germany, 2Department of Infectiology, Medical
Clinic III, J. W. Goethe University, Frankfurt, Germany
and 3Ernst-Moritz-Arndt-Universität, Institut für
Mikrobiologie, Greifswald, Germany
<lipophilic of MICs the on influence their about
known is little Only medium. casitone or MuellerHinton (AM3), three Medium Antibiotic like media
complex especially used, widely are medium RPMI
than other testing susceptibility antifungal> Seventytwo clinically documented isolates of Candida albicans
were tested for their susceptibility to fluconazole
(FCZ), itraconazole (ICZ), voriconzole (VCZ) and
posaconazole (PCZ) using RPMI medium and microtiter plates made of polystyrene and glass. Selected
isolates were tested with AM3 and casitone medium
additionally. The tests were done based on the NCCLS
M27-A guidelines, but reading was made after 24 h.
MICs of FCZ and VCZ were influenced less than one
titer step on the average by the media and materials
used. MICs of ICZ and PCZ were markedly affected by
the medium, RPMI medium showing higher MICs
compared to other media. The material of the
microtiter plates showed an effect on the MICs of
the extremely lipophilic azoles, ICZ and PCZ, only.
Differences in MICs up to16-fold compared with the
NCCLS method could be observed for ICZ, for PCZ the
effect was somewhat smaller. The MICs of extremely
lipophilic azoles may be artificially elevated. Thus the
potency of such substances could be underestimated
and would lead to a false evaluation of cross
resistance in Candida albicans.
63
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P030
P031
Efficacy of micafungin combined with amphotericin b in a murine model of disseminated
blastoschizomycosis
Candida glabrata strains resistance to
itraconazole
C. Serena, M. Marine, B. Fernandez, J. Pastor and
J. Guarro
University Rovira i Virgili, Unitat de Microbiologia,
Reus, Spain
Background: Blastoschizomyces capitatus is an
uncommon, but frequently fatal, cause of invasive
infection in immunocompromised patients, particularly those with haematological malignancies.
Elimination of the underlying predisposing factors
is crucial for resolution of the infection. No
satisfactory antifungal treatment exists. We have
developed a murine model of disseminated blastoschizomycosis to evaluate the activity of the new
echinocandin, micafungin (MFG) in combination
with AMB.
Materials and methods: Mice were immunosupressed with cyclophosphamide i.p. and 5-fluorouracil i.v. and infected i.v. with 2 · 106 CFU/mouse
with two clinical isolates of B. capitatus FMR 8720
and FMR 8722. One day after challenge, four
groups of 15 mice for each strain received the
following treatments: AMB at 1 mg Kg)1 d)1 i.p.,
MFG at 5 mg Kg)1 twice)1 a day s.c., MFG plus
AMB at the mentioned doses and controls received
no treatment. All drugs were given for 6 days.
Survival was recorded for 30 days and mean
survival time (MST) for each group was calculated
using the Kaplan–Meier method. Five mice from
each group were sacrificed 7 days after challenge.
Liver, kidneys and spleen were aseptically removed,
homogenized and cultured on Sabouraud dextrose
agar. The number of CFU g)1 tissue was then
calculated.
Results: MFG combined with AMB prolonged
significantly the MST respect to control group
(P < 0.0001) and respect to treatments with a
single drug (P < 0.05) in both strains. Combined
therapy reduced significantly the kidney, spleen
and liver fungal loads respect to control group
(P < 0.0001) and respect to monotherapies
(P < 0.05).
Conclusions: The results suggest that micafungin
in combination with amphotericin B may have a
clinical role in the treatment of life-threatenin.
64
E. Swoboda-Kopeć,1,2 M. Da˛browska,1
B. Sulik-Tyszka,2 D. Kawecki,1 M. Łuczak1,2 and
S. Błachnio1
1
Medical University, Microbiology, Warsaw, Poland
and 2Central Clinical Hospital, Microbiology
Labolatory, Warsaw, Poland
Aim of the study: Analysis of susceptibility of
C. glabrata strains isolated from surgical patients
hospitalized in the Central Clinical Hospital (CCH) in
Warsaw.
Material and methods: The study comprised surgical patients hospitalized in The CCH (1200 beds) in
2004–2005. Standard mycological cultures were
done on the following clinical samples: peritoneal
swabs taken during surgery, peritoneal fluid, blood,
bronchial washings, bile, drains, catheters, pus and
urine. Isolated yeast-like fungi were identified by
ID32C assay (bioMerieux, France). Susceptibility to
antifungal agents was tested by Etests (AB Biodisk).
Results: Of 2352 specimens yielding fungal growth,
2422 fungal strains were isolated. There were 874
fungal isolated cultured from surgical patients.
Among them there were 278 strains of C. glabrata.
Resistance to itraconazole was detected in 111
strains (40%) of C. glabrata. The majority of itraconazole-resistant isolates were cultured from peritoneum – 44 (39.6%) and blood cultures – 23 (20.7%).
Conclusions: 1. Surgical patients are at risk for
infections caused by C. glabrata. 2. An emergence of
itraconazole-resistant strains of C. glabrata is
observed in the analysed group of patients.
Acknowledgement: This work is supported by
Polish State Committee for Scientific Research (grant
No. 3PO5A 028 25)
P032
Iatrogenic Pleural aspergillosis treated with
combination of systemic and intrapleural
amphotericin B
Y. Tezer, O. Memikoglu and H. Kurt
Ankara University Facultyof Medicine, Clinical
Bacteriology and Infectious Disease, Ankara, Turkey
Pleural aspergillosis was first described around
170 years ago but remains relatively rare entity
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
when compared with other Aspergillus infections.
This uncommon condition predominantly affects
patients with a bronchopleural fistula complicating
thoracic surgery or tuberculosis. Less commonly
pleural aspergillosis has been reported on a complication of a open or closed pleural instrumentation following extension of established pulmonary
aspergillosis into the pleural space. Although
commonly employed intravenously, new strategies
are necessary to reduce the high mortality rate of
pleural apergillosis. Few reports have investigated
the efficacy of pleural antifungals in human beings.
We report a man with a pleural aspergillosis
without parenchymal involvement and a bronchogeniccarcinoma. and we present our experience on
pleural lipid complex amphotericin B on pleural
aspergillosis fallowing lung surgery.
Case report: A 57-year-old man was referred to
our hospital with diagnosis of lung carcinoma.
The patient was admitted to thorax surgery
department of tertiary hospital. After initial examinations and routine scanning he had been
operated for resection of mass. The frozen result
was adenocarcinoma. After the operation, he
complained of fever, dyspnea and right sided
pleuritic chest pain. During postoperative course
chest tube continually drained purulant fluid.
Fever and leucocytosis were predominant at that
time. Aspergillus fumigatus was grown on cultures
from pleura. Then caspofungin therapy was started. After 14 days, cultures were repeated. A
14-day course of intravenous caspofungin failed
to sterilize the cavity. In repeated cultures again
A. fumigatus were grown. So we decided to
attempt intrapleural antifungal treatment and
systemic amphotericin B. Conventional amphotericin B caused allergic reactions. So have changed
the conventional form to lipid complex form. As
described in literature, a 20-ml solution of 5%
dextrose containing 5 mg of amphotericin B lipid
complex infused during 5 min as an initial dose,
which was gradually increased to 50 mg dl)1 in a
week. The solutions remained in the pleural cavity
with a catheter clamped. After 20 min or 30 min
which was tolerated by him, the catheter was
unclamped and then the residual solution mixed
with purulent fluid was permitted to drain out.
After 35 days therapy, there were no grown on
repeated cultures. Fever and dyspnea like complaints disappear in second week.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
P033
Testing of the in vitro susceptibilities of
Madurella mycetomatis to six antifungal agents
by using the sensititre system in comparison
with a viability-based 2,3-Bis(2-methoxy)4-nitro-5-sulfophenyl)-5-{(phenylamino)carbonyl}2H-tetrazolium hydroxide (XTT) assay and a
modified NCCLS method
W.W.J. van de Sande,1 A. Luijendijk,1
A.O.A. Ahmed,2 I.A.J.M. Bakker-Woudenberg1 and
A. van Belkum1
1
Erasmus MC, Medical Microbiology and Infectious
Diseases, Rotterdam, Netherlands and 2Mycetoma
Research Group, University of Khartoum, Khartoum,
Sudan
Patients suffering from madura mycosis or mycetoma are usually treated by surgery in combination
with postsurgical chemotherapy. In order for the
chemotherapy to be effective, antifungal susceptibility patterns for the fungus should ideally be known.
The in vitro susceptibilities of 36 clinical isolates of
Madurella mycetomatis, prime agent of eumycetoma
in Sudan, for ketoconazole, itraconazole, fluconazole,
voriconazole, amphotericin B and 5-flucytosine were
determined by the sensititre YeastOne system. The
Sensititre YeastOne system is a commercial microdilution method that uses the oxidation–reduction
indicator Alamar blue to determine the in vitro
susceptibilities to the various antimycotic agents.
This system provided a rapid and easy test, and by
using hyphal suspensions it generated results comparable to those of a modified NCCLS method. The
reproducibility of the system ranged from 88.2 to
97.1% depending on the antifungal. The agreement
with the NCCLS method was excellent with percentages agreement between 88.2 and 100.0%, but was
less when compared to the XTT assay (67.6 to
100.0% agreement). Overall, the XTT assay resulted
in relatively higher MICs than the sensititre method,
with a two- or three-step dilution difference. The
MICs of the different antifungal agents appeared
variable and dependent on the M. mycetomatis
isolate. The antifungal activity of the azoles ketoconazole (MIC90 of 0.125 mg ml–1), itraconazole
(MIC90 of 0.064 mg ml)1) and voriconazole
(MIC90 of 0.125 mg ml)1) appeared superior to
that of fluconazole (MIC90 of 128 mg ml)1) and
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amphotericin B (MIC90 of 1 mg ml)1), MIC values
being in the clinically relevant range. All isolates
were resistant to 5-flucytosine (All MICs above
64 mg ml)1). In this study, we found that the azoles
itraconazole and ketoconazole are in vitro very
effective in inhibiting the fungal growth. The current
treatment of eumycetoma patients with these antifungals seems justified. Based on the relatively broad
range of MICs for the antifungal agents obtained,
routine antifungal susceptibility testing for M. mycetomatis isolates seems to be relevant for adequate
therapeutic management.
P034
Antifungal and cytotoxic activity of two
inhibitors of 2,3-oxidosqualene cyclase
S. Voyron,1 P. Forni,2 F. Rocco,3 M. Ceruti3 and
V. Filipello Marchisio1
1
Università di Torino, Biologia Vegetale, Torino, Italy,
2
Università di Torino, Anatomia, Farmacologia e
Medicina Legale, Torino, Italy and 3Università di
Torino, Scienza e Tecnologia del Farmaco, Torino,
Italy
In the present study, we determined the antifungal
properties of two inhibitors of 2,3-oxidosqualene
cyclase (OSC): squalene bis-diethylamine (SBD) and
squalene bis-diethylmethylammonium iodide (SBDI).
OSC is a widely distributed enzyme that catalyse the
cyclization of (3S)-2,3-oxidosqualene (OS) to lanosterol in mammals and yeasts and to cycloartenol or
to a variety of tetracyclic and pentacyclic triterpenes
in higher plants. Effective inhibitors have been
obtained by mimicking the carbocationic intermediates formed during cyclization of OS, by designing
squalene-derived structures, in which the positively
charged carbocation was replaced by nitrogen.
Among this series, SBD and SBDI displayed a high
inhibition activity towards fungal OSC. Fungistatic
and fungicidal activity towards dermatophytes and
other fungi involved in cutaneous and systemic
infections was tested (eight isolates from six species).
For each mycelial isolate, the minimum inhibitory
concentration (MIC) and the minimum fungicidal
concentration (MFC) of both compounds were determined by the broth dilution method. MIC was
evaluated by inoculating 10 ll of mycelial homogenate in 1 ml of Sabouraud glucose liquid medium,
containing serial dilutions from 100 to 0.25 lg ml)1
66
of the substance, and incubated at 24 C in triplicate.
The inoculum for Cryptococcus neoformans was 10 ll
of 1.5 · 105 cells ml)1 suspension. Five days after
the appearance of visible growth in the controls, the
MIC was determined, defined as the lowest concentration of compound giving zero growth in all three
replicates. The MFC was then determined, defined as
the minimum concentration of compound able to
impede further growth on drug-free medium. For this
purpose, the contents of tubes that proved negative
(absence of fungal growth), the first positives and the
controls were filtered through sterile cellulose nitrate
filters, and washed twice with sterile distilled water in
order to eliminate drug residues. The filters were then
placed on drug-free Sabouraud glucose agar in Petri
dishes at 24C for 10 days. SBDI was more active
(MIC range 1.5–12.5 lg ml)1) than SBD (MIC range
6.25–100 lg ml)1). SBDI also showed fungicidal
action towards the dermatophytes tested (MFC range
3–25 lg ml)1) at lower concentration than SBD
(MFC range 12.5–100 lg ml)1). However, SBD was
the only one to show fungicidal activity against
Aspergillus fumigatus and C. neoformans (MFC
100 lg ml)1). Regarding the possible use of these
compounds as antimicotic drugs, we tested the
toxicity of SBD and SBDI on MDCK dog kidney cells.
Our preliminary results showed that both compounds exerted toxic effects at high concentrations
of SBD (12.5 lg ml)1) and SBDI (25 lg ml)1) while
they seemed to be innocuous at lower doses. Because
of the dose dependent toxicity of both compounds on
MCDK cells, further studies on different epithelial cell
lines and in vivo treatments are needed to further
investigate possible pharmacological uses of these
two new antifungal molecules.
P035
Evaluation of the commercial colourimetric
microdilution method (YeastOne) for determining caspofungin minimum inhibitory
concentrations for 70 bloodstream isolates of
Candida species
T.A. Vyzantiadis,1 A. Milioni2 and A. Velegraki2
1
A’ Microbiology Department, Medical School,
Aristotelian University of Thessaloniki,
Thessaloniki,Greece and 2Microbiology Department,
Medical School, University of Athens, Athens, Greece
YeastOne (Trek diagnostic systems, West Sussex, UK)
is a microdilution-based system for testing of seven
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
antifungal agents (amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, voriconazole and
recently caspofungin). Caspofungin, an echinocandin-class antifungal agent, is approved for primary
treatment of candidaemia and other invasive Candida
infections. As it is with all antifungals, caspofungin
susceptibility testing requires careful monitoring of
all test variables to ensure reliability and reproducibility of results. The performance of YeastOne for
testing susceptibility of caspofungin was assessed
against the NCCLS M-27A2 broth microdilution
(BMD) method by testing 70 bloodstream isolates
comprising 23 C. albicans, 10 C. parapsilosis, eight
C. tropicalis, eight C. glabrata, four C. famata, four
C. rugosa, two C.guilliermondii, seven C. lusitaniae and
four C. krusei. Trials were repeated three times on
independent occasions and all plates were incubated
at 35 C for 24 and 48 h before reading. Complete
(MIC-0 at 48-h incubation time) and partial (MIC-2
at 24-h incubation time) endpoint inhibition criteria
were used to read the MICs generated by BMD and
YeastOne. The NCCLS recommended quality control
strains were used. For both methods the lowest
caspofungin concentration that caused significant
reduction (£50%) of growth, below control growth
levels, was taken as the MIC (lg ml-1) value. The
range of MICs recorded for C. albicans was 0.03–1,
C. parapsilosis 0.12–2, C. tropicalis 0.06–2, C. glabrata
0.03–1, C. famata 0.12–2, C. rugosa 0.03–1, C. guilliermondii 1–8, C. lusitaniae 0.5–2 and C. krusei
0.25–1. The percentage agreement between the two
methods varied according to the species tested and
that was attributed to the varied degree of trailing
displayed by the different Candida species in the RPMI
medium. The highest percentage agreement between
BMD and YeastOne was recorded for C. lusitaniae, C.
krusei, C. gilliermondii and C. glabrata (100%) followed
by C. albicans (99%), C. tropicalis (89.9%), C. famata
(88.5%) C. parapsilosis (87.8%) and C. rugosa
(86.5%). There is intraspecific variation in the degree
of susceptibility of certain Candida species such as
C. albicans, C. parapsilosis, C. tropicalis, C. famata and
C. guilliermondii. This emphasizes the importance
performing MICs on every bloodstream and systemic
Candida isolate, and certainly the need for determining the clinical significance of each set of MICs in
respect to outcome. There is good agreement
between the NCCLS M27A2 BMD method and
YeastOne at 24 h incubation using the partial
inhibition criterion. Overall, both methods demonstrated that caspofungin is potent against a wide
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
range of Candida spp. YeastOne proved to generate
reliable and reproducible caspofungin MICs, provided
the major test variable, as the inoculum concentration is rigorously monitored, and trailing phenomena
are carefully interpreted in routine clinical laboratory
practice.
P036
In vitro antifungal drug susceptibilities of clinical isolates of Candida albicans in Tehran Iran
F. Zaini, M. Gerami-Shoar, P. Kordbasheh, M. Safara
and E. Khedmati
1
Medical Mycology Unit, School of Public Health,
Tehran University of Medical Sciences, Tehran, Iran
For the first time from Iran, The antifungal susceptibility pattern of 199 clinical C. albicans isolates were
studied against fluconazole (FL), ketoconazole (KE),
itraconazole (IT), amphotericin B (AP), nystatine (NY)
and terbinafine (TE) using the broth microdilution
method as recommended by NCCLS (m27_A) the end
point was defined as the drug concentration resulting
in ‡90% inhibition of growth relative to control
growth. Triplicate testing of reference and test isolates
performed in each test. MICs range for control
reference strains determined as follow: FL < 0.125–
0.5 lg ml)1 KE < 0.0625–0.125 lg ml)1, IT < 0.125–
0.5 lg ml)1, AP < 0.125–1 lg ml)1, NY < 0.5–1 lg
ml)1, TE < 0.125–1 lg ml)1.
Thus isolates in this study were classified as
susceptible to different antifungal of the MICs were
on-scale, and resistant of the MICs were off-scale of
the clinical isolates tested, 95%, 98%, 92%, 98.5%,
73.6% and 75.4%, were found to be susceptible to
FL, KE, IT, AP, NY and TE respectively. In conclusion, this study documents an excellent agreement
with findings of other investigators.
P041
Distribution of Aspergillus species from clinical
material during the 10 years of period
S. Akçağlar S, C. Evci, B. Ener and O. Töre
University of Uludağ, Microbiol and Infect, Bursa,
Turkey
1
In this retrospective study, species distribution of
Aspergillus was evaluated. All the clinical material in
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which Aspergillus like colonies grow were sent to
mycology section and identified according to coloni
morphology and microscopic morphology. After
identification the importance of this growth was
discussed with the clinician and the ones which were
thought as ethologic agents were appraised in this
study. Total of 602 isolations were detected and
approximately, A. fumigatus constitute half of the
isolations (40.9%). A. flavus (22.9%), A. niger (13%),
A. terreus (1.5%), A. candidus (0.5%) and A. nidulans
(0.2%) were the other species isolated. Thirteen
percentage of isolates could not be identified to
species level. Haematological wards were the main
place, following chest diseases and long-term hospitalisation was the most important factor for aspergillosis.
P042
Surveillance of airborne Aspergillus in
a Portuguese University Hospital
R. Araujo, C. Pina-Vaz and A.G. Rodrigues2
Faculty of Medicine, Microbiology, Porto, Portugal
and 2Faculty of Medicine and Hospital S. Joao,
Anesthesiology, Porto, Portugal
1
Aspergillus species cause serious infections among
immunocompromised and transplant patients.
A. fumigatus is responsible for >90% of all invasive
aspergillosis diagnosed infections. Our objective was
to quantify airborne Aspergillus in wards, intensive
care units and operating rooms of a University
Hospital in Porto during the summer (four consecutive weeks from 23 of June to 22 of July of 2004) and
the autumn (four consecutive weeks from 22 of
September to 15 of October of 2004). Sixteen rooms
were evaluated in this study: two operating theatres
(no visitors; use of sterile clothes), one support room
adjacent to operating room (no visitors; use of sterile
clothes), four intensive care units (eight visitors per
day each; use of sterile clothes), two rooms presenting negative pressure (four visitors per day; no use of
sterile clothes), two haematological units (eight
visitors per day; use of sterile clothes), four intermediate care units (12 visitors per day each; no use of
sterile clothes) and one control room (no limitation of
visitors; no use of sterile clothes). HEPA filters are
used on operating rooms and intensive care units.
Detection of Aspergillus in the air was performed by
analysis of 1512 litres of air using an Andersen
68
impactor (flow of 28 l min)1). Air samples were
grown in DG18 culture medium during 10 days, at
37 C. A. fumigatus was the most frequent species
among clinical units, followed by A. flavus, A. niger
and A. glaucus. Operating theatres, the support room
adjacent to operating theatre and intensive care units
presented none or rare isolates of Aspergillus species
(never higher than 3 cfu m)3 for any species). Wards
with negative pressure presented lower values of
Aspergillus species than intermediate care units and
haematological units, but higher values comparing
with operating rooms and intensive care units (never
higher that 13 cfu m)3 for A. fumigatus and presenting considerable lower values for other species).
Haematological units and intermediate care units
showed a similar level of air contamination by
Aspergillus comparing with the control room. The
maximum detected values in haematological units
were: A. fumigatus 76 cfu m)3, A. flavus 9 cfu m)3,
A. niger 6 cfu m)3 and A. glaucus 6 cfu m)3. Other
Aspergillus species like A. terreus, A. nidulans and
A. versicolor were also occasionally detected in much
smaller amounts. No significant differences were
found when comparing the two observational periods. HEPA filters and negative pressure reduce
significantly Aspergillus species from medical facilities. Limitation of visitors and the use of sterile clothes
may also influence significantly the air quality of
medical care units. Continuous air monitoring of
medical facilities should be performed frequently for
preventive reasons.
Acknowledgement: This study was supported by
grant n 60901 from Fundaçao Calouste Gulbenkian.
P043
Invasive aspergillosis in Children
E. _Ince,1 E. Çiftçi,1 H. Güriz,1 S. Fitöz,2 S. Öncel,1
N. Dalgıç,1 N. Taçyıldız,1 M. Ertem1 and Ü. Doğru1
1
Department of Pediatrics, Ankara University,
Ankara, Turkey and 2Department of Radiology,
Ankara University, Ankara, Turkey
Objective: To evaluate the clinical features and
outcome of children with invasive aspergillosis in a
Turkish children’s hospital.
Methods: The children with invasive Aspergillosis
between 2000 and 2005 in Ankara University
Medical School, Department of Pediatrics were retrospectively investigated.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
Results: During the study period, 14 children with
invasive aspergillosis were detected. Nine patients
had invasive pulmonary aspergillosis, two patients
had disseminated aspergillosis, one patient had
hepatic aspergillosis, one patient had sino-orbitalcerebral aspergillosis, and one patient had catheterrelated infection. All patients had an underlying
illness, including leukemia (12 patients), hemophagocytic lymphohistiocytosis (one patient), and
systemic lupus erythematosus (one patient). Two
patients with disseminated aspergillosis had also
lung involvement. Main symptoms and findings
seen in 11 children with lung involvement were
fever, chest pain and dyspnea. In 4 of 11 children,
there were no respiratory symptoms or findings at
the time of the diagnosis, and fever was the only
finding in three children. Hemorrhagic nasal discharge and nasal soft tissue necrosis were observed
in two children with sinus involvement. Fever was
the only finding in a child with catheter-related
infection, whereas fever and hepatomegaly were
detected in a child with liver involvement. The time
between initial symptoms and diagnosis were 1–
30 days. Chest plain radiograms were normal 4 of
11 children with invasive pulmonary aspergillosis.
The diagnosis of invasive aspergillosis was confirmed by cultures in eight patients (Aspergillus
fumigatus in four children, Aspergillus niger in three
children, and Aspergillus flavus in a child). The
diagnosis of invasive aspergillosis was established by
histological (two patients) and radiological (four
patients) tests in the remaining patients. Serum
Aspergillus galactomannan antigen test were performed in only three patients and results were
negative. Initial therapy included amphotericin B in
all patients. Some patients received itraconazole or
caspofungin in combination with amphotericin B.
Surgical intervention was required in three patients.
Three patients died during acute infection. All
patients, whose primary infections have resolved,
have survived. Relapse has not been observed in
children who have recovered.
Conclusion: Invasive aspergillosis may present
with various clinical findings. Prognosis depends
mainly on underlying illness. Amphotericin B, in
combination with itraconazole or caspofungin is
effective in severe infections. After acute infection
resolves, prolonged treatment with oral itraconazole
is effective for prevention of relapses.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
P044
Effect of thyme and ajowan essential oils on
growth control of Aspergillus parasiticus in
Pistachio
Maskooki,1 Mortazavi,2 Mehraban2 and
Poorazarang2
1
Khorasan Science and Technology Park (KSTP), Food
Tech., Mashhad, Iran and 2Ferdowsi University of
Mashhad, Food Tech., Mashhad, Iran
In a laboratory research for evaluating effects of
essential oils against growth of aflatoxin-producing
strain of Aspergillus parasiticus on pistachio as
fungicidal agents, thyme and ajowan oils at
200 ppm and 300 ppm respectively on two type of
pistachio (with green peel and with dry peel) and
control were studied . Also organoleptic tests for
evaluating odor and flavor residual of mentioned
essential oils on pistachio were evaluated. The data
were analyzed by RCBD and DMRT mean separation.
The results showed that in spite of more thymol
content in ajowan oil compared to thyme oil, thyme
oil in 200 ppm can better than ajowan oil in
300 ppm control growth of Aspergillus parasiticus
and completely can prevent the growth of mycelium.
Evaluating of organoleptic tests by 13 panelists
statistically showed that the samples that treated by
ajowan oil more accepted than the others. We
concluded that both thyme and ajowan oils are
natural promising fungistatical agents and can be
used instead of harmful synthetic fungicides.
P045
Release of glucan compared to other surrogate
markers by Aspergillus fumigatus during
in vitro growth
M.A.S.H. Mennink-Kersten, D. Ruegebrink,
R.R. Klont and P.E. Verweij
1
Radboud University Nijmegen Medical Centre,
Medical Microbiology, Nijmegen, Netherlands
Surrogate markers, such as the detection of circulating genomic DNA or antigen, are becoming increasingly important for the early diagnosis of invasive
aspergillosis (IA). The kinetics of release of these
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markers, however, is largely unknown. We investigated the release 1,3-ß-D-glucan (BG) (Glucatell)
compared to galf-antigens (Platelia Aspergillus) and
DNA (PCR) during in vitro growth of A. fumigatus on
different media. The results showed that release of
surrogate markers is correlated to the growth phase
of the fungus, which depends on available nutrients,
i.e. glucose. Whereas BG and galf-antigens are
released during logaritmic growth, DNA is only
released during the lytical phase. BG seems to be
detected somewhat earlier than galf-antigens but the
medium BG background is variable, which makes it
difficult to estimate the exact time when a culture
supernatant turns positive. During the lytical phase,
concentration of BG in the culture supernatant
decreased after 48 h, while concentration of galfantigens decreased after 64 h of growth. The activity
of b-galactofuranosidase, an extracellular enzyme
that is able to destroy galf-epitopes, fluctuated in the
culture filtrates in parallel with galf-component
concentration. Furthermore, the pH of the culture
supernatant influenced the b-galactofuranosidase
activity and the maximum galf-component concentration. Decrease of BG is probably due to b-glucanases produced by the fungus. In conclusion, several
factors clearly influence the detection of surrogate
markers in vitro. These same factors might also play a
role at the infection site in the clinical situation, i.e.
the human lung.
P046
Cutaneous aspergillosis treatment with
voriconazole and local LIPOSOMAL
amphotericin-B: a case experience
R. Orlando,1 D. De Blasi,1 A. Gagliardi,1 L. Ziello,1
P. De Cristofaro2 and E. Miraglia1
1
U.O.C. Ematologia, Ospedale San Giovanni Bosco,
Naples, Italy and 2Laboratorio Patologia Clinica,
Ospedale San Giovanni Bosco, Naples, Italy
Skin infection by Aspergillus is often reported in
patients with oncohaematological diseases such as
acute leukaemias. Cutaneous aspergillosis is defined
primary, involving sites exposed to mechanical
trauma injuring cutis integrity, and secondary,
caused by spreading for contiguity from a neighbouring infection area or by infection haematogenous dissemination to the skin from a primitive
extracutaneous infection source (often the lung).
70
We describe a case of cutaneous aspergillosis in a
patient with acute lymphoblastic leukaemia. In the
deep and lengthy granulocytopenic period following
anti-leukaemic chemotherapy the patient developed
several ulcerative-necrotic cutaneous lesions resembling ecthyma gangrenosum. Nevertheless specimen
culture of these skin lesions revealed Aspergillus
flavus, so systemic antifungal therapy with voriconazole i.v. was started. In addition, cutaneous lesions
local care (i.e. debridment plus liposomal amphotericin-B smearing) was performed. This combined
approach allowed after two weeks Aspergillus disappearance in the cultures obtained from skin ulcerations.
P047
Postoperative aspergillosis
A.C. Pasqualotto1 and D.W. Denning2
1
School of Medicine, University of Manchester,
Manchester, UK and 2Wythenshawe Hospital,
Manchester, UK
Introduction: While invasive aspergillosis typically
occurs in immunocompromised patients, cases of
troublesome surgical site infections have been reported in immunocompetent patients. The purpose of
this article is to describe 3 cases of post-operative
aspergillosis, followed by literature review.
Cases: The first case was a 70-year-old patient who
underwent elective aortic valve replacement and died
because of Aspergillus endocarditis. The diagnosis
was not suspected premortem. The second case was a
16-year-old girl submitted to elective neurosurgery
for Chiari I malformation who died despite medical
and surgical therapy. The third case was a 43-yearold woman who recovered from cerebral aspergillosis
following treatment with voriconazole.
Methods: Medline search and review of references
in articles and in our own files for cases meeting the
definition of aspergillosis occurring in the postoperative setting, not related to primary lung infection.
Cases of primary cutaneous aspergillosis were not
considered for this review, as well as infection
associated with intravascular devices.
Results: Postoperative aspergillosis is usually an
indolent infection. After the publication of the first
case in 1933, many additional patients with this
condition were reported. More than 100 well-documented cases of endocarditis and aortitis have been
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
reported to date. These cases were characterized by a
low rate of antemortem diagnosis (Aspergillus is
rarely isolated from blood cultures) and an extremely
high mortality. Preautopsy diagnosis was usually
associated with the examination of the valve or
emboli to large peripheral arteries. Most of the
reports have associated the infection with contaminated grafts, contaminated sutures or intraoperative
shedding of spores. More than 30 cases of wound
aspergillosis were reported; different from patients
with endocarditis, the interval between surgery and
infection was measured in days, not months. Wound
aspergillosis can progress to profound or disseminated infection, so all patients need to be aggressively
treated with combined medical and surgical therapy.
A. flavus is more common than A. fumigatus in many
of these wound infections. Other presentations of
postoperative aspergillosis, including mediastinitis,
bronchial infections, aspergillosis following breast
surgery, abdominal surgery, orthopedic surgery,
neurosurgery, ophthalmological surgery, and surgical dental procedures are reviewed. The optimal
treatment of Aspergillus surgical infections has not
been specifically studied. Survival seems to depend on
the excision of the infected tissue. Concomitant use
of a systemic antifungal agent is also crucial.
Prophylaxis of these infections should include special
care with the ventilation system in the operating
room.
Conclusions: Postoperative aspergillosis is relatively common compared with many other manifestations of disease and rarely affects the lungs.
Mortality is high in non-cutaneous infections.
P048
Non-surgical endocarditis by aspergillus with
mould isolation in blood culture and negative
galactomannan
J. Pemán,1 R. Ortı́z,1 F. Osseyran,2 C. Pérez-Bellés,1
M. Crespo,2 J. Garcı́a,2 J. Frasquet1 and M.
Gobernado1
1
Servicios de Microbiologı´a y Reanimación, Hospital
Universitario La Fe,Valencia, Microbiology, Valencia,
Spain and 2Servicios de Microbiologı´a y Reanimación,
Hospital Universitario La Fe,Valencia, Critical Care,
Valencia, Spain
Introduction: Infectious endocarditis caused by
Aspergillus spp. is rare and usually affects heart
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
surgery or immune-suppressed patients. In the cases
described in the literature, the isolation was obtained
postmortem or from cultures of the valve and only in
very few cases had the mould also been isolated in
the blood culture. To our knowledge, this is the third
case described of Aspergillus endocarditis in which the
mould was isolated in the blood culture. Paradoxically, in he present case serum galactomannan
antigen (GM) was negative.
Clinical report: A 58-year-old male, ex-smoker of
20–30 cigarettes day)1 without other toxic habits;
diagnosed of chronic obstructive pulmonary disease
in treatment with oxygen and inhaled b-agonist
therapy and without history of immune-suppression. Before admission, he presented brief episodes
of amaurosis fugax, embolism in lower members
and abdominal pain which brought him to consultation in the Emergency Area of the hospital.
After admission, several examinations were
performed with the following results: (i) Echocardiogram: vegetation in the mitral valve, (ii) Abdominal Angio-CT: obstruction of a branch of the
mesenteric artery and renal bilateral infarction
areas, and (iii) Abdominal arteriography: mycotic
aneurysm in the ileocecal branch of the mesenteric
artery. Consequently, urgent valve replacement
was indicated and a metallic prosthesis was
implanted. The native valve and the vegetation
were sent to the Microbiology Laboratory for
microscopic examination and culture. In the Gram
stain o the vegetation, branched structures were
observed and confirmed as fungal by calcofluor
white stain. Thus, antifungal therapy was started
with voriconazole (250 mg day)1). In the vegetation culture a filamentous fungi was isolated and
later identified as Aspergillus fumigatus. After surgery, the patient presented fever and A. fumigatus
was also isolated from both blood and urine
cultures even though two serum samples for GM
were negative (OD < 0.5). Due to poor evolution
of the patient, 10 days later caspofungin
(70+50 mg day)1) was added to treatment, but,
in spite of double antifungal therapy, the patient
died 45 days after his admission.
Conclusions: (i) This is the third case described of
Aspergillus endocarditis with isolation in blood
culture. (ii) The vision of hyphae in the direct
microscopic examination allowed early treatment.
(iii) Paradoxically, GM determinations were negative.
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P049
Bronchopulmonary aspergillosis – role of
selective culture media in its rapid laboratory
diagnosis.
H.S. Randhawa, A. Chowdhary, K. Preeti Sinha and
T. Kowshik
Department of Medical Mycology, V. P. Chest
Institute, University of Delhi, India
Background: The isolation in culture of the etiologic agent remains the mainstay in the laboratory
diagnosis of aspergillosis, notwithstanding the various advances in serologic and molecular diagnostic
techniques. We have recently reported that Aspergillus fumigatus, the principal etiologic agent of
aspergillosis, is inhibited in vitro by Candida albicans
(1), a frequent commensal or colonizer of the
oropharynx, gastrointestinal and respiratory tract
of humans. These observations were corroborated by
Becker and coworkers from Germany (2). The in vitro
antagonism between A. fumigatus and C. albicans as
well as other species such as C. glabrata has clinical
significance in that it may result in under-diagnosis
of aspergillosis especially in patients whose respiratory tract is colonized by C. albicans. Consequently,
such patients may receive inappropriate chemotherapy and suffer from avoidable complications. With a
view to overcoming this difficulty, a series of
experiments were conducted in our laboratory,
demonstrating that incorporation of fluconazole
(5 lg ml)1) in peptone glucose agar (PGA) facilitated
rapid and enhanced isolation of A. fumigatus from
aqueous suspensions and sputum specimens seeded
with predetermined conidial inoculum and graded
concentrations of C. albicans yeast cells (3). In this
communication, we shall highlight the relevance of
selective culture media such as peptone glucose
fluconazole agar (PGFA), peptone glucose egg albumin agar (PGEA) and yeast phosphate agar (YPA) for
rapid and enhanced isolation of A. fumigatus from
sputum specimens of patients clinically diagnosed as
invasive pulmonary aspergillosis (IPA), allergic bronchopulmonary aspergillosis (ABPA) and aspergilloma.
Material and methods: Three grades of mixed
saline inocula of the two antagonistic fungi were
prepared with relative population density of A. fumigatus vis-à-vis C. albicans in the ratios of 1 : 10,
1 : 20 and 1 : 100. They were inoculated on PGFA,
PGEA, YPA and PGA plates and incubated at 28 C
72
for up to 8 days. In addition, freshly expectorated
sputum specimens were collected from patients
clinically diagnosed as IPA, ABPA, aspergilloma or
other bronchopulmonary diseases with colonization
of their oropharynx/respiratory tract by C. albicans.
The specimens were homogenized, streaked liberally
over plates of the afore-mentioned media and incubated at 28 C for up to 8 days. Evaluation of the
A. fumigatus growth was done by enumerating the
number of its colonies appearing on a medium plate
and measuring their radial spread.
Results: It would be apparent from the data to be
presented that PGFA is the best selective medium for
rapid and enhanced isolation of A. fumigatus from
sputum of patients with C. albicans colonization in
the oropharynx or respiratory tract. This was
followed by YPA and PGEA which also served as
efficacious selective media. The superiority of the
selective media over PGA as control was determined
by the number of A. fumigatus colonies isolated and
the incubation time required for their macroscopic
growth and sporulation to be readily recognizable.
Conclusions: PGFA, followed by PGEA and YPA, is
recommended as the best selective medium for rapid
and enhanced isolation of A. fumigatus from sputum
or other clinical specimens colonized by C. albicans in
patients suspected of aspergillosis. The use of these
selective media would facilitate early diagnosis and
timely initiation of appropriate antifungal therapy
that is so vital for successful treatment of aspergillosis. The role PGEA and YPA as selective media for
the isolation of Histoplasma capsulatum, Blastomyces
dermatitidis and dermatophytes such as Trichophyton
mentagrophytes is already well known (4–6).
References
1. Randhawa HS, Sandhu RS, Kowshik T. In vitro inhibition of
Aspergillus fumigatus by Candida species, especially C. albicans
and C. glabrata. Curr Sci 2002; 82: 860–865.
2. Becker A, Akkad N, Forster DH, Kniehl E. Growth of Aspergillus
fumigatus is inhibited by Candida species on sealed Sabouraud
agar plates but not sealed Columbia agar plates. Clin Microbio.
Infect 2004; 10(Suppl. 3): 507.
3. Randhawa HS, Kowshik T, Preeti Sinha K, Sandhu RS,
Chowdhary A. Peptone glucose fluconazole agar, a selective
medium for rapid and enhanced isolation of Aspergillus
fumigatus from aqueous suspensions and sputum seeded with
Candida albicans. Curr Sci 2005; 88: 449–454.
4. Fisher JB Kane J. The laboratory diagnosis of dermatophytosis
complicated with Candida albicans. Can J Microbiol 1974; 20:
167–182.
5. Kane J, Blakeman JM, Fischer JB. Tinea cruris: diagnostic
condition due to isolation of Candida albicans alone. Can Med
Assoc J 1976; 114: 797–798.
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6. Chaturvedi S, Randhawa HS, Chaturvedi VP, Khan ZU. Efficacy
of brain heart infusion-egg albumin agar, yeast extract
phosphate agar and peptone glucose agar media for isolation
of Blastomyces dermatitidis from sputum. Mycopatholgia 1990;
112: 105–112.
were not reached. These data indicate that low local
complement synthesis and activation may represent
a central reason for the insufficient antifungal
defense in the brain and the high mortality rate of
cerebral aspergillosis.
P050
Innate immunity in cerebral aspergillosis
G. Rambach,1 I. Mohsenipour,2 H. Maier,3 G. Vago,4
M.P. Dierich1 and C. Speth1
1
Department of Hygiene, Microbiology and Social
Medicine, Innsbruck Medical University, Innsbruck,
Austria, 2Department of Neurosurgery, Innsbruck
Medical University, Innsbruck, Austria, 3Institute of
Pathology, Innsbruck Medical University, Innsbruck,
Austria and 4University of Milano, Milan, Italy
Spreading of Aspergillus hyphae into the brain of
immunocompromised patients is a complication of
invasive aspergillosis that leads to death in nearby
100% of the cases. To study the reasons for the
antifungal immune failure we analyzed the efficacy of
cerebral complement to combat Aspergillus fumigatus,
the most common species in cerebral aspergillosis.
Incubation of fungal hyphae in non-inflammatory
cerebrospinal fluid (CSF) revealed that complement
levels were sufficient to obtain a deposition on the
surface, but opsonization was much weaker than in
serum. Consequently complement deposition from
normal CSF on fungal surface stimulated a very low
phagocytic activity of microglia, granulocytes, monocytes and macrophages compared to stimulation by
conidia opsonized in serum. Similarly, opsonization
of Aspergillus by CSF was not sufficient to induce an
oxidative burst in infiltrating granulocytes whereas
conidia opsonized in serum induced a clear respiratory signal. Thus granulocytes were capable to
considerably reduce the viability of serum-opsonized
Aspergillus conidia, but not of conidia opsonized in
CSF. The limited efficacy of antifungal attack by
cerebral complement can be partly compensated by
enhanced synthesis, leading to elevated complement
concentrations in CSF derived from a patient with
cerebral aspergillosis. This inflammatory CSF was
able to induce (i) a higher complement deposition on
the Aspergillus surface than non-inflammatory CSF,
(ii) an accumulation of complement activation
products and (iii) an increase in phagocytic and
killing activity of infiltrating granulocytes. However,
levels and efficacy of the serum-derived complement
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
P051
Efficacy of caspofungin against Aspergillus
terreus
E. Spreghini, G. Scalise and F. Barchiesi
Institute of Infectious Diseases and Public Health,
Universita Politecnica delle Marche, Ancona, Italy
Background: Invasive aspergillosis (IA) is the most
common filamentous fungal infection in immunocompromised patients. Although Aspergillus fumigatus and A. flavus are the species most commonly
associated with IA, the frequency of IA caused by
A. terreus (At) is increasing. In vitro and in vivo
studies as well as clinical data show that IA due to At
may be resistant to amphotericin B (AMB) and it is
associated with a high rate of mortality. However,
the optimal therapy for infections caused by this
emerging pathogen is uncertain.
Study aim: In this study, we evaluated the in vitro
and in vivo activity of caspofungin (CAS) against two
clinical isolates of At (At)1 and At-2).
Materials and methods: AMB and itraconazole
(ITC) MICs and CAS MECs were determined by the
NCCLS methodology. The MFCs of each drug was
also determined. Hyphal damage was analysed by
the MTT-assay with each drug utilized at concentrations of 1, 5 and 10 times their respective median
MICs (or median MECs). For in vivo studies, a murine
model of systemic aspergillosis was established by
intravenous injection of conidia in cyclophosphamide
pre-treated mice. CAS was administered i.p. at
doses of 0.5, 1, 2.5 and 5 mg kg)1 day)1. AMB
was administered i.p. at 2.5 mg kg)1 day)1. ITC was
administered by oral gavage at 30 and 100
mg kg)1 day)1. Efficacy was determined by survival
curves, tissue burden (brain, kidneys and lungs),
galactomannan serum levels and histology. CAS
serum drug levels were performed by HPLC.
Results: CAS MECs ranged from 0.25 to 1.0 lg ml)1,
AMB MICs ranged from 0.5 to 4.0 lg ml)1 while ITC
MICs ranged from 0.06 to 0.25 lg ml)1. Both CAS
and AMB MFCs were >16 lg ml)1, while ITC MFCs
6 ranged from 4.0 to >16 lg ml)1. By MTT assay, CAS
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exerted significant lower levels of hyphal damage than
those observed for AMB and ITC against both isolates
of At. Survival studies conducted with At)1 showed
that CAS 2.5 and 5, but not CAS 0.5 and 1, were
effective than and superior to ITC 100 and AMB. In
survival studies conducted with At-2 both CAS 1 and
CAS 2.5, but not AMB, were active. Although neither
CAS 1 nor 5 were able to reduce significantly the CFUs
in the brain tissue, histology studies showed that
hyphal length was significantly shorter in mice treated
with CAS 5 than in controls. In the lung, CAS showed a
dose-dependent activity with 5, but not 1, being
effective at reducing the fungal burden against
controls. Finally, both CAS doses were effective at
reducing the kidney burden. Although CAS serum
levels were in the expected ranges, serum galactomannan performed after 5 days of CAS treatment
showed no significant differences between treated and
untreated mice.
Conclusions: Although we observed a lack of
correlation between in vitro and in vivo activity of
CAS against At, our study supports its role against
infections due to this emerging pathogen.
P052
The role of serotonin in antifungal host defense
Unterdorfer,1 H. Niederegger,2 M.P. Dierich1 and
C. Lass-Floerl1
1
Hygiene, Microbiology and Social Medicine, Medical
University Innsbruck, Innsbruck, Austria and 2Section
for Experimental Pathophysiology and Immunology,
Medical University Innsbruck, Innsbruck, Austria
Serotonin (5-hydroxytryptamine, 5-HT) shows antifungal activity against Aspergillus spp. and Candida
spp. in vitro. To determine the mode of action, the
extra- or intracellular 5-HT accumulation and possible cell membrane damage in Aspergillus hyphae
were investigated. Clinical isolates of A. fumigatus
(n = 1) and A. flavus (n = 2) were examined. Detection of 5-HT was performed using indirect immunofluorescence (IF) staining and laser scanning
confocal microscopy (LSCM). 5-HT concentrations
from 3.7 lmol L)1 to 235 mmol L)1 were used and
incubated for 30, 60 and 90 min as well as for 6
and 12 h. Detection was performed using an unlabelled primary and a FITC conjugated secondary
antibody. To clarify possible cell membrane damage
caused by 5-HT, a double staining method with
74
propidium iodide (PI) and fluorescin diacetate (FDA)
was used. IF and LSCM showed specific fluorescent
signals in the cytoplasm of 5-HT sensitive hyphae
after 30 min of incubation for all tested 5-HT
concentrations. Likewise PI/FDA displayed 5-HT
concentration dependent impairment of Aspergillus
membranes. 5-HT is stored in dense granules of
platelets at 65 mmol L)1 and shows antifungal
activity. Therefore the role of platelets in defense
against Aspergillus spp. needs to be further investigated.
P061
Microsporum canis a dermatophyte with special
habitat
S.R. Aghili,1 T. Shokohi,1 H.R. Naseri1 and
A.R. Khalilian2
1
Medical Mycology & Parasitology, Mazandaran
University of Medical Sciences, Sari, Iran and 2Statics,
Mazandaran University of Medical Sciences, Sari, Iran
Microsporum canis (M. canis) is the leading cause of
ringworm in cats. This is very contagious and able to
spread from pet to pet, pet to human or human to pet.
In spite of abundance of stray cats in Sari city, during
15 years (1990–2005), no case of fungal lesion due to
M. canis was observed in the patients referring to the
mycology reference laboratory (affiliated to the Mazandaran University of Medical Sciences). This issue
provoked us to study the existence of M. canis in cats
living in Sari. In order to determine the prevalence and
characterize the carriage of M. canis, we examined 100
stray cats which were captured in different parts of
city. None of them had mycotic lesions. Different
mycological examinations were performed on all
animals, including culture obtained by using the
Mack Kenzie’s tooth brush technique, scraping, contact of surface of media to the lip and ear loop which
are supposed to be the main sites of colonization and
direct examination with KOH and Calcofluor white
method (Cw). M. canis was not isolated from all
samples; however, saprophytic fungi were isolated
from them. Four dermatophytes including M. gypseum
in three and Trichophyton mentagrophytes variety
mentagrophytes were isolated. Ten species of saprophytic fungi were isolated from all cats. Aspergillus spp.
(59%), Cladosporium spp. (46%), Alternaria spp. (44%),
Penicillium spp. (39%) and Mucor spp. (24%) were the
most frequently isolated. The sampling was done in fall
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
P062
of Malassezia spp. yeasts grew in Dixon’s medium. The
patient was treated orally with Itraconazole (200 mg
o.d.) for 2 weeks: the rash cleared totally. Malassezia
yeasts have been involved with fungal folliculitis,
although this is not widely accepted. It is known that
imidazoles could have not only a fungistatic or fungicidal antiyeast action but they could also act due to
the anti-inflammatory properties inherent to these
compounds. This case would appear to be related to a
lipophilic yeast (Malassezia spp.) although the mechanism by which Erlatinib would induce the growth of
this yeast needs clarification. Although this is a case
report based on a single patient, we recommend to
plant skin biopsies also in rich lipid media which
permit the growth of Malassezia spp. yeasts.
Rosacea like folliculitis in a erlotinib treated
patient: possible relationship with lipophilic
yeasts (Malassezia spp.)
P063
and winter in 2003 and spring season in 2004 with
average temperature of 14.4 C, humidity of 76% and
rain amount 57.3 mmHg. No significant differences
were found in sex, hair length, hair color, temperature,
humidity, rain amount and flora of the hair coat
between all cats. According to this study, stray cats in
Sari are not the reservoir and vector of M. canis. The
results are similar to medical mycology reference
laboratory reports that there are no lesions in man due
to M. canis. However, M. canis were isolated in other
regions of Iran. This different isolation may be due to
difference of climates in different parts of Iran.
M.S. Cuetara,1 G. Flox,2 A. Aguilar,2 L. Martin,3
A. Del Palacio,4 C. Aspiroz5 and I. Wilhelmi1
1
Microbiology, Hospital Severo Ochoa, Leganes,
Madrid, Spain, 2Dermatology, Hospital Severo
Ochoa, Leganes, Madrid, Spain, 3Anatomic
Pathology, Hospital Severo Ochoa, Leganes, Madrid,
Spain, 4Microbiology, Hospital 12 De Octubre,
Madrid, Spain and 5Microbiology, Hospital Royo
Villanova, Zaragoza, Spain
Recently introduced antitumoral drugs for the treatment of solid tumours such as Gefitinib (ZD-1839) and
Erlotinib, inhibit the epidermal growth factor receptor.
The most frequent undesirable side effects are gastrointestinal and cutaneous disturbances (acneiform
rash, skin dryness, paronychia nasal and oral ulcers,
urticariform rash and seborrheic dermatitis). We
herein present a rare cutaneous rosacea like side effect
due to Erlotinib in a 70-year-old male with renal
carcinoma who was treated with radical nephrectomy
2 years before. When seen again due to carcinoma
lung metastasis he was treated with combined chemotherapy and radiotherapy. The patient was treated
with inhaled corticosteroids and Erlotinib; 1 week
later an intense papulopustular erythematous itchy
facial rash appeared, which was topically treated with
clindamycin solution without improvement. A skin
biopsy (PAS stain) showed multiple round small yeasts
with unipolar and broad-based budding in the infundibulum of the hair follicules. Biopsy material was
planted in several medium (namely for bacteria,
mycobacteria and fungi including DIXON agar
medium and Sabouraud agar medium). A pure culture
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
Identification of dermatophytes isolated from
tinea capitis in Western China using rDNA ITS
sequencing
S. Deng,1,2 R. Summerbell,1 G.S. de Hoog1 and
G.S. Bulmer3
1
1Centraalbureau voor Schimmelcultures, Utrecht,
The Netherlands, 2The Department of Dermatology,
First Hospital Xinjiang Medical University, No. 8 Liyu
Road, Urumqi, 830054, Xinj, Urumqi, China and 368
Beverly Hills, Ave Beverly Hills Subdivision Taytay,
Rizal, 1920, Philippines, Riza, Philippines
Objective: Tinea capitis is a common dermatophyte
infection of the scalp in children in Western China.
Over the past 20 years, the most common etiologic
agent initially was Trichophyton schoenleinii, followed
later by T. violaceum. Until recently, identification of
dermatophytes was mainly based on the cultivation of
fungal isolates on special media and on microscopic
morphology. Molecular methods such as sequencing
of the internal transcribed spacer (ITS) region of the
ribosomal DNA have proven to be useful for identification of dermatophytes. The aim of this study is to
identify the spectrum of species causing tinea capitis by
molecular methods and to establish epidemiological
trends in the Western China.
Methods: Molecular identification: A total of 94
isolates was collected from clinically suspected tinea
capitis patients in 2003. The entire ribosomal DNA
internal transcribed spacer (ITS1–5.8S-ITS2) region
was sequenced. T. violaceum was identified by PCR
using a microsatellite primer set (T1). Conventional
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identification: Colony and microscopic morphology.
Physiological testing.
Results: Molecular identification: Five species of dermatophytes were identified by ITS sequencing down
to the species level. The remaining species were
identified by specific PCR using the T1 primer set.
The tree was constructed by the neighbor-joining
method. The results showed that T. violaceum (37
strains) and T. schoenleinii (33 strains) are the
preponderant species in the area investigated. Other
organisms included M. ferrugineum (10 strains),
T. verrucosum (nine strains) and T. tonsurans (five
strains). With microsatellite markers, among the
strains of T. violaceum, just one type of polymorphism
was found. It was type C. Conventional identification:
There was a greater proportion of cases in the age
range from 4 to 14 years and predominantly of the
male sex. Gray-patch was the most common clinical
form. No significant differences were found between
the strains regarding the physiological features.
T. verrucosum as the only exception. T. verrucosum
var. autotrophicum are vitamin independent.
Conclusion: 1. Species causing tinea capitis in Xinjiang are totally different from those found in other areas
of China (REF). This may reflect different genetic
predispositions. 2. Trichophyton violaceum and T. schoenleinii are endemic in Western China. 3. Incidence of
some species appeared rather different from literature
data; this may be due to differences in diagnostic
methods, classical vs. molecular. 4. T. verrucosum has
increased remarkably in incidence. Other apparent
changes in frequency may be due to (3).
P064
Asymptomatic scalp carriage with zoophilic
dermatophytes in school children in Adana,
Turkey
M. Ilkit,1 H. Demirhindi,1 M. Yetgin,2 A. Ates,1
A. Turaç-Biçer1 and E. Yula1
1
Microbiology, University of Çukurova Faculty of
Medicine, Adana, Turkey and 2Public Health,
University of Çukurova Faculty of Medicine, Adana,
Turkey
The aim of this study was to verify the prevalence of
asymptomatic dermatophyte scalp carriage and
index cases of tinea capitis in Adana Province,
Çukurova region, Turkey. For this purpose, a screening study was performed in five schools, between
January 2004 and May 2005, covering a total of
76
5143 children with 2740 (53.3%) boys and 2403
(46.7%) girls, aged 7–14 years. The diagnosis was
made by using cotton swab method with inoculation
onto Sabouraud glucose agar amended with cycloheximide, chloramphenicol and gentamicin. Among
10 (0.2%) cases, six asymptomatic carriers and four
index cases were detected, all of who were boys and
immigrated from south-eastern and eastern region of
Anatolia, Turkey. Zoophilic dermatophytes, namely
Microsporum canis (40%) and Trichophyton mentagrophytes var. mentagrophytes (40%) were the most
commonly isolated species, followed by anthropophilic Trichophyton tonsurans (10%). No causative agent
was detected in a case (10%) with tinea capitis
superficialis. As a conclusion, the prevalence of
asymptomatic carrier state was found to be higher
than that of the index cases in our study, and we
found a significant predominance of zoophilic species.
P065
Intrafamilial transmission of Microsporum canis
in Adana, Turkey: an epidemiological filiation
study
M. Ilkit, A. Turaç-Biçer, A. Ates, M. Polat, F. Koksal
and K. Ozcan
Microbiology, University of Çukurova Faculty of
Medicine, Adana, Turkey
Tinea capitis is uncommon in Çukurova region,
Turkey. The most common clinical form is tinea
capitis superficialis, and the most frequent causative
agent is Trichophyton violaceum. This study presented
the results of a screening performed at the family
background of a kerion Celsi case in Adana province
and at the school attended, with the aim of
determining the infection source and route of
transmission. Tinea capitis superficialis was detected
in the two siblings of the index case, with tinea faciei
in one sibling. Microsporum canis was isolated in all of
the cases. The screening performed at the primary
school revealed tinea capitis superficialis in three
(60%), and asymptomatic carriage in two (40%)
among five (0.5%) of 961 students. While Microsporum canis was isolated in four of the five cases, no
causative agent was detected in the other tinea
capitis superficialis case. As a conclusion, this study
presented an outbreak of Microsporum canis in a
family. This is also the first report of an asymptomatic carriage in Turkey, and suggesting the need for
a more detailed research.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
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P066
Novel extracellular keratinolytic protease from
Trichophyton rubrum
R. Kano,1 T. Yamada,2 K. Makimura,2
Y. Yamaguchi,2 S. Watanabe3 and A. Hasegawa1
1
Pathobiology, Nihon University School of Veterinary
Medicine, Kanagawa, Japan, 2Medical Mycology and
Genome Research Center, Teikyo University, Tokyo,
Japan and 3Dermatology, Teikyo University School of
Medicine, Tokyo, Japan
The novel keratinolytic protease of Trichophyton rubrum
was isolated and characterized. The cDNA sequences of
the T. rubrum keratinolytic protein (1010 bp) were
found to contain a single long open reading frame of
651 bp coding a protein of 216 amino acids. The
amino acid sequences of the conserved region in the
cDNA shared the low sequence alignment similarity
(less than 50%). The protein mRNA was detected in
T. rubrum cultured with or without keratin by RT-PCR
analysis. The molecular weight of the recombinant
keratinolytic protein expressed in E. coli was approximately 30 kDa. This 30 kDa protein was active against
keratin azure in Tris–HCl buffer. The apparent optimum temperature of keratinolytic activity was 30 C.
The activity was week at 20 C and was inactivated at
50 C. The optimum pH value of this enzyme activity
was pH 7.5. The enzyme activity was inhibited by serin
protease inhibitors, but not by EDTA. After 1- to 3-h
co-culture of epidermal keratinocytes with the keratinolytic protein, cytokine levels in the supernatants
were determined by ELISA. IL-6 and IL-8 levels in the
supernatant increased as the time of co-culture past,
although IL-1 alpha and TNF-alpha levels were very
low or undetectable.
P067
Insulin-independent diabetes mellitus patients
with foot onychomycosis
V.G. Kornisheva,1 S.G. Belova1 and G.A. Sokolova1
1
Dermatology, Medical Academy of Postgraduate
Education, St Petersburg, Germany, 2Dermatology,
Academy of Postgraduate Education, St Petersburg,
Germany and 3Endocrinology, Academy of
Postgraduate Education, St Petersburg, Germany
The posterior nail fold microcirculation of all the
toenails in 30 patients with insulin-independent diabetes mellitus (26 females, four males, with a range of
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
45–76 years) was examined. The number of infected
nails varied from 2 to 10. The total onychomycosis was
revealed in 26 patients. The control group consisted of
10 healthy people. Apparatus ‘Minimax-Doppler-K’
with 25 Hz sensor was used to investigate the posterior
nail fold microcirculation, the blood velocity in arteria
dorsalis pedis and arteries tibialis posterior was determined
with 10 Hz sensor, with patients being kept in supine
position at the air temperature of 24 C. The diabetic
patients were divided into three groups according to the
duration of diabetes: the first group – less than 5 years,
the second one – from 5 to 10 years, the third one –
more than 10 years. The gradual decrease in the blood
velocity in groups 1 and 2 compared with the control
group due to the increasing of diabetic microangiopathy and hyalinosis and capillary contraction was
revealed. The predominance of yellow color in spectrogrammes in group 1 showed evidence of perivascular
edema, and early stage of microcirculation disorders.
The blood velocity in group 3 increased and approached
to the blood velocity in the control group due to the
obliteration of some parts of lateral artery and the
activation of arteriovenous anastomosis, initial manifestation of obliterating atherosclerosis accompanying
with tissue hypoxia. In comparing blood velocity in
intact and affected nails in group 2 and groups the
volume rate in affected toenails tended to become
slower because of the exposure of toxic fungal products.
The blood velocity in toenails in group 3 was the same
as the one in group 2 due to generalized damage of
vascular bed which developed after 10 years duration
of diabetes mellitus. Thus, significant microcirculation
disorder was found out in type 2 diabetic patients.
Severity of angiopathy increased together with the
duration of diabetes. Antifungal therapy must be added
to vascular medicine. Vascular therapy must be more
intensive if obliterating atherosclerosis is revealed.
P068
Tryptophan-metabolism of the yeast Malassezia
furfur: impairment of skin pigmentation
by both apoptosis of melanozytes and
tyrosinase-inhibition
H.J. Krämer,1 K. Dahms,1 M. Podobinska,1 W. Hort,1
A. Bartsch,2 W. Steglich2 and P. Mayser1
1
Zentrum für Andrologie und Dermatologie, Giessen,
Germany and 2Department Chemie LMU, München,
Germany
Tryptophan as nitrogen source induces the synthesis of fluorochromes and pigments in cultures of
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Malassezia (M.) furfur (1). Pityriasis versicolor (PV)
is characterized by the occurrence of depigmentation with reduced melanin-synthesis (PV alba) in
the course of the disease. We report here an
inhibition of melanogenesis by toxic influence on
melanocytes as well as tyrosinase-inhibition both
induced by tryptophan-metabolites of the yeast.
Both mechanisms could contribute to the pathogenesis of PV alba. M. furfur (CBS1878) was
cultured as described in (1,2) and the resulting
brown coloured agar was extracted with ethylacetate. Separation of the extract was achieved by
chromatography (Sephadex LH20, HPLC). Resulting subfractions were incubated with cultures of
primary human melanocytes as well as in preparations of human epidermis, which were stained
with L-dopa (selective melanocyte-specific, tyrosinase-dependent staining). Observed changes in both
systems were used as indicator for the responsible
agents. Structural elucidation of the obtained
compounds gave as toxic principle malassezin {2(1H-indol-3-ylmethyl)-1H-indol-3-carbaldehyd}, a
potent agonist of the Ah-receptors (EC50 =
1.57 lmol L-1) (2), which induced apotosis in
melanocytes (IC50 5 lmol L)1). Apotosis was
detected by FACS-Analysis, activation of caspase)9
and characteristic changes of the nucleus (apoptotic bodies) as well as DNA-degradation. Immunohistochemical staining showed a reduction of the
actin cytoskeleton (3). The tyrosinase-inhibition
was caused by two compounds, identified as
3-keto-Malassezin and malassezione (1,3-bis(1Hindol-3-yl)propan-2-on) (4). Both mechanisms
could be involved in the depigmentation in the
course of Pityriasis versicolor.
References:
(1) Mayser P, Wille G, Imkampe A, Thoma W, Arnold N, Monsees
T. Synthesis of fluorchromes and pigments in Malassezia furfur
by use of tryptophan as single nitrogen source. Mycoses 1998;
41: 265–71.
(2) Wille G, Mayser P, Thoma W, Monsees T, Schmitz H-J, Schrenk
D, Zeitler K, Steglich W. Isolation and synthesis of malassezin –
an agonist of the arylhydrocarbon receptor from the Yeast
Malassezia furfur. Bioorg Med Chem 2001; 9: 955–60.
(3) Krämer H-J, Podobinska M, Bartsch A, Battmann A, Thoma W,
Bernd A, Kummer W, Irlinger B, Steglich W, Mayser P.
Malassezin, a novel agonist of the aryl hydrocarbon receptor
from the yeast Malassezia furfur, induces apoptosis in primary
human melanocytes. ChemBioChem 2005; 6(5): 860–5.
(4) Irlinger B, Bartsch A, Krämer H-J, Mayser P, Steglich W. New
tryptophan metabolites from cultures of the lipophilic yeast
Malassezia furfur. Helv Chim Acta 2005 (accepted for publication).
78
P069
Pityriarubins A, B and C: novel putative
anti-inflammatory agents from cultures of
Malassezia furfur. Inhibition of respiratory
burst in human granulocytes
H.J. Krämer,1 D. Kessler,1 C.H. Hipler,2 B. Irlinger,3
W. Steglich3 and P. Mayser1
1
Department of Dermatology, Giessen, Germany,
2
Klinik für Hautkrankheiten, Jena, Germany and
3
Department Chemie LMU, München, Germany
Tryptophan as nitrogen source induces the synthesis
of fluorochromes and pigments in cultures of
Malassezia (M.) furfur (1). Pityriasis versicolor shows
a significant less pronounced inflammatory response
in affected skin areas as compared to other mycoses.
We isolated three metabolites of M. furfur (Pityriarubins A, B, C), which have a common bis-indolylcyclopenten-1,3-dione moiety and resemble the
PKC-inhibitor acyriarubin A (bis-indolymaleimide).
M. furfur (CBS1878) was cultured as described in (1)
and the resulting brown coloured agar was extracted
with ethylacetate. Separation of the extract was
achieved by chromatography (Sephadex LH20,
HPLC). Three red coloured compounds were isolated
and structurally elucidated by HR-MS and HR-NMR.
The obtained substances were used for burst-experiments. Granulocytes were obtained from whole blood
of healthy volunteers. Burst was induced by incubation with calcium-ionophore A23187, N-formylMET-LEU-PHE (fmlf), 1,2-dioctanoyl-glycerole, Phorbole-12-myristat-13-acetate, Zymosan, IL3 and
aluminium fluoride. O2-radical-release was measured by reduction of cytochrome C. The pityriarubins
A, B and C were able to suppress burst in case of the
stimuli A23, IL3 and fmlf (IC50 ~ 2 lmol L-1). No
inhibition was observed in cases of the other stimuli,
contrasting to the influence of the unspecific PKCinhibitor acyriarubin A. We postulate a highly
specific impact on the signal transduction of the
agents. All compounds could be involved in the
minimal granulocytary activation in areas of Pityriasis versicolor.
References:
(1) Mayser P, Wille G, Imkampe A, Thoma W, Arnold N, Monsees
T. Synthesis of fluorchromes and pigments in Malassezia furfur
by use of tryptophan as single nitrogen source. Mycoses 1998;
265–71.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
P070
P071
In vitro activities of amphotericin B, itraconazole, voriconazole and caspofungin against
Scytalidium dimidiatum and Scytalidium
hyalinum isolates
Causative agents of dermatomycosis in horses
and their diagnostic features
C. Lacroix and M. Feuilhade de Hauvin
Mycology laboratory, Hospital St Louis, Paris, France
Background: Scytalidium dimidiatum (Sd) and
S. hyalinum (Sh) are moulds responsible for dermatomycoses localized especially on feet and hands.
Clinical presentation is indistinguishable from dermatophytosis due to Trichophyton rubrum. Scytalidiosis are mostly diagnosed in patients living in
tropical or sub-tropical areas. To date, most topic or
systemic antifungal drugs available in dermatology
are ineffective to treat these infections. Antifungal
susceptibility testing was performxed on clinical
isolates of Sd and Sh, to compare the in vitro activity
of the new antifungal agents voriconazole and
caspofungin to amphotericin B and itraconazole.
Materials and methods: A total of 29 clinical
isolates were tested including 14 S. dimidiatum and
15 S. hyalinum. These isolates were cultured from
various clinical specimens: soles, interdigital toe
webs, palms and nails. Minimal inhibitory concentrations (MIC) of amphotericin B, itraconazole,
voriconazole and caspofungin were determined using
the E-test method on RPMI 1640 medium after
7 days of incubation at 27 C.
Results: The MICs to amphotericin B for Sd and Sh
are similar and ranged from 0.016 to 0.38 mg L)1.
The MICs to itraconazole ranged from 0.002 to
>32 mg L)1 for Sd and Sh. The MICs to voriconazole
ranged from <0.002 to 0.094 mg L)1 for Sd and
from <0.002 to 0.006 mg L)1 for Sh. The MICs to
caspofungin are similar for Sd and Sh and ranged
from 0.003 to >32 mg L)1.
Conclusions: Voriconazole exhibited the highest in
vitro antifungal activity against S. dimidiatum and S.
hyalinum with very low MICs. S. hyalinum seems to be
more susceptible to azoles than S. dimidiatum. Clinical
studies should be developed to investigate the potent
activity of voriconazole in the treatment of human
scytalidiosis.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
M.G. Manoyan, R.S. Ovchinnikov and A.N. Panin
Laboratory of Mycology, The All-Russia State Center
for Quality of Animal Medicines (VGNKI), Moscow,
Russia
This study was concerned on the prevalence of
dermatomycoses in horses and their etiologic pattern. Clinical skin samples were collected from the
thoroughbred horses presenting skin lesions and
then were performed for further mycological examination. Identification of cultured isolates was performed according to routine diagnostic procedures
accompanied by additional physiological tests. Data
presented is focused on diagnostic features in identification of closely related dermatophyte species
because of their significant phenotypic similarity.
Eighty-two skin samples were investigated. Dermatophytosis was diagnosed in 17 cases (20.7%).
Causative agents were presented by Microsporum
gypseum (35%), M. canis (23%), Trichophyton tonsurans (syn. T. equinum) (23%), T. mentagrophytes
(11%), M. equinum (6%). In two cases Dermatophilus
congolensis was identified as causative agent. This is
the first case of dermatophytosis occurred in our
practice. Distinctive morphological features of
T. tonsurans were cerebriform colony folding, vinered reverse. Microscopically 2–3-celled rudimentary
macroconidia and chlamydospores were revealed in
this species. In physiological tests T. tonsurans and
T. mentagrophytes demonstrated positive urealytic
activity, gelatin liquefaction, milk peptonization,
and negative citrate utilization. In hair perforation
test both species revealed positive results, but
T. tonsurans was able to produce perforations in hair
shafts, while T. mentagrophytes provoked mainly
erosive destruction in hair. Another distinctive
feature of T. tonsurans was its inability to grow on
Czapek’s medium. M. canis and M. equinum are
closely related species and in last years they are
treated as synonyms (S. de Hoog et al., 2000). In
spite of such conception and macroscopic similarity
this ‘species’ revealed distinctive morphological and
physiological features. M. equinum characterized by
slow growth rate, irregular colony folding and
revealed very low sporulation, but abundant bamboo-like hyphae. Single spindle-shaped 3–4-celled
macroconidia were detected comparing with
79
xxx
abundant 5–8-celled macroconidia in M. canis.
Physiologically both strains produced urease, evoked
milk peptonization and were negative in citrate
utilization. Unexpectedly, M. canis strains did not
provoke gelatin liquefaction, while M. equinum did.
Conversely, M. canis showed positive results in
human hair perforation test in contrast to M. equinum. When we used horsehair in perforation test,
both species caused strong destruction of the hairs.
Dermatophilus congolensis was identified according to
appropriate bacteriologic criteria. It was able to urea
hydrolyze, milk coagulation and gelatin liquefaction.
Overall, five dermatophyte species were recognized as
etiologic agents in horses with prevalence of M. gypseum. Additionally, D. congolensis was diagnosed as
skin pathogen; such appearance is unusual for
temperate climate regions.
P072
Epidemiological study of superficial and
cutaneous fungal infections in Tonekabon, Iran
2004
N. Ayat Nasrollahi Omran,1 H. Jamal Hashemi,2
K. Alireza Khosravi,3 G. Alinaghi Ghiaci4 and
N. Sahereh Mohammadi Nakhjiri5
1
Medical Mycology, Iau of Toekabon-Faculty of
Medical Science, Toekabon, Iran, 2Vet. Mycology, Iau
of Tehran-Faculty of Specialised of Veterinary,
Tehran, Iran, 3Iau of Tehran-Faculty of Specialised
Vet. Mycology, Tehran, Iran, 4Dermatology, Iau of
Eonekabon-Faulty of Medical Science, Tonekabon,
Iran and 5Microbiology, Iau of Tonekabon-Faculty of
Science, Tonekabon, Iran
The objective of this investigation was to determine
the prevalence and the aetiological agent of these
diseases in a geographically restricted area in the
northern Iran, Tonekabon. Skin mycological specimens were taken for 12 months in the period from
April 2004 to April 2005 by scraping from 500
patients suspected to these mycosis referred to clinics
dermatology. Diagnosis was confirmed by direct
microscopy culture specimens, dermatophyte and
Malassezia spp. diagnosis tests for taxonomic identification of species. Totally our results from this
survey showed that 350 patients suffered from
superficial and cutaneous mycosis. Sixty-five per cent
of these patients were male and the rest 35% female.
The group at the ages of 15–25 specially students
80
had the most fungal infections; the rest were officers,
collar workers, farmers, thrifty. Eighty per cent of
patient living in cities rest 20% were villagers.
Dermatophytosis 48.5%, tinea versicolor 34%, candidiasis 12.5%, saprophytic infections composed the
mycosis. The frequency of clinical types according to
anatomic site involvement of dermatophytosis: tinea
cruris 50%, tinea manum 20%, tinea pedis 17.6%,
tinea captitis 4.76%, tinea corporis 4.76%, tinea
ungium 1.9%, tinea barbae 0.8%. Epidermophyton
floccosum 28.5% was most common etiological agent
of dermatophytosis, the rest were: T. rubrum 28.8%,
T. violaceum 7.14%, T. mentagrophytes 14.28%,
T. verrcosum 4.76%, M. canis 4.76%. The frequency
of clinical types according to anatomic site involvement of versicolor was trunk 35%, arm 15%, leg
10%, back 25%, chest 15%. Malassezia furfur 65%,
M. sympodialis 15%, M. sllofiae 15%, M. globosa 5%
etiological agents of tinea versicolor. Sixty per cent
nail infection, 40% intertrigo candidiasis clinical of
candidiasis. The study highlights a common problem
in many areas of the northern Iran and suggests that
further measures regarding public health and specially personal hygiene be undertaken in order to
reduced of superficial and cutaneous.
P073
Utility of potato dextrose agar medium in the
identification of dermatophytes
A. Rezusta,2 M. Arias,1 A. Betran,2 Y. Martin,2
M.J. Revillo2 and M.C. Rubio3
1
Microbiologia, Hospital Miguel Servet, Zaragoza,
Spain, 2Microbiologia, Hospital Miguel Servet,
Zaragoza, Germany and 3Microbiologia, Hospital
Universitario Lozano Blesa, Zaragoza, Germany
Introduction: Dermatophytes are keratinophilic
and infect three cutaneous areas (skin, hair, nail) of
humans and animals, producing a disease called
dermatophytosis. The objective of this study is to
evaluate the utility of the Potato Dextrose Agar
(PDA) in the identification of T. rubrum and M. canis.
Material and methods: Retrospectively, we studied 148 samples with M. canis and T. rubrum isolates.
They were obtained during routine clinical practice
at our Hospital. We cultured on Sabouraud agar
(SDA), Dermatophyte Test Medium and incubation at
30 C for 28 days. Cultures were examined every
Monday and Thursday. The dermatophytes isolated
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
in DTM but did not isolate in SDA, and we made a
subculture in PDA and SDA; if they isolated in both
media we subcultured only in PDA. Identification
was realised according to Rebell and Taplin form.
Results: Of all strains isolated 34 were identified as
M. canis and 114 as T. rubrum. In the study of the
subcultures of PDA and SDA, PDA showed that
M. canis and T.rubrum had been identified at least 3
or 4 days before at 77.9% and 69.2%, respectively.
When the identification was realised on the original
SDA and the subculture on PDA, PDA showed to be a
faster procedure to identify M. canis and T. rubrum in
96% and 100%, respectively.
Conclusions: PDA medium favoured the identification of these dermatophytes species, M. canis and
T. rubrum.
P074
T. soudanense and M. audouinii caused tinea
capitis in adult. Case report
A. Rezusta,1 A. Betrán,1 I. Querol,2 M. Arias,3
M.J. Lavilla3 and M.J. Revillo3
1
Microbiologia, Hospital Miguel Servet, Zaragoza,
Spain, 2Dermatologia, Hospital Miguel Servet,
Zaragoza, Germany, 3Microbiologia, Hospital Miguel
Servet, Zaragoza, Germany,
Introduction: Tinea capitis is a common dermatophyte infection of the scalp in children but is
uncommon in adults. In Europe the most common
agent of tinea capitis are Microsporum canis and
Microsporum audouinii. In Central and West Africa
there are M. audouinii and Trichophyton soudanense
and in North East Africa, Trichophyton violaceum. In
Spain, M. audouinii practically disappeared after
1960s and the most common dermatophytes are
M. canis, Trichophyton mentagrophytes and Trichophyton tonsurans. T. soudanense had been rarely isolated
in Spain. We are going to present in this report a case
of tinea capitis in an adult caused by two organisms
(T. soudanense, M. audouinii).
Case report: Over a period of years, a 31-year-old
black woman from Senegal suffered atrophic lesion of
the skin producing cicatricial alopecia and scarring.
A medical examination revealed scaly lesions of
10 cm in diameter on the parietal scalp with atrophy
and cicatricial alopicia. We also found severe desquamation inside the lesions. Other members of her
family showed no signs of any type of infection or of
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
being affected. Cultures in Sabouraud Agar with
chloramphenicol and Dermatophyte Test Medium
showed a large number of T. soudanense and a small
quantity of M. audouinii on the patient. The patient
was treated with Terbinafine 250 mg day for
56 days.
Discussion: The most predominant agents in Europe are M. canis, M. audouinii, T. tonsurans and
T. soudanense in rural areas and T. tonsurans, T. soudanense and T. violaceum in urban areas. T. soudanense
is a typical African dermatophyte affecting in particular Senegal, Ghana, Nigeria and Chad. M. audouinii
and T. soudanense are increasing, particularly in
France, but are uncommon in Spain at the moment.
M. audouinii produces pre-puberty ringworm of the
scalp; suppuration is rare. Infections produced by
T. soudanense are inflammatory, scarring tinea capitis.
Conclusions: Clinical lesion suggests infection by
T. soudanense but no other family member was
affected in this particular case. As a consequence of
the immigration increase, the isolation of anthropophilic species in tinea capitis has also increased.
P075
Incubation time of dermatophytes cultures
A. Rezusta,1 M. Arias,1 Y. Martin,1 M.J. Lavilla,1
M.J. Revillo1 and M.C. Rubio2
1
Microbiologia, Hospital Miguel Servet, Zaragoza,
Spain and 2Microbiologia, Hospital Universitario
Lozano Blesa, Zaragoza, Spain
Introduction: Dermatophytes are a group of fungi
that have the capacity to invade keratinized tissues
(hair, nails, skin) of humans and animals, producing a disease, called dermatophytosis (tinea).
Several aspects of routine fungal cultures should
be evaluated in order to implement appropriate
necessary changes. There are several recommendations that mycology cultures be incubated for
28 days. However, there is no recent evidence
supporting this practice. The objective of this study
is to evaluate for 28 days incubation for dermatophyte cultures.
Material and methods: Dermatophytes specimens
were cultured on Sabouraud Dextrose Agar (SDA),
Dermatophyte Test Medium (DTM) and incubated at
30 C for 28 days. Microscopic examinations was
realised by KOH mounts. Cultures were examined
every Monday and Thursday. The clinic report was
81
xxx
carried out after 10 days of incubation only if the
culture was negative at that moment. The plates
continued incubation for another 18 days, the report
was modified if dermatophyte grew during this time.
Results: Out of 333 cultures of dermatophytes, 310
(93.1%) were isolated for 10 days of incubation
followed by 19 additional isolations over the next
18 days, and during the fourth week four dermatophytes were isolated and these four showed positive
under direct microscopic examination.
Conclusions: Three weeks suffice for detecting the
majority of dermatophytes, and we need only to
incubate the plates for 4 weeks when the specimens
show positive during a microscopic examination.
P076
Onychomycosis of the great toenail caused by
Chaetomium globosum
A. Rezusta,3 M.C. Aspiroz,1 J. Gene,2 L. Charlez4 and
R.C. Summerbell5
1
Microbiologia, Hospital Royo Villanova, Zaragoza,
Spain, 2Microbiologia, Facultad de Medicina, Reus,
Spain, 3Microbiologia, Facultad de Ciencias de la
Salud y del Deporte, Hospital Univesitario Miguel Ser,
Huesca-Zaragoza, Spain, 4Dermatologia, Grande
Covian, Zaragoza, Spain and 5Mycology,
Centraalbureau voor Schimmelcultures, Utrecht, The
Netherlands
Introduction: Chaetomium species have been isolated from infections involving blood, lung, brain, skin,
and nails in both healthy and immunocompromised
patients. We report a case of onychomycosis caused
by C. globosum with an excellent response to
terbinafine.
Case report: The patient was visited by a dermatologist sent by his family practitioner. The patient
had a history of 4 years with repeatedly loose nail
(four episodes in 4 years: two spontaneous and two
surgical ablations). The patient was a 23-year-old
white man otherwise healthy. He was an amateur
football player and his job obligated him to stay
many hours in pedestation. He showed subungual
hyperkeratosis thickening and yellow brown discoloration in the first toenail on his left foot. All the
fingernails and the other nine toenails were normal
and the surrounding skin was not affected. Toe web
spaces were normal. The feet were normal and any
signs or symptoms of tinea pedis were observed or
82
recorded by the patient himself. Scrapings from the
affected toenail were examined microscopically and
cultured. Hyaline, septate filaments and irregular
hyphae were seen on light microscopy. Culture onto
Sabouraud dextrose agar added with chloramphenicol yielded rapidly growing colonies (3 days), which
first appeared white but soon became dark grey to
brown. Repeated cultures and microscopic observation of the toenail yielded identical mycologic results
on two separate occasions (23/01/04 and 06/02/
04). The count of colonies in Sabouraud dextrose
agar (SDA, Oxoid) of 7 CFU on SDA plates in the first
nail sample and 9 CFU on second SDA plate). Media
for recovery of dermatophyte fungi were negative
(Mycobiotic) and Taplin-DTM (dermatophyte test
medium, Oxoid). The organism was identified as C.
globosum. Patient was treated with topical and oral
terbinafine for 12 weeks. The patient was seen with
almost complete clinical cure, microbiological resolution and without evidence of fungal relapse
(November 2004).
P077
Quality of life in patients suffering from toenail
onychomycosis
J.C. Szepietowski,1 A. Reich,1 P. Pacan,2
E. Garlowska3 and E. Baran1
1
Department of Dermatology, Venereology and
Allergology, University of Medicine, Wroclaw,
Poland, 2Department of Psychiatry, University of
Medicine, Wroclaw, Poland and 3Novartis Poland,
Warsaw, Poland
Background: Onychomycosis is the most frequent
nail disease, which could impair the patient’s quality
of life.
Objective: The presented study was undertaken to
evaluate the impact of toenail onychomycosis on
quality of life among Polish population.
Material and methods: 3904 (2269 females and
1635 males) individuals fulfilled an international
onychomycosis-specific quality of life questionnaire
consisting of statements regarding social, emotional
and symptoms problems. All patients had toenail
onychomycosis confirmed by the positive direct
microscopic examination and/or by the positive
mycologic culture. 767 patients suffered simultaneously from fingernail onychomycosis. All patients
were divided into subgroups according to sex, age,
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
education level, place of living, type of onychomycosis, number of involved toenails, fingernails involvement, duration of illness and previously used
antimycotic therapy.
Results: Most of the patients demonstrated significantly reduced quality of life. The degree of life
impairment varied between analyzed subgroups.
Patients with more advanced toenail onychomycosis
and with fingernail involvement were more seriously
affected. Both social and emotional impairments
were more pronounced in females than in males,
although there were no differences according to
symptoms. Moreover, patients with better education
level and people living in towns or cities were more
emotionally and socially affected by onychomycosis,
although people living in the country or with poorer
education level presented with significantly more
severe symptoms.
Conclusions: Toenail onychomycosis is still a serious medical problem, which can significantly reduce
the patient’s quality of life.
P078
patient, a renal transplant recipient, who developed
scaly plaques in the right knee. Histological analysis
revealed diffuse inflammatory infiltration of the
dermis with hyphae, while cultures of biopsy and
skin scales grew Alternaria alternata. Susceptibility
testing showed resistance to itraconazole and susceptibility to amphotericin B, but this was not
administered because of potential renal toxicity.
Hyperthermic therapy was applied using daily direct
warmth on the lesions, achieving clinical cure and
negative cultures after 12 months. Biopsy did not
show fungal elements and 18 months after finishing
the treatment there was no relapse. Alternariosis is
infrequent mycoses in Spain. From 1980 to 2004 19
cases were published, only three in non-immunocompromised patients. Treatments were very variable but in any case thermotherapy was employed.
We present here the first case of subcutaneous
alternariosis that was healed without the use of
antifungal or surgical treatment. This was achieved
by applying only persistent thermotherapy. Hyperthermic treatment should be considered a safe and
conservative sole therapy in cases where antifungal
drugs are contraindicated.
Alternaria alternata subcutaneous infection
treated with local thermotherapy in a renal
transplant recipient
P079
J.M. Torres-Rodrı́guez,1 M. Pérez-González,1
M. Sellart-Altisent,1 J.M. Corominas,2 R.M. Pujol3
and P. Saballs-Radresa4
1
Experimental and Clinical Mycology Research Unit
(URMEC/IMIM), Hospital del Mar. Autonomous
University of Barcelona, Barcelona, Spain, 2Pathology
Department, Hospital del Mar. Autonomous
University of Barcelona, Barcelona, Spain,
3
Dermatology Department, Hospital del Mar.
Autonomous University of Barcelona, Barcelona,
Spain and 4Infectious Diseases Department, Hospital
del Mar. Autonomous University of Barcelona,
Barcelona, Spain
Cutaneous and subcutaneous alternariosis are usually opportunistic mycoses that occur in immunocompromised hosts. At present there are more than
90 published case reports of cutaneous alternariosis
most of them in Europe, including Spain. There is not
consensus on how to treat these mycoses, oral
itraconazole, seems to be the most active antifungal
agents against Alternaria but the treatment failed in
several cases. In some patients surgery were needed
to achieve success. We describe a 55-year-old male
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
In vitro antifungal activity against Fusarium
spp. isolates from skin and nail infections
refractory to treatment
A.M. Tortorano,1 C. Gianni,2 C. Ossi,3 E. Biraghi,1
G. Dho1 and M.A. Viviani1
1
Istituto di Igiene e Medicina Preventiva, Università
degli Studi di Milano, Milano, Italy, 2Dermatologia,
IRCCS Ospedale San Raffaele, Milano, Italy and
3
Laboratorio di Microbiologia Diagnostica e Ricerca,
IRCCS Ospedale San Raffaele, Milano, Italy
Treatment of skin and nail infections caused by
Fusarium spp. is frustrating. Failure of systemic and
local treatment is common and relapses frequently
occur. In the last 8 years we have observed 20
patients (13 women and seven men) with superficial
infection caused by Fusarium spp. refractory to
systemic and topical treatment: 17 onychomycoses,
one intertrigo of feet and two with both of them. We
observed a typical clinical aspect of onychomycosis
caused by Fusarium spp.: in nine cases total or
traversal deep leuchonychia associated, or not, with
painful perionyx; in eight cases total onycholisis
without perionyx. Patients received systemic
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antifungals – terbinafine (TERB, 250 or
500 mg day)1 for 2–3 months) and/or itraconazole
(ITR) as continous (200 mg day)1 for 2–3 months)
or pulsed therapy (400 mg day)1 for 1 week per
month for 4 months) – associated with topical
treatments with amorolfine, bifonazole (BIFO) or
ciclopiroxolamine (CPX). All the patients did not
respond or relapsed. The in vitro antifungal susceptibility of 20 Fusarium isolates was investigated by
broth microdilution following the NCCLS guidelines
(M38-P) in RMPI 1640 broth and in Antibiotic
Medium 3. Besides BIFO, CPX, ITRA and TERB, other
systemic antifungals – amphotericin B (AMB), caspofungin (CASP), posaconazole (POSA) and voriconazole (VORI) – were tested. MICs (mg L)1) at
which 90% (MIC90) of the strains were inhibited and
range of MICs for each antifungal are the following:
MIC90 (range)
RPMI
BIFO 0.5
CPX 8
ITRA 8
TERB 2
P091
MIC90 (range)
AM3
(0.03–1)
1
(4–8)
16
(0.25->16) 16
(0.25–2)
1
RPMI
(0.03–1) AMB 2
(8->16)
CASP 16
(0.12–>16) POSA 1
(0.03–2) VORI 2
AM3
(1–4)
8
(1->16)
16
(0.03–>16) 2
(0.5–4)
2
(0.5->16)
(0.25->16)
(0.03–4)
(0.5–4)
Cherckerboard tests were employed to evaluate
combination of TERB-BIFO, TERB-CPX, and TERBITRA. All the interactions were interpreted as
indifferent on the basis of the Fractional Inhibitory
Concentration Index. No correlation between in vitro
data and clinical and mycological outcome was
demonstrated.
Further studies are warranted to identify an
effective treatment for those cases of nail and skin
infections due to Fusarium spp. refractory to the
current therapies.
P080
A survey on dermatophytosis of cattles in
Research and Breeding Center of Veterinary
Medicine, University of Tehran
R. Yahyaraeyat, A.R. Khosravi, H. Shokri and
M. Ghiyasi
Mycology Research Center, University of Tehran,
Tehran, Iran
In this study 70 cattle suspected of having ringworm
were examined clinically between March and April
2003 in Breeding and Research Center of Faculty of
84
Veterinary Medicine University of Tehran. Samples
were collected from suspected lesions by scrapping
method. Direct microscopic examination and culture
were carried out. The mean of infection duration was
2 weeks. Forty-three cases were appeared with
positive clinical signs of ringworm and 27 cases
had no clinical symptoms. The etiologic agent of the
infection was only Trichophyton verrucosum. A significant relation was observed between frequency of
head and neck lesions and other sites and also there
was a significant difference between frequency of
head lesions and other sites. Since human dermatophyte infections due to T. verrucosum have been
reported repeatedly in Iran, it is necessary to design a
preventive method, like developing an effective
vaccine against dermatophytosis into the future.
Airborne fungi in indoor and outdoor of
asthmatic patients’ home in Sari city – Iran
M.T. Hedayati, S. Mayahi and R. Aghili
Health Faculty, Department of Medical Mycology and
Parasitology, Sari, Iran
Despite growing evidence of the importance of
exposure to fungi as an environment risk factor for
asthma, there is not any report on the identification
of airborne fungi of asthmatic patients living in Iran
inner cities. The aim of this study was identification
of fungi in indoor and outdoor of asthmatic patients’
home. Opened plates (containing of malt extract agar
media) were used for isolation of fungi in the air of
indoor (n = 360) and outdoor (n = 180) of 90
asthmatic patients’ home living in the city of Sari
at the level of breathing height. Plates were incubated in room temperature for 7–14 days. Then grown
fungi were identified by standard mycological techniques. Totally, 1866 colonies with 31 different
genera of fungi and 1683 colonies with 27 different
genera of fungi were isolated and identified from
indoor and outdoor of asthmatic patients’ home,
respectively. The most common isolated genera were
Cladosporium (29.20%), Aspergillus (19.03%), Penicillium (18.27%), Sterile hyphae (11.26%) and
Alternaria (6.65%) in indoor air of the houses of
asthmatic patients; whereas, Cladosporium (44.50%),
Aspergillus (12.42%) and Alternaria (11.11%) had the
most frequencies in the outdoor of the houses of
asthmatic patients. Among the Aspergillus species,
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
Aspergillus flavous had the most frequency. CladospoP093
rium, Aspergillus, Penicillium, and Alternaria as the
Cryptococcus neoformans in pigeon exreta in
most common allergenic moulds had the most
Kermanshah
frequencies in indoor air of the houses of asthmatic
patients. Since in cities, people spend the most of
7,8
7 Mikaeili, Nazari somayeh, Mikaeili Asal
their times in indoor environment, therefore, indoor
airborne fungi has an important role in fungalCryptococcosis caused by Cryptococcus neoformans is
induced diseases like asthma.
an opportunistic fungal infection which is becoming a
very common clinical entity in patients with AIDS. It
is believed that the patients are being infected by
P092
inhaling the aerosolized yeasts in excreta of pigeons.
Abundance of Pseudallescheria boydii in urban
This study was conducted to estimate the rate of
and natural habitats
infection of excreta of 1000 pigeons by C. neoformans
in Kermanshah at 1999–2000. Droppings of 1000
J. Kaltseis and J. Rainer
pigeons were collected in aluminum foils. The specInstitute of Microbiology, Leopold-Franzens
imens were incubated at 30 C for 3–10 days. The
University, Innsbruck, Austria
colonies were examined macroscopically and microscopically. Encapsulated yeasts of C. neoformans were
Pseudallescheria boydii is an opportunistic fungus
present in eight of the specimens (0.8%). Lower rate of
causing diverse clinical pictures in humans. Approxiinfections is identified in Kermanshah study in
mately 50% of P. boydii infections affect the CNS.
comparison with Isfahan (8.1%) and Tehran (2.1%)
Common routes of infection with P. boydii are the
studies. We recommend similar studies in other areas.
aspiration of polluted water after near-drowning and
traumatic inoculations after accidents with polytrauKey words: Cryptococcus neoformans, pigeon.
matisation. As the source of infection is always
located in the environment, an exact knowledge of
the habitats of P. boydii is important for epidemiolP094
ogy. We know from literature that P. boydii is found
preferably in eutrophic habitats e.g. agricultural- and
A survey on the keratinophilic fungi in sewage
ornithogenic soil, manure and polluted water. In this
sludge from Mazandaran, a north Province of
study six different habitats were sampled: soil from
Iran
three nature protection areas and three urban and
industrial locations in Austria. These locations were
A. Mohseni-Bandpei, M.T. Hedayati and
a raised bog, two mountain ranges, industrial areas,
M. Mirzakhani
agricultural lands, urban parks and playgrounds. We
Health Faculty, Department of Medical Mycology and
used a soil corer to sample the soil down to a depth of
Parasitology, Sari, Iran
over 30 cm. The soil cores were split into segments
representing 0–15, 15–30 and >30 cm soil layers to
In Iran there is not any research on the keratinoobtain information on the depth distribution of
philic fungi in sewage sludge. So, in this study our
P. boydii. From each habitat type we took 15 samples.
purpose was isolation of keratinophilic fungi in
The soil samples were homogenised and diluted with
sewage sludge from wastewater treatment plants.
sterile Tween 80 Solution 0.1% (w/v). The dry
Sludge samples were taken from seven wastewater
weight, the ammonium- and pH–values of the soil
treatment plant with different sewage treatment
samples were measured. Selective media for Scedostechnologies (n = 35) from center of Mazandaran
porium were inoculated with the soil suspension and
Province (Sari city), north of Iran. At each plant,
incubated for 1 week at 37 C. From natural habitats
3 kg of sludge were collected in clean plastic bags.
no P. boydii were isolated. The highest concentration
Each sample was taken from five points of sludge
of P. boydii was found in industrial areas, urban
drying bed or lagoon (corner and middle). The
samples were delivered to the laboratory within
parks and playgrounds. No marked differences were
4–5 h. The contents of the plastic bags were
found between soil layers. In this presentation
possible correlation between the abundance of P.
thoroughly mixed to prepare average samples for
boydii and pH- and ammonium–values are discussed.
fungal analysis. The hair baiting technique (HBT)
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
85
xxx
was used for examination of keratinolytic fungi in the
sludge. For each sludge sample five hair supplemented Petri dishes were set up. Also, each of sludge
samples was cultured in five plates containing
Sabouraud’s dextrose agar with cyclohexamide and
chloramphenicole (SCC). Of 35 sludge samples cultured in SCC medium, 28 produced fungal growth.
Totally, 326 fungal colonies belonging to seven
species
were
isolated.
Geotricum
(59.5%),
Cladosporium (13.8%), Alternaria (11.3%) and Penicillium (10.7%) species were the most prevalent. No
growth of keratinophilic fungi was observed in SCC
medium. On the other hand, in HBT, Microsorum
gepseum, Chrysosporium spp. and Geotricum spp. were
isolated. Our and other results showed sewage sludge
contain pathogenic fungi and possible health risk
problems that may arise in the use of sludge for land
reclamation and fertilization.
P095
Spores of Aspergillus spp. in the air of the
Department of Haematology in Ljubljana
Clinical Center
R. Ocvirk,1 S. Zver,2 T. Matos1 and M. Sever2
Institute of Microbiology and Immunology, Medical
Faculty Ljubljana, Slovenia, Mycology, Ljubljana,
Slovenija and 2Clinical Center Ljubljana, Haematology
Department, LJubljana, Slovenija
1
Invasive aspergillosis is the most frequent and the
most dangerous infectious complication in neutropenic hematological patients. In order to prevent
exogeneous aspergillus infection in such patients,
their environment should contain as little as possible Aspergillus spores, i.e. considerably below
1.0 CFU m)3. With air sampling at different sites of
the Haematology Department in Clinical Center
Ljubljana we intended to compare concentration
spores of Aspergillus spp. and to define the efficiency
of hygienic-sanitary measures. We also wanted to
compare two incubation temperatures (30C and
37 C) and two culture media (Sabouraud agar and
Czapek Yeast Exctract agar). Results of air sampling
revealed, that there is no essential difference in spores
concentration of Aspergillus spp. between external
and internal air and also among different room settle
at Haematology Department. The concentration of
86
spores of Aspergillus spp. varied between 0 and
18 CFU m)3. These concentrations were extremely
high and unacceptable. This is probably due to
different external and internal environmental factors;
the essential one is non-filtered air of the department.
The statistical data processing showed that the type
of medium and the temperature of incubation did not
essentially influence the frequency of isolation of
Aspergillus spp. from air.
P096
Protein pattern of Penicillium species isolated
from natural sources in Iran
A. Sabokbar1 and A.R. Khosravi2
1
Department of Mycology, Islamic Azad University,
Tehran, Iran and 2Department of Mycology, Faculty
of Veterinary Medical University, Tehran, Iran
Objective: Fractionation of different Penicillum species based on protein bands.
Procedure: In this study Penicillium citrinium, Penicillium oxalicum, Penicillium notatum and Penicillium
frequentes isolated from air in Iran have been
compared for their protein pattern antigens. All first,
the isolates were cultured on Sabouraud dextrose
agar medium and then subcultured on czapexagar
and were maintained on 300 C for 48–72 h. Then
they were cultured on Sabouraud broth medium for
preparing protein extracts and Braudford method
were used for measuring the level of protein. The
protein were differentiated using SDS_PAGE with
10% separating gel. Coo massie blue G250 were used
for staining.
Results: 34 protein band with molecular weight of:
19.5, 24, 26, 27, 28.5, 32, 36, 39, 45, 48, 50, 52,
53, 55, 56.5, 59.5, 63, 65, 66.5, 68, 76, 84, 88, 90,
92, 93, 94, 95, 97, 107, 116, 123, 128 and
158 kDa were observed. The bands 19.5, 24, 28.5,
45, 52, 53, 56.5, 59.5, 76, 84 and 97 kDa were
present in all 30 isolates under study.
Clinical implications: The results, indicate that
there are inter-species and intra-species differences
but there is no significant difference in protein
patterns of the isolates.
Keywords: Penicillium, protein, SDS_PAGE.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
P097
Effect of amino acids on the growth of
Epidermophyton floccosum and Microsporum
gypseum
M.R. Sarasgani and M. Firozrai
Biochemistry Department, Iran Medical University,
Tehran, Iran
Amino acids have different effects on the growth of
some dermatophytes. Some may increase and the
others inhibit their growth. The concentration of some
amino acids are also an important factor for their
effect. To investigate the effects of amino acids on the
growth of six Iranian species of dermatophytes
(include Epidermophyton floccosum and Microsporum
gypseum). In this study two concentrations (1 and
0.1 gr dL)1) of the 23 amino acids were added to sabro
glocos agar media of these dermatophytes. The
experiment was carried out three times. After 2 weeks
the means of the colonies were compared with the
control without adding any amino acids to Sabouraud
glucose media. The results showed that L-cysteine
hydrocholoride, L-cysteine, L-aspartic acid, L-glutamic acid and DL-tryptophan had more inhibitory
effects on the studied dermatophytes and the rest of
amino acids had less inhibitory on even stimulatory
effects on the growth of the dermatophytes and
Microsporum gypseum has more sensitivity to amino
acids in contrast of Epidermophyton floccosum.
P101
Combined pulmonary infections due to fungi
and other opportunistic pathogens in severely
immunocompromised patients in a tertiary care
hospital
A.D. Argyropoulou, E. Perivolioti, Z. Psaroudaki,
M. Nepka, K. Foundoulis, M. Anagnostopoulou,
G. Margaritis and O. Paniara
Clinical Microbiology, "Evangelismos" Hospital,
Athens, Greece
Invasive fungal infections are important causes of
morbidity and mortality among immunocompromised patients. Their incidence is continuing to rise,
partly due to the growing number of patients with
impaired host defenses. We present eight patients
with immunosuppression, different predisposing risk
factors and underlying diseases who developed
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
pulmonary infection due to more than one opportunistic pathogens. All cases with clinical symptoms,
chest X-ray or CT suggestive of invasive fungal
infection were confirmed by microscopic examination and culture of repeated clinical specimens.
Pneumocystis jiroveci was identified by an indirect
qualitative immunofluorescence method.
Isolated pathogens
Age
Sex
Underlying diseases
Aspergillus sp.,
Nocardia sp.
40
M
CML, allogeneic BMT, GVHD
77
F
25
M
74
F
23
M
Crohn’s disease, long-term
glucocorticoid and
immunosupressive
treatment
AML, allogeneic
BMT, GVHD
Temporal arteriitis,
long-term systemic
glucocorticoid treatment
Aplastic anaemia
25
M
48
57
M
M
Nocardia sp.,
Pneumocystis
jiroveci
Aspergillus sp.,
Mucorales sp.
Accident, traumatic
fungal inoculation caused
by contaminated soil
NHL, allogeneic BMT, GVHD
CML, allogeneic
BMT, GVHD
Opportunistic infections such as nocardiosis,
mucormycosis, aspergillosis or pneumocystosis should
be suspected in immunocompromised patients presented with pulmonary symptoms. Laboratory confirmation of suspected diagnosis is essential to early initiation
of prompt therapy. The identification of one opportunistic agent is not sufficient evidence to rule out the
possibility of other clinically significant opportunistic
co-pathogens. Awareness and rapid intervention diagnostically and therapeutically is of utmost importance
for the successful treatment of the patients.
P102
Microvariation and recombination as genetic
basis for diversity in Pneumocystis jiroveci
J. Beser, M. Lebbad, P. Hagblom and V. Fernandez
Swedish Institute for Infectious Disease Control,
Parasitology, Mycology and Environmental
Microbiology, Stockholm, Sweden
The infectious fungus Pneumocystis jiroveci remains
an important cause of pneumonia (PCP) in the
87
xxx
immunocompromised host. Several studies highlight
the widespread genetic diversity among P. jiroveci
organisms recovered from patients and sporadically
from environmental samples. It is unclear what the
biological significance of this diversity is and how it
is generated. To study the heterogeneity of P. jiroveci
isolates in Sweden a segment containing the ITS1,
5.8S and ITS2 sequences of the rDNA was amplified, cloned and sequenced from a set of BAL
diagnostic specimens obtained from 64 PCP patients. The analysis of 5–13 randomly picked clones
per specimen (408 in total) resulted in the identification of a large number of ITS variants. Among
these, 18 ITS1 and 14 ITS2 established genotypes
and previously described sequences were found.
Seventy-two ITS haplotypes were determined with
Eg being the most common as it was found in 39%
of the specimens, followed by Ne (15%), Bi (14%)
and Eb (14%). A new ITS2 genotype was identified
in four patients forming novel haplotypes with types
I and E of ITS1. Mixed infections, as defined by
the finding of more than one ITS haplotype in
the specimen, were found in 64% of the patients.
In the total material the average number of ITS
haplotypes per patient was 2.9, with up to eight
genotype combinations per patient. In addition to
mixed infections, the diversity in P. jiroveci in the
individual host was found to be further increased by
the occurrence of microvariation of the ITS
sequences. In this context, microvariation was
defined as minor changes in the sequences that do
not warrant the definition of new genotypes.
Examination of the 408 sequences suggested the
occurrence of microvariants of ITS genotypes in
89% of the clinical samples. Up to six variants from
a consensus ITS1 or ITS2 haplotype, displaying one
or two base substitutions, could be found in one
single specimen. The genetic diversity in the P.
jiroveci isolates was further increased by what
appeared to be recombination between parental
haplotypes. In 46% of the specimens with mixed
infections ITS genotype combinations seemingly
representing parental clones and recombinant offspring were found. Hence, microvariation and
recombination in the ITS sequences underlie the
extent of the diversity in P. jiroveci that can be
found in both individual patients and in the total
population studied. The heterogeneity in the ITS
locus is discussed in the light of the genetic diversity
observed in intronic and coding DNA sequences of
this fungus.
88
P103
Food-deriver fungi as possible pathogens for
hematologic patients
S. Błachnio,1 E. Swoboda-Kopeć,1,2 B. Sulik-Tyszka,2
E. Stelmach,1,2 M. Jaworska,1 and M. Łuczak1,2
1
Microbiology, Medical University, Warsaw, Poland
and 2Microbiology Laboratory, Cantral Clinical
Hospital, Warsaw, Poland
Introduction: Fungi are microorganisms being part
of human endogenous intestinal flora. One of the
main sources of fungi is nutrients. Some species are
used in food production, presence of others results
from food contamination. Food deriver fungi may
overgrow alimentary tract, which may result in
opportunistic infections in immunocompromised
patients.
Aim of study: Analysis of relation between prevalence of fungi in alimentary tract immunocompromised patients and fungi occurrence in nutrients.
Study included patients hospitalized in Hematology
Department of Central Clinical Hospital of Warsaw
Medical University between years 2001 and 2004.
Materials and methods: Mycologic tests were
performer instool specimens drawn from 1160
patients. Specimens were inoculated on Sabourauda
Grodnu supplemented with gentamycine and chloramphenicol (Becton Dickinson). Isolated tribes were
identified by using CHROMagar Candida ground and
automatic test ID32C (bioMerieux). Review of fungi
occurring in nutrients was based on literature data.
Results: Material elaboration resulted in isolation of
1390 tribes of yeast belonging to types: Candida –
1266 szczepów (91%), Geotrichum – 90 (6.5%),
Saccharomyces – 24 (1.7%), Zygosaccharomyces – 5
(0.4%), Trichosporon – 3 (0.2%), Rhodotorula – 1
(0.1%), Cryptococcus – 1 (0.1%). There were particular domination of Candida albicans – 558 of strains
(40%) and Candida glabrata – 456 (32.8%). The
biggest number of strains was isolated in 2003 –
445, 2002 – 369, 2004 – 320, 2001 – 256.
Saccharomyces, Geotrichum, Candida, Penicillium,
Aspergillus, Fusarium, Mucor, Rhizopus are the most
often isolated from nutrients.
Conclusion: Nutrients contaminated with fungal
spores may be dangerous for hematologic patients’
health.
Acknowledgment: Supported by Polish State Committee for Scientific Research (grant no. 3PO5A 028
25).
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
P104
P105
Secondary prophylaxis after invasive fungal
infection of the central nervous system or
retinitis
Bloodstream fungal infections at a Single Cancer Center Institute in Latin America, Colombia,
2000–2003
O.A. Cornely,1,2 G. Maschmeyer,2 S. Cesaro,2 D.
Arenz,1,2 J. Effelsberg,1,2 H. Eimermacher,2 A. Haas,2
J. Maertens2 and R. Martino2
1
Klinik I für Innere Medizin, Universität Köln, Köln,
Germany and 2DGHO, Infectious Diseases Working
Party, Köln, Germany
J.A. Cortes,1,2 J.P. Rivas3 and S.I. Cuervo1,4
1
Infectious Diseases, Instituto Nacional de
Cancerologia, Bogota, Colombia, 2Microbiology,
Universidad Nacional de Colombia, Bogota,
Colombia, 3Mycology Laboratory, Instituto Nacional
de Cancerologia, Bogota, Colombia and 4Medicine,
Universidad Nacional de Colombia, Bogota,
Colombia
Background: The long-lasting treatment of invasive fungal infection (IFI) delays consolidation therapy in patients with hematologic and oncologic
disease. Prognosis is especially worse in the subgroup
of patients with central nervous system (CNS) IFI.
Less toxic modern antifungals may allow simultaneous antineoplastic and antiinfective treatment.
Secondary prophylaxis (SP) has evolved as a current
practice, but its efficacy has not been evaluated.
Methods: A survey on SP regimens currently in use
in 60 tertiary care centers in 16 countries. Inclusion
criteria: proven/probable IFI (EORTC/MSG) during
the most recent neutropenia with subsequent chemotherapy to follow. For this analysis prior IFI must
have been CNS IFI.
Results: Nine patients from six tertiary care centers
in four European countries were enrolled. Forty-four
per cent acute myelogenous leukaemia, 56% ALL,
median age 44 years, range 4–65, 22% female, 22%
allogeneic SCT, 78% conventional chemotherapy.
Prior IFI were proven in three (33%; two Aspergillus
spp., one Candida sp.) and probable in six (67%).
Organ distribution of IFI was as follows: four
cerebral, five retinal, eight lung, two liver, two
spleen, two blood stream, two skin. Secondary
prophylactic regimens were: six (67%) voriconazole,
three (33%) itraconazole, one (11%) fluconazole, one
(11%) ABLC. Three (33%) patients received more
than one SP antifungal, of these none received
combination, but all three sequential therapy. Incidence of breakthrough IFI was one (11.1%), i.e. a
probable pulmonary aspergillosis. No proven IFI and
no patient death occurred.
Conclusions: In this high risk population the
success rate of secondary antifungal prophylaxis
after CNS IFI was 89% and no patients in this
population died. Selection of patients for prognosis
cannot be ruled out in our register.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
Background: Fungal infections are common complications found among patients with cancer. Disseminated infections have a higher morbidity and
mortality.
Materials and methods: A retrospective review of
cases of bloodstream infections caused by a fungal
agent detected at Instituto Nacional de Cancerologı́a,
the major center for the treatment of cancer in
Bogota, Colombia, was made. Known risk factors,
treatment and species variables and their influence
over mortality were analysed.
Results: 78 patients developed bloodstream infections during this period; 56% were male, 61.5% had
hematological cancer, 2.4% had a bone marrow
transplant. The median age was 28 years old.
Candida sp. was 88% of the isolates. C. tropicalis was
the more frequent yeast found (46%), followed by
C. albicans (26%). General mortality was 49%. There
was no significant difference in mortality between
treatment with amphotericine B or fluconazole.
Mortality was higher for C. albicans and C. tropicalis,
but not statistically significant differences were found
in a multivariate analysis. In univariate analysis
recuperation from neutropenia and treatment with
an antifungal agent were significant for decreasing
mortality. Mortality for fungal infections not susceptible to fluconazole and treated with this antifungal was 80% in contrast to those susceptible, in
which mortality was 27.8% (P < 0.05 in univariate
analysis, 23 patients). Among 41 patients treated
with amphotericine B, mortality was 65% for those
who received a dose lower than 400 mg, and 0% for
those who had a higher dose (P < 0.05 in univariate
analysis). In the multivariate analysis, only recuperation of neutropenia was significant for decreasing
mortality.
89
xxx
Conclusion: Patients with cancer and bloodstream
infections by fungal pathogens in Latin America
have a high mortality. Outcome is determined by the
immune recovery of the individual. Impact of
treatment and susceptibility was not statistically
significant in our series, and a higher number a
patients may be required to understand the relations
between these two variables.
P106
Fungal infections in lung cancer
M. Dinulescu
INCDMI Cantacuzino, Laboratory of Mycology,
Bucharest, Romania
Abstract: The infection in cancer is the cause of the
morbidity and mortality. Mycoses are frequent
opportunistic infections in immunodeficiency
patients. Alteration if cellular immunity is a predisposing factor in development of certain fungal
infections.
Objectives: The aim of the study was to determine
the fungal infections in: blood culture, sputum,
bronchoalveolar lavage fluid and pleural liquid of
48 patients with lung cancer. This study was
designed to evaluate the influence of radiotherapy
on the increasing incidence of fungal infections
(especially opportunistic infections).
Results: The total number of isolated strains was
639, distributed as follows: Candida albicans and nonalbicans 334, Zygomycetes 115, Aspergillus spp. 141
and other fungi 49. Candida species and other yeasts
represented 52.26% of all. Candida albicans was the
most frequent species 15.8%, followed in prevalence
by Geotrichum capitatum 8.92%, G. candidum 7.19%
and Candida tropicalis 17.19%. Filamentous
fungi were: Aspergillus fumigatus 7.66%, A. flavus
4.69%, A. niger 3.59%. A. nidulans 2.34%. Zygomycetes represented 17.99% of all isolated strains (Absidia
corynbifera 5.94%, Mucor hiemalis 4.38%). The
presence and frequency of the fungi are discussed
in relation to the type of carcinoma.
Conclusions: After radiotherapy fungal infections
(55.79%) were increased (yeast and filamentous
fungi). The patients with microcellular carcinoma
have a polymorphic and abundant mycoflora.
90
P107
Fungal infections in HIV infected adults
S.M. Erscoiu,1 M. Nica,1 E. Mozes,1 G. Tardei,2
T. Biolan,2 P. Calistru1 and E. Ceausu1
1
HIV Department, Clinical Hospital of Infectious and
Tropical Diseases V. Babes, Romania and
2
Biochemical Laboratory, Clinical Hospital of
Infectious and Tropical Diseases V. Babes, Romania
Background: Candida albincans (CA), C. sp. and
Cryptococcus neoformans (CN) are the most frequent
causes of fungal infections in HIV infection, involving
superficial and subcutaneous or systemic infection.
Aim of the study was to identify the etiology of fungal
infection in HIV infected adults admitted in our clinic
during a 10-month period.
Methods: From August 2004 to May 2005 12
isolates of CA, 13 of C. sp., six of CN, one of
Saccharomyces cerevisiae, one of Geotricum sp. were
recovered. Identification was performed by standard
microbiological procedures. The MICs for the isolates
was determined according the NCCLS methods. The
antifungal agents tested were: amphotericin B
(AMB), itraconazol (IZ), fluconazol (FL), 5-fluorocitozin (5-Fc) for 25 isolates and voriconazol (VZ) for one
isolate.
Results: The fungal isolates were identified in 26
patients. The origin of isolates per patient was: two
CA and one C. glabrata from oropharingeal swab; one
CA, one C. tropicalis and one C. sp. from vaginal
secretions; one CA from urine; one CA from CSF; five
CN from CSF; one CN from blood cultures to a patient
without other criptococcal clinical manifestations;
one C. parapsilosis from cutaneous lesions; one CA
from ocular swab; six CA, two C. krusei, one C.
inconspicua, two C. tropicalis, two C. sp., one C. sake,
one C. norvegiensis, one Geotricum sp. and one
Saccharomyces cerevisiae from stools in patients with
or without antibiotic associated diarrhoea. The
susceptibility of different isolates showed two CN,
three C. sp high resistant to AMB, three CA and nine
C. sp high resistant to FL, six CA, 10 C. sp., one CN
high resistant to IZ. CDC classification of HIV
infection showed 16 patients of class C and 12
patients of class B.
Conclusion: Fungal infections are frequent in all
stages of HIV infection. Both antibiotic therapy and
diarrhoea itself are responsible for increased faecal
count of Candida. CN infection must be tested,
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
especially in patient treated previous by antifungal
agents.
P108
A case of fulminating fungal sinusitis due to
Valsa sordida, a plant pathogen, in an immunocompromised patient
A. Kalkanci,1 S. Kustimur,1 G. Sucak,2 E. Senol,3
T. Sugita,4 G. Adams,5 G. Verkleij6 and
R. Summerbell6
1
Microbiology, Gazi University Faculty of Medicine,
Ankara, Turkey, 2Haematology, Gazi University
Faculty of Medicine, Ankara, Turkey, 3Infectious
Disease, Gazi University Faculty of Medicine, Ankara,
Turkey, 4Microbiology, Meiji Pharmaceutical
University, Tokyo, Japan, 5Plant Pathology, Michigan
State University, East Lansing, MI, USA and
6
Centraalbureau voor Schimmelcultures, Ultrecht,
The Netherlands
Most cases of acute sinusitis are viral in etiology,
however, acute fungal sinusitis is most often fulminating. In this case report an unusual etiologic agent,
a plant pathogenic fungus in the genus Valsa
(anamorph Cytospora), is identified as involved in
the sinusitis that resulted in mortality. A 55-year-old
woman with a past medical history significant for
chemotherapy for cancer presented to our medical
center complaining of weakness and lethargy. The
patient was hospitalized September 10th 2003 in the
haematology unit with a diagnosis of acute myeloid
leukemia (AML). Four weeks after the start of antileukemic treatment, fever, headache and sinusitis
was noted. The collected material was biopsied,
examined directly by microscopy, and cultured.
Nasal lesions revealed fungal mycelium and Valsa
sordida is cultured from biopsied samples. On October
8th antifungal treatment was initiated using a lipid
preparation of amphotericin B at 1 mg kg)1 day)1.
On October 15th the patient developed pneumonia.
On October 20th voriconazole was added to the
antifungal therapy. The patient died on October
22nd. The fungus is identified based on morphology
in culture, homology of DNA sequence and phylogenetic analysis. The sequence is of the internal
transcribed spacers of the nuclear ribosomal DNA
operon. Delimitation of species of Valsa is currently
under re-assessment because morphological features
are often unreliable for identification. More precise
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
concepts of species are being inferred from phylogenetic analysis of DNA sequence. The accessions of
DNA sequences in the National Center for Biotechnology Information (NCBI) repository, GenBank, are
not comprehensive for Valsa species. Therefore, this
report includes a phylogenetic evaluation of the
etiologic agent to support its identity as Valsa sordida
Nitschke. The fungus is a ubiquitous pathogen of
trees in the genera Populus and Salix, worldwide.
Koch’s Postulates are performed on a neotropenic rat
model. This is the first substantiated report of a
clinical isolate of Valsa (anamorph Cytospora) being
identified in sinusitis.
P109
Fungal involvement in patients with paranasal
sinusitis
P. Kordbacheh,1 F. Zaini,1 M. Emami,1 H. Borghei,2
M. Khaghanian1 and M. Safara1
1
1 Department of Medical Parasitology and
Mycology, School of Public Health, Tehran University
of Medical Sciences, Tehran, Iran and 2Amir Alam
Hospital ENT Ward, Amir Alam Hospital, Tehran
University of Medical Sciences, Tehran, Iran
Fungal involvement of the paranasal sinuses is
frequently observed in the immunocompromised host
and it can become life-threatening if it is not
diagnosed. Definitive diagnosis is made by tissue
biopsy and culture. In this study biopsy materials of
maxillary, ethmoidal and frontal sinuses of 60
patients with clinical manifestation of sinusitis and
no response to medical therapy were assessed by
mycological and pathological methods for the presence of fungi. Invasive fungal sinusitis was diagnosed
in three patients and etiologic agents were Candida
albicans, Rhizopus sp. and Aspergillus fumigatus.
Predisposing factors in these patients were leukemia,
diabetes mellitus and previous sinus and polyp
surgery, respectively. Allergic fungal sinusitis also
was seen in one patient and Alternaria sp. isolated
from the biopsy material. Only the patient with
allergic form of disease survived but all the patients
with invasive form of fungal infection were expired.
This clearly underscores the need of early recognition
of fungal sinusitis in at risk population in order to
start urgent treatment. In this study Nocardia asteroids also was isolated from the biopsy sample in a
patient with sinunasal adenocarcinoma.
91
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P110
Pulmonary embolism in a child with AML and
angioinvasive aspergillosis
F. Athanassiadou,1 M. Kourti,1 A. Tragiannidis,1
T. Papageorgiou,1 A. Haritanti,2 V. Kaloutsi3 and
A. Velegraki4
1
Division of Pediatric Hematology and Oncology,
Second Department of Pediatrics, Aristotle University
of Thessaloniki, Thessaloniki, Greece, 2Radiology
Department, Aristotle University of Thessaloniki,
Thessaloniki, Argentina, 3Pathology Department,
Aristotle University of Thessaloniki, Thessaloniki,
Greece and 4Microbiology Department, Athens
Medical School, University of Athens, Athens, Greece
Invasive fungal infection can cause life-threatening
infections in immunocompromised children. We
present a case of invasive pulmonary aspergillosis
(IPA), which occluded the right pulmonary artery
resulting in pulmonary embolism with a fatal
outcome in a 10-year-old girl with acute myelogenous leukemia (AML). A 10-year-old girl with secondary AML after aplastic anemia was admitted to the
Hematology–Oncology unit and started chemotherapy according to the 7 + 3 treatment regimen. In a
course of severe and prolonged neutropenia while on
induction treatment she started having fever and
cough and she received broad-spectrum antibiotics.
The persistence of symptoms after a week prompted
antifungal treatment. Diagnosis of aspergillosis was
established by computerized tomography (CT) scan of
the lung and by PCR that detected Aspergillus
fumigatus DNA in blood and bronchoalveolar lavage.
She received intravenous liposomal amphotericin B
(5 mg kg)1 day)1). Two months after initiation of
antifungal treatment the patient deteriorated with an
episode of acute dyspnea, pleuritic chest pain and
rales. The contrast enhanced CT scan of the chest
was compatible with pulmonary embolism demonstrating embolus in pulmonary artery. Pulmonary
scintigram confirmed the diagnosis of pulmonary
embolism and the patient was started on LMW
heparin. In a 2 weeks time and after a downhill
course the patient died. Postmortem histopathologic
examination of the lung revealed aspergillosis of the
lung and arterial occlusion due to aspergillus hyphae
growth. Pediatric oncologists must be aware of this
rare complication of angioinvasive aspergillosis,
which has not been described so far in children.
Radiological investigations and serologic markers
92
should be utilized early for confirmation and prompt
therapy.
P111
Oral posaconazole use in recipients of allogeneic hematopoietic stem cell transplantation
with graft-vs.-host disease: a pharmacokinetics
analysis
G. Krishna, D. Wexler, A. Shah, M. Martinho and
H. Patino
Schering-Plough Research Institute, Kenilworth, NJ,
USA
Background: Posaconazole (POS), an extendedspectrum triazole, is currently in development for
the treatment and prophylaxis of invasive fungal
infections (IFIs). The pharmacokinetics of POS in 246
hematopoietic stem cell transplantation (HSCT)
recipients with graft-vs.-host disease (GVHD) was
evaluated.
Methods: As part of a phase 3 trial evaluating POS
prophylaxis (200 mg p.o. tid) in high-risk allogeneic
HSCT recipients with acute (grades 2–4) or chronic
GVHD and intensive immunosuppressive therapy,
blood samples were collected before and after POS
dosing. POS plasma levels were determined by a
validated LC/MS-MS method. Because POS levels at
steady state are relatively constant, average concentration (Cav) was calculated for each subject from
multiple time points once steady state was reached.
Cav and maximum plasma levels (Cmax) were analyzed to evaluate POS pharmacokinetics in this highrisk population.
Results: While on treatment, median POS Cav and
Cmax for subjects without IFI were 922 and 1360
(n = 241) vs. 611 and 635 ng mL)1 for those with
proven/probable IFI (n = 5). POS Cav and Cmax were
not clinically influenced by race, sex, body weight, or
age. Further stratification by body weight and age
revealed POS concentrations were slightly higher in
patients aged >45 years vs. those aged 18–45 years
(median Cmax 1510 vs. 1290 ng mL)1) and in
patients with a lower body weight (<65 kg) vs. those
who weighed 65–80 kg and >80 kg (median Cmax
1510 vs. 1320 and 1170 ng mL)1, respectively).
POS levels were higher for patients with chronic
GVHD (n = 82) compared with those with acute
GVHD (n = 158; Cmax 1790 vs. 1130 ng mL)1; Cav
1410 vs. 814 ng mL)1; P < 0.05). No differences in
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
ALT, AST, and bilirubin with respect to Cav or Cmax
were noted. Overall, POS was well tolerated and
effective in preventing IFIs.
Conclusions: In high-risk allogeneic HSCT patients
with GVHD, oral administration of 200 mg POS tid
exceeded efficacious plasma concentration in subjects
with acute or chronic GVHD without compromising
safety. POS concentrations were generally unaffected
by demographic variables.
P112
Cryptococcosis and Cryptococcus neoformans in
Cuba
G.M. Martinez, M.T. Illnait, C.M. Fernandez,
I. Valdes, M.R. Perurena and M. Torres
1
Bacteriology–Mycology, Institute of Tropical
Medicine "Pedro Kouri" IPK, Havana, Cuba
The incidence of cryptococcosis has increased dramatically in the last 20 years, being closely related to
the AIDS pandemic and our country has not been
excluded from this phenomenon. The two objectives
of this study were: (1) To describe the clinical
features and laboratory findings in Cuban’s AIDS
patients suffering cryptococcosis prior to the widespread use of HAART. We study 83 patients (72
AIDS and 11 HIV negative). All of them had
mycological evidences of cryptococcosis (positive
India ink stain and/or culture of CSF) and were
hospitalized at the Pedro Kourı́ Tropical Medicine
Institute, between January 1997 and June 2002. In
both groups, the frequency of clinical symptoms and
signs was similar except for the neurological signs,
which prevailed in AIDS patients. Curiously, the HIVnegative patients did not have an obvious predisposing illness and they have a normal CD4 counts.
Cryptococcus. neoformans infection was found to be
the initial AIDS-defining illness in 45.8% of our AIDS
patients. Thirty-three per cent of the HIV+ infected
patients died within the first 2 weeks of diagnosis. A
fatal outcome, related to treatment failure was
associated (P < 0.001) to abnormal mental status,
convulsions, and low glucose concentration in CSF.
(2) To identify species, varieties and serotypes of
Cryptococcus strains isolated from clinical source. A
total of 86 C. neoformans strains belonging to the
collection of the National Reference Laboratory of
Mycology at the Tropical Medicine Institute were
studied. Of these, 76 strains were isolated in our
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
institution from CSF of AIDS and non-AIDS patients
from 1988 through 1997; another nine strains (also
from human sources) were kindly supplied by
Institute Rafael Rangel, Venezuela (seven) and the
North Caroline University (two) and one strain was
isolated from a Cheetah at the National Cuban’s Zoo.
The identification of C. neoformans strains was
established by conventional procedures. To determine the biovariety two methods were used: The
capability to grow on CGB medium and the D-proline
assimilation test, finally the serotyping studies were
carried out using Crypto Check agglutination test
(Iatron Labs Inc., Tokyo). It was found that 95.3% of
strains were var. neoformans and the 4.7% remaining
var. gattii. Curiously the total of Cuban’s strains
isolated from humans (56 from AIDS patients and 20
from non-AIDS patients) were C. neoformans var
neoformans serotype A (var grubii) and the other one
isolated from a Cheetah was serotype B but we
cannot assure that it is an autochthonous strain
because the Cheetah came from South Africa.
Finally, four strains serotype D, two serotypes C
and one serotype B were identified among the
Venezuelan and North American isolates.
P113
Rhino-orbital-cerebral zygomycosis due to
Rhizopus spp. in a insuline dependent diabetes
mellitus
C. Rodriguez,1 A.J. Rodriguez-Morales,2 A. Garcia,1
B. Pastran1 and P. Meijomil1
1
Microbiology, West General Hospital, Caracas,
Venezuela and 2JWT Center for Research,
Universidad de Los Andes, Trujillo, Venezuela
Rhinocerebral zygomycosis is usually an aggressive,
fulminant and, at times, fatal disease most often
affecting poorly controlled diabetics of all ages. We
report the case of a 31-year-old white female, 3 years
previously diagnosed as insulin dependent diabetic.
She came to our hospital with necrotic lesions on
right nasal wing, consciousness alteration and
volume increase in periorbitary and infraorbitary
region up to nasal region with an important nose
morphology alteration. Bilateral palpebral edema
was evident and presented also total occlusion of
right eye. Initial evaluation revealed that the patient
was in diabetic ketoacidosis. Aggressive medical and
surgical treatment was made, and her condition
93
xxx
improved rapidly. Initial anatomopathological evaluation indicated a suspected infection due to Mucor
spp.; treatment with amphotericin B 1.5 mg kg)1 d)1
IV (up to 1400 mg) is begun. Additionally, topical
amphotericin B was applied (spray, 40 mg OD).
Patient was HIV negative. NMRI showed nasal
septum destruction in its cartilaginous portion without bone affectation, without extension to skull base.
Our mycological study revealed hyphae thick septed
and in the culture growth Rhizopus spp. Surgical
debridement was made for those necrotic lesions,
showing clinical improvement in the following
2 weeks, outcoming without any other complication.
Conservative debridement with local amphotericin B
irrigations is an effective adjunct in the control of
sino-orbital fungal infections, especially in patients
with reversible immunosuppression and good preoperative visual acuities. The effect of treatment has
improved since the disease was first described, but
the condition still has a high mortality, especially if it
is not diagnosed early.
P114
Fungal colonization/infection in patients with
hematological diseases
G. Mircevska,1 M. Petrovska,1 N. Panovski,1
N. Siljanovski2 and Z. Zafirovic1
1
Institute of Microbiology and Parasitology, Skopje,
Macedonia, Germany and 2Clinic for Haematology,
Skopje, Macedonia, Germany
The number of patients predisposed to infections with
opportunistic microorganisms such as Candida species
has increased significantly over the last decade. The
isolation of Candida species has been associated with
infections, as well as colonization, in both immunocompromised and immunocompetent patients. The
aim of this study was to determine fungal colonization/
infection in upper and lower respiratory tract, urinary
tract, blood and other body sites of 73 patients with
hematological diseases (29 patients with acute leukemia, nine patients with non-Hodgkin lymphoma, eight
patients with morbus Hodgkin, six patients with
myeloma multiplex and other). Qualitative mycological investigation over the oral cavity swab, nasopharyngeal swab, sputum, urine, blood and other specimens, was carried out. All specimens were examined
for presence of fungi by using the standard microbiological methods – microscopic examination and
94
culture on blood agar and commercially available
CAN medium (bioMerieux, France) for detection of
Candida species. Non-albicans Candida species were
further identified with YBC VITEK biochemical card
(bioMerieux, France). A total of 141 isolates of Candida
spp. were detected in 1–3 clinical specimens of each
patient, most frequently in sputum, urine, oral cavity,
pharyngeal swab and in blood culture. Among these,
59.6% were C. albicans, while the others were nonalbicans Candida species, out of which the following species were identified: C. tropicalis, C. kefyr, C. glabrata, C.
parapsilosis, C. kruzei, C. lusitaniae (51%, 18.4%,
12.3%, 7%, 7% and 4%, respectively) and C. lambica,
as well as non-Candida yeasts (Geotrichum candidum,
C. neoformans, C. humicolus and Saccharomyces cerevisiae) with one isolate each. Association of fungi with
bacterial pathogens was found in 38 patients. Patients
suffering from hematological diseases are frequently
colonized with fungi that may show variable degree of
susceptibility to commonly used antifungal agents.
Besides C. albicans, C. tropicalis and other non-albicans
Candida species are being recognized with increasing
frequency and may be fatal in immunocompromised
patients. Mycological investigations in these patients
have to be systematically performed.
P115
Survey the prevalence of fungal infections in
patiented dialysis
N. Ayat Nasrollahi Omran,1 H. Jamal Hashemi,2
N. Sahereh Mohammadi Nakhjiri3 and H. Jawad
Hakimi4
1
Medical Mycology, Iau of Tonekabon-Faculty of
Medical Science, Tonekabon, Iran, 2Vet. Mycology,
Iau of Tehran, Tehran, Iran, 3Microbiology, Iau of
Tonekabon, Tonekabon, Iran and 4Division
Microbiology, Amol Emamreza Hospital, Amol, Iran
The objective of this investigation was to determine
the prevalence and identification of yeast infections
in patiented dialysis for 6 months in the northern
Iran, 2004. Thus with sampling of 300 urine
patiented dialysis and diagnosis confirmed by microscopy examination, culture of the sample on
differentional media and supplementary tests. Our
results showed that 75 case culture positive cause
yeast agent colonization were seen, and only in 30
were positive case after direct examination. Thus
25% form patient suffered from yeast infections that
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
70% were male the rest were female and majority
age was 30–55 years. Yeast agent were observed
isolated yeast were included: Candida albicans 60%,
C. glabrata 5%, C. parapsilosis 5%, Trichosporn sp.
5%, Cryptococcus laurentii 2%, Rhodotorula sp. 7%,
C. guilliermondii 6%, C. kruisii 10%. Furthermore
dialysis and other immunosuppressive agent increased risk of fugal infections especially disseminated (urinary) candidiasis as commonalism and
opportunistic fungi.
P116
Uncommon fungal infections in immunocompromised paediatric patients
P.E. Papakonstantinou,1 S.F.V. Sidi-Fragandrea,1
R.E. Roilides,2 K.A. Karakoli,1 H.E. Hatzipantelis,1
B.E. Bibashi,3 S.D. Sofianou3 and K.E.D. Koliouskas1
1
Hippokration, Paediatric Oncology, Thessaloniki,
Greece, 2Hippokration, 3rd Department of
Paediatrics, Thessaloniki, Greece and 3Hippokration,
Department of Microbiology, Thessaloniki, Greece
Invasive fungal disease is an important cause of
mortality among immunosuppressed patients,
receiving chemotherapy. We report six cases of
uncommon fungal infections that occurred in our
department over a 10-year period. The patients were
three boys and three girls aged 4–15 years, three
ALL, one NBL, one NHL, one S. Ewing’s sarcoma. All
patients were neutropenic after chemotherapy. A
boy with B–ALL developed pulmonary zygomycosis
of Cunninghamella bertholletiae isolated from bronchoalveolar lavage fluid. A boy with NBL and a girl
with ALL developed Acremonium. A boy with T-ALL
developed trichosporonosis from Trichosporon asahii
(fever, fungemia, pulmonary invasion and cutaneous
infection). A girl with Burkitt NHL developed
fungemia from Candida famata isolated from multiple
blood cultures taken from both the Hickman catheter and peripheral veins. A girl with Ewing’s
sarcoma developed Candida glabrata isolated from
multiple urine cultures. Three of the patients were
treated with liposomal amphotericin B (LAMB)
5 mg kg)1 d)1, two patients with combined LAMB
and voriconazole and one with LAMB initially and
subsequently voriconazole. Three patients were
treated successfully. The patient with C. bertholletiae
died of invasive zygomycosis, the patient with
disseminated trichosporonosis died of gram–negative
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
sepsis that subsequently developed and the patient
with C. glabrata died of her disease. Immunosuppressed patients receiving chemotherapy sometimes
present with fungal infections due to less common
pathogens. Successful management usually warrants aggressive systemic antifungal therapy.
P117
Rhizopus arrhizus (Oryzae) isolated from
sputum of a patient with a myelodysplastic
syndrome – a case report
M.D. Pinheiro,1 S. Xerinda,2 L. Santos2,3 and A. Mota
Miranda2,3
1
Laboratory of Microbiology, Hospital de S. João,
Porto, Portugal, 2Service of Infectious Diseases,
Hospital de S. João, Porto, Portugal and 3School of
Medicine, Service of Infectious Diseases, Porto,
Portugal
Rhizopus arrhizus is a filamentous fungus belonging
to the class of Zygomycetes and the order of Mucorales.
The manifestations of human disease caused by
Zycomycetes have evolved from primarily rhinocerebral, pulmonary and disseminated to include gastrointestinal, cutaneous/subcutaneous and even asymptomatic colonization. Within Zygomycetes class, the
Rhizopus spp. is the most common cause of human
zygomycosis. Its various species have a worldwide
distribution and Rhizopus arrhizus was identified as
the most common environmental isolate. We report
the case of a 77 years old man with a myelodysplastic syndrome, a past history of type 2 diabetes and
samples from the respiratory apparatus positive for
Rhizopus arrhizus. On June 2nd 2004, the patient
came to the emergency department complaining
with fever and productive cough for 7 days. He had
type 2 diabetes and myelodysplastic syndrome.
Haemolytic anaemia was diagnosed 1 month before
and he began prednisone. On physical examination,
the patient was febrile (38.5 C) and tachypnoeic.
Analytical
study revealed haemoglobin of
9.7 g dL)1, leukocyte count of 16.03·109 L)1 with
polymorphonuclears
77%
and
platelet
of
63·109 L)1; blood glucose was 627 mg dL)1 and
liver and kidney tests were normal. The chest X-ray
revealed a right lower lobe consolidation with
cavitation and air-fluid levels. Arterial blood gas
analysis showed hypoxemia. He was admitted to a
medical unit and started treatment with imipenem.
95
xxx
Samples of blood, sputum and urine were negative
for bacteriological exam and the search for acid-fast
bacilli was also negative. Meanwhile, a computerized
tomography of the chest displayed a cavitation with
air-fluid level and extensive condensation of the right
upper and lower lobes. On June 8th, a sputum
sample was sent to the laboratory for bacteriological
and mycological exam. As the patient’s respiratory
status continued to deteriorate, despite treatment
with imipenem, he was admitted to the Intensive
Care Unit of Infectious Disease Service for ventilatory
support, on June 10th and scheduled for bronchoscopy. Endobronchial and bronchoalveolar washings
were collected for culture and while waiting for the
results the treatment was changed to piperacillintazobactam associated to vancomycin and voriconazole. In these cultures, sputum included, there was a
rapid-growth filamentous fungus with an erect,
cotton candy-like aerial mycelium, covering the
entire culture medium. On microscopic examination
of a ‘tease prep’ stained with lactophenol cotton blue,
a Rhizopus arrhizus was identified. Despite the antifungal therapy, the patient died on June 12th. The
case we report emphasizes the potential for severe
fungal infection in the setting of diabetes and
additional/iatrogenic immunodepression. A higher
degree of awareness is thus required, because most of
the times the final diagnosis is not established while
the patient is alive.
P118
Beta-1,3-D-glucan (BGL) antigenemia in
neutropenic cancer patients (Pts) with invasive
aspergillosis (IA) and candidiasis (IC)
L. Senn,1 O.J. Robinson,1 S. Schmidt,2 M. Knaup,1 B.
Duvoisin,2 N. Asahi,3 S. Satomura,3 S. Matsuura,3 T.
Calandra1 and O. Marchetti1
1
CHUV, Infectious Diseases, Lausanne, Switzerland,
2
CHUV, Radiology, Lausanne, Switzerland and
3
Wako, Osaka, Japan
Background: IA and IC are associated with high
morbidity and mortality in neutropenic cancer Pts.
New diagnostic tests are needed for early diagnosis of
invasive mycosis. BGL, a fungal cell wall antigen, can
be detected in the systemic circulation of Pts with IA
or IC. The aim of the study was to evaluate the utility
of BGL monitoring in neutropenic cancer Pts at high
risk of IA and IC.
96
Methods: Prospective study of 125 consecutive
episodes of neutropenia (median duration 23 d) in
Pts with acute leukemia. IA and IC were defined
according to EORTC-MSG criteria. Blood was collected 2· weekly before onset of fever and daily
thereafter. BGL was measured by turbidimetric or
colorimetric assays (Wako, Japan). Positive tests
defined by two consecutive BGL values >6 and
>5 pg mL)1, or >11 pg mL)1 for both tests (two cutoff values).
Results: 24 episodes of invasive mycoses occurred
during 125 neutropenic episodes: 13 IC (one proven,
12 probable) and 11 IA (three proven, eight
probable). Sixteen samples/episode (3–35) were analysed over 35 days (17–122).
BGL (turbidimetric) BGL (colorimetric)
Cut-off (pg mL)1)
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
>6
50
98
92
84
>11
32
100
100
79
>5
86
86
70
94
>11
50
100
100
84
Colorimetric BGL with cut-off 2· > 5 provided the
best performance for diagnosis of IA and IC. BGL was
detected before or at onset of fever in 41% Pts.
Median time (3 d) between fever onset (as first sign of
IFI) and BGL positivity was significantly shorter than
that between fever onset and conventional diagnosis
of IFI (12 d) (P = 0.005).
Conclusions: Twice weekly monitoring of blood b1,3-D-glucan is a promising non-invasive tool for the
diagnosis of invasive aspergillosis and candidiasis in
neutropenic cancer patients.
P119
Etiological agents of infections in organ transplant recipients with diagnosed diabetes
I. Netsvyetayeva,1 E. Swoboda-Kopeć,1
J. Ke˛dzielska,1 S. Błachnio,1 M. Łuczak1 and I. Fiedor2
1
Microbiology, Medical University, Warsaw, Poland
and 2Transplantology, Medical University, Warsaw,
Poland
Aim of the study: The aim of the study was
analysis of pathogenic microorganisms from organ
transplant recipients with diabetes. All patients
were constantly monitored in Transplantology
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
Department, Medical University of Warsaw in the
years 2000–2002.
Materials: Total of 1301 patients were investigated,
including 213 with diagnosed diabetes. In most
patients three-drugs immunosuppression was administered: cyclosporin (CsA) or tacrolimus (FK),
glicocortysteroids (GS) and antipolipheration drugs mycophenolate mofetil (MMF) or azathioprine (AZA).
Methods: Standard microbiological bacterial and
fungal cultures were done on the following clinical
samples: blood, urine, stool, intravenous lines,
wound swabs, pharyngeal and oral cavity swabs.
Isolated microorganisms were identified with automated system ATB Expression (bioMerieux). Viral
infections were estimated by detecting viral nucleic
acid directly by PCR technique or by indirect
detection of specific antibodies by ELISA method.
Results: From all patients different bacteria and fungi
were cultured: Gram-negative bacilli, Gram-positive
cocci, Coryneforms, Anaerobes, Candida spp., Trichosporon spp., Geotrichum spp., Cryptococcus neoformans.
Many viral infections were reactivated (EBV, CMV,
HCV, HBV) or primary present (CMV, HSV-2, VZV,
HCV, HBV).
Conclusion: Organ transplant recipients are highly
predisposed group for infections of different etiology.
Fungal infections dominate from diabetes individuals.
Acknowledgment: Supported by Polish State Committee for Scientific Research (grant no. 3PO5A
02825).
P120
Fungemia as a real threat for haematological
patients
1
1,2
P121
2
S. Błachnio , E. Swoboda-Kopeć , B. Sulik-Tyszka ,
E. Stelmach1,2, M. Jaworska1 and M. Łuczak1,2
1
Medical University, Microbiology, and 2Central
Clinical Hospital, Microbiology Laboratory, Warsaw,
Poland
Introduction: Fungi are microorganisms being part
of human endogenous intestinal flora. In case of
haematological colonization of fungi may result in
opportunistic infections. Dialysed and immunosuppressive patients with vascular and urethral catheters are most often susceptible to blood infections.
Aim of study: Analysis of fungi prevalence in blood
of patients hospitalised in Haematology Department
of Central Clinical Hospital of Warsaw Medical
University in 2004.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
Material and Methods: Fungi were isolated from
number of 485 clinical materials including four
samples of arteria blond and 13 samples of venous
blond. All samples of blood were examined by
automatic equipment Bact/Alert (Organon). Specimens were inoculated on Saburauda ground supplemented with gentamycine and chloramphenicol
(Becton Dickinson). Isolated tribes were identified
by using CHROMagar Candida ground and automatic
test ID32C (bioMerieux). Sensitivity of strains on
amfoterycine B, itraconazol, flukonazol, voriconazol
were examined by using Etest (AB Biodisk), but
Candida parapsilosis were examined by using automatic test ATB FUNGUS 2 (bioMerieux), which
included: 5- fluorocytozine, amfoterycine B, miconazol, ketoconazol, itraconazol, fluconazol.
Results: Material elaboration resulted in isolation of
17 strains of fungi belonging to species: C. parapsilosis
– 11 strains (64.7%), C. glabrata – 2 (11.7%), C. krusei
– 1 (5.9%),
C. lusitaniae – 1 (5.9%), C. kefyr – 1 (5.9%). Also
Fusarium oxysporum was isolated – 1 strain (5.9%).
One strain was sensitive on fluconazol, one on
voriconazol, all studied strains (12) were sensitive
on amfoterycine B by using Etest. Also one mediumsensitive strain on itraconazol was observed. The rest
four strains weren’t sensitive on itraconazol and on
fluconazol (one strain). All strains of Candida parapsilosis were sensitive on antymycotic preparations.
Conclusions: Candida parapsilosis was common etiological factor of fungemii In case of haematological
patients. Fungi may be dangerous for haematological
patients’ health
Combination of PCR / single – stranded
conformation polymorphism analysis for
identification and typing of Candida species
clinical isolates
N. Tegos1, P. Menounos2, A. Mitroussia-Ziouva1 and
A. Velegraki1
1
Mycology Reference Laboratory, Microbiology,
Medical School and 2Molecular Biology Laboratory,
Military School of Nursing, Athens, Greece
Candidosis remains the most frequent infection among
hospitalized patients. This study aimed to evaluate the
potential of the ITS (Internal - Transcribed Spacer)
regions of the nuclear rDNA complex as molecular
markers for reliable typing of Candida nosocomial
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isolates. The 5S rDNA genomic region was included as
an example of a conserved region and potential
candidate for molecular typing. 81 immunocompromised patients hospitalised in the NIMTS Army
Veterans Hospital in Athens, within two years (2003
- 2004), were studied. The 97 clinical isolates used
for DNA extraction comprised 50 C. albicans, 20
C. tropicalis, 11 C. glabrata, six C. krusei, four C. parapsilosis, three C. kefyr, one C. dubliniensis, one C. sake
and one C. sphaerica. The ITS 1, ITS 2 and 5S target
regions were amplified by PCR, followed by SSCP.
Sequencing was performed on Candida species amplicons that showed distinct SSCP patterns. The results
displayed a different degree of hypervariability of the
ITS regions, for each of the Candida species studied. The
ITS 1 region distinguishes two C. tropicalis SSCP
subtypes and three C. parapsilosis subtypes. The ITS 2
region distinguishes eight C. albicans SSCP subtypes. In
addition, SSCP of the amplified 5S region identified
eight out of nine Candida species. In conclusion, PCR SSCP proved to successfully subtype Candida species.
Also, it was possible, in one working day, to identify
Candida species using the 5S rDNA region (even in
mixed Candida species DNA samples), whereas Candida
intraspecific differences can be determined by SSCP of
the ITS 1 and ITS 2 regions.
P122
Invasive fungal infections in children with
haematological malignancies: a 5-year study
F. Athanassiadou1, A. Tragiannidis1,
T. Papageorgiou1, M. Kourti1, A. Velegraki2,
A. Kalogera3 and A. Drevelengas3
1
Aristotle University of Thessaloniki, 2nd Pediatric
Department, Thessaloniki, 2Athens Medical School,
Mycology Laboratory, Athens and 3Aristotle
University of Thessaloniki, Radiology Department,
Thessaloniki, Greece
Invasive fungal infections (IFI) represent an important
cause of morbidity and mortality in immunocompromised
children,
particularly
those
with
haematological malignancies and stem cell transplantation. The objective of our study was to evaluate the
frequency of IFI and to report our results regarding
patient’s characteristics, diagnosis, efficacy of treatment and outcome. Of 90 children with haematological malignancies admitted in our department the last
5 years 14 (15.5%) were enrolled and analysed for IFI:
two (2.2%) had a documented (proven) fungal
98
infection, six (6.6%) a highly probable and six (6.6%)
a possible IFI. Mean age of patients was 6.92 ± 2.1
years. Among the 14 patients ten had acute lymphoblastic leukemia (ALL), three acute myeloid leukemia
(AML) and one non-Hodgkin lymphoma (NHL). All
patients developed an IFI while on induction chemotherapy treatment for ALL or during the first two
months of chemotherapy for AML and NHL. Moreover,
among the 14 patients of our study we found that eight
(57.1%) presented an IFI during or after an episode of
severe febrile neutropenia (ANC < 500/mm3/duration of febrile neutropenia > 10 days). Another factor
associated with development of an IFI was prolonged
(>3 weeks) corticosteroid administration (eight patients had discontinued corticosteroid treatment
<2 weeks before diagnosis of the IFI). Of 14 children
five (35.7%) had a complete resolution and six (42.9%)
presented deterioration resulting in death. Regarding
antifungal treatment, ten children received liposomal
amphotericin B and combined antifungal treatment
(liposomal amphotericin B and voriconazole) was
administered in four children because of the unresponsiveness in conventional antifungal monotherapy. We
conclude that there is a high rate of IFI in children
receiving chemotherapy for haematological malignancies. The early initiation of empiric antifungal
treatment and the prolonged use of antimycotic
therapy may increase survival in these patients.
P123
Cryptococcoma in a child with acute
lymphoblastic leukemia.
F. Athanassiadou1, T. Papageorgiou1,
A. Tragiannidis1, A. Lefkopoulos2 and A. Velegraki3
1
Aristotle University of Thessaloniki, 2nd Pediatric
Department2Aristotle University of Thessaloniki,
Radiology Department and 3Athens Medical School,
Microbiology Laboratory, Athens, Greece
We report a case of a 5-year-old boy with acute
lymphoblastic leukemia treated with ALL BFM’95
protocol. While on induction chemotherapy treatment, the patient had a sudden onset of CNS
symptoms with headache, nausea and vomiting. On
clinical examination the boy was afebrile, didn’t have
focal neurological deficits or signs of meningeal
irritation. Standard laboratory tests were normal.
Tests for Candida and Aspergillosis were negative. On
the basis of a lumbar puncture result {300/mm3
leukocytes, mild increase in CSF protein levels
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
(181 mg/dl) and hypoglycorrachia (15 mg/dl)} and
the ineffectiveness of the antibiotic therapy, he was
started on liposomal amphotericin B and ceftazidime.
MRI findings revealed the presence of two focal
lesions. Diagnosis of cryptococcal infection was based
on laboratory data (serum and CSF antigenic titers,
positive detection of cryptococcal DNA by PCR,
isolation of the microorganism in the urine). Radiological findings on MRI scans 2 weeks later have
demonstrated the eradication of the parietal lesion
and the permanence of the occipital one. The
patient’s clinical status deteriorated rapidly and was
admitted in an ICU were he died of pulmonary and
renal insufficiency 4 weeks later. CNS cryptococcosis
is mainly associated with manifestations of meningitis and encephalitis such as headache, nausea,
irritability and vision disturbances. Both fever and
nuchal rigidity are usually absent as in our patient.
Lumbar puncture (CSF findings similar to that found
in tuberculosis meningitis) and serological antigenic
titers are useful diagnostic criteria and confirmed the
diagnosis of cryptococcosis in our case. Many studies
in literature have described a series of findings related
to poor prognosis such as corticosteroid treatment,
low glucose levels in CSF and high titers of cryptococcal antigen, findings compatible with ours. To our
knowledge this is the first case of cryptococcoma in a
child with acute lymphoblastic leukemia reported in
literature.
P124
Penicillium marneffei infection in a HIV-positive
male in Greece
M. Toutouza1, A. Tsiringa1, X. Trelogianni1,
A. Xanthaki1, A. Filiotou2, A. Velegraki3 and
C.H. Kontou-Kastellanou1
1
Hippokration hosp, Microbiology, 2Hippokration
hosp, Internal Medicine 2nd dept and 3University of
Athens, Microbiology, Athens, Greece
Introduction: Penicillium marneffei is a dimorphic
fungus that can cause systemic mycosis in humans.
It is endemic in Southeast Asia. It is an important
disease among HIV - infected persons. The incidence
of this fungal infection has increased markedly
during the past few years, paralleling the incidence
of HIV infection.
Objective: We report the case of a 45-year-old
Greek male with sepsis of Penicillium marneffei. First
case in Greece.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
Case report: We describe a 45-year-old Greek
homosexual male who presented in our hospital
with fever, anemia and pronounced weight loss. He
returned from China, 4 days ago and he stayed there
for 10 days. He was diagnosed HIV positive in our
hospital, for the first time, with average number of
CD4 T lymphocytes at presentation: 50/cells/mm3.
In our laboratory were sent ten bottles of blood
cultures (BACTEC 9240,Becton-Dickinson). The four
of ten bottles of blood cultures were microscopically
positive for Penicillium and grew pigment - producing fungus, Penicillium marneffei on the Sabouraud
dextrose agar. P.marneffei produced a distinctive red
diffusible pigment. The identification of P. marneffei
was done microscopically and from the biochemical
profile. The isolates were sensitive to amphotericin B,
flucytosine, itraconazole, ketoconazole, voriconazole
and fluconazole. The patient started antifungal
treatment with amphotericin B for 2 weeks, followed
by itraconazole for the next 10 weeks. The response
to antifungal treatment was good, because the
treatment started early. The fever declined in 6 days
and the blood cultures were negative. After the initial
treatment the patient was given itraconazole as
secondary prophylaxis for life.
Conclusion: P.marneffei is an emerging pathogen,
potentially life threatening in immunocomprised
individuals, which have geographic proximity in
Southeast Asia. History of travel and immigration
from the endemic areas are major clues to diagnosis
as illustrated in this case. The early recognition and
diagnosis of P.marneffei and susceptibility testing are
important for the appropriate antifungal treatment in
these infections.
P125
Fungal endocarditis, a rare manifestation of
invasive fungal infection in an
immunocompromised patient
B. Hendriks2, A.L. Van de Velde1, E. Steel1, T. Van
den Wyngaert4, B. Ogunjimi4, T. Panis4, M.M. Ieven3
and A.P. Gadisseur1
1
Antwerp University Hospital, Hematology, 2Antwerp
University Hospital, Tropical Medicine, 3Antwerp
University Hospital, Microbiology, Edegem and
4
Antwerp University, Medicine, Antwerp, Belgium
Background: Fungi are an uncommon cause
(1–6%) of infective endocarditis with aspergillus
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xxx
species being demonstrated in 25% of fungal
endocarditis (FE). In the absence of positive blood
cultures or accessible peripheral emboli for biopsy,
consecutive aspergillus antigen detection may represent an additional, non invasive and rapid
diagnostic tool orienting the clinician to a fungal
etiology, thereby initiating antifungal therapy in an
earlier stage, prior to a thoracotomic biopsy of the
vegetation.
Case report: We present a 63-year-old man with
chronic obstructive pulmonary disease (COPD) and
low-grade small cell B-cell non-Hodgkin lymphoma,
on low dose corticosteroid maintenance therapy. He
was admitted because of subfebrilitas, progressive
dyspnea, weight loss and a productive cough. Routine
blood analysis showed normal white blood cell count
with neutrophil predominance and slightly elevated C
reactive protein. There were no pulmonary infiltrates
on chest X ray. CT scan revealed non specific
interstitial infiltrates.Re-entry tachycardia was succesfully treated farmacologically. A tentative diagnosis of infectious exacerbation of COPD was made.
However, transthoracal echocardiography showed a
right atrial tumor. The vegetation (2 · 3 cm) at the
junction of the vena cava inferior and the right atrium
was confirmed by transesophageal echocardiography
(TEE) and magnetic resonance immaging. Repeated
blood cultures remained negative. Broncho-alveolar
lavage was not performed because of respiratory
distress. Galactomannan testing was repeatedly
strongly positive (> 3 ng/ml). Despite a nonspecific
clinical presentation and CT scan findings, absence of
culture proven or histopathologic evidence of IA, the
diagnosis of a fungal endocardiditis was made. Tapering of corticosteroids and empirical initiation of
antifungal therapy with voriconazole resulted in a
gradual improvement of the patients overall health,
with a rapid decrease in serum galactomannan levels.
After two months the vegetation was no longer visible
by TEE. Although the literature supports an improved
outcome by a combined medical and surgical
approach, our patient was deemed ineligible for
surgery for medical reasons. Fungal endocarditis
recurs in 30% of cases. Therefore, life-long suppressive
antifungal therapy has been advocated.
Conclusion: The addition of the recently validated
Galactomannan ELISA test to the existing microbiological criteria in the definition of invasive aspergillosis, next to the clinical and host criteria, may result
in an earlier diagnosis of this potentially fatal
complication and consequently to an improved
100
outcome of the underlying hematological disease.
Galactomannan testing in culture negative endocarditis may focus the attention to a fungal etiology in
an earlier stage.
P126
Prevention of contagious fungal infections with
permanent nation-wide Internet campaign
V.Y. Sergeev, B.I. Shpigel and A.Y. Sergeev
All-Russian National Academy of Mycology, Moscow,
Russia
The incidence of the only known contagious fungal
infections, caused by dermatophytes, is growing in
Russia and Europe, as reported by recent prevalence
studies. Searching for patients and treating them
effectively appears to be the only way to stop the spread
of tinea pedis/onychomycosis that now affects at least
5% of Russian adult population. With current motivation of patients to seek for medical care being low,
and conventional approaches for prevention (mass
media campaigns, foot care days, etc.) being very
expensive, we need a new, reliable tool for permanent
informational prophylaxis of contagious tinea infections. The Internet as an open, constantly available
information media provides new solution for prevention of tinea infections. In a Russian-speaking segment
of Internet, search engine queries with keywords
relevant for tinea infections approach at least 30 000
per month. For establishing a nation-wide Internet
campaign for prevention of tinea pedis and onychomycosis, the Gribok.RU project (http://www.gribok.ru website) was started by All-Russian National
Academy of Mycology. The goal of Gribok.RU project is
informational prophylaxis of tinea infections and
raising the patient’s motivation to seek medical advice.
Main tasks are: providing evidence-based data on tinea
infections and onychomycosis, its impact on health
and life-quality; offering the possibility to self-diagnose
the fungal disease; teaching the basics of sanitary
measures of prophylaxis and hygiene; and finally –
describing the modern treatment options with possibility of cure. The website includes the special online
test for preliminary self-diagnosis of onychomycosis
and online consultations web-engine. After 2 years of
the project activity, the attendance of Gribok. RU
website has reached 69 100 unique visitors per year
(average 5340 visitors with 33 157 hits per month),
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
about half of the visitors being from Moscow region.
About 20% of visitors reads a page with addresses of
medical centers offering treatment. That statistics is
comparable to reported yearly incidence of tinea pedis
in Moscow (331 per 100 000 of population in a 12million megalopolis). Web-based campaigns are a
modern, cost-effective approach for fighting contagious tinea infections. Compared with recent Hotline
project, where TV and other mass-media sources were
used, new Internet campaign attract almost the half of
target audience per year, with expenditures decreased
more than tenfold.
P131
Cryptococcus neoformans – a unique molecular
type from India: detection, confirmation and
epidemiology.
Banerjee1, N. Jain1 and K.N. Prasad2
1
AIIMS, Microbiology, New Delhi and 2Sanjay Gandhi
Post Graduate Institute, Microbiology, Lucknow,
India
Cryptococcus neoformans is an ubiquitous opportunistic
pathogenic yeast. It is an important etiological agent
causing chronic meningitis both in immuno-suppressed and immuno-competent patients. The tropical
climate of the Indian subcontinent offers a suitable
environment for survival of C. neoformans in nature.
The onslaught of HIV disease pandemic since the early
1990s have led to a sharp increase in the number of
reported cases of cryptococcosis in the past decade. The
wide variety of clinical presentations of the disease
seen in India suggests the possibility of occurrence of
strain variation. Genetic differences among C. neoformans strains have been detected by several typing
methods. The existence of genetic variation for a
pathogen is important because it could translate into
differences in virulence and/or response to therapy. It
also has important role in vaccine design. Furthermore, knowledge of genetic variation is essential for
understanding the population structure and evolution
of a microorganism. In a recent study on molecular
epidemiology of C. neoformans strains in India revealed
some unique molecular pattern of some of the strains
isolated in different geographic location wide apart
(AIIMS-Delhi, Lucknow-U.P & Jabalpur-M.P). Strains
were typed by PCR fingerprinting using M13 core
sequence as a single primer (Meyer et al.). After
molecular typing of 125 freshly isolated strains tested,
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
three types of band pattern were observed. They were
named as ANI, ANII and ANIII. The band pattern of
ANI and ANII were similar to the VNI and VNII group
respectively, already established by Meyer et al. A
smaller but significant proportion, 4.6% were ANIII,
which showed unique pattern. Details of this will be
9 discussed.
P133
Development of a rapid molecular method for
the identification of fungal agents causing
mycetoma
S-J. Miles1, C.J. Linton2, A.M. Borman2,
C.K. Campbell2 and E.M. Johnson2
1
Dept. Pathology and Microbiology, University of
Bristol and 2Mycology Reference Laboratory, Health
Protection Agency, Bristol, United Kingdom
Mycetoma is a destructive infection of the skin and
subcutaneous tissues that progresses to involve
muscle and bone. Antifungal drugs are often ineffective in treating fungal mycetomas, forcing clinicians
to resort to surgical methods, including amputation.
Of the causative fungi, Madurella grisea, Madurella
mycetomatis, and Pyrenochaeta romeroi pose particular diagnostic problems, frequently failing to produce structures by which they can be identified in
culture, and the genetic inter-relationships remain
obscure. The aim of this study was to establish a
molecular method for the identification of these
organisms. Isolates from 24 cases of eumycetoma
were cultured and DNA was extracted using Whatman FTA filter papers. PCR amplification and
sequencing of the nuclear ribosomal repeat regions
enabled discrimination between the causative agents.
M. mycetomatis was the only fungus to sporulate and
was also clearly genetically distinct. Of the group
previously identified as M.grisea by classical methods
there were three separate genetic groups. One group
was identical to P.romeroi and may represent isolates
of P.romeroi that were mis-identified because they
produced no spores. The two other groups were
genetically different from P. romeroi. Genetic similarities with other sequenced fungi suggest that these
other groups are likely members of either the
Pleosporales or Leptosphaeraceae and Sordariomycetaceae orders. Finally, antifungal susceptibility
profiles were found to differ significantly between
the various groups of organisms studies here. In
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conclusion, these initial findings suggest that
molecular methods are an essential adjunct for
identification of non-sporing fungi, and should be
pursued wherever possible to aid informed therapeutic decisions. Moreover, our data indicate that M.
grisea is in fact poly-specific and comprises at least
two genetically distinct and possibly geographically
constrained species.
P134
Typing of Candida isolates from patients with
invasive infection and concomitant
colonisation.
A. Brillowska-Dabrowska1, O. Bergmann2,
I.M. Jensen3, J.O. Jarløv4 and M.C. Arendrup5
1
Statens Serum Institute, Unit of Mycology and
Parasitology, Copenhagen, 2Herlev University
Hospital, Department of Hematology, Herlev, 3Herlev
University Hospital, Department of Clinical
Microbiology, 4Herlev University Hospital,
Department of Clinical Microbiology, Herlev and
5
Statens Serum Institute, Unit of Mycology and
Parasitology, Copenhagen, Denmark
Invasive candidiasis is on the rise in Denmark and
has been so over the latest decades. In a recent
survey the annual incidence was found to be as high
as 11/100 000 population. It has been shown that
invasive candidiasis rarely occurs without preceding
colonisation, however the variation in molecular
genotypes of invasive and colonising strains have not
been thoroughly studied. Various methods have been
applied for the differentiation and genotyping of
Candida species. These include: RFLP, RFLP followed
by probe hybridization, PFGE karyotyping, RAPD,
AFLP, microstaelite analysis and sequencing of
house-keeping genes by multi-locus sequence typing
- MLST. The different methods differ in strength of
discriminatory power. In the time period 1997–2000
we prospectively enrolled patients at high risk of
invasive candidiasis in a study of different diagnostic
methodologies. From all of 11 patients blood cultures
were obtained by colorbact (1997–1998) or Bactec,
by Bactec mycosis and Isolator 10 lysis centrifugation system and colonisation specimens from throat,
feces and urine was obtained. Isolates from these
patients are included in this study of molecular
characterisation of invasive and colonising isolates.
The comparative genotyping of 70 Candida isolates
102
(50 of Candida albicans, 16 of Candida glabrata and 4 of
Candida krusei) was preformed by two methods. 1)
One of the described RAPD (random amplified
polymorphic DNA) methods as this is widely used
and has proven suitable for typing and identification
in microbiology and 2) by MSLT as this one that has
the highest discriminatory power of all of the above
mentioned methods and allow comparison with
previously performed studies in other laboratories
(http://calbicans.mlst.net). Seven genome regions
AAT1a, ACC1, ADP1, MPIb, SYA1, VPS13 and
ZWF1b of each isolate were sequenced. Alignment
of obtained results enables us to define the relation
between colonising and invasive Candida isolates.
P135
RAPD typing and phylogeny of pythium
insidiosum clinical isolates in Thailand
A. Chaiprasert1, S. Pannanusorn1,
C. Prariyachatigul2, T. Krajaejun3,
W. Wanachiwanawin4 and B. Stapatayawong5
1
Mahidol University, Microbiology, Faculty of
Medicine Siriraj Hospital, Bangkok, 2Khon Kaen
University, Clinical Microbiology (CMDL), Faculty of
Associate Sciences, Khon Kaen, 3Mahidol University,
Pathology, Faculty of Medicine Ramathibodi Hospital,
4
Mahidol University, Medicine, Faculty of Medicine
Siriraj Hospital and 5Mahidol University, Medicine,
Faculty of Medicine Ramathibodi Hospital, Bangkok,
Thailand
Background: Pythium insidiosum is the etiologic
agent of pythiosis that caused disease in both animals
and human. Human pythiosis is found predominantly in Thailand. Although its occurrence is rare,
the disease causes severe morbidity and has a high
rate of mortality. There are three types of clinical
manifestation: cutaneous/ subcutaneous, systemic/
vasculitis, and opthalmic pythiosis. The differences in
severity of these clinical forms might be associated
with host immune response, interaction between
host and pathogen, pathogenicity, and/or virulence
of the agent. Molecular typing is a beneficial tool to
preliminary examine these possibilities.
Objective: Random amplified polymorphic DNA
method (RAPD) was used to type and to characterize
the clinical isolates of Pythium insidiosum.
Materials and Methods: A total of 43 Pythium
insidiosum clinical isolates recovered from human
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xxx
pythiosis cases in different geographical regions of
Thailand were characterized. Five primers namely
OPU6, OPU14, OPW11, OPW12, and OPX13 were
used to generate bands in RAPD analysis. The
obtained patterns were further analysed with GelCompar software for dendrogram construction
using P. aphanidermatum and P. middletonii as out
groups.
Results: Two primers; OPU6 and OPU14 were excluded in RAPD analysis, due to their poor priming ability.
The other primers; OPW11, OPW12 and OPX13
exhibited high variability among isolates. Two isolates; MCC15 and MCC16 isolated from diferent
patients in the northern part of Thailand, showed
identical patterns using all three primers. In addition,
identical DNA profiles were demonstrated in isolates
obtained from the same patients as well. Reproducibility of these primers were 95.5%, 88.8%, and 93.3%
and discriminatory powers were 0.83, 0.82, and
0.77,respectively. The analysis based on the combination of three primers displayed the genetic diversity
of this microorganism with 39 RAPD patterns.
Conclusion: All 43 clinical isolates of P. insidiosum
were grouped into the specific clusters after denfrogram construction. Members in each cluster were
varied in both of clinical forms and/or geographical
locations. The strains from the same patients (SIMI
7873 and 7874) and (SIMI 9642–9743) revealed
more than 90 to 100% similarity of band patterns.
There was identical RAPD patterns of isolates MCC15
and MCC16 by amplification with all three primers
which indicated the genetic identity of these strains
owing to these markers. RAPD pattern No.15 was
predominate and found in 13.95% of studied isolates.
Five from six isolates in this pattern were the
causative agent of ophthalmic infection. Our results
revealed very high genetic heterogeneity among
Pythium insidiosum isolates in Thailand by analysis
with RAPD method.
P136
RAPD-PCR heterogeneity analyzing of
Malassezia pachydermatis genomic DNA
B. Dworecka-Kaszak and M. Lesniak
Warsaw Agricultural University, Faculty of Veterinary
Medicine, Warsaw, Poland
Yeast-like fungi of genus Malassezia have a medical
importance. Most of previously created species have
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
lipid-dependent growth, except M. pachydermatis.These fungi are frequently isolated from skin
diseases basing on Atopic Dermatitis, Seborrhoeic
Dermatitis and Pityriasis Versicolor in human and
animals. In principal Malassezia pachydermatis is
recognize as zoophilic species, but occasionaly maybe
isolating from human fungemia cases, mostly in
newborn. Strains isolating from different animals
hosts and sites mostly belongs to M.pachydermatis
species but frequent have different phenotypes. None
differentiation in biochemical properties was shown
among the species, but existing subpopulations of
M.pachydermatis was suggested. In our study we use
RAPD-PCR technique with arbitrary primers ERIC
IR, ERIC2 and BG2. Genomic DNA of 50 isolates
M.pachydermatis strains obtained from clinical cases
of otitis externa in dogs was prepared using MasterPure Yeast DNA Purification Kit (EPICENTRE). For
analyzing phylogeneity of nucleotide sequences the
Quantity one VersaDoc (BioRad) and Statgraphics
plus 4.1 programs were employed. BG2 primer
doesn‘t differentiate the investigated population.
Most successful results were obtained when ERIC IR
and ERIC2 primers were used commonly, but using
separately also shown some differences between DNA
isolates. Phylogenic tree showed that in investigated
strains of Malassezia pachydermatis ten different
phylogenic types occurred, what we are planning
to confirm by another molecular technique. These
results seem to be very useful in epidemiologic
enquiry and in analyzing of increasing drug resistance.
P137
Multilocus sequence typing (MLST) for Candida
albicans: a useful typing method to investigate
transmission from donor to transplant
recipients
J.P. Gangneux1, L. Favennec2, C. Camus1, I. Etienne2,
M. Théraud1, C. Guiguen1, C. D’Enfert3 and
M.E. Bougnoux3
1
CHU Pontchaillou, Rennes, 2CHU Charles Nicolle,
Rouen, 3Institut Pasteur, Unité Biologie et
pathogénicité fongiques, Paris, France
Two transplant recipients from two distant French
hospitals developed Candida albicans fungemia probably procured in a same donor. The first patient was
hospitalized in the Rennes teaching hospital (Brittany,
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west-part of France) for alcoholic and hepatitis C virus
cirrhosis and underwent liver transplantation. Two
blood cultures were positive for C. albicans in the
immediate post-transplantation period. The second
patient was hospitalized in the Rouen teaching
hospital (Normandie, north part of France) for kidney
transplantation. A septic syndrome associated to an
acute renal failure indicated a transplantectomy. The
culture of a kidney biopsy evidenced C. albicans. A
rapid investigation showed a unique donor for both
organs (Etablissement Français des greffes, Pr D
Houssin). The donor was not diagnosed as candidemic
and the mycological culture of organ transport
medium showed no fungal growth. We performed
several phenotypic tests in order to compare the three
isolates. The API 32 gallery (BioMérieux) showed
similar carbohydrate assimilation patterns. The sensitivity to antifungal showed similar CMI using E-tests
(AB Biodisk). We then performed a multilocus
sequence typing of the three isolates. Previous studies
demonstrated that this technique is applicable to a
diploid species such as Candida albicans and can
characterize sets of related isolates. We sequenced
the internal region (loci) of nine housekeeping genes
(AAT1, ACC1, ADP1, GLN4, MP1b, RPN2, SYA1,
VPS13, ZWF1b) and showed that the 3 isolates were
related. The high discriminatory power of 100%
previously evaluated (Bougnoux et al, 2003) and the
unique diploid sequence type (DST) found for these
strains compared to other DSTs described in the
international database (http://calbicans.mlst.net)
support the transmission of a strain of C. albicans from
a single donor (i.e. liver and kidney) to both recipients.
P138
provide this method, but it has not yet been used to
differentiate C. glabrata isolates. A typing method based
on polymorphic microsatellite marker (PMM) analysis
was assessed. Six loci from the GenBank database were
found to harbor microsatellites (3 trinucleotide and 3
dinucleotide repeats: Cg4, Cg5, Cg6, Cg7, Cg10,
Cg11); they were investigated using fragment size
analysis of PCR products. Typing of 103 C. glabrata
strains, including three reference strains and 100
independent clinical isolates, identified a total of 54
alleles (5 to 12 per locus) giving 37 different patterns
with a discriminatory power (D) of 90.2%. The
combination of several of the most discriminant loci
showed a D between 86.6% and 89.2%, with two or
three loci, and 90.2% with four loci (Cg4, Cg5, Cg6,
Cg10). PMM profiles for three serially subcultured
isolates (4 weeks, approximately 300 generations)
were identical to the original culture, demonstrating
short-term stability and reproducibility of the methods. A clinical study of 21 epidemiologically related
isolates (blood culture and peripheral site isolates from
eight patients with C. glabrata candidemia) showed
colonizing and infecting isolates with a similar genotypic profile in each case, confirming that candidemia
usually originates from the colonizing isolate. Due to
its good discriminatory power, reproducibility, ease of
use and general availability, PMM analysis may
provide a good typing method for epidemiological
and surveillance investigations of C. glabrata.
P139
Short-term responses to the loss of the cell
integrity induced by cell wall drugs in the yeast
Saccharomyces cerevisiae
F. Grenouillet1, L. Millon1,2, S. Roussel2, I. Biot1,
J.M. Bart2, J. Knapp2, G. Reboux1 and R. Piarroux1,2
1
University Hospital, Mycology and Parasitology and
2
University of Franche-Comté, Serf, Besancon, France
K. Kuranda1, V. Le Berre3, S. Sokol3, G. Palamarczyk1
and J. François2
1
Institute of Biochemistry and Biophysics,
Department of Fungal Glycobiology, Warsaw,
Poland, 2Centre Bioingenierie Gilbert Durand and
3
Biochips-Plateform Toulouse Génopole, Toulouse,
France
Since the 1990s, C. glabrata has emerged as a major
fungal pathogen, causing mucosal and deep infections. Although several typing methods for the study of
nosocomial colonization, infection and transmission of
C. glabrata already exist, there is a need for one which is
easy to perform, reproducible, and applicable to a large
number of isolates. Microsatellite analysis could
Recently, a new class of antifungal drugs has emerged,
which is directed against a seemingly easy target, the
fungal cell wall. However, it is clear that when the cell
wall is impaired fungi activate defense mechanisms to
prevent cell lysis. In order to study these mechanisms,
we used genome-wide DNA microarrays. We investigated the early response of a model fungus, the yeast
Polymorphism of microsatellite markers for
rapid typing of candida glabrata isolates
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2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
Saccharomyces cerevisiae, to Congo red, Calcofluor
white and caffeine. These compounds are known to
interfere with cell wall biogenesis and assembly. In this
work, we found them to activate the cell wall integrity
pathway and to alter the cell wall architecture,
measured as a level of dually phosphorylated Mpk1
kinase and changes in Zymolyase 20-T sensitivity,
respectively. However, transcriptomic analysis
showed strong discrepancy in both the type and the
timing of the expression profiles elicited by Congo red,
Calcofluor white and caffeine. Significant qualitative
and quantitative changes in gene expression induced
by the first two drugs were obtained just after 90 min
of treatment and to some extent they were doseindependent. Majority of affected genes fell into
functional categories of organelle organization and
cell wall biogenesis. In contrast, transcriptional changes in response to caffeine were fast and dosedependent. Moreover, the expression profiles obtained
in response to caffeine resembled that of cell having a
reduced activity of TOR kinases. We also showed that
caffeine caused a transient decrease in the cellular
cyclic AMP, which was followed by a change in the
expression of genes involved in the regulation of the
adenylate cyclase activity and genes controlled by
cyclic AMP signaling cascade. Altogether, we propose
that the main target of caffeine is the TOR pathway
whose inhibition leads to activation of the cell wall
integrity pathway and eventually induces cell wall
remodeling as it happens normally in the stationary
phase of growth. Hence, contrary to Congo red and
Calcofluor white, which trigger the cell wall integrity
pathway directly through the cell wall damage, the
effect of caffeine on this process is indirect.
P140
Evaluation of different molecular approaches
for direct identification of fungal agents in
clinical samples
M. Perotti, C.M. Ossi, M. Sampaolo, S. Carletti, M.
Sassi, R. Burioni, M. Clementi and N. Mancini
Diagnostica e Ricerca San Raffaele; Università "VitaSalute" San Raffaele, Laboratorio di Microbiologia,
Milano, Italy
The increasing diffusion of immunosoppression conditions and the widespread use of antimycotic drugs
has led to a higher incidence of fungal infections. In
addition to well-documented opportunists, new emer-
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
ging agents are now causing of life-threatening
infections in immunocompromised patients. Rapid
and specific diagnosis represents a crucial element in
the clinical management of these conditions. Firstly,
standard microbiological tests are certainly necessary,
but often lack rapidity usually taking longer than a
week for growth and definitive identification of the
aetiologic agent. Secondly, the identification of infectious agent is not just a mycological curiosity, but
could be important for the choice of an effective
therapy. Thirdly, a conventional mycological identification approach could be elusive in some cases,
especially when morphological features are not easily
differentiated. In this context, and in particular
focusing our attention on the management of suspected fungal keratitis, we recurred to an already
described molecular strategy including the direct
sequencing of an amplified portion of the genome
encompassing the highly divergent internal transcribed spacers 1 and 2 (ITS1-2). Along with classical
cultural approaches, DNA extracted from corneal
samples of 20 patients was amplified with a couple of
universal fungal primers. Sequences of amplified
products were submitted to BLAST database and the
sequence with the highest bit score was considered for
identification. The approach resulted absolutely concordant to culture reducing to only one day the time
needed for identification and helping classical approach in one case of uncommon Microsporum canis
keratomycosis in which identification was not
straightforward. Moreover, in order to evaluate the
reliability of this approach we performed a phylogenetic analysis of ITS1 and ITS2 sequences of
reference strains of fungal isolates described in different ocular infections. A multiple-sequence alignment
and the construction of a phylogenetic tree were
performed. This approach showed that ITS1 and ITS2
genetic divergences allow identification at the species
level. Given this background, we devised a speciesspecific real-time PCR targeting fungal agents usually
described in ocular infections. From sequences alignment, we designed molecular beacon probes recognizing, along with a ribosomal target conserved among
fungi, ITS1-2-specific targets for fungal species commonly involved in keratomycosis (Aspergillus spp.,
Scedosporium apiospermum., Scedosporium prolificans,
Fusarium spp., Candida spp). This approach presents
some advantages if compared to sequence analysis of
amplified ITS1 ITS2 regions, as reduced risk of false
positives due to carryover, improved sensitivity and
reproducibility and lowering of processing time.
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Moreover, the molecular specificity of this approach
could also allow its use on other non-ocular samples
even if contaminated by resident flora.
P141
A37kDa serin proteinase from Trichophyton
vanbreuseghemii
H. Moallei1, G. Larcher2, J.P. Bouchara2 and F. Zaini3
1
Sabzevar School of Medical Science, Dept. of Basic
Science, Sabzevar, Iran, 2Centre Hospitalier
Universitaire, Groupe d’Etude des interaction HoteParasite, Laboratoire de Parasitologie-Mycologie,
Angers, France and 3Tehran University of Medical
Sciences, Department of Medical Mycology and
Parasitology, Tehran, Iran
An extra cellular proteinase produced by geophilic
Dermatophyte Trichophton vanbreuseghemii has been
purified and characterized.The fungus was cultivated
in modified Czapek -Dox liquid medium supplemented
with 0.1% bacteriological peptone and 1% (w/v)
glucose as the nitrogen and carbon sources. Purification to homogeneity of the proteinase was accomplished by (NH4) 2So4 precipitation, followed by Ion
exchange chromatography through Diethylaminoethyl (DEAE). Analysis of the purified enzyme by
SDS-PAGE revealed a single polypeptied chain with an
apparent molecular mass of 37kDa. Proteinase activity was optimum at 37 oc and pH 8, but remained high
with a range of PH values between 8 and 10. The
inhibition profile confirmed that this enzyme belongs
to the subtilisin family of serin-proteinases. In agreement with this, the specefic synthetic substrate Nsuccinyl-Ala-Ala - pro- Phe-p-nitroanilide proved to be
an excellent substrate for the proteinase.
P142
Detection of three virulence genes in a clinical
Candida krusei isolate from Nigeria
F.I. Okungbowa1,2, R. Chowdhury2 and K. Pal2
1
University of Benin, Botany, Benin City, Germany
and 2Indian Institute of Chemical Biology, Infectious
Diseases, Kolkata, India
The expression of three Candida albicans virulence
genes (Tec1, Tup1 and Rad6) was determined in a
106
C. krusei strain with cell morphology similar to that
of C. albicans. These genes are known to play a role in
morphogenesis. The C. krusei strain was isolated
from a human male urethra in Benin City, Nigeria.
Chromosomal DNA was isolated from 18 hour
shaken cultures of the strain and amplification was
done with 5microlitres DNA sample at Polymerase
Chain Reaction (PCR) conditions of 94oC: 30 seconds, 57oC: 30 sec,72oC: 45 sec, for 40 cycles.
Reverse-Transcriptase Polymerase Chain Reaction
(with a single RT-PCR kit, INVITROGEN) was also
performed. No amplification of Tec1 and Rad6 genes
was detected when chromosomal DNA was used. But
the RT-PCR analysis indicated that substantial rRNA
synthesis occurred under the growth conditions
employed but the virulence genes Tup1 and Rad6
were not expressed in both the C. krusei strain and
the clinical C. albicans strain that was used concurrently. However, Tec1 was detected in the C.krusei
but not in the C. albicans. A possible explanation of
these results is that an intron may be present within
the Tec1 gene in the C. krusei strain. Further
investigation is needed to elucidate this. Also, the
RT-PCR seems to be a more effective method of
detecting these genes in Candida strains.
P143
Genotyping of turkish environmental
cryptococcus neoformans variety neoformans
isolates by pulsed field gel electrophoresis and
mating type
M.A. Saracli1, S.T. Yildiran1, K. Sener2, A. Gonlum1,
L. Doganci2, S.M. Keller3 and B.L. Wickes3
1
GMMA, Medical Mycology, 2GMMA, Medical
Microbiology, Ankara, Turkey and 3UTHSCS,
Microbiology and Immunology, San Antonio, United
States
A total of 26 environmental Cryptococcus neoformans
var. neoformans strains isolated from 634 samples of
pigeon droppings collected from 54 different provinces of Turkey in 1996 and 1997 were included in
this study. The results of pulsed field gel electrophoresis (PFGE) showed that the 26 strains could be
separated into 24 different PFGE patterns. In a
mating type study, of 26 strains, 20 were MATalpha,
four were MATa, one was MATa/alpha and one was
nontypable by STE20 specific primers. By the PCR
typing, all the isolates were serotype A. The extensive
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
heterogeneity among these isolates suggests that a
single clonal population may not be present in
Turkey. Additionally, the presence of an AMATa/
DMATalpha hybrid may indicate the existence of
strains that are AMATa mating type in Turkish
environment.
P144
Molecular typing of Iranian cryptococcus
neoformans isolates – a preliminary study
T. Shokohi1, L. Campbell2, T. Bui1 and D.A. Carter1
1
Sari Medical School, mycology and Parasitology,
Sari, Iran and 2School of Molecular anobial
Biosciences, Discipline of Microbiology, Sydney,
Australia
To date, cryptococcosis is a relatively uncommon
disease In Iran, Changing human behaviour and
rising numbers of immunosuppressed people are
likely to increase this mycosis in the future,
however. This study examined three environmental
isolates (from pigeon guano) and two clinical
isolates of Cryptococcus obtained from Iran. The
aims were: (1) to identify the varieties and molecular types of the isolates by DNA fingerprinting; and
(2) to compare the genetic diversity of the Iranian
samples with Australian isolates using AFLP. All
isolates gave positive urease tests and produced a
dark brown pigment on Niger Seed Agar. Molecular
typing found the two environmental isolates and
one of the clinical isolate to be VNII (=C. neoformans
serotype A). The third environmental isolate was
C. neoformans VNIV (serotype D), and the remaining
clinical isolate was C. gattii, molecular type VGI.
Surveys of guano and soil contaminated with
guano from various regions of Iran have
found between 0.8 and 17.8% to be positive for
C. neoformans, and it is likely that this is the source
of C. neoformans infection. Eucalyptus trees known
to harbour C. gattii have been imported and
cultivated in Iran. However, the analysis of decaying wood from the hollows of 200 trees belonging
to 21 different species failed to find any viable
C. gattii cells. In addition, AFLP analysis found the
Iranian C. gattii strain to be genetically distinct
from Australian strains, suggesting they are not
epidemiologically linked. This study is the first
molecular analysis of pathogenic cryptocococci from
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
Iran, and it extends the molecular epidemiologic
survey of Cryptococcus to the Middle East, where
data are currently scarce.
P145
Optimization of random amplified polymorphic
DNA fingerprinting methods for isolates of
candida parapsilosis
I. Wojciechowska-Koszko1, B. Krasnode˛bskaSzponder1, U. Kołodziej-Czekajło2, M. Mnichowska1,
L. Szymaniak1 and S. Giedrys-Kalemba1
1
Pomeranian Medical University, Microbiology and
Immunology and 2Agriculture University, Food
Microbiology, Szczecin, Poland
Introduction: Recently methods based on analysis
of arbitrarily amplified target sites of microorganism
genomes have been extensively applied in microbiological studies especially in epidemiological study.
The range of their applications is limited by problems
with discrimination and reproducibility resulting
from lack of standardized and reliable methods of
optimization.
Objective: The aim of study was obtained optimal
parameters RAPD reaction by using ortogonal –
array according to a protocol Taguchi and Wu,
which modified by Cobb and Clark for Candida
parapsilosis isolates.
Material and methods: Yeasts were isolated from
four different body sites of twins and their family
members: oral cavity, rectum, navel, interdigital
space. After species identification (ATB ID32 C,
BioMerieux) we have chosen 39 isolates of Candida
parapsilosis from 22 people and genetic analysis by
optimized PCR-RAPD was made. Genomic DNA was
isolated as described by Graham at el. with some
modifications. Decamer primers: RP 2 (5 - AAG GAT
CAG A - 3) and 1247 (5 - AAG AGC CCG T - 3)
(Symbios, Poland) were chosen from nine highly
discriminatory combinations. Optimization was performed according to protocol of nine reactions for
variable concentrations four components (MgCl2,
dNTP, primers, DNA). The optimal concentration of
reagents was determined by SNL(signal to noise
ratio)- calculating the number amplicons per each
lane. The PCR was performed with Gene Amp PCR
System 9 600 termocycler (Perkin – Elmer). The
ampilification products were analyzed by horizontal
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electrophoresis in 2% agarose gel, in 1x TBE running
buffer at 120 V by 1 h. The similarity values of DNA
profiles were computed on the basis of band positions
by using the Dice coefficient in BioGenProfil software
(BioProfil – BioGen Windows Application Version
99.04) and dendrograms (UPGMA) were generated.
The discriminatory power of RAPD method determined using Simpson index.
Results: The increased discriminatory power of
RAPD method was detected applying a set of two
primers. Optimal RAPD profiles were obtained using
25 ll PCR initial reaction mixture containing components: MgCl2 – 2.5 mmol L)1, dNTP – 1,5 mmol L)1,
each primer – 30 pM, DNA – 10 ng. Cluster analysis of
Candida parapsilosis strains revealed 22 types: nine
identical, nine similar (70–100%) and four unique.
The discriminatory power according to Simpson index
was 0.92. Similarly SAB coefficient for all pairs of
Candida parapsilosis strains between the twins family
members genotypes were detected – 0.66.
Conclusion: High Simpson index value and low
Dice coefficient indicate a high level of intraspecies
DNA polimorphism among Candida parapsilosis
strains. Optimised RAPD method can to be useful in
microbiological and epidemiological studies.
& RNA) were isolated from obtained mycelial mass
by standard methods. The sequences of known Betatubulin genes in other fungi were aligned and pairs of
21 nt primers were designed from highly conserved
regions. Using mentioned primers; we amplified
predicted molecules by using genomic DNA as well
as cDNA of T.rubrum as PCR templates.
Results: 1863 nucleotides have been sequenced
from Beta-tubulin Gene (Accession AY763789),
which encodes a polypeptide with 447 amino acids
(Accession AAV33733). Nucleotide sequence comparison in gene data banks (NCBI, NIH) for both DNA
and its deduced amino acid sequence revealed
significant homology with the Beta-tubulin genes of
eukaryotes. The amino acid sequence of the encoded
protein was also about 82% identical to the sequence
of Beta-tubulin proteins of the other fungi.
Discussion: In summary, we report the complete
genomic sequences of Beta-tubulin Gene of T. rubrum.
Considering the role of Tubulin protein in formation
of mitotic spindle structure and the cell architecture,
this sequence may be applied in antifungal research
fields and phylogenic studies.
P147
P146
Molecular characterization of beta-tubulin
gene in dermatophyte pathogen Trichophyton
rubrum
K. Zomorodian, P. Kordbacheh, M.R. Khorramizadeh,
M. Rezaian, B. Tarazooie and S. Rezaie
Tehran University of Medical Sciences, Medical
Mycology & Parasitology, Tehran, Iran
Introduction: Trichophyton rubrum (T.rubrum) is a
cosmopolitan arthropophilic dermatophyte which
causes skin and nail infection. The most of antifungal
drugs available for clinical use are targeted four
molecules including: beta-glucan, sterol-alpha-demethylase, ergosterol and DNA/RNA synthesis. The
limited selection of effective antifungal agents combined with the emergence of drug resistance, has
resulted in a critical need for development new drugs
directed against novel molecular targets. In the present
study we tried to characterize the Gene encoded Betatubulin protein in Trichophyton rubrum.
Methods: Clinical isolated of T.rubrum were cultured on liquid medium and the Nucleic Acids (DNA
108
Protein patterns of Alternaria alternata isolated
from Tehran–Iran
M. Saghazadeh1 and A.R. Khosravi2
1
Department of Microbiology, Faculty of Basic
Sciences, Qom Azad University, Qom, Iran
and2Center of Medical Mycology, Faculty of
Veterinary Medicine, University of Tehran, Tehran, Iran
Fungal conidia are spread in environment and some of
them have an important role in appearing signs of
different allergies. Alternaria alternata is one of the
allergenic fungi which exists in different climate of the
world such as different areas of Iran. The airborn
fungal conidia can receive to respiratory tracts by
inhalation, which resulted allergic reaction (asthma),
specially in atopic patients. In this study, 10 isolates of
A. alternata which was separated from different parts of
Tehran were achieved from the Mycology Collection,
Mycology Center, Faculty of Veterinary Medicine.
They were cultured on Sabouraud broth for 10–
12 days at 25 C and shacked in specific incubator,
regularly. Then colonies were harvested, washed and
disrupted with ultrasound homogenizer. After ultra
centrifuging, supernatant was separated and kept as a
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
crude antigen. Using PAGE and SDS_PAGE techniques, protein components of different isolate extractions were identified. It is concluded that there are
different patterns between PAGE proteins pattern of all
isolates. In SDS_PAGE, the most protein bonds were
12 proteins ranging from 14.7 to 69.5 kDa molecular
weight. Among these isolates, number 8 (12 bonds)
and number 4 (5 bonds) had the most and least bonds,
respectively. All fungal extracts had shown common
proteins with molecular weight 14.7, 48.5 and
63 kDa bonds, but a protein with 26 kDa was found
only in number 8 isolate and also, 44 kDa bond were
observed in number 1, 8 and 9 isolates. In this study,
the isolates under study were different on the antigenic
diversity point of view. On the base of some researches,
allergenic components of A. alternata (alt1, alt2) are
(15–20 kDa), (35–40 kDa), (44–45 kDa), (55–
60 kDa), respectively. It is concluded that the A. alternata isolated in Iran, have the most important
allergenic components and they should be considered
in allergic tests into the future.
Duplicate isolates from same patients at the same
periods are not recorded. The isolation rate of Candida
spp. was <1% among the 101.511 specimens obtained
in 3-years period being Candida albicans predominant
(47.2% 47.6% 45.3% of Candida spp. in 2002, 2003
and 2004 respectively). Most of the Candida isolations
(55%–70%) were from urine specimens followed by
blood (6.6%–17.9%). Candida tropicalis, Candida glabrata and Candida keyfr were other common serotypes
with isolation rates between 9%to 15%. Candida krusei
consisted only 2%–3%. Isolation rate of Candida
dubliniensis was 2.2% in 2002 and 2004 with no
isolation in 2003. Distribution of isolates did not differ
for all Candida spp. in the 3-year period. There was
significant increase in Trichosporon spp. 1,3% in 2002
and 2003 to 8% in 2004. Ten percent of the Candida
spp. remained unidentified with our system. Candida
albicans is still the predominant yeast isolate in our
hospital.
P152
P151
Candida species isolated at Ankara University
Hospital during 2002–2004
Ö.A. Akan and S. Uysal
Ankara University Medical School, Ibni Sina Hospital
Central Laboratories, Ankara, Turkey
Candida species, especially Candida albicans is the most
common cause of hospitally acquired fungal infections. Interest in non-albicans Candida has been
increasing since in the last decades there has been
apparent shift in candida infections with increased
recognition of infections due to species other than
Candida albicans at some centers. This caused therapeutic challenges in candida infections as well. This
study is performed to show the distribution of Candida
spp. isolated from hospitalised patients at Ankara
_
University Ibni
Sina Hospital during 3 years period
between 2002 –2004 by evaluating the culture
records of patients sent to the central bacteriology
laboratory. This is one of the largest University
Hospitals in Turkey with 1450 beds. Only clinically
significant specimens (blood, urine, lower respiratory
tract secretions, pus, catheter tip, fluids from closed
spaces such as CSF, peritoneal fluid etc.) were counted.
Vaginal specimens are excluded. Fungal identification
was performed by miniAPi (Biomerieux) system.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
Yeast
S. Akçağlar, C. Evci, B. Ener and O. Töre
University of Uludağ, Microb and Infect, Bursa,
Turkey
The aim of this retrospective analaysis was the
examine of the distribution of yeast species isolated
from blood specimen at our laboratory during the ten
years of period (1995–2005). Total 1337 growth
during the study period was evaluated. All the yeast
colonies were identified by germ tube test, chalamidospor formation and assimilation tests using API C
AUX and/or API ID 32 (Biomerioux). Candida albicans
39.3(%), C parapsilosis (23.9%), C krusei (10.9%),
C tropicalis (9.3%) and C glabrata (2.0%) were the
Candida species most prevelently isolated. The ratio of
other rare encountered Candida species was 10.7 %,
inculuding one isolation of C dubliniensis confirmed
by API ID 32. Among the other yeasts, Trichosporon
spp (2.8%), Rhodotorula spp (0.7%), Sacchoromyces
cereviciae(0.1%), Cryptococcus neoformans (0.1%) were
isolated. Thus the shift from C albicans to non-abicans
species was also seen at our documentation.
The isolation of yeasts were mostly seen from blood
specimens of internal medicine patients (especially
from haemotologia) (58%) who stayed long at
hospital wards and used wide spectrum antibiotics.
Reanimation and intensive care units (26.3%) were
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the second place where fungemia has been seen.
Major disposing factor in these wars was the use of
intravaenous cathaters. The least isolations were
from surgical wards (15.6%) where the turnover of
the patients were highest
P153
Identification of secretory aspartyl proteinase in
different isolate of Candida albicans by sodium
dodesyl sulfate poly acrylamide gel
electrophoresis and identification dy using sera
from infected mice wite Candida albicans
A. Qomi, A. Ghadjari and M. Shams
Arak Islamic Azad University, Medical sciences, Arak,
Iran
Candidiasis is one of the most important and the
common opportunistic mycosis which predisposing
factors such as immune sustem disorders play a role
in disease. Various factors like adhesins,toxins and
enzymes as virulence factors have been described.
The aim of this study was the identification of Candida
albicans secretory aspatyl proteinase by using sodium
dodesyl sulfate poly acrylamid gel and immunoblotting.By using human serum for production of germ
tube and chlamidoconidia production corn meal agar
containing tween 80 candida albicans isolate was
identified from other Candia isolate.
Candida albicans isolates were cultured in plate media
consisted of 1.2% yeast carbone base, 0.2%agarose
and 0.2% bovine serum albumin for 4 days at 30 C
Plates were subsequently stained with 1% amidoblack
in 3.5 M acetic acid for 30 min and destained with
1.2M acetic acid. Zones of bovine serum albumin lysis
were observed as discolored hoalos around colonies.
Isolates subcultured for 4 days at 30 C into a liquid
medium containing 1.2% yeast carbone base, 0.2%
bovine serum albomin and sediment was detected by
centrifugation and its supernatant was precipitated by
actone and liophilized.
The enzymes molecular weight was estimated by
sodium dodesyl sulfate polyacrylamid gel and identified as a single band of 42 KDa. Then 40BALB/c
mice were injected by sub lethal dose of Candida
albicans and on days 10 and 24 blood samples were
collected from mice and detected the mice sera. At
final stage the enzyme was identified by
immunoblotting but enzyme specific band was not
identified.
110
P154
Activity of hydrolytic enzymes of Candida
strains isolated from oral cavity of caries
susceptible and caries resistant subjects
E. Baran1, A. Majewska2, Z. Sozańska2,
E. Plomer-Niezgoda1, A. Hryncewicz-Gwozdz1 and
A. Czajczyńska-Waszkiewicz2
1
University of Medicine, Department of
Dermatology, Venerology and Allergology, University
of Medicine, 2University of Medicine, Department of
Conservative Stomatology, Wroclaw, Poland
Determination of hydrolytic enzymes activities of
Candida albicans strains and other Candida species
isolated from the oral cavity of youth subjects susceptible to dental caries as compared to those caries
resistant. Investigations were carried out in 64 healthy
subjects (three groups), from 16 to 32 years old, with
no sings of oral mucous membrane diseases. Group I
included 26 subjects with DMF-T (decayed, missing and
filled teeth) index = 0–4, group II included 18 subjects
with DMF-T = 5–10, group III included 20 subjects
with DMF-T = 11 and above. Activity of hydrolytic
enzymes was examined using ApiZym test (BioMerieux). Results: Mycological examinations showed
positive cultures in 42 samples (group B) while in 22
negative (group A). Mean values of DMF-T in group B
was10.21 and was statistically significant higher than
in group A DMF-T = 4.77 (P = 0.001). No statistically
significant correlation was fund between activity of
hydrolytic enzymes of Candida albicans and other
Candida species and dental caries intensity. Occurrence
of Candida albicans strains and other Candida sp. in oral
cavity may be risk factor for dental caries.
P155
Structural analyses and enzymic characteristics
of the secreted aspartic proteinases of Candida
albicans
C. Borelli1, E. Ruge1, K. Maskos2, W. Bode2,
M. Monod3, H.C. Korting1 and R. Huber2
1
Ludwig-Maximilians-University of Munich,
Dermatology and Allergology, Munich, 2Max-PlanckInstitut für Biochemie, Strukturforschung, Martinsried,
Germany and 3Centre Hospitalier Universitaire
Vaudois, Laboratoire de Mycologie du Service de
Dermatologie et Venerologie, Lausanne, Switzerland
Candida albicans, the most virulent of the Candida spp.,
can cause severe mucosal and life-threatening sys-
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
temic infections in immunocompromised hosts. In
particular, the secreted aspartic proteinases (Saps),
encoded by the SAP gene family with ten members,
appear to play a major role in Candida albicans
virulence. It has been shown that SAP1-3 contributes significantly to tissue damage and invasion of
oral epithelium and cutaneous epidermis, while
SAP4-6 is important for systemic infections. The
crystal structures of the two closely related Saps,
Sap2 and Sap2X, respectively, complexed with a
potent inhibitor have already been defined by two
different groups in 1995/1996. We started crystallization trials on the isoenzymes Sap1 and 3.
Expression of both Saps in the methylotrophic yeast
Pichia pastoris was followed by several purification
steps. Protein concentrations were calculated from
the absorption of protein at 280 nm and protein
extracts were analyzed by SDS-PAGE and stained
with Coomassie brilliant blue R-250. Samples recovered from buffer exchange were concentrated to
about 10 mg/ml and pepstatin A was added to
samples in a 1.5 fold, ATBI in a 4 fold and Ritonavir
in a 10 fold molar excess. Five crystals appeared in
the presence of Zn2+ within a few days after adding
100 ll 5 M NaCl to the buffer reservoir (300 ll) and
reached full size after a few weeks. Determination
and investigation of crystal structures of further Saps
will give implication for the structure-based inhibitor
design of novel anticandidal agents. Identifying
regions of similarity can provide a basis for the
development of inhibitors, which cross-react with the
different Sap enzymes without inhibiting human
aspartic proteinases.
P156
Enzymatic biotypes of Candida albicans strains
isolated from the sputum of lung cancer
patients
B. Brajer and H. Batura-Gabryel
University of Medical Sciences, Dept. of Pulmonary
Diseases, Poznań, Poland
Introduction: One of the crucial clinical problems
in lung cancer patients is the occurrence of infections, including fungal infection. The mycosis occurrence worsens the patients’ general condition and
inhibits further anticancer treatment. In the author’s
previous studies carried out the positive Candida
albicans mycological examination were observed in
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
over 70% of lung cancer patients. In 25% of subjects
in this group the oral mycosis was diagnosed,
statistically more frequently than in the healthy
population. One of the Candida virulent features is
their enzymatic activity. It determines the occurrence
and the course of infection. It seems important to
describe the enzymatic profile of strains isolated from
these patients and healthy people and describe their
biotypes. It would allow isolating virulent strains and
using antifungal prevention.
Aim: The determination of enzymatic biotypes of
Candida albicans strains isolated from the sputum of
lung cancer patients and healthy persons.
Material and methods: 46 Candida albicans isolated from the sputum of 33 lung cancer patients and
13 healthy people were assessed. The hydrolytic
activity was estimated using API ZYM test (bioMérieux). The biotyping of all strains was performed
according to Williamson, Kurnatowska and Kurnatowski, Krajewska-Kułak et al. and the author’s
previous study classification.
Results: Among 46 strains of Candida albicans
isolated from both groups, 76.09% conformed to
Williamson’s classification (69.26% of lung cancer
patients and 92% of healthy persons). 21.74% of
strains conformed to Batura-Gabryel et al. A further
biotype was described for the strain isolated from the
lung cancer patients, which made up 2,17% of
isolated strains.
While describing the enzymatic biotypes it was
affirmed that in the group of lung cancer patients the
biggest percentage of strains belonged to biotype E
(36.36%) and C and T (each 24.24%), then biotypes
belonging to biotype A, F, H, U and additional biotype
Y (each 2%) followed. Among the strains isolated
from the healthy group, the predominant biotype
was F (69.23%), the remaining biotypes belonged to
biotypes A, D, G and S (each 7.69%).
Conclusions: The Candida albicans strains isolated
from the lung cancer patients and healthy persons
had different enzymatic biotyping. The more frequent
occurrence of biotypes E, C and T in lung cancer
patients and more frequent Candida infection including the symptomatic one may confirm their considerable virulence in this group of patients. It seems
necessary to continue further biotypes description as
well as the continuation of the search for estimating
enzymatic biotypes depending on the progression of
the lung proliferative process and the method of the
disease treatment.
Keywords: Candida albicans, biotypes, lung cancer
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P157
P158
The role of glutaredoxin and superoxide
dismutase genes in virulence of
Candida albicans
Candida albicans adhesion to titanium powder:
modulation by lactoperoxidase systems
G.M. Chaves, S. Bates, C.A. Munro,
D.M. MacCallum, N.A.R. Gow and F.C. Odds
Institute of Medical Sciences, Department of
Molecular and Cell Biology, Aberdeen, United
Kingdom
Candida albicans has developed a complex antioxidant
response in order to repair the damage caused by
reactive oxygen species (ROS) produced by phagocytes to destroy the pathogen. The ROS include
superoxide radical anion (O2-), hydrogen peroxide
(H2O2) and hydroxyl radical (OH) and affect many
essential cell components, including DNA, lipids and
protein. An expression analysis comparing a strain
naturally attenuated in virulence of C. albicans
(RV4688) with the virulent strain SC5314, revealed
that genes related to oxidative stress (TTR1, SOD2,
SOD1.3, GPX1, GPX2, GPX5, IPF6629 and
IPF10842) were down-regulated in RV4688. To
investigate whether those genes actually play a role
in virulence of C.albicans, we made the Dttr1 mutant
and phenotypically characterized the strains Dsod1
and Dsod2. The Dsod1 and Dttr1 mutants were
attenuated in virulence in an animal model of
systemic infection. All three strains had a delay in
hyphal evagination in some or all conditions tested
when compared to SC5314 and were defective in
hypha formation except in serum. In tests with
oxidative stress inducers, the Dsod1 and Dsod2 null
mutants were more sensitive to menadione than the
Dttr1 null mutant. Unexpectedly, the Dttr1 mutant
was more resistant to diamide than the wild type,
recovering sensitivity in the Dttr1+TTR1 reintegrant
stain. Subsequently, we constructed a double mutant
for SOD1TTR1 genes and single reintegrants. The
double mutant and reintegrates also showed a
delayed hypha formation and other defects in all
the conditions tested. They were all sensitive to
menadione and resistant to diamide, suggesting
different gene regulate responses to different oxidative stresses.
112
M. Ahariz1 and P. Courtois2
CHU Saint-Pierre, Stomatology and 2Free University
of Brussels, Lab.Experim.Horm., Brussels, Belgium
1
The present study aimed at describing an experimental design to investigate the effect of peroxidase
systems on Candida albicans adhesion to titanium.
Titanium powder (0.5 g, ~ 325 mesh) was suspended in Sabouraud liquid medium contaminated by
Candida albicans ATCC 10231. After continuous
stirring during 2 days at room temperature, titanium
powder was separated by sedimentation from liquid
medium and washed thrice with Sabouraud broth.
Yeast growth in the supernatant (planktonic phase)
was monitored by turbidimetry at 600 nm. Titanium-adherent yeast biomass was evaluated by the
tetrazolium salt MTT (coefficient of variation £ 12.0
%, analytical range from 0.5 to 10 · 106 blastospores
/ ml). Two different peroxidase systems were tested in
this model of yeast biofilm: hydrogen peroxide (H2O2)
/ lactoperoxidase (L, 48 U/ml) / iodide (I-, 1.2
mmol l)1) system generating hypoiodite (OI-) and
H2O2/ L (48 U/ml) / thiocyanate (SCN-, 1.2
mmol l)1) producing hypothiocyanite (OSCN-).
H2O2 was enzymatically produced by glucose-oxidase
(GOD, 1.3 mU/ml or 0.2 mU/ml) and glucose present
in the culture medium (115 mmol l)1). The washing
process was enough to decrease supernatant turbidity of controls (contaminated just before washing
process) to less than 6.0 ± 0.9 % (n = 18) of the
initial value. In opposition, the last washing liquid
phase turbidity of the contaminated assays still
yielded 110.1 ± 15.0 % (n = 26) of the initial value.
MTT-formazan measurement attested then the presence of a residual adherent biomass equivalent to
6.66 ± 0.55 · 106 blastospores (n = 6) per gr of
titanium powder. In the presence of 1.3 mU/ml GOD,
both G/GOD/I-/L and G/GOD/SCN-/L systems prevented Candida growth in the planktonic as well as the
attached phase during at least 4 days. In contrast, G/
GOD (0.2 mU/ml)/I-/L kept both planktonic and
attached phases sterile during 4 days while G/GOD
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
(0.2 mU/ml)/SCN-/L delayed the Candida adhesion
onto titanium powder without effect on the planktonic growth. Indeed, a 2-day incubation in the
presence of OSCN- decreased the attached biomass to
31.7 ± 2.5 % (n = 3) of the paired control
(P < 0.01,Dunnett’s test). However, after a 4-day
incubation, the attached biomass reached
95.5 ± 6.6 % (n = 3) of the control. This model
demonstrated that Candida albicans growth could be
modulated by lactoperoxidase-generated OI- and
OSCN-. Moreover, OSCN- at non-antifungal concentration was able to delay Candida albicans adhesion to
titanium surfaces, which is frequently used as
biomaterial for implantable devices.
ations in group with diabetes mellitus were infected.
Mycological examination proved that the most
frequent pathogenic fungi were Candida species. The
analysis of such factors proved that diabetes mellitus
often coexists with crural ulcerations and has an
influence on their course. The groups of patients with
diabetes mellitus are prone to bacterial and fungal
infections, which prolonged the treatment of the
crural ulcerations and increased the hospitalization’s
costs.
P159
E. Dósa1, I. Dóczi1, D. Wilhelms1, A.S. Kekulé1,
E. Nagy2,3
1
Martin-Luther-University, Institute of Medical
Microbiology, Halle-Wittenberg, Germany, 2Institute
of Clinical Microbiology, Faculty of Medicine,
University of Szeged and 3Microbiological Research
Group of Hungarian Academy of Sciences, University
of Szeged, Szeged, Hungary
Mycological flora of crural ulcerations in
patients with diabetes mellitus
M.D. Deja1,2, A.Z. Zawirska1,2, K.W. Wierzejska1 and
Z.A. Adamski1,2
1
Provincial Hospital, Department of Skin Diseases and
2
University of Medical Sciences in Poznan,
Department of Medical Mycology at the Department
of Dermatology, Poznan, Poland
Patients with diabetes mellitus are a risk group of
patients especially susceptible to trophic skin changes. Lesions like crural and pedis ulcerations usually
promote further bacterial and fungal infections. The
aim of the research was to analyze the effect of
diabetes mellitus and fungal infections of the ulcerations on the occurrence and course of the crural
ulcerations. The investigated group was composed of
40 patients with established diagnosis of diabetes
mellitus type II. The control group consisted of 20
patients who were not suffering from diabetes
mellitus. Mycological investigation of the flora from
the ulceration was performed. The fungal species
isolated from the crural ulcerations among patients
with diabetes mellitus and without this disease were
analyzed. Secretions from crural ulcerations were
taken as specimens. The material was cultured on
the Sabouraud’s dextrose Agar with chloramphenicol. The cultures were performed according to
mycological rules. The patients had suffered from
diabetes mellitus for 1 to 20 years (mainly of DM
type II). Most patients were treated with oral drugs.
The majority of patients were overweight or obese
(BMI>25). We observed that the majority of ulcer-
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
P160
Detection of biofilm production of Candida
isolates
The prevalence of different microorganisms in
catheter-related bloodstream infections and the
biofilm production of Candida spp originating from
such infections were investigated. During the period
January-December 2002, intravascular catheterrelated sepsis cases were analysed in two institutions: the 1208-bed University Hospital in Halle,
Germany, and the 1240-bed University Hospital in
Szeged, Hungary. There were 877 specimens in
Halle and 1551 specimens in Szeged, submitted to
the laboratories with the suspicion of intravascular
catheter-related infections. The most prevalent
causative agents isolated from the blood cultures
or from the catheter tips were coagulase-negative
staphylococci, including S. epidermidis (39–32.2%)
and S. aureus (21–19.5%). Unusual pathogens, such
as Enterobacter spp. and non-fermentative Gramnegative rods, were also isolated; these accounted
for 21.5–20.2% of all the positive isolates. C.
albicans, together with the non-albicans Candida
isolates, constituted the third most common causative agents (14.4–14.0%). In 11.7% of the specimens in Halle and 10.6% of those in Szeged, the
existence of an intravascular catheter-related bloodstream infection was proven. The biofilm production
of 38 Candida species originating from such infec-
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tions was investigated by two methods. A screening
method was used to demonstrate the slime production of the isolated Candida spp. Production of slime
was quantitatively determined as previously described. The optical densities of the biofilm were read
by a Microplate Reader using a wavelength of
570 nm on the single-wavelength mode. Confocal
laser scanning microscopy (CLSM) was applied to
visualize the biofilm, together with the Candida cells.
20 of the 32 C. albicans isolates and none of the six
non-albicans Candida isolates obtained from catheter
tips or from blood cultures of patients with suspected catheter-related infections exhibited biofilm
production with clear double-staining images after
the use of a combination of O-safranin and FITCconcanavalin-A staining, which allowed differentiation of the Candida cells from the matrix by means
of CLSM. This study showed the frequent occurrence of the biofilm-producing Candida isolates in
intravascular catheter-related bloodstream infections.
P161
RAPD differentiation of Candida sp. isolated
from animals
B. Dworecka-Kaszak, M. Bieganska and M. Lesniak
Warsaw Agricultural University, Dept. of Preclinical
Sciences, Faculty of Veterinary Medicine, Warsaw,
Poland
Dimorphic fungi from Candida genus are very
frequent aethiological agent of superficial and
systemic diseases of animals. The frequency of
isolation of these yeasts in Warsaw region was
increasing for last years and have reached in our
study 15% of mycological positive results. The aim
of this study was to estimate the genetic diversity of
Candida sp. animal isolates. We have used RAPDPCR method with ERIC IR and ERIC2 primers. The
strains was grown on Sabouraud medium (bioMerieux) and for all of them ID32C microtests
(bioMerieux) were done. The DNA from different
clinical isolates was prepared with MasterPure Yeast
DNA Purification Kit (Epicentre) according to the
manufacturers procedure. For analysis of obtained
sequences the Quantity one VersaDoc (BioRad) was
used. These tests have started further experiments
on establishing the possibility of crosspecies transmission of these dimorphic fungi.
114
P162
Cost-effectiveness analysis of pre-emptive
strategy to prevent severe candidiasis in critically ill surgical patients
L. Bene1, F. Grenouillet2, A. Boullot3, C. Menat1, G.
Blasco3, L. Millon2, R. Piarroux2 and M.C. WoronoffLemsi1,4
1
University Hospital, Pharmacy, 2University Hospital,
Mycology, 3University Hospital, Surgical Intensive
Care, 4University Hospital, Besancon, France
Candidiasis has been widely reported in surgical
intensive care units (SICU). Pittet et al. (Ann Surg
1994; 220:751–58) showed that the intensity of
colonization by Candida spp. was significantly related
to the risk of proven candidiasis. They proposed a
calculation of two indexes: a colonization index (CI)
and a corrected colonization index (CCI). Since
December 2000, a care strategy, associating systematic assessment of colonization indexes of all patients
admitted to the SICU and antifungal pre-emptive
therapy in highly colonized patients (CCI ‡ 0.4) has
been implemented. A before/after intervention study,
with 2-year prospective and two-year historical
control cohorts, demonstrated a decrease in incidence of SICU-acquired proven candidiasis (2.2%
before versus 0% after, (P < 0.001) (Crit Care Med
2004; 32:2443–2449). The aim of the study was to
assess the cost-effectiveness of this pre-emptive
strategy versus no strategy. A matched case study
(SICU patients with proven systemic or deep candidiasis versus control non-infected patients) was
conducted to determine the additional costs of
hospitalization attributed to Candida infections. The
matching criteria were diagnosis-related groups,
period of hospitalization (same year), age (±5 years).
The calculation of the pre-emptive strategy’s direct
costs (costs of the systematic mycological screening
for the determination of CCI and costs of the preemptive treatment in highly colonized patients) was
performed during the after-intervention period, from
December 2000 to July 2002. Candidiasis produced
an estimated additional cost of 25 603€ per case. The
pre-emptive strategy’s total cost amounted to 44 500
€ per year. Per year, this strategy prevented five
SICU-acquired proven cases of candidiasis, thus
saving the University Hospital 16 700 € per prevented infection. A sensitivity analysis demonstrated
that for the most unfavorable cases, the strategy still
saved 861 € per prevented infection. Thus, the
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
combination of systematic assessment of Candida
colonization and antifungal pre-emptive therapy is a
dominant care strategy, i.e. more effective and less
expensive than no strategy.
positive or negative correlation with the virulence of
the isolates. 3) Comparison of the mean of the
esterase activity of the human and animal isolates as
well as isolates from patients and healthy hosts
showed minor but significant differences against
some of the substrates.
P163
Comparison of the virulence of Candida albicans isolates and evaluation of the Esterase
activity and electerophoretic pattern of cellular
proteins
A.R. Khosravi and M. Riazipoor
University of Tehran, mycology research center,
Tehran, Iran
In this study 17 isolates of Candida albicans isolated
from healthy and patient hosts were used.Virulence
was determined in animal model of systemic candidiasis by inoculating the isolates through lateral
tail vein to Balb/c moce groups.The survival time of
the animals within each group was recorded and the
results were analyzed with appropriate statistical
methods such as Life Table and Kaplan-Meier. PAGE
and SDS-PAGE were used to determine the electerophoretic patterns of the cytoplasmic and cell wall
proteins of the isolates. The esterase activity of the
isolates against five synthetic substrates was assayed
by a colourimetric method,and pure 1-Naphtol used
as standard. The results of the virulence determining
showed: 1) the virulence of Candida albicans isolate is
considerably different from each other. 2) There is no
significant difference between the virulence of human and animal isolates. 3) There is no significant
difference between the virulence of the isolates from
patients with candidiasis and the isolates from
healthy hosts and the high and low virulent isolates
are found in each group. The results of the electrophoresis of the cellular proteins indicated that: 1)
There is many differences between the electrophoretic patterns of the isolates and in average; defferences in PAGE are more than SDS-PAGE. 2) The
protein patterns detected in both methods are not
useful differentiate : high virulent isolates against low
virulent isolates, human isolates against animal
isolates, and isolates from pateints with candidiasis
against isolates from healthy hosts. The results of the
esterase activity showed: 1) There is quantitative and
qualitative differences in the intracellular esterase
activity of the isolates. 2) The esterase activity
against each of the five substrates didnt show any
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
P164
Correlation between in vitro development of
resistance against Candida albicans to fluconazole and phenotypic switching
N. Kiraz, A. Koygun, A. Kiremitci and Y. Akgün
Osmangazi University, Faculty of Medicine
Department of Microbiology, Eskisehir, Turkey
A collection of 24 isolates of Candida albicans were
tested. Of 24 isolates, 13 were obtained from patients
with manifest candidiasis (infecting agents), remainder were obtained from patients colonized with
Candida albicans (colonizing agents). All isolates tested
had same phenotype and all were susceptible to
fluconazole previously. Overnight cultures of each
isolate were serially subcultured into yeast nitrogen
base (YNB) broth with or without fluconazole (8 lg /
ml). After each passage, all isolates were tested to
determine whether they developed phenotypic
switching and fluconazole resistance or not. To
observe phenotypic variation, pholoxine B agar was
used. The susceptibility organism to fluconazole were
determined by broth microdilution method described
elsewhere. All isolates serially passage in YNB broth
containing low concentration of fluconazole developed
high-level
of
fluconazole
resistance
(MIC ‡ 256 microgram/ml) and most developed
phenotypic switching (P < 0.0001). However, isolates passage into YNB broth without fluconazole
developed neither fluconazole resistance of any level
nor phenotypic switching. High level of resistance to
fluconazole was detected after five to twelve passages,
corresponding to 14 to 24 days of exposure to
fluconazole. Also, phenotypic switching was
observed after one to five passages, corresponding
to two to fourteen days. In addition, phenotypic
variation was more frequent among infecting agents
than colonizing ones (P < 0.01). Smooth colonies
had most variable phenotypes, followed by stipple
and fuzzy colonies. After this investigation of
development of resistance and phenotypic changing,
all isolates developed high-level of fluconazole
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resistance and phenotypic switching were subcultured serially into YNB broth without fluconazole to
determine the stability of resistance. Passages were
continued until organisms had returned to the
baseline. All isolates tested eventually reverted to
the original phenotype and fluconazole susceptibility
forms. Reversion to the normal form of each isolate
was detected after nine to nineteen passages,
corresponding to 18 to 40 days.
P165
Treatment of fungal peritonitis without
removal catheter
N. Kiraz1, B. YIdIz2, H. _Ilhan3 and N. Kural2
1
Osmangazi University Faculty of Medicine,
Department of Microbiology, 2Osmangazi University
Faculty of Medicine, Department of Pediatric
nephrology and 3Osmangazi University Faculty of
Medicine, Department of Pediatric surgery, Eskisehir,
Turkey
Fungal peritonitis is an uncommon but potentially
life-threatening complication for patients undergoing
continuous ambulatory peritoneal dialysis. Comprehensive treatment is early removal of the CAPD
dialysis catheter in most cases. But some treatment
options may be mandatory such as without removal
of the catheter. We report the successful treatment of
refractory fungal peritonitis in two girls with and
without removal of the catheter. Case 1, an episode
of fungal peritonitis produced by Candida parapsilosis
in a 10-year-old girl with chronic renal failure
secondary to scar nephropath and who was treated
with CAPD. She was successfully treated with
intravenous loading dose of fluconazole (5 mg/kg)
followed by intraperitoneal (75 mg/L) and also
catheter was removed. Dialysis cathether was reinserted after eight weeks later but peritoneal adhesions and bleeding diatesis due to disorders of
thrombocyt function was seen (including prolonged
bleeding time and aggregation with ristocetin). Case
2; 9-year-old girl presented with a history of
abdominal pain. Since 4 years the patient had been
treated with CAPD due to tubulopath. Peritoneal
culture was shown Candida parapsilosis. She was
successfully treated with intraperitoneal and systemic fluconazole without removal CAPD catheter
because of refused hemodialysis from her family.
Treatment of the fungal peritonitis was difficult but it
116
may treated without removal of the catheter like as
our patients.
P166
On the role of farnesol mediated quorum
sensing in candidal biofilm formation
B.P. Krom1, M.A.S. Alem2, H.C. van der Mei1,
H.J. Busscher1, L.J. Douglas2 and R.L. Cihlar3
1
University Medical Center Groningen, Biomedical
Engineering, Groningen, Netherlands, 2University of
Glasgow, Division of Infection and Immunity,
Glasgow, United Kingdom and 3Georgetown
University Medical Center, Microbiology and
Immunology, Washington DC, United States
Quorum sensing (QS) plays an important role in
candidal biology and to date two quorum sensing
molecules (QSM) have been identified; tyrosol and
farnesol (or farnesoic acid). Farnesol inhibits the
yeast-to-hypha transition and, at very high concentrations, prevents biofilm formation. Using a mutant
that is refractory to farnesol we studied the biological
role of quorum sensing in candidal biofilms. Biofilms
were grown using the catheter disk model and
different aspects of biofilm biology were assessed
using a set of microscopic and microbiological
techniques. Farnesol inhibited adhesion of yeast cells
to both biotic and abiotic surfaces. The QS mutant
formed biofilms that were different in hyphal composition as shown with confocal laser scanning
microscopy and scanning electron microscopy. In
addition, significantly more biomass was present
after 48 and 72 h when compared to both the
parental strain and the reconstructed heterozygote.
A cell release assay showed that significantly less
cells were released from the QS mutant biofilm as
compared to the parental and reconstructed strain.
From our results we propose the following hypothesis
for the role of farnesol mediated QS in candidal
biofilm formation. Biofilm formation starts with
adhesion of cells, followed by growth including the
yeast-to-hypha transition. Growing cells will produce
farnesol and the concentration of farnesol will
increase in time. Upon reaching the threshold level
of farnesol in the medium hyphal cells will start to
produce yeast cells. These yeast cells will have
reduced adhesive potential due to the presence of
farnesol and are prone to be released from the
biofilm. These changes will gradually disappear in
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
time when the yeast cells have floated away from the
farnesol containing medium and the farnesol concentration in the surrounding medium is below the
threshold level. The adhesive properties of the yeast
cell have now changed and the cell is capable of
adhering to a new biotic or abiotic surface and start a
new biofilm.
P167
Utility of Bichro-Dubli, a new technique for
rapid identification of candida dubliniensis
M.J. Linares, A.D. Garcia-Mayorgas, F. Solis,
R.M. Gordillo and M. Casal
School of Medicine H. U. Reina Sofia, Microbiology,
Cordoba, Spain
Candida dubliniensis was described in 1995 as a new
pathogen. Identification and phenotypic differentiation between C. albicans and C. dubliniensis is based
on the inability of C. dubliniensis to develop at 45C
and to assimilate methyl-D-glycoside and xilose.
Bichro-Dubli (Fumouze) is a new method for the
rapid identification of C. dubliniensis. It is based on the
agglutination of its blastospores with latex particles
covered with monoclonal antibodies, which allow the
specific detection of C. dubliniensis antigens located on
the cell surface. Our goal with this work was to study
the utility of this method in daily practice evaluating
199 yeasts isolated from clinical samples in our
Hospital; 78 strains were isolated from blood samples, 85 from oropharingeal samples, 29 from
respiratory samples and seven from purulent exudates. Among these 199 strains, 176 were Candida
albicans, ten C. tropicalis, ten C. parasilopsis and three
C. dubliniensis. These yeasts were previously identified
using API-ATB ID32 C (Bio-Merieux) and Chromagar Candida (Chromagar Microbiology) (with
Chromagar Candida we could not appreciate differences between C. albicans and C. dubliniensis. We also
performed seven negative controls with C. albicans
(ATCC 90028) and seven positive controls with
C. dubliniensis (Control SEIMC M-2/04) All of the
C. dubliniensis tests were positive, and all non-C.
dubliniensis were negative. This high sensitivity and
specificity (both 100% in our study) and the easiness
and quickness at performing the test validate it as a
very useful tool in clinical laboratories. C. dubliniensis
has the ability to develop an early resistance to azole
antifungal agents, which justifies the need to
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
correctly identify it from clinical samples; perhaps a
part of the previously related azole resistant C. albicans
strains is really due to a bad identification of
C. dubliniensis.
P168
Vaginal carriage of Candida species – especially
regarding Candida dubliniensis
D. Niemann1, K. Tintelnot2 and W. Mendling1
1
Vivantes Klinikum Am Urban, 2Klinik für
Gynäkologie und Geburtsmedizin and Robert
Koch-Institute, Berlin, Germany
Background: For more than 20 years there was no
prospective study on the epidemiology of the vaginal
colonization with yeasts in Germany. Other specialities – f.e. intensive care medicine – report an
increase of non-C. albicans Candida species. Furthermore the importance of Candida dubliniensis - first
described in 1995 – as a pathogen of Vulvovaginal
infections is yet unknown.
Patients/Methods: From November 2001 till
March 2003 a vaginal swab was taken from 600
women (81 of them HIV-seropositive) between 16
and 80 years old in four hospitals and two practices
in Berlin and Cologne. The smear cultures were
extensively differentiated in the Robert Koch-Institute, Berlin. In addition the susceptibility to fluconazole was determined on 75 isolates. The results were
compared to former studies in Germany and current
international reports.
Results: In 173 of 600 swabs (28.8%) Candida
species were found, 85.5% of them C. albicans, 8.7%
C. glabrata, 1.2% C. krusei. C. albicans is isolated in
90–95% in healthy premenopausal or pregnant
women as well as patients with symptoms of
vaginal Candidosis. Surprisingly only in barely
40% of women with symptoms like "itching",
"burning" or vaginal discharge yeasts were recovered. Among postmenopausal and HIV-seropositive
probands the rate of C. albicans recovery was 66.7%
respectively 64.5%. C. glabrata was significantly
more frequently seen in postmenopausal women
(13.3%) than in premenopausal as well as in HIVseropositive women (25.8%) than in HIV-seronegative women. C. dubliniensis was isolated only once
from a non-symptomatic HIV-seronegativ pregnant
proband. There was no isolate resistant to fluconazole.
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Conclusion: There is no evidence for a change of
the distribution pattern of Candida species in vaginal
yeast colonization. C. dubliniensis is insignificant as a
pathogen in gynaecology and obstetrics. With reservation by the small number of isolates tested,
Fluconazole-resistance in vaginal yeast isolates is
not being expected.
P169
Epidemiological typing of Candida albicans
strains and their adherence to culture cells
M. Mnichowska, L. Szymaniak, B. Klimowicz,
B. Krasnode˛bska-Szponder, I. WojciechowskaKoszko and Giedrys-Kalemba
Pomeranian Medical University, Microbiology and
Immunology, Szczecin, Poland
Introduction: The most common species of Candida
isolated from symptomatic vaginal candidosis (VC)
patients is Candida albicans. The differentiation of
Candida albicans strains isolated from different body
sites and in different time clarifies the epidemiology of
VC. It can be performed by enzymatic biotyping and
genotyping. The high incidence and involvement of
Candida albicans in symptomatic VC is due to the
ability to adhere to human cells. Adhesion plays a
crucial role as first, initial step of infection.
Objective: The aim of the study was to determine enzymatic and genetic similarity of Candida
albicans strains and to assess their ability to adhere to
in vitro cell line.
Material and methods: Candida albicans isolates
(n = 170) were collected three times at 2-month
intervals from vagina, anus, and oral cavity from 20
women (mean age–33) suffering from single episode
of VC. Biotyping by enzyme profiles was performed
with API-ZYM commercial system. Molecular analysis was performed by the Random Amplified Polymorphic DNA (RAPD) assay, with the primers set
1290 (5–GTG GAT GCG A-3) /1247 (5-AAG AGC
CCG T-3) and with Molecular Analyst software
application. Strains relatedness were generated on
basis of a similarity coefficients (SAB = 0–1). Adhesion of Candida albicans to human epithelial-like
cervix carcinoma cells (HeLa cells) was tested as
described by Hazen with some modifications and was
determined quantitively by percentage of adhered
cells and by adherence index. To screen the presence
of cell surface-associated structures in Candida albicans strains scanning electron microscopy was used.
118
Results: The majority of Candida albicans strains
belonged to biotype C (54.1%) regardless of ontocenosis and the period of sampling. The genotyping by
PCR-RAPD divided Candida albicans strains into 27
(1–27) major RAPD genotypes at 82% similarity. 22
of them were represented by 2–9 identical strains
(SAB = 1) and 2–7 similar strains (SAB = 0.82–0.99),
further five include similar strains only. Eighteen
strains with unique patterns (SAB lower than 0.82)
were detected. SEM showed the ability of Candida
albicans isolates to adhere to in vitro cell line. All of
the tested Candida albicans strains adhered to the
HeLa cells, however at different rates. Adherence
index was ranged 2 to 266 ± 15 and percentage of
adhered cells was 0.1–15.9%.
Conclusion: Application of typing methods for epidemiological purposes is essential tool for monitoring
of variability of strain-types. Similarity of Candida
albicans strains isolated from different body sites and in
different period of time indicates the possibility of
transmission and persistence of strains. Analyzed
ontocenoses can be a potent reservoir for VC. The
same genotype-strains can express different adherence
index values and enzymatic activity what enable
persistent colonization - a requisite for the VC.
P170
What about ‘‘rafting’’ in Candida albicans?
G. Molero1, M. Insenser1, C. Nombela1, O. Jensen2
and C. Gil1
1
Fac. de Farmacia, Univ. Complutense de Madrid,
Microbiologı´a II, Madrid, Spain and 2University of
Southern Denmark, Department of Biochemistry and
Molecular Biology, Odense, Denmark
Lipid rafts are membrane microdomains enriched in
saturated phospholipids, sphingolipids, cholesterol
and proteins. These microdomains have been implicated in diverse cellular processes including signal
transduction pathways, apoptosis, cell adhesion and
migration, organization of the citoskeleton and protein
sorting during both exocytosis and endocytosis. (1,2)
The presence of lipid rafts in the yeast Saccharomyces
cerevisiae has already been described by Bagnat (3),
where lipid-rafts are involved in the polarization of
proteins at the cell surface during mating (4). In
Candida albicans, a different distribution of these
structures has been described for hyphal cells (5).
We are interested in the study of the proteins
associated with lipid-rafts from Candida albicans. The
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
first step of this work was the generation of a lipid
rafts protein reference map of C. albicans. The
biochemical definition of lipid rafts is that they are
resistant to detergents such as 1% Triton X-100.
After Triton extraction, they float to the top of an
ultra centrifugation density gradient. The proteins
were separated using SDS and two-dimensional
polyacrylamide gel electrophoresis (SDS-PAGE and
2-D PAGE, respectively). The proteins were excised,
digested in gel with trypsin and identified by MALDITOF-TOF (Matrix-Assisted Laser Desorption/Ionization–Time of Flight) mass spectrometry. This work
led to the identification of 18 proteins from 1D gel
and 40 proteins from 2D, including known rafts
proteins, glycolytic enzymes, and heat shock proteins
and proteins involved in several other processes. We
have started complementary strategies in order to
analyse raft proteins during morphological transition
of yeast and hyphae. The study of the differential
expression of these proteins in yeast and hyphal
forms of C. albicans cells could be crucial for the study
of virulence determinants or signalling proteins
involved in the interaction with the host.
Acknowledgement: This work was supported by
grants BIO2003-0030 from the Comisión Interministerial de Ciencia y tecnologı́a (CICYT), Spain and
from the Fundación Ramón Areces.
References
1. Simons K, Toomre D. Nat Rev Mol Cell Biol. 2000; 1: 31–9.
2. London E, Brown DA. Biochim Biophys Acta 2000; 23: 182–
195.
3. Bagnat M, Keranen S, Shevchenko A, Shevchenko A, Simons K.
Proc Natl Acad Sci U S A 2000; 97: 3254–9
4. Bagnat M, Simons K. Proc Natl Acad Sci U S A. 2002;
99:14183–8.
5. Martin SW, Konopka JB. Eukaryot Cell. 2004; 3:675–84.
an important role in clinical manifestation of allergic
reactions especially in atopic patients . Identification
of its allergenic components is valuable because these
components can have plenty of applications in
allergen diagnostic tests.
In this study we identified the allergenic components of Candida albicans using the serum samples
from asthmatic and atopic dermatitis patients. For
this purpose various components of this microorganism were separated by SDS-PAGE. The obtained
protein bands were used to detect specific IgE in the
serum samples from 95 atopic dermatitis and 85
asthmatic patients separately by immunoblotting. 70
serum samples from normal none atopic persons
were used as negative control. SDS-PAGE revealed
several protein bands in the molecular weight range
of 13 to 135 KD. Specific IgE was detected against 33
of these bands in immunoblotting. 25,34 and 57 KD
protein bands were the more important antigenic
bands in atopic dermatitis patients, which, react with
63.3, 50 and 42 percent of serum samples respectively. In asthmatic patients 22,25,32 and 34 KD
bands were more important. They reacted with
59.5,59.5, 45.2 and 66.7 percent of patients serum
respectively. No protein band was stained in control
group. By gel filtration chromatography we separated 4 fractions from antigenic extract. Which most
antigenic bands were in fraction number 3 and this
finding was true about both groups of patients.
The results of the present study showed that
Candida albicans has several allergenic components,
which can play a role in clinical manifestation of
allergic reactions. So the mentioned components
must be included in diagnosis of relevant allergen.
P172
Acid proteinase production in clinical Candida
albicans isolates
P171
Identification of allergic components of
Candida albicans
A. Naseri1, A. Khosravi2, P. Mansori3 and
M. Moazzeni4
1
Birjand University of Medical Sciences, Microbiology,
Birjand, Iran, 2Tehran University, Mycology3Emam
Khomaini Hospital, Dermatology and 4Tarbiyat
Modarres University, Immuonology, Tehran, Iran
Candida albicans is a member of the normal flora of
mucus membranes, which with high probability has
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
U. Nawrot1, K. Wlodarczyk1, P.-.A. Fonteyne2,
J. Skala3 and N. Nolard2
1
Wroclaw Medical University, Department of
Microbiology, Wroclaw, Poland, 2Scientific Institute
of Public Health, Mycology Section, Brussels,
Belgium, 3Wroclaw University, Department of
Genetics, Institute of Microbiology, Wroclaw, Poland
The extracellular acid proteinase is regarded as one
of the most important virulence factors of C. albicans.
The higher proteinase production has been found in
strains isolated from mucosa-associated infections
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comparing to bloodstream isolates. Recently, the
association between the presence of CaLSU intron
and high level of acide proteinase has been observed.
CaLSU is a selfsplicing intron located in the gene
encoding large rRNA subunit in this study the
proteolytic activity of strains obtained from blood,
and isolated from respiratory tract was compared.
Additionally, the correlation between the CaLSU and
protease production was analysed.
Strains: The study was performed on 115 C. albicans
isolates, of them 58 blood isolates were collected in
Belgium (BCCM-IHEM Collection), and 57 isolates
were obtained from throat swabs (40 isolates) and
sputum (17) in Poland (Wroclaw Medical University). The presence of CaLSU intron was previously
tested in all strains. Among blood isolates 30 strains
belong to type A (without intron), 20 to type B (with
intron), and eight to type C (intron present partially).
The respiratory tract isolates included 35 type A, 14
type B, and eight type C. For 34 BCCM-IHEM strains
the sequence of the internal transcribed spacer
regions of the rRNA gene were available.
Method: The protease activity was tested in supernatants of the 7 days Candida cultures in Staib’s
medium supplemented with 0.5% casein. As a
substrate for protease the 0.5% haemoglobin was
used. The protein concentrations were measured at
280 nm and the enzyme activity was express in
arbitrary units, calculated for 1 l of the medium (U/
L). The Student’s test was used for statistical analysis.
Results: We did not find any significant difference in
protease production between strains with and without CaLSU intron, in any of tested groups of
microorganisms. The protease activity of respiratory
tract isolates ranged from 75–2150 U/L
(432.6 ± 313 U/L) and was significantly higher
than the activity of strains from blood
(315 ± 202U/L) (P < 0.05). The comparison between blood and respiratory isolates performed for
particular genotypes revealed that significantly higher activity displayed only the strains of genotype B
(P < 0.05) and C (P < 0.005) whereas the level of
protease activity of genotype A was very similar in
blood and respiratory tract isolates (328.8 ± 223 U/
L and 385 ± 339 U/L, respectively (P > 0.05). We
did not find any relationships between diversity of
ITS sequence and the level of protease production.
Conclusion: The results obtained indicated that
strains with CaLSU intron isolated from respiratory
tract produced higher protease level than strains
with intron but isolated from blood.
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P173
Cloning and sequence analysis of the high
affinity iron permease (FTR1) gene from Rhizomucor miehei, a basis for functional analysis
Nyilasi1, T. Papp2, E. Nagy1 and C.S. Vagvolgyi2
1
Hungarian Academy of Sciences-University of
Szeged, Microbiological Research Group, Univ.
Szeged, Faculty of Sciences. and 2University of
Szeged, Faculty of Sciences, Dept. Microbiology,
Szeged, Hungary
Siderophores and various iron-transport proteins
have been suggested to function as virulence factors
because the acquisition of iron is a crucial pathogenetic event. Patients on dialysis with elevated
levels of available serum iron who are treated with
the iron chelator deferoxamine have an increased
susceptibility to invasive zygomycosis. The high
affinity iron permease (FTR1), which encodes a
protein required to scavenge iron from the environment, is likely to be a critical determinant of
virulence. The zygomycete Rhizomucor miehei may
be agent of frequently fatal mycotic diseases in men
and animals. The aim of this study was to isolate and
characterise the gene encoding high-affinity permease of this species. A short region was amplified from
R. miehei (CBS 360.92) genomic DNA by polymerase
chain reaction (PCR) with degenerate primers
designed according to the conserved regions of
Saccharomyces cerevisiae and Candida albicans FTR1
gene. The sequence analysis of the amplified region
showed significant homology with other FTR1
sequences. In contrast with known fungal FTR1
genes the resulting DNA fragment of R. miehei had a
length of 716 bp and contained two introns.
Remaining parts of the 3’ and 5’ region of the gene
was amplified and cloned by using inverse-PCR
method. Further analysis is in progress to determine
the complete sequence of the gene including the
promoter and terminal regions. Our purpose is to use
this gene in transformation studies to elucidate the
role of FTR1 in the pathogenesis of zygomycosis.
Experiments with mutant C. albicans strains suggest
that the siderophore transporter system (ARN) is
required for epithelial invasion and penetration
across the mucosal epithelium, while the highaffinity iron permease is indispensable for C. albicans
to establish systemic infection. A vector containing
the
orotidine-5’-monophosphate
decarboxylase
(pyr4) casette inserted into the FTR1 gene was
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
constructed. Experiments are in progress to transform a R. miehei pyr4 mutant strain and to disrupt
the FTR1 gene by homologous recombination. The
virulence of the yielded FTR1– mutant strain would
be investigated in a mouse model of systemic
infection.
Acknowledgement: This research was supported
in part by grants from the Hungarian Scientific
Research Fund (OTKA T37471, F046658 and
D048537).
P174
The distribution frequency of Candida species
isolated from the genitourinary tract in some
Nigerian cities
F.I. Okungbowa1, A.P.O. Dede1 and
M.O. Okungbowa2
1
University of Benin, Botany and 2University of Benin
Teaching Hospital, School of Medical Laboratory
Sciences, Benin City, Germany
A clinical survey was carried out in seven cities in the
southern part of Nigeria to determine the relative
distribution of genitourinary Candida species in
symptomatic patients reporting for diagnosis and
treatment. Seven Candida species were identified by
the CHROMagar Candida and the API 20C methods,
represented by C. glabrata (33.7%), C. albicans
(20.1%), C.tropicalis (18%), C.guilliermondii (17.8%),
C.parapsilosis (5%), C. pseudotropicalis (4.2%) and
C. stellatoidea (1.2%). The distribution of these species
among the various age groups (15–25,26–30,31–
35,36–40 and 41 plus years) was statistically
insignificant. Out of the 517 positive samples, 182
(35%) were found in the age group 26–30 years,
while age 41 plus had the lowest frequency (1.2%)
The distribution pattern reported here supports
recent findings that several non-albicans Candida
species are now frequently isolated. The level of
social activities such as sexual promiscuity and drug
abuse may account for the distribution frequency of
Candida species among different age groups.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
P175
Comparison of phospholipase D sequences
from clinically important Candida species
T. Papp1, A. Csernetics1, I. Nyilasi2, E. Nagy2 and
C.S. Vagvolgyi1
1
University of Sciences, Faculty of Sciences, Dept.
Microbiology and 2Hungarian Academy of SciencesUniversity of Szeged, Microbiological Research
Group, Faculty of Sciences, Szeged, Hungary
Phospholipase D (PLD) catalyzes the hydrolysis of
phosphatidylcholine to phosphatidic acid and choline. Mammalian PLD is one of the key enzymes of
the intracellular signaling. Studies on the C. albicans
PLD activity suggest that PLD plays an important
role in the dimorphic transition, which is thought to
be a virulence determinant in Candida (1) PLD genes
contain highly conserved regions interspaced with
more or less variable segments raising the possible
applicability of these genes for phylogenetic and
taxonomic investigations and for strain identification. This work is a first attempt to test the value of
PLD sequences from different Candida species in this
aspect. Conserved regions of the known C. albicans
and C. glabrata PLD genes obtained from international genome databanks were analyzed to design
degenerate primers for polymerase chain reaction.
The constructed primer pair amplifies a fragment
containing parts of the conserved regions I and II
separated with a short variable sequence. Six
different Candida isolates representing C. albicans,
C. parapsilosis, C. krusei, C. guillermondii, C. lusitaniae
and C. zeylanoides were involved in the study. The
resulted fragments have various lengths between
544 and 609 bps. Variable region of the sequence
from the C. albicans strain involved in our study
differed from those of the C. albicans SC5314 strain
deposited in GenBank (accession number:
AB010810). The analyzed PLD sequences could
differentiate all of the studied isolates. Putative
sequences of the respective protein regions were
determined. Phylogenetic trees were constructed on
the basis of the aligned DNA and protein sequences.
Results of the phylogenetic analysis are in good
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agreement with previous studies such as the analysis
of the ITS regions or the 26S rRNA genes (2).
Acknowledgement: This research was supported
in part by grants from the Hungarian Scientific
Research Fund (OTKA T37471, F46658, D48537)
and GVOP-3.1.1.-2004-05-0471.
Reference:
1. McLain, N. and Dolan, JW (1997) Phospholipase D activity is
required for dimorphic transition in Candida albicans. Microbiology 143: 3521–26.
2. Chen, YC., Eisner, JD., Kattar, MM., Rassoulian-Barrett, S.L.,
Lafe, K., Bui, U., Limaye, A.P. and Cookson, B.T. (2001)
Polymorphic internal transcribed spacer region 1 DNA
sequences identify medically important yeasts. J Clin Microbiol.
39: 4042–51.
P176
Catheter-related Candida biofilms observed in
scanning electron microscopy
Paulitsch1, E. Stabentheiner2, B. Willinger3,
H.J. Dornbusch4, E. Marth1 and W. Buzina1
1
Institute of Hygiene, Medical University, 2Institute
for Plant Sciences, Karl Franzens University, Graz,
3
Institute of Hygiene and Medical Microbiology,
Medical University, Vienna, 4Clinical Department for
Haematology-Oncology, Medical University, Graz,
Austria
Catheter-related infections due to Candida species are
frequently encountered in the clinical routine. We
attempted to investigate whether these yeasts are
involved in biofilm formation. Therefore, 37 catheters
which were positive in Candida culture were collected
from intensive care unit patients from October 2004
to April 2005; most of the samples being central
venous catheters. A total of five different Candida
species could be identified. The identification of
C. albicans was performed by using Candida-id agar
(Biomérieux). Other species were identified by using
commercial biochemical panels such as Vitek II and/
or API 32C (Biomérieux). As expected, C. albicans was
the most common species, it was collected in 28 cases.
C. parapsilosis was found on four catheters, C. glabrata
on three, and C. krusei and C. tropicalis were both
detected only once. For further investigations, the
organisms were fixed directly on the catheter with 2%
paraformaldehyde. After washing steps, they were
dried in increasing concentrations of ethanol and
finally air-dried in a drying chamber. The dry
catheter-pieces were cut along into two halves and
122
sputter-coated with gold. Then, the lumen of the
catheters were observed with a scanning electron
microscope (Philips XL30 ESEM), using the high
vacuum mode, which offered the best resolution. The
focus of interest was to observe, whether there is a
biofilm formed on the catheters or not. On all of the 37
catheters at least some sort of film was found.
However, these films were morphologically very
different. Additionally, some of them turned out not
to be produced from any fungal growth stage. On
closer examination, yeast forms could be identified in
20 cases, hyphae were also found on 20 samples.
Different growth stages of Candida biofilms were
detected in 19 cases. C. albicans showed hyphae in
15 cases, yeasts in 16 and biofilm formation was
observed on 15 catheters. C. parapsilosis hyphae,
yeasts and biofilm formation were detected on two
catheters each. Hyphae, yeasts and biofilm formation
of C. glabrata were found simultaneously on one
catheter. No stage of C. krusei growth could be
observed. Some yeast cells and many hyphae were
found on the only catheter which was culture-positive
in C. tropicalis culture. In summary, a Candida biofilm
was found in the lumen of the catheters in 51.4% of
all cases - hence C. albicans is still the leading cause of
Candida biofilms. But also the other species, primarily
C. parapsilosis, should be kept in mind. Candida
biofilms are known for their higher resistance to
antifungal agents. Therefore, further research will be
necessary to show the effects of different antifungal
agents on the morphology of biofilms in detail.
P177
Esterase activity in Candida albicans and some
of its properties
M. Riazipour1, A.R. Khosravi2, L. Mousavi3 and
A. Lotfi4
1
Baqiyatallah Medical Sciences University, Faculty of
Medicine, Microbiology, 2Tehran University,
Mycology, 3Imam Hossein University, Biologycal
Science and 4Tarbiat Modares University,
Biochemistry, Tehran, Iran
Introduction: Candida albicans is the most common
agent of fungal infections as well as the main cause of
candidiasis. The opportunistic fungal infections including candidiasis are increased in recent years and
management of affected patients necessitate using more
excite diagnostic methods and more effective treatments. Identification and characterization of different
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
traits of C.albicans may introduce new potential targets
for diagnosing and treating the infections due to this
pathogenic yeast and eventually may lead to more
effective methods for managing of candidal infections.
Objective: Esterase activity in C.albicans and characterization of some of its properties was the aim of
the present study.
Materials and Methods: C.albicans was grown in
different conditions and using a colorimetric method,
the esterase activity of the medium supernatant, as
well as cell wall and cytoplasmic extract of the
harvested yeast cells were assessed. Also, the effect of
some variables was tested and optimal condition for
enzyme activity determined.
Results: The results indicated that after growing on
YPG medium, the cytoplasmic extract of C.albicans
shows an esterase activity that incubation at 37C, pH
7.5, phosphate buffer 10 mmol l)1, and incubation
time equal to 90 min provide its optimal activity. The
maximal activity observed against a-Naphthyl acetate
flowed by b-Naphthyl acetate, and by increasing the
substrates carbon atoms number the enzymatic activity
was decreased against a-Naphthyl caprilate, a-Naphthyl laurate, and a-Naphthyl palmitate respectively.
Conclusion: C.albicans has a kind of intracellular
esterase activity that its expression is not affected by
the lipase induction medium. Evaluation of the
enzyme activity as a diagnostic or therapeutic target
may be helpful.
Keywords: Candida, Esterase, Enzyme
P178
Revertion of resistance of Candida due to
efflux: a phenotype and genotype study
C. Pina-Vaz, S. Costa-de-Oliveira, E. Ricardo and
10 A.G. Rodrigues
Objective: Resistance to antifungal relates to efflux
pumps exporting drugs with several modulators
11 block them, reverting resistance. Verapamil, ?estradiol, progesterone, known efflux pumps inhibitors of human neoplasic cells, and ibuprofen were
tested as potential modulators of resistance of
Candida spp.
Methods: 42 clinical isolates of Candida (38 fluconazole-resistant), two ATCC and two C. albicans strains
with known resistance mechanisms to fluconazole
resistance were incubated with sub-inhibitory concentrations of such modulators. After exposure, MICs
to fluconazole, itraconazole and voriconazole were
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
re-determined. Simultaneously, yeasts exposed to
modulators were stained with FUN-1 and analysed
by flow cytometry. 3H-labelled itraconazole was used
to study efflux in the presence and absence of
modulators. The expression of CDR1, CDR2 and
MDR genes, related with efflux on C. albicans were
evaluated by real time PCR.
Results: Fluconazole MICs decreased in most
strains after exposure to modulators, including
control strains with documented efflux over expression. No significant MIC variation was noticed on all
C. krusei strains tested, on the resistant strain by
target change, on susceptible strains, and in a very
few clinical isolates. Fluconazole resistant strains
showing a revertant phenotype showed additionally
cross-resistance to itraconazole and to voriconazole,
which was also reverted by the modulators. On
these strains, both an increase of FUN-1 staining
and an increased accumulation of 3H-labelled
itraconazole after incubation with modulators were
noticed. An over expression of efflux genes was
evident on resistant strains which phenotype was
reverted.
Conclusions: Resistance related to overexpression
of efflux pumps is common among clinical isolates
and could be reverted by the assayed modulators,
particularly ibuprofen. The mechanism of resistance
in a few Candida strains and in all tested C. krusei
seems, however, to be of a different nature.
Ibuprofen is a promising compound in association
with azoles deserving future clinical trials. Additionally, a good correlation between phenotype and
genotype regarding efflux mechanisms was found,
validating phenotype evaluation of resistance by
efflux mechanisms.
P179
Evaluation of the effects of Zataria multiflora,
Geranium pellargonium roseum, Myrth and
Lemon shell essences on immune system function in experimental animals
H. Shokri,1 A.R. Khosravi,1 M. Franco2 and R.
Yahyaraeyat1
1
University of Tehran, Centre of Mycology, Tehran,
Iran and 2chool of Medicine and Immunology Division,
UNESP, Department of Pathology, Botucatu, Brasil,
In this study, effects of some Iranian herbal essences
have been evaluated on the function of immune
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system using experimental animals. Rabbits were
divided into 10 groups, consist of five animals, each
group was administrated orally and or subcutaneously by Zataria multiflora, Geranium pellargonium
roseum, Myrth and lemon peel essences and normal
saline (control group), six times with six days of
interval. One day before the first administration and
three days after the last one, rabbits were bleeded
from marginal vein. Candida albicans antigens were
prepared using standard techniques. Five days after
the last injection of the essences, Candida antigens
were injected subcutaneously (SC) and intramuscularly (IM) in all animals. Then ELISA test carried out
using Candida antigens, sera before and after
immunization. Also, phagocytosis and killing assays
and lymphocyte transformation test (LTT) carried
out on blood samples. The results indicated that
cellular immunity were stimulated against Candida
albicans antigens and con-canavalin A (Con-A)
mitogen significantly in animals were injected subcutaneously by Zataria multiflora and Geranium
pellargonium roseum in compare with control group,
whereas myrth essence had no considerable effect
and lemon peel essence suppressed the cellular
responses. Zataria multiflora, myrth and lemon peel
essences stimulated innate immunity when injected
subcutaneously, whereas Geranium pellargonium
roseum essence had no significant effect. Humoral
responses to Candida antigens were significantly
decreased in animals injected by lemon peel essence
comparing to other essences (P £ 0.05). From the
present study, it is concluded that Zataria multiflora
and then myrth and Geranium pellarginium roseum
essences have immuno-stimulatory effects, and they
may be used in certain immunocompromised
patients.
P180
Susceptibility to fluconazole, itraconazole and
voriconazole of clinical isolates of Candida
species
D.T. Trojanowska, A.B. Budak and P.N. Nowak
Jagiellonian University Medical College,
Pharmaceutical Microbiology, Poland
Introduction: The increase of patients from risk
groups predisposed to development of fungal infections of Candida and Aspergillus etiology, caused the
124
increased used of anti-fungal drugs, both in prophylaxis and in therapy itself. Due to frequent incorrect
prophylaxis and prolonged therapy with low doses of
anti-fungal drugs, there appeared a danger of
selection from patients resistant strains, leading to
failure in therapy of mycoses. Results of laboratory
and clinical investigations confirmed the occurrence
of resistance to fluconazole among Candida albicans
strains.
Material and methods: Investigation included
134 fungal isolates: Candida albicans (46), Candida
glabrata (42), Candida tropicalis (26), Candida parapsilosis (six), Candida guilliermondii (three), Candida kefyr
(three), Candida krusei (three) and five strains from
different species of Candida. Strains were isolated
from BAL, blood, wounds and urine from patients of
specialized hospital in Kraków. The susceptibility of
strains to fluconazole, itraconazole and voriconazole
was estimated by marking of minimal inhibitory
concentration /MIC/ using Etest method (AB Biodisk, Sweden).
Results: Among strains of Candida albicans, Candida
glabrata, Candida tropicalis, Candida parapsilosis the
highest percentage of isolates was sensitive to
voriconazole, respectively: 87% (MIC 0.023–
0.125 mg ml)1), 64.3% (MIC 0.004–1 mg ml)1),
96.2% (MIC 0.012–1 mg ml)1) and 100% (MIC
0.016–0.094 mg ml)1). Following places as far as
susceptibility of isolates was concerned were taken
by: fluconazole and itraconazole. Resistance to
fluconazole was started only among Candida glabrata strains (23.8%; MIC >256 mg ml)1) and Candida albicans strains (21.7%; MIC > 256 mg ml)1).
However, the lowest resistance to voriconazole was
observed among Candida tropicalis strains (3.8%),
then higher to Candida albicans (13%) and Candida
glabrata isolates (36%). The lack sensitivity to all
tested drugs was noted both in six of Candida
albicans and Candida glabrata strains (MIC for
fluconazole > 256 mg ml)1, MIC for itraconazole
> 32 mg ml)1, MIC for voriconazole > 32 mg
ml)1).
Conclusions: 1. Investigations confirmed the highest susceptibility to voriconazole among tested isolates from different Candida strains.
2. Applying of anti-fungal drug to prophylaxis or
treatment of Candida infections, should be preceded
by identification of species, marking sensitivity in vitro
and monitoring of drug during therapy, especially in
patients from high risk group.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
P181
Prevalence of Candida dubliniensis in pregnant
women with vulvovaginal candidiasis
E. Us1 and S.A. Cengiz2
1
Ankara University School of Medicine, Microbiology
and Clinical Microbiology, Ankara, Turkey and 2Gazi
University School of Medicine, Medical Microbiology,
Ankara, Turkey
Candida dubliniensis is recently described pathogenic
Candida species, which was originally identified in
cases of recurrent oral candidosis in human immunodeficiency virus (HIV)-infected and AIDS patients.
C. dubliniensis is very similar to Candida albicans in
terms of genotypic and phenotypic characteristics. Its
importance based on rapidly developing resistance to
fluconazole, a common anti-fungal drug used for the
treatment of mycoses. In retrospective analyses of
stock collections, C. dubliniensis was found to account
for at least 1.2% to 2% of the yeast initially identified
as C. albicans. It is becoming clinically relevant yeast
due to its worldwide distribution and its association
to both mucosal and systemic candidiasis. The yeast
has been isolated not only in the oral cavities of
immuno compromised patients but also in lungs,
vagina, blood, sputum, and gastrointestinal tract. It
is very important to identify C. dubliniensis correctly
in clinical specimens for clinical and epidemiological
evaluations. In this study, investigation for isolation
C. dubliniensis in vaginal discharges of pregnant
women with vulvovaginal candidiasis were made.
The hormonal mileau of the vagina during pregnancy enhances Candida colonization and serves as a
risk factor for symptomatic expression. Moreover,
normal pregnancy is characterised by a lack of
maternal cell-mediated immunity. Although C. albicans isolates were more adherent to vaginal epithelial
cells, C. dubliniensis isolates also showed high levels of
adherence to this epithelium. Seventy seven vaginal
samples positive for vulvovaginal candidiasis
obtained from 218 pregnant women in different
weeks of gestation in a 1.5 year period from Microbiology Laboratory of Ankara University School of
Medicine were investigated for C. dubliniensis subsistence. The diagnosis for vulvovaginal candidiasis was
made not only regarding to vaginal cultures wet
mount and gram-stained preparations of vaginal
discharges (positive for blastospores and pseudohyphae) were also made. A total of 41 Candida species
phenotypically identified as C. albicans on the basis of
a positive germ tube test (germ tubes without
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
constriction at the point of origin were taken positive
for C. albicans) and carbohydrate assimilation tests
were screened for the presence of C. dubliniensis,
using a variety of phenotypic characteristics. Phenotypic tests for differentiation C. dubliniensis from C.
albicans, such as growth at 42 and 45 degree Celsius
on Sabouraud dextrose agar (SDA), appearance on
CHROMagar Candida medium and colony morphology on Cornmeal-Tween 80 agar (CMA) and Staib
agar (SA) were carried out. Only one strain (2.43%)
was phenotypically identified as C. dubliniensis. There
is no other report possessing isolates concerning with
the frequency of C. dubliniensis in vaginal samples of
pregnant women. Large-scale studies of pregnant
women are required to discover the etiologic importance of this yeast.
P182
Four-year persistence of a Candida albicans
genotype causing bloodstream infections in a
surgical ward proven by multi-locus sequence
typing
M.A. Viviani, M. Cogliati, M.C. Esposto, A. Prigitano
and A.M. Tortorano
Università degli Studi di Milano - IRCCS Ospedale
Maggiore, Istituto di Igiene e Medicina Preventiva,
Milano, Germany
Prevention of candidemia within hospital setting
depends not only on surveillance and treatment of
mucosal colonisation, but also on epidemiological
surveillance aimed to identify the origin of the
infecting organism. Typing methods have shown
that outbreaks of Candida bloodstream infections
(BSIs) were caused by strains endemic in the
hospitals. Among the several typing methods used
to characterise C.albicans isolates in epidemiological
studies multi-locus sequence typing (MLST) was
proven to be a highly discriminant and stable
method. Recently, a set of seven housekeeping gene
fragments has been recommended for typing C.
albicans isolates. We have applied this method to
retrospectively investigate a suspected outbreak of C.
albicans BSIs, involving six patients that occurred in a
surgical ward in the period November 1988–January
1989. Analysis of the laboratory database showed
that other 10 cases of C. albicans BSI occurred in the
same ward between July 1987 and October 1991. A
total of 14 strains cultured from blood and/or
125
xxx
vascular catheter tip or residual parenteral nutrition
fluid of 11 patient were investigated. In addition, 14
C. albicans strains isolated from blood of patients
hospitalised in other wards/hospitals were analysed
as control strains. All the isolates were genotyped by
PCR fingerprinting using (GTG) 5 as single primer.
Seven C. albicans gene fragments were sequenced for
all 14 isolates from patients of the surgical ward and
seven control strains. The same fingerprint (B
genotype) was identified in isolates from nine of the
11 patients hospitalised in the surgical ward and in
none of the 14 control strains. MLST analysis
showed that eight out of 12 isolates presenting the
B genotype had also the same allelic profile, Ia. Of the
remaining four genotype B strains, one had an
unrelated allelic profile (IV), whereas three presented
a sequence type highly related to the profile Ia, that is
profile Ic (97% similarity) and profile Ib (99%
similarity). The other strains, beside not presenting
the B genotype, presented also different MLST
profiles. On the basis of the molecular analysis of
the available strains, we demonstrated that the
suspected candidemia outbreak as well as five
additional cases identified in an extended analysis
were caused by the same strain endemic during a
four-year period.
P183
Tryptophan-dependent pigment synthesis by
Candida glabrata
M. Wenzel,1 G. Haase,2 P. Mayser,1 H.J. Krämer3 and
P. Spiteller4
1
Justus-Liebig-University, Dermatology, Giessen,
2
University of Aachen, Medical Microbiology,
Aachen, 3Justus-Liebig-University, Clinical
Pharmacology, Giessen, Germany and 4Technical
University of Munich, Organic Chemistry, Munich,
Germany
With regard to new virulence factors we analysed
the pigment synthesis in Candida glabrata. This
present study is the first to demonstrate the tryptophan dependent secondary metabolism (we found
earlier in Malassezia furfur) now as well in ascomycetes. When tryptophan is added as the only
nitrogen-source to cultures of this yeast, it induces
the production of numerous indole alkaloids with
biological activity.
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P184
Evaluating the phenotypic diagnostic methods
to distinguish between Candida dubliniensis
and Candida albicans from clinical specimens in
I. R. IRAN
M.H. Yadegari,1 F. Katiraei2 and M. Shams
Ghahfarrokhi3
1
Tarbiat Modares University, Medical Mycology,
Tehran, Iran, 2Tarbiat Modares University, Medical
Mycology, Tehran, Iran and 3Tarbiat Modares
University, Medical Mycology, Tehran, Iran
Candida species are the most important and common
fungal agents of human infections, and Candida
albicans has more incidences among these fungi.
Candida dubliniensis is similar to Candida albicans in
many characteristics. The present study evaluated
520 isolates obtained from vagina, urine, mouth,
faeces and skin through five phenotypic methods of
differential diagnosis. The methods were performed in
two stages of probable diagnosis and absolute diagnosis (supplementary test). In the first stage, three
methods of colony colour in CHROMagar candida
medium, creating chlamydoconidia in casein agar and
growth at 45C were used. The isolates that demonstrated the characteristic of Candida dubliniensis at this
stage were tested in the second stage in terms of
assimilation of terehalose and xylose and intercellular
B-D-glucosidase activity. Finally, five out of 520 isolates
were diagnosed to be Candida dubliniensis, of which four
isolates were vagina and one from urine. The present
study determined the incidence of Candida dubliniensis
in vagina, urine and total of the clinical isolates as
1.33%, 0.74%, and 0.96%, respectively. It is to be
noted that it has rare incidence in human infections.
According to the finding of this study, among the
phenotypic methods of differential diagnosis,
using CHROMagar candida medium with intercellular
B-D- glucosidase activity for the fresh isolates obtained
from clinical specimens is suggested, whereas the
isolates obtained from subcultures, genotypic methods
are not recommended. It seems that the use of methods
such as casein agar and growth at 45C along with
other methods could be useful.
P185
Tekst Volgt
Abstract not found
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
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P186
Candidemia in Antalya University Hospital:
Species distributions and anti-fungal
susceptibility patterns
M. Yaman, G. Mutlu, D. Ogunc, G. Ongut, D. Colak
and M. Gultekin
Akdeniz University Medical Faculty, Medical
Microbiology, Antalya, Turkey
Introduction: Candida species are the major pathogens of invasive fungal infections. Candidemia also
carries the highest mortality (40%) rate of all nosocomial bloodstream infections (BSI). Epidemiologic
surveys reveal that Candida sp. are now the fourth
most common pathogen isolated from the blood of
hospitalized patients, especially in intensive-care
units (ICU). Candida albicans used to be the predominant cause of candidemia and in general has
remained susceptible in vitro to both amphotericin
B and fluconazole. However, it is important to
monitor for any increase in azole resistance among
bloodstream isolates of C. albicans and non-albicans
species. Our aim was to define the rate of candidemia,
predominant isolation locations, identification of
species and anti-fungal suspectibilities of Candida sp.
causing BSI in during two-year period.
Materials and Methods: All patients with candidemia were identified retrospectively from 01
January 2003 to 31 December 2004. Automated
blood culture system BACTEC (Becton Dickinson
Microbiology System) were used for blood cultures.
Positive cultures sub cultured on Sabouroud Dextrose Agar. The yeasts were identified by examining
colony and microscopic morphology, germ tube and
chlamydoconidia production and API (ID 32C)
systems (Biomerioux France). Candida isolation from
blood cultures at least one month intervals were
considered as an episode. Anti-fungal susceptibility
tests were performed by the E-test method for
amphotericin B and fluconazole. Chi-square test
was used for statistical analyses.
Results: A total of 103 (2.4%) Candida species were
isolated from the 4141 positive blood cultures.
Candida sp. was the eighth most frequent microorganisms among all the isolates from blood cultures.
C.albicans (45%) was the leading cause of candidemia
followed by C.parapsilosis (15.6%). Three isolates
(two C.albicans and one C.parapsilosis strains) were
accepted as separate episodes. The rate of candidemia
varied according the patient’s location in the wards
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
of hospital; 40% from ICU, 19% from hematologyoncology, 17% from pediatric hematology-oncology.
Amphotericin B MIC of <1 lg ml)1 was demonstrated for all Candida species. Fluconazole resistance
(MIC ‡ 64 lg ml)1) was found for one C.albicans. No
statistically significant differences in the rates of
fluconazole and amphotericin B resistance for Candida species were detected in the two-year periods.
Discussion and Conclusion: This study demonstrated that there was no increase in the rate of
candidemia. Candidemia was the common in ICUs.
Significantly there was no change between Candida
species. Fluconazole resistance among bloodstream
isolates of C.albicans remained substantially low.
P187
The prevalence rate of candidiasis in recurrent
vaginities in pregnant women in Isfahan–Iran, a
phenotypic study on Candida albicans isolates
M.A. Zia1 and S.J. Hashemi2
1
Islamic Azad University of Khorasgan, Department
of Medicine, Isfahan, Iran and 2Medical Sciences
University of Tehran, Department of Health, Tehran,
Iran
One of the most common vaginal infections is
candidiasis, mainly caused by Candida albicans.
Pregnancy is one of the predisposing factors of
recurrent vulvovaginal candidiasis. The mechanism
by which pregnancy favors colonization by Candida is
complex. Some investigators believe the elevated
frequency of colonization is related to the high levels
of reproducing hormones. These hormones have
been found to stimulate the germination and formation of pseudohyphae, which correspond to the
invasive form of the yeast. In this study an effort
has been made to rank prevalence rate of vaginal
candidiasis and phenotype of C. albicans strains in
pregnant women with previous vaginit symptoms. In
total one hundred pregnant women in different age
groups have been involved in this research. Vagin
secretions was collected by sterile swab and then the
samples were subjected to direct examination with
20% KOH and also samples inoculated on Petri
dishes containing Sabouraud-dextros agar supplemented with 100 mg ml)1 chloramphenicol. Then in
order to confirm the identification of C.albicans, the
isolates have been examined in terms of production
of germination tubes in serum and chlamidospores
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on corn meal agar containing Tween 80. Finally
Aims: To evaluate the performance of CS4 medium
using a swab the yeast suspentions were inoculated
in terms of selectivity for the differentiation of
on Petri dishes containing 6% malt agar and 2%
mixtures of yeast species, as well as the presumptive
agar, and incubated at 25 degrees centigrade for
identification of medically important yeasts. The
10 days. The results are as mentioned below: Yeast
performance of CS4 was compared with that of the
cells were observed in 21 cases (21%) of direct
chromogenic medium, CHROMagar Candida
examination. Yeast growth was observed in 14 cases
(Becton Dickinson, CA, USA).
of total inoculated samples in SDA media suppleMaterials and methods: CS4 and CA media were
mented with chloramphenicol (66/6% of positive
used according to the manufacturer’s instructions.
cases in direct examination).Candida albicans strains
The reference procedure for identifying yeast species
were isolated from 10 malt agar media (47/61% of
involved morphological tests (RAT medium), rapid
positive cases in direct examination and 71/42% of
identification tests (Bichrolatex albicans, glabrata
positive cases of yeast growth in SDA media).The 15 RTT), and API 32C assimilation tests (BioMérieux).
morphotype obtained was recorded using a four-digit
This prospective study was carried out at the
code, based on the macro morphological aspects of
University Hospital Lille during a 4-month period.
colony fringe and surface as mentioned in Hunter
Three types of specimen were analysed: (i) 1549
et al C. albicans typing model. The phenotypes
samples sent directly to the mycology laboratory
include: five cases of code 000 0 (with no fringe
(primary cultures); (ii) strains sent from the bacterioand smooth surface), three cases of code 754 0 (with
logy laboratory for identification (88 subcultures);
wide-fine fringe and smooth surface), one case of
and (iii) subcultures of reference strains (n = 18)
code 733 0 (with narrow-fine fringe and smooth
(particularly moulds). The media were incubated at
surface) and one cases of code 732 0 (with narrow37 C and the cultures were examined at 24, 48 and
coarse fringe and smooth surface). It is concluded
72 h. The following criteria were assessed: (i) the
that recurrent vaginal candidiasis is caused by the
culture properties of the media (speed and extent of
various acromorphological phenotypes of C. albicans.
development of fungal colonies); (ii) number of
isolates of C. albicans that were identifiable immediately; and (iii) the extent to which the chromogenic
P188
media could differentiate between yeast species
(appearance of yeast colonies other than C. albicans).
Prospective evaluation of chromogenic CandiResults: Primary cultures: five-hundred and two of
Select 4 medium for the direct identification of
the 1549 (32%) samples were positive. A total of 542
Candida albicans, differentiation and presumpyeasts were isolated including 465 pure cultures and
tive identification of the major pathogenic
37 mixed cultures: 392 C. albicans, 60 C. glabrata, 25
Candida species
C. tropicalis, 12 C. krusei, 53 other species of Candida.
The percentage of strains of C. albicans that could
B. Sendid, N. François, A. Fievez, A. Vitse-Standaert,
be identified directly after 24, 48 and 72 h culture
D. Poulain and D. Camus
12 Parasitologie-Mycologie, CHRU de Lille
was 31.6%, 82.9% and 92.1%, respectively, for CS4,
and 32.9%, 82.9% and 91.1% for CA. The presump13 Invasive Candida infections are a serious cause of
tive identification of C. glabrata, C. tropicalis and
C. krusei was evaluated after 48 h incubation. The
morbidity and account for more than 9% of all
percentage of strains with morphologically typical
nosocomial infections. Despite recent advances in
colonies was 80%, 68% and 84.62%, respectively for
antifungal therapy, mortality directly attributed to
CS4 compared with 75%, 76% and 76.92% for CA.
Candida infection remains high (20-39%). In this
Pure cultures (subcultures of bacteriology strains and
context, the rapid identification of pathogenic yeasts
collection strains): From 24 h, all strains of C. albicans
is a crucial step in ensuring that antifungal treat(n = 21) were directly identifiable on the two chromment is started as early as possible. The chromogenic
14 agar, CandiSelect 4 (CS4) (Bio-Rad), is a new medium
ogenic media, CA and CS4. At 48 h, the proportion
of typical strains observed on the two chromogenic
for the isolation of fungi, the rapid direct identificamedia was identical for C. glabrata (85%) and C. krusei
tion of Candida albicans and the presumptive identi(100%). A slight difference in favour of CS4 was
fication of the major pathogenic Candida species
observed for C. tropicalis (100% vs. 95%).
(C. glabrata, C. tropicalis and C. krusei).
128
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
Medium CS4 also allowed the growth of several
other fungi including: Cryptococcus neoformans,
Geotrichum spp. Trichosporon, Scedosporium apiospermum, Fusarium oxysporum, Aspergillus fumigatus, A. flavus, A. niger, Mucor spp. and Rhizopus spp.
Conclusion: Compared with conventional identification methods, the new chromogenic medium CS4
can be recommended as a primary isolation medium
for the identification of isolates of C. albicans, and for
the rapid and effective differentiation of the principal
pathogenic Candida species.
P191
Ultra-rapid preparation of total genomic DNA
from yeasts and moulds using Whatman FTA
filter paper technology – a reusable DNA
archiving system
A.M. Borman,1 C.J. Linton,1 S.J. Miles,2
C.K. Campbell1 and E.M. Johnson1
1
Mycology Reference Laboratory, Health Protection
Agency, Bristol, UK and 2Department of Pathology
and Microbiology, University of Bristol, Bristol, UK
Conventional methods for purifying PCR-grade fungal
genomic DNA typically require cell disruption (either
physical or enzymatic) coupled with laborious organic
extraction and precipitation stages, or expensive
column-based technologies. Here we present an easy
and extremely rapid method of preparing yeast and
mould genomic DNAs from living cultures using
Whatman FTA filter matrix technology. Aqueous
suspensions of yeast cells or hyphal fragments and
spores (in the case of moulds) are applied directly (or
after freeze thawing) to dry FTA filters. Inoculated
filters are then subjected to brief microwave treatment, to dry the filters and inactivate the organisms.
Filter punches are removed, washed rapidly, dried and
placed directly into PCR reactions. We show that this
procedure inactivated all of the 38 yeast and 76
mould species tested, and generated PCR-grade DNA
preparations in around 15 min. A total of 218 out of
226 fungal isolates tested on FTA liberated amplifiable
DNA after application to FTA filters. Detection limits
with yeasts were approximately 10 colony-forming
units per punch. Moreover, we demonstrate that filter
punches can be recovered after PCR, washed and used
to programme fresh PCR reactions without detectable
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
cross-contamination. FTA technology thus represents
a cheap, ultra-rapid method of fungal genomic DNA
preparation, and a powerful fungal DNA archiving
and storage system.
P192
Fungal infections in intensive care unit: a
retrospective study
A.M. Carvalho,1 M.J. Ferreira2 and E. Fernandes3
1
Hospital São Teotónio, Patologia Clı´nica, Viseu,
16 Portugal2
We perform a retrospective study of three patients
with severe fungal infections who we treated in
intensive care unit (ICU) of Hospital São Teotónio,
Viseu, in Portugal. We are presenting a case with
postoperative status of severe abdominal sepsis that
developed systemic mycosis. The patient is an
undernourished, alcoholic, 53-year-old oligofrenic
female admitted in the ICU after abdominal surgery
for perforated duodenal ulcer, who presented a
clinical state of severe sepsis and circulatory shock.
We isolated Trichosporon pullulans, Candida albicans
and Candida krusei from blood cultures, ascitic liquid
and central venous catheter. Aspergillus fumigatus
was also isolated from bronchial aspirates. We are
reporting a rare case of bronchial aspergillosis due to
Aspergillus fumigatus in a 49-year-old white male
who was a farmer and lived in a rural environment.
We state that our patient was immunocompetent
because despite continual alcohol abuse there were
no laboratory changes or prior history suggestive of
immunodeficiency. He had successive isolates of
Aspergillus fumigatus from bronchial aspirates
obtained in a way that can be considered aseptic,
as they were obtained through an endotracheal tube.
and now we are describing a case of endocarditis
caused by Candida parapsilosis on a previously healthy
aortic valve in a 31-year-old man who was intravenous-drug dependent for ten years. Candida parapsilosis was isolated from all blood samples. The
patient was vegetations larger, a valve failure
causing moderate congestive heart failure, which is
a strong indication for urgent surgery. The patient
undergoing aortic-valve replacement surgey. All of
these patients were successfully treated with liposomal Amphotericin B.
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P193
Rapid detection, identification and monitoring
of yeast species by analysis of PCR products
with denaturing high performance liquid
chromatography
O. Goldenberg,1 S. Herrmann,1 T. Adam,1
G. Marjoram,1 G. Hong,2 U.B. Göbel1 and B. Graf1
1
Institute of Microbiology and Hygiene,
Universitätsmedizin Berlin, Charite, Mycology, Berlin,
Germany and 2Transgenomic Ltd, Omaha, NE, USA
Basic observation: Detection and identification of
clinically relevant yeast species based on classical
methods is often time-consuming and may lead to
ambiguous results. Molecular methods are able to
rapidly identify clinical isolates or detect fungal mono
infections of sterile body compartments; however,
common molecular methods are unable to monitor
colonization of mucous surfaces by several distinct
yeast species or distinguish yeast strains on the
subgenus level.
Objective: Our aim was to establish a DNA-based
procedure for detection, identification and monitoring of clinically relevant yeasts with resolution
capability on the subgenus level.
Methods and Results: We first developed a
method based on PCR amplification of the intergenic transcribed spacer region 2 (ITS2) and subsequent resolution of the PCR products by denaturing
high performance liquid chromatography (DHPLC)
on the WAVE Microbial Analysis System (Transgenomic, Omaha, NE) to characterize reference
yeast strains by their retention profiles. Using this
PCR/DHPLC approach we could unequivocally
identify all 12 common yeast species tested. We
next studied blood culture samples from patients or
spiked with different yeast strains showing applicability of the method for identification of yeast
infections of sterile body compartments. Taking
profit from the capability of the method to simultaneously identify several yeast species in a single
sample, we applied the PCR/DHPLC approach to
monitor yeast colonization of complex mucosaassociated microbiota, i.e. stool. Finally, we could
show that the method allows identification of intraspecies variants providing a novel tool for future
epidemiological studies.
Conclusion: We present a PCR/DHPLC-based
method for detection, identification and monitoring
130
of clinically relevant yeast species with additional
potential as a tool for epidemiological studies.
P194
Comparison of two chromogenic media for
presumptive identification of pathogenic
yeast’s
P. Hamal,1 J. Cankarova,1 V. Raclavsky2 and
D. Koukalova1
1
Palacky University, Faculty of Medicine, Institute of
Microbiology, Olomouc, Czech Republic and 2Palacky
University, Faculty of Medicine, Institute of Biology,
Olomouc, Czech Republic.
Introduction: Systemic mycoses due to Candida sp.
still represent the most serious problem in mycology
connected with high mortality rate. Moreover, some
pathogenic yeast species are known to be resistant
to systemic anti-fungals frequently used in clinical
practice. Therefore, rapid yeast identification is
imperative for prompt initiation of appropriate
therapy. Aim of this study was to compare the
usefulness of two chromogenic media – the wellestablished CHROMagar? Candida (Becton Dickinson)
and the new chromogenic medium CANDISelect?4
agar (BioRad) – for detection and rapid presumptive
identification of pathogenic yeast species from
clinical materials in routine practice. The following
characteristics were compared: (1) recovery of
yeasts directly from clinical samples (2) detection
of mixed cultures and (3) inter-species discrimination.
Materials and methods: In the first part of the
study, 243 samples were taken from mouth, throat,
sputum, stool and perianal region. All samples were
inoculated on one quadrant on plates of both
chromogenic media and streaken for single colonies.
Numbers of yeasts were compared semi-quantitatively based on colony counts. In the second part of
the study, 193 strains of 13 yeast species, all fresh
clinical isolates, were identified based on distinctive
colour and morphology, mostly within 2 and 4 days
after inoculation. Such presumptive identification
was confirmed by presence of chlamydospores on rice
agar and biochemically by assimilation and fermentation tests including the commercial kit ID32C
(BioMérieux).
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
Results: Yeasts were detected in 87 of total 243
clinical samples. In 11 samples yeast growth was
detected on CandiSelect but not on CHROMagar,
whereas in six samples yeast growth was detected on
CHROMagar but not on CandiSelect. However, only
very few colonies were mostly detected in these
cases. Mixed cultures were recovered from 23
samples. CHROMagar enabled unequivocal differentiation of such mixtures in 19 cases (82.6%) and
CandiSelect in 21 cases (91.3%), respectively. Using
both chromogenic media, it was easy to presumptively identify C. albicans, C. tropicalis, C. glabrata and
C. krusei, i.e. the target species declared by the
manufacturers. For identification of C. albicans,
CHROMagar was found to express more different
phenotypes, whereas CANDISelect was more stable in
colony colour and morphology. Differentiation of
other yeast species was slightly easier on CHROMagar because of better color variability. Interestingly,
when contamination of yeast cultures with multiresistant gramnegative rods occurred, their growth
was always inhibited markedly better on CANDISelect.
Conclusions: Our results indicate that performance
of the new chromogenic medium CANDISelect4 is
fully comparable to the traditional CHROMagar
Candida and represents the best alternative for
presumptive identification of yeast’s in a clinical
mycology laboratory.
Ministry of Health, Czech Republic supported this
17 work (NR/8365).
P195
Specific oligo nucleotide primers for identification of Candida nivariensis, a recently described
Candida species
J. Julia Alcoba-Flórez,1 M.P. Mª Pilar Arevalo
Morales,1 F.J.F. Javier González-Paredes,1 J. Josep
Cano,2 J. Josep Guarro,2 E. Eduardo Pérez-Roth1 and
S. Sebastian Méndez-Alvarez1
1
University of La Laguna, Microbiology, Santa Cruz
de Tenerife, Spain and 2University Rovira i Virgili,
Microbiology, Tarragona, Spain
Introduction: Molecular studies have shown that it
is not uncommon that different strains of a species
vary somewhat in their fermentation and assimilation profiles, which can lead to misidentifications.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
Molecular approaches are more promising than such
phenotypic methods for the rapid detection and
identification of pathogenic organisms. Candida nivariensis is recently described pathogenic yeast closely
related to Candida glabrata.
Objective: Identification of C. nivariensis by the new
PCR method. We developed a specific set of oligo
nucleotide primers based on the internal transcribed
spacer regions of ribosomal DNA for the rapid
identification of C. nivariensis.
Material and méthods: A total of 35 yeast isolates
including three isolates of C. nivariensis, another
medically pathogenic yeast and four reference strains
were examined.
Results: In the present study, definitive species
identification of C. nivariensis was achieved by the
new PCR-method. We developed a specific set of oligo
nucleotide primers based on the internal transcribed
spacer regions of ribosomal DNA for the rapid
identification of C. nivariensis. (NIVF/NIVR). Forward
and reverse species-specific primers were designed by
the authors and were derived from a comparison of
the sequences of ITS1-5.8S-ITS2 genes (ribosomal
DNA) in the GenBank database (NCBI). The species
identity of the isolates was subsequently confirmed
by specific amplification of rDNA targeting the
internally transcribed spacer 1 (ITS1). The ITS1
region was automatically sequenced from the PCR
products by using previously described primers. All
sequencing reactions were performed twice from
independent PCR amplifications. Phylogenetic analyses using the Neighbor-joining (NJ) method and
based on Kimura’s 2-parameter corrected nucleotide
distances were performed with MEGA 2.1 software,
including both transitions and transversions, and
with pair wise deletion used to handle gaps and
missing data. Confidence values for individual branches were determined by bootstrap analyses (1000
replicates).
Conclusions: The species specific primers for C. nivariensis presented here provide a molecular diagnostic
method that can be used, in conjunction with current
clinical tools, to diagnose cases of C. nivariensis
infections with greater confidence and accuracy.
Rapid identification is essential so that an appropriate
clinical decision can be made concerning the identity
of the isolate, the therapeutic approach to be used,
and the dosage and duration of the appropriate
therapy.
131
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We report four cases of histoplasmosis that are
represented in the following table:
P196
Fast and sensitive detection of dermatophytes
in dermatological specimens by polymerase
chain reaction – restriction fragment length
polymorphism
1
2
2
H. Mirzahoseini, M. Razaghi Abianeh, G. Sadeghi,
F. Bayat,1 R. Arabi Mianroodi1 and A. Khaksar2
1
Pasteur Institute of Iran, Biotechnology, Tehran, Iran
and 2Pasteur Institute of Iran, Mycology, Tehran, Iran
Early and specific diagnosis of dermatophyte species,
permitting prompt and targeted initiation of antifungal therapy, is an essential step in the successful
management of patients. In this study we describe a
rapid and sensitive protocol to detect dermatophyte
species in dermal specimens from affected patients by
polymerase chain reaction–restriction fragment
length polymorphism (PCR-RFLP). Two fragments
of conserved gene coding internal transcribed spacer
(ITS) rDNA were amplified, using two primer pairs.
This procedure was successfully applied to 120
Iranian human specimens, and the diagnosis was
confirmed with direct microscopic examination of
cellular morphology, macroscopic observation of
in vitro cultures and tests of metabolic features. In
addition, the RFLP patterns of Iranian dermatophyte
strains were compared with standard strain patterns.
P197
Histoplasmosis: our experience
V.M.L. Monteiro Lopes,1 I.C. Calhim,2 O.L. Lima,2
D.S.A. Sá Leão,3 R.C. Carvalho,4 J.A. Abrunhosa,5
C.C. Cardoso5 and J.M.M. Amorim1
1
Microbiology, 2Pathology, 3Surgery, 4Endocrinology
and 5ORL, Hospital Geral Santo António, Porto,
Portugal
Introduction: Histoplasmosis is an endemic mycosis that has a great possibility to be misdiagnosed.
In Portugal the majority of cases are related to
African patients or patients that had been in Africa
for some time. Clinical manifestations in African
histoplasmosis (non-pulmonary) are different from
the American histoplasmosis (pulmonary), being in
most of the cases chronic localized infections in the
cutaneous and subcutaneous tissue, bones and
lymph nodes.
132
Patient 1
Patient 2
Patient 3
Patient 4
Age
50
46
56
60
Sex
Male
Female
Male
Male
Race
Caucasian
Black
Caucasian
Caucasian
Endemic contact
Guinea (army)
Guinea (home)
Guinea (army)
Guinea (army)
Clinical
symptoms
Odynophagia
Mesenterial
mass in
Addisońs disease
Dysphonia
Pharynx/larynx
CT scan
Tumor/suprarenal
Vocal cord
lesions
First diagnosis
Histoplasmosis
gland
Neoplasia
Neoplasia
lesion
Neoplasia
relapse
Final diagnosis
Histoplasmosis
And tuberculosis
And tuberculosis
Mesenterial
Adrenal
of the
Main clue
histoplasmosis
pharynx/larynx
histoplasmosis
Patient history
Oval formations in
Yeasts in histology
Larynx
histoplasmosis
Yeasts in
histology
the acid fast smear
Basis for diagnosis
Clinical clues
Yes (Diagnosed and
No
No
No
Histopathology
treated in 1987)
Yes
No
Yes
Yes
Microbiology
Yes: Culture
Direct observation
No: no specimen
Yes: culture
and culture
All the male patients had been in the endemic
area many years ago while the female patient lived
in Guinea. All the patients had a chronic evolution
and localized disease. Only in the first case the
patient history was the basis for the diagnosis,
while in all other cases the laboratorial work
played a major role. Although these cases have
coursed as a localized disorder one must be aware
that the disease can disseminate and become fatal,
particularly in the immuno compromised individuals. Clinical awareness, epidemiologic clues, microbiological experience and a good clinical/laboratorial relation are essential to reach an accurate and
rapid diagnosis for an adequate therapeutic
approach.
P198
Molecular differentiation of most frequent
Candida species causing blood-stream infection
F. Somogyvari,1 J. Serly,1 I. Doczi1 and E. Nagy1,2
1
Institute of Clinical Microbiology, University of
Szeged, Szeged, Hungary and 2Microbiological
Research Group, Hungarian Academy of Sciences
and University of Szeged, Szeged, Hungary
Candida species are important fungal pathogens
particularly when causing blood-stream infections.
These infections are now regarded as the fourth
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
most frequent cause of septicemia, with the mortality rate about 50%. Although the C. albicans is
the most commonly isolated, other, anti-fungal
resistant Candida species, such as C. parapsilosis, C.
tropicalis, C. glabrata, C. kefyr, C. inconspicua and C.
krusei are increasingly common. This is the reason
why it is important for clinical laboratories to
discriminate between Candida species. Several phenotypic methods have been used for their differentiation, but molecular methods are considered most
reliable in their diagnostics; however, some tests
require considerable technical efforts. Our aim was
to introduce a rapid and reliable real-time PCR
assay with which the problem of detection and
differentiation can be solved simultaneously. The
amplification was carried out with the ITS1, ITS4
consensus primer pair and discrimination was
achieved through consecutive melting-point analysis with the help of the non-specific fluorescent dye
SybrGreen. First we set out a rapid DNA preparation method which is adaptable for the whole
blood and the blood-culture vessels. The melting
point analysis following the amplification was
suitable to discriminate the seven most frequent
Candida species due to the extreme variability of the
amplified region. The species and the melting points
were Candida albicans (87.1C), C. tropicalis
(84.5C), C. glabrata (84.9C), C. parapsilosis
(85.4C), C. kefyr (86.7C), C. inconspicua (88.0C)
and C. krusei (90.8C), respectively. This method
offers a simple and rapid tool for the differentiation
of two closely related germ tube positive Candida
species, the C. dubliniensis from C. albicans as well.
The measured melting point for C. dubliniensis is
86.0C. These differences are sufficient for the
accurate differentiation. The results perfectly match
those of the previously used phenotypic and
molecular (PCR-RFLP) differentiation. The whole
procedure can be completed within 2 h with the
rapid mini preparation of the fungal DNA. This
method is not adaptable as a general identification
method but useful when some of the fungal species
are expected from clinical samples.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
P199
Comparison between PCR and detection of
antigen in whole blood samples for early
diagnosis of invasive Aspergillosis
J.C. Palomares,1,2 M. Ramirez,2 A. Aller,3
M.J. Torres,1 C. Castro,2 E. Jean-Paul,3 J. Aznar,4
E. Nartin-Mazuelos2
1
School of Medicine, 2H. de Valme, Microbiology,
Seville, Spain, 3H. de Jerez, Microbiology, Jerez de la
Fra., Spain and 4H.Virgen del Rocı´o, Microbiology,
Seville, Spain
We evaluated prospectively the usefulness of PCR
and antigen detection for the early diagnosis of
invasive fungal infection, in a series of 259
consecutive sera and whole blood samples from
31 patients in high risk of having Aspergillosis and
stratified according to the probability of IA based
on recently accepted case definition sets. To detect
circulating Aspergillus galactomannan in sera samples, the commercialised enzyme-linked immunosorbent assay method (Platelia test) was used
following the manufacturer instructions. For DNA
detection, we used the method developed by J.
Loeffler et al (1). DNA was extracted from whole
blood samples by using recombinant lyticase (Sigma, Madrid, Spain) and the QIAmp Tissue Kit
(Qiagen, Barcelona, Spain). The Light Cycler PCR
and detection system Roche Diagnostics, (Mannheim, Germany) has been used. Primers bind to
conserved regions of the fungal 18S rRNA gene
and for amplicon detection, the light cycler DNA
master hybridization probes kit was used as
described by the manufacturer. Antigen detection
was positive (‡1 ng ml)1) in 11 patients during 14
episodes. Two patients were classified as having
probable infection with clinical, radiological pulmonary signs or micrological positive cultures plus
GM detection positive (two or more consecutive
samples above 1 ng ml)1). On the nine patients
stratified as with possible infection, two were
133
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receiving piperacillin/tazobactam during the episode, and six were receiving anti-fungal prophylaxis when the samples for PCR were obtained.
None of the whole blood samples had a positive
PCR result, even using a method with sensitivity as
low as five conidial cells. According to these
results, the GM positive results anticipated to
clinical signs of infection. In this type of patients,
the PCR method used is not useful for early
diagnosis of fungal infection, and it can be due to
that most of the samples tested were obtained after
the initiation of prophylaxis.
P200
Evaluation of a new differential culture medium
for rapid identification of clinical specimens for
important Candida species
A. Taillandier, M.T. Baixench and A. Paugam
Cochin, Parasitology-Mycology, Paris, France
OCCA (Oxoı̈d, Basingstoke, UK) is a novel differential
culture that allows selective isolation of yeasts and
simultaneously identifies colonies of C. albicans,
C. tropicalis and C. krusei. We evaluated this novel
medium and compare it with a reference medium:
CHROMagar Candida (Becton Dickinson, Meylan,
France) for the presumptive identification of yeast
species isolated directly on the medium from clinical
specimens (i.e. urine, stool and respiratory sources).
A total of 142 clinical specimens showed growth
(30C) on either CHROMagar Candida or OCCA and
97.8% on both media. The overall agreement
between two observers reading the OCCA plates
and the CHROMagar Candida were respectively
96.3% and 99.2%. OCCA identified C. albicans with
a sensitivity of 95.5% (86/90) and a specificity of
100% (52/52). A total of five C. tropicalis and one
C. krusei were isolated, all identified correctly with
OCCA. We confirmed the absence of significant
difference in growth development on both media by
an experimental study. Regarding these results,
OCCA is a satisfactory isolation medium allowing
correct identification of the commonly encountered
yeasts and easy recognition of mixed cultures in
clinical specimens.
P201
Specificity of CPL5 and CPR8 primers to Pythium
insidiosum by polymerase chain reaction
C. Prariyachatigul,1 A. Chaiprasert2 and
A. Jetrasrisupab3
1
Khon Kaen University, Center for research and
Development of Medical Diagnostic Laboratories,
AMS, Khon Kaen, 2Mahidol University, Faculty of
Medicine, Siriraj Hospital, Bangkok, and 3Khon Kaen
University, Faculty of Medicine, Srinagarind Hospital,
Khon Kaen, Thailand
Pythium insidiosum is only fungus species in genus
Pythium, which reported causes mammals diseases
that called pythiosis insidiosi. Clinical manifestation of
this disease includes cutaneous/subcutaneous pythiosis, ophthalmic pythiosis and systemic pythiosis. The
present treatments of pythiosis patients are vaccines,
drugs therapy and surgical excision. However, vaccines and drugs are still under developing and
researching. Therefore the more rapid diagnosis and
identification of P. insidiosum is the better advantage
for patients. In this study we identified P. insidiosum
from isolated stock cultures by using polymerase
chain reaction (PCR) method, a rapid technique for
molecular biology diagnosis. In order to evaluate
specificity and sensitivity of this method. We used CPL
5 and CPR 8 primers, which had base sequence
complementary with 18S rRNA gene of P. insidiosum
strain AF 442497 but not to other 23 fungi strains
and 10 bacteria strains. The result showed that 31 of
32 strains of P. insidiosum could be detected by PCR
(97%), and the primers used in this study were not
specific for other fungi and bacterias. In addition, the
result showed that the least DNA volume, which
could be detected by PCR method was 100 pg.
P202
Dwelling surveys in hypersensitivity
pneumonitis management
S. Roussel,1 G. Reboux,1 L. Millon,1 J.C. Dalphin2 and
R. piarroux1
1
Department of Mycology, 2Department of
Respiratory Disease, University Hospital, Besançon,
France
Hypersensitivity pneumonitis is a pulmonary disease
with symptoms of dyspnea and cough resulting
134
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
from repeated exposure to high concentrations or
prolonged exposure to low concentrations of inhaled
antigens from mouldy matter, or antigenic compounds from avian droppings, both of which lead to
sensitisation and development of this disease. Finding the offending antigen sources and keeping
patients from exposure is the only treatment. The
home and professional environments, bird presence
and tobacco consumption render etiological
research difficult. If a bird is present in the
environment, the bird fancier lung hypothesis
should be studied first and special bird antigens
produced. Few antigens are found to be in common
to differing bird species, and microorganisms isolated from droppings have little immunological influence on bird fancier lung patients. To discover the
antigenic source, one must bear in mind new home
hobbies (Sauna, carpentry, gardening…) responsible
for antigens dispersion. Several media are necessary
to isolated micro organisms present in patient
environment including the cultivation of bacteria.
The production of several microorganism extracts is
useful to demonstrate the presence of precipitins.
Patient serological results must be compared to
results from a large control group. Some patients
may suffer from bird fancier lung and domestic
hypersensitivity pneumonitis, other may initially
have had a bird fancier lung and then relapsed with
hypersensitivity pneumonitis from indoor molds. In
our series, only half of etiological agents seem to
have been discovered by correlation between environmental and serological analyses, but in all cases
presence of precipitins have furnished a supplementary argument in favour of hypersensitivity
pneumonitis diagnosis. New tools for environmental
measurement with species identification and without cultivation, are necessary. Haugland and coll.
recommend using real time PCR to quantify more
than 130 fungal species in environment. For the
year our team has been experimenting successfully
the quantification of six fungi species with real time
PCR. The next step is to extend the number of
species sought to produce selected new antigens on
the basis of real time PCR results.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
P203
Rapid detection of Candida species directly
from blood
F. Somogyvari,1 I. Doczi,1 J. Serly,1 E. Hajdu1 and
E. Nagy1,2
1
Institute of Clinical Microbiology, University of
Szeged and 2Microbiological Research Group,
Hungarian Academy of Sciences and University of
Szeged, Szeged, Hungary
Diagnosis of fungemia or hematogenous candidiasis
has been problematic due to the low positivity of
blood cultures. Therefore non-culture methods including polymerase chain reaction (PCR), galactomannan antigen detection and western blot have being
developed for systemic fungal infections. Recently,
the amplification of short internal transcribed spacer
(ITS) region has been described as a rapid and
reliable method to detect fungi in different clinical
specimens. After amplification further analysis was
carried out with restriction fragment length polymorphism (RFLP), sequence analysis or enzyme
immunosorbent assay. All of these investigations
are time consuming and labour intensive. Newly
developed method is the rapid real-time PCR assays,
which generally work with hybridisation probes. Our
aim was to introduce a rapid, sensitive and specific
method for the detection of Candida species causing
invasive infections on the basis of the amplification of
the ITS2 region and the consecutive melting-point
analysis as described previously1. Forty-seven blood
samples originated from nine patients in different
time were examined. The basis of the patient
selection was the positivity of more than one
specimen (trachea, urine, catheter etc.) for Candida
by using the routine laboratory culture methods.
DNA was extracted from blood samples by using
Helix pomatia gastric juice and QIAmp Tissue Kit
(Qiagen) purification kit after the lysis of red blood
cells. Fungal DNA has been amplified by the
LightCycler (Roche) real-time PCR. One patient
samples were negative with PCR method and with
blood culture too. This patient got over the illness
without the anti-fungal therapy. Two patient samples were positive with both investigation. Despite of
negative blood culture results we could confirm the
presence of Candida DNA in six patient blood samples
by using PCR method. The patients got well after the
anti-fungal therapy. Our results suggest that the
newly developed real-time PCR method may provide
135
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Supported by Polish State Committee for Scientific
good solution in the identification of clinically
important Candida species. This method is more rapid 18 Research (grant No.3PO5A 028 25
and more sensitive than culture methods, so, it may
help to diagnose invasive fungal infections.
References:
1. Somogyvari F, Serly J, Doczi I, Nagy E. Molecular differentiation
of most frequent Candida species causing blood-stream infection.
Poster, Trends in Medical Mycology 2, Berlin, 2005: 23–6.
P204
Activity of hydrolytic enzymes of Candida
albicans in urine and from different clinical
materials from hospitalized patients.
M. Dabkowska, E. Swoboda-Kopec, D. Kawecki,
S. Błachnio and M.Łuczak
Medical University, Microbiology, Warsaw, Poland
Objectives: The aim of our study was to perform
enzymes analysis of urine samples from the five
patients undergoing abdominal surgery in Central
Hospital, Medical University of Warsaw. Analysis of
hydrolytic enzymes of the fungal strains cultured
from clinical samples from hospitalized patients
without UTIs.
Methods: Specimens sent for culture comprised
swabs from the peritoneum, swabs from the drains,
urine and other. The specimens were inoculated into
Sabouraud’s medium with chloramphenicol and
gentamicin. The isolation and identification of cultures was tested with the use of API ID32C system
(bioMerieux) and then all strains were examined for
enzymatic activity by API ZYM system (bioMerieux).
The urine analysis was negative in microbiological
cultures in five patients.
Results: In total sixteen strains of Candida albicans
were isolated. A value of enzymatic activity ranging
from 0–5: (0) corresponds to the negative reaction,
(5) to the maximum intensity. In our examination
the activity of hydrolytic enzymes showed the highest
activity in urine of acid phosphatase (XI), naphtholAS-BI-phosphohydrolase (XII) and cystine arylamidase. Candida albicans isolated from other clinical
samples showed the difference of activity of examined
enzymes between urine and another site of fungal
infections.
Conclusions: Detections of hydrolytic enzymes in
urine with negative culture seem to be a useful
method for identification of systemic fungal infections.
136
P205
The true causative agents of onychomycosis:
new insights from molecular methods
N.E. Zharikova,1 Y.V. Sergeev,2 A.Y. Sergeev,2
S.N. Scherbo,3 V.M. Leschenko2 and N.V. Nekhaeva2
1
Central Clinical Hospital, Microbiology, 2All-Russian
National Academy of Mycology, 3NPO Genetech,
Moscow, Russia
Prevalence of certain causative agents of onychomycosis may be important for selecting an agent for
systemic anti-fungal therapy. Yet the exact etiology
of onychomycosis remains a subject of discussion.
Estimation of proportions of major causative agents:
dermatophytes, yeasts and moulds by conventional
methods are limited by low sensitivity and specificity
of both microscopy and culture. A little agreement is
met in establishing the ‘true’ non-dermatophyte
onychomycosis. In our recent study, we used the
new Russian PCR duplex probes for Trichophyton
rubrum and T. mentagrophytes var. interdigitale for
detection of dermatophytes in nail specimens to
evaluate the etiology of onychomycosis and verify
the results of conventional methods. The sub-sample
of the large multi-center study for clinical testing of
PCR method consisted of 94 culture-positive toenail
specimens verified by PCR. The causative agents
obtained were: T. rubrum 79.2%, T. var. interdigitale
2.1%, Candida spp. 5.2%, non-dermatophyte mould
12.5%, T. rubrum + Candida 1%. More accurate PCR
duplex method, covering more than 98% of agents of
Tinea unguium in Russia, brings the new concept of
PCR-positive and PCR-negative onychomycosis. In
this context, almost all PCR-negative cases of onychomycosis may represent the true non-dermatophyte
onychomycosis (21% of KOH-positive samples in our
study). Among PCR-negative cases occurrence of
Candida spp. is 15.4%, moulds 46.2%, but also 38.5%
of T. rubrum, possibly representing other dermatophyte species (such as T. violaceum) erroneously
identified as T. rubrum. Among PCR-positive samples,
yeast and mould cultures were obtained in 11.1%,
possibly representing the mixed infection (12.3% in
KOH-positive samples). Among all PCR-positive
cases, 88.9% were positive for T. rubrum and 11.1%
for T. mentagrophytes var. interdigitale, showing
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
difference from culture results (97.4% vs. 2.5%).
Advent of PCR for direct identification of fungi in
clinical specimens may change our views on etiology
of onychomycosis. New data confirm the significance
of non-dermatophyte fungal infections of the nails.
The prevalence of yeast and mold onychomycosis
appears to be significant, bur may differ from
previous reports. This may have relevance for antifungal treatment.
P211
Anti-fungal susceptibility testing of clinical and
non-clinical Trichoderma longibrachiatum
isolates
L. Kredics,1 Z. Antal,1 I. Dóczi,2 L. Manczinger,3
C. Vágvölgyi3 and E. Nagy1,2
1
Hungarian Academy of Sciences and University of
Szeged, Microbiological Research Group, 2Institute of
Clinical Microbiology, 3Department of Microbiology,
University of Szeged, Szeged, Hungary
The genus Trichoderma is already on the growing list
of potential fungal pathogens in immunocompromised hosts. Most of the Trichoderma infections are
reported from patients undergoing peritoneal dialysis
and from transplant recipients. The most frequent ethiologic agent reported within the genus is
T. longibrachiatum. As data about the anti-fungal
susceptibilities of opportunistic fungi are crucial for
the choice of adequate therapeutic interventions, we
studied the anti-fungal susceptibilities of 12 clinical
and nine non-clinical Trichoderma longibrachiatum
isolates. The E-test and the agar dilution methods
were compared in the case of Trichoderma strains for
the determination of MICs of fluconazole and amphotericin B. MICs for fluconazole were in agreement
while MICs for amphotericin B were higher with one
or two steps of two-fold dilutions for most of
Trichoderma strains by the agar dilution method.
These data indicated differences between the two
susceptibility testing methods. As the E-test is easier
to perform, less labour-intensive and much simpler to
set up than the broth microdilution or agar dilution
methods, it provides the flexibility to test anti-fungal
agents against other mould species as well. To
investigate the overall anti-fungal susceptibility of
the Trichoderma longibrachiatum isolates, the E-test
method was used for the determination of MIC values
for further two anti-fungal drugs, itraconazole and
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
ketoconazole as well. On the whole, most of the
strains had high MIC values for fluconazole. In the
case of itraconazole, most of the clinical isolates
proved to be resistant and just some of them were
susceptible. Similar results were obtained for the nonclinical isolates as well. The MIC values of the strains
for ketoconazole and amphotericin B were lower and
variable. Interestingly, the clinical isolates were the
most susceptible to ketoconazole, while the soilderived ones had the highest inhibition zone in the
case of amphotericin B or ketoconazole. In conclusion, there were no significant differences between
strains derived from clinical and soil samples,
suggesting that Trichoderma longibrachiatum strains
may be naturally resistant to anti-fungal drugs. The
low susceptibility level of some clinical Trichoderma
isolates to anti-fungal drugs may cause difficulties in
the therapy of infected patients.
This work was supported financially by grant
F037663 of the Hungarian Scientific Research Fund.
P212
Taxonomic investigations within the species
Trichoderma longibrachiatum, an emerging
fungal pathogen
Z. Antal,1 J. Varga,2 L. Kredics,1 A. Szekeres,2
L. Hatvani,2 L. Manczinger,2 C. Vágvölgyi2 and
E. Nagy1,3
1
Hungarian Academy of Sciences and University of
Szeged, Microbiological Research Group,
2
Department of Microbiology, 3Institute of Clinical
Microbiology, University of Szeged, Szeged, Hungary
More and more common airborne and soil-borne
organisms are continually being added to the list of
potential fungal pathogens. Infections may occur
when normal barriers of the human body are broken
down (traumatic inoculation, surgery), host defenses
are weakened by medical conditions or treatment, or
in-patient with chronic rhinosinusitis. The emerging
incidence of these fungal pathogens is primarily the
result of the increasing number of immunocompromised patients. Among the recently emerging fungal
pathogens, Trichoderma strains were detected on the
skin, in the lung or as causative agents of peritonitis
in peritoneal dialysis patients, moreover disseminated
in the liver, brain, heart or stomach of immunocompromised patients. The majority of these pathogenic
Trichoderma isolates belong to the T. longibrachiatum
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species, which is related to Hypocrea orientalis and
H. cerebriformis. To study the taxonomic relationships
among T. longibrachiatum strains derived from clinical or soil samples, molecular characters were
examined by the use of three molecular methods:
restriction fragment length polymorphisms (RFLPs)
of the mitochondrial DNA, cellulose acetate electrophoresis of isoenzymes, and sequence analysis of the
internal transcribed spacer (ITS) region. Afterwards,
the molecular data were used to prepare dendrograms. The ITS sequences of the strains do not have
enough variability to investigate the relationships
among strains of the T. longibrachiatum species.
Cellulose acetate electrophoresis of seven enzyme
systems (glucose-6-phosphate-dehydrogenase; glucose-6-phosphate-isomerase; 6-phosphogluconatedehydrogenase; phosphoglucomutase; peptidases A,
B, and C) was useful for dividing the strains into 10
electrophoretic types belonging to two separate
clusters on the phylogenetic tree. Regarding the
RFLP profiles of mitochondrial DNA, the examined
isolates exhibited seven and 10 different patterns by
using the restriction enzymes BsuRI and Hin6I,
respectively, and let us divide the strains into four
groups on the dendogram. The discriminating power
of these two latter methods was higher than that of
ITS sequence analysis, and produced more consistent
profiles than the PCR-based RAPD technique. Nevertheless, the saprophytic and clinical isolates did not
form separate clusters on the dendrograms, suggesting that every environmental isolate may have the
capacity to cause infection.
Acknowledgement This work was supported financially by grant F037663 of the Hungarian Scientific
Research Fund.
P213
Disseminated Penicillium marneffei infection in
AIDS patients outside endemic regions: case
report and review of the literature
S. Antinori,1 E. Gianelli,1 F. Croce,1 C. Parravicini2
and A.L. Ridolfo1
1
Infectious Disease and Tropical Medicine, Clinical
Science ‘L.Sacco’ Hospital, Milan, Italy and
2
Pathology, Clinical Science ‘L.Sacco’ Hospital, Milan,
Italy
Objective: To describe a case of disseminated penicilliosis in an Italian HIV-positive patient and to
138
review all cases reported in the literature among
AIDS patients outside endemic regions.
Methods: We performed a search (years 1988–
2004) through Medline using the following terms:
penicilliosis, imported, Europe, USA, AIDS.
Results: On August 14, 2004 a 36-year-old Italian
man came to our attention by chance since his wife,
a 32-year-old Thailandese woman, was referred to
our department for a coma secondary to a spaceoccupying CNS lesion. Both subjects had HIV infection disclosed 1 month previous in Thailand. During
the interview, several maculo-papular lesions some
of which had a molluscum-like appearance were
noted on the face of the man. Clinical examination
revealed the presence of similar lesions on the arms,
trunk and limbs. The clinical picture and the
geographic residence of the patient (northern Thailand) were consistent with a presumptive diagnosis of
penicilliosis; the skin biopsy specimen stained with
hematoxylin-eosin, periodic acid-Schiff and GrocottGomori revealed numerous rounds to oval thinwalled yeast-like organisms some of which had a
central septation; a few similar organisms were
observed in the bone marrow aspirate. Penicillium
marneffei was isolated from blood, bone marrow and
skin .The patient was treated with liposomal amphotericin B for 2 weeks subsequently switched to
itraconazole and was discharged in good health with
almost complete disappearance of skin lesions and
negative blood cultures. From 1988 (when the first
AIDS-associated case outside endemic region was
described) to December 2004 (comprising the present
report) a total of 37 case reports of HIV-associated
penicilliosis were identified .Twenty-nine (76.3%)
occurred in male patients with a median age of
34 years (range 22–55 years) and all but one were
diagnosed during life (97%). Diagnosis was made by
means of histology plus culture in 25 patients
(67.6%), by culture alone in nine patients (24.3%)
and by histology alone in the remaining three
patients. No differences were observed in the number
of cases reported in the period before (1988–96 = 19
cases) and after the introduction of HAART (1997–
2004 = 19 cases). Except for two African subjects all
the remaining patients where either natives or
residents or had traveled in Asia. Skin lesions were
observed in 52%, anemia in 60%, weight loss in 63%
and lymphadenopathy in 34% of cases. Countries
reporting the cases were France (nine cases); Switzerland and United Kingdom (five cases each); USA,
Netherlands and Germany (three cases); Australia,
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
Belgium, Italy (two cases); Denmark, Japan, Spain
and Sweden (one case each).
Conclusion: A review of the literature shows that
penicilliosis albeit unusual may represent an emerging opportunistic infection among HIV-positive
people traveling to or coming from endemic areas.
In this review skin lesions were less frequently
observed compared with reports from Thailand.
P215
Paeilomyces lilacinus endophthalmitis an
immunocompetent patient
M.S. Cuetara,1 F. Romero,2 J. Gene,3 Y. Gil,1
J. Guarro,3 E. Cortes2 and I. Wilhelmi1
1
Microbiology, 2Hospital Severo Ochoa,
Ophthalmology, Leganes. Madrid and 3Universitat
Rovira I Virgil, Reus, Spain
P214
Cases of funguria in hospitalized patients
A. Rokosz, A. Sawicka-Grzelak, E. Swoboda-Kopeć,
I. Serafin and M. Łuczak
Medical University, Microbiology, Warsaw, Poland
Objectives: Isolation, identification and susceptibility testing to antifungal agents of uropathogenic
fungal strains cultured from urine samples of hospitalized patients.
Methods: Urine specimens were collected from
patients hospitalized in different departments of a
university hospital in Warsaw during 18 months in
2001–2002. fungal strains were isolated in cases of
significant funguria (‡104 CFU ml–1). Fungal uropathogens were identified in an automatic ATB system
(bioMerieuxSA) with the use of ID32C tests. Susceptibility of strains to antifungal agents: flucytosine,
amphotericin B, miconazole, ketoconazole, itraconazole
and fluconazole were determined by means of Fungitest (BIO-RAD, France). Reference Candida albicans
ATCC 90028 strain was used as control stain in each test.
Results: Fifty-two strains of fungal uropathogens
were cultured during 18 months. All strains belonged
to the group of yeast-like fungi (genera: Candida and
Trichosporon). Cases of funguria concerned about 0.5%
of patients treated in our hospital. Twenty-nine fungal
uropathogens were isolated from patients of transplantology departments. Strains of genus Candida (33/52)
dominated among fungi causing urinary tract infections (UTIs). Remaining uropathogens belonged to the
species Trichosporon asahii (19/52) and were mostly
isolated from patients of transplantology departments
(16/19). All Candida spp. Strains were susceptible (17)
or intermediately susceptible (2) to fluconazole.
Conclusions: Trichosporon asahii (19/52) and
C. albicans (17/52) strains dominated among fungal
uropathogens isolated from patients of our hospital in
Warsaw. Fluconazole was the most active in vitro
agent against fungal strains causing urinary tract
infections in patients.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
Forty-three-year-old woman was first seen in the
Emergency Department of a tertiary hospital due to
perforated injury with wood in her left eye. An
anterior vitrectomy with a lense implant was done,
with implementation of local wide spectrum antibiotics and topical corticosteroids. She was immediately
transferred to the Department of Ophthalmology of
our hospital, where the above treatment was continued for 2 weeks. Two months later the patient
returned with hypopion and corneal opacity. Direct
examination of paracentesis (anterior chamber and
vitreous) permitted the diagnosis of fungal endophthalmitis; intravitreus conventional amphothericin B
was implemented. Cultures allowed the identification
of Paecilomyces lilacinus. Progressive visual loss and
intense pain required eye enucleation. MIC values
obtained later showed in vitro resistance to amphotericin B. Fungal endophthalmitis, prevalence of
offending species and management of this condition
is discussed.
P216
Fatal paracoccidioidomycosis as first clinical
manifestation of HIV/AIDS. Case report and
review
M.V. Ferrer,1 M. Arreaza,1 A.J. Rodriguez-Morales,2
C.N. Rodriguez3 and P. Meijomil3
1
Salud Nueva Esparta, 1Hospital Armando Mata
Sanchez, Punta de Piedras, 2Universidad de Los
Andes, Center for Research JWT, Trujillo and 3JGH
West General Hospital, Microbiology Lab, Caracas,
Venezuela
Introduction: Paracoccidioidomycosis (PCM) has
been rarely reported among HIV patients, despite
being an endemic mycosis in South America. For this
reason we report a fatal clinical case of PCM
presenting as first clinical manifestation of HIV/AIDS.
Methods: Case report and review. Appropriate
mycological studies were made.
139
xxx
site with exophthalmus and paralysis of the 7th
Results: A female patient, 34-year-old, born in
cranial nerve. Histological examination and culture
Porlamar, Margarita Island, Venezuela, presented
of specimens obtained by endoscopy from the nasal
persistent cough with dyspnea and asthenia, of
cavity showed granulomatous suppurating inflam5 months of evolution, sometimes fever (39 C); she
mation with large, non-septate, irregularly branched
received previously different antimicrobial treatments.
hyphae suggesting zygomycosis. Identification of the
No significant personal medical history, including
mycete as Rhizopus oryzae was based on culture on
non-i.v. drug user. At income, physical examination:
Sabouraud dextrose agar with chloramphenicol.
BP 110/70 mmHg, HR: 90 bpm, RR: 25 bpm,
Despite therapy with amphotericin B, the infection
T.37 C. She presented in regular general conditions,
eroded the orbit and reached the brain. The patient
hydrated, mild mucocutaneous paleness, caquetic.
died of meningoencephalitis after surgical enucleaCardiopulmonary evaluation revealed respiratory
tion of the left eye and areas of necrotic brain tissue.
sounds in both hemithorax, with ronchus and bulous
Case 2: A 70-year-old male presented with a painful
sounds in both sides. Rest physical examination
erythematous edematous patch on the left knee,
without apparent alterations. Income diagnosis: Bronattributed to cellulitis. Despite systemic antibiotic
chopneumonia; TB to be rule out. Patient begun to
therapy and surgical drainage, nodules associated
receive Azithromycin 500 mg p.o. o.d. · 5d. IgG
with fistulas formed. Biopsy showed chronic granuloserology for Chlamydophila pneumoniae was positive
matous suppurating inflammation containing hyphae
(1:32 dil), IgM for Mycoplasma pneumoniae negative.
similar to those found in case 1. Culture led to isolation
TB studies were all negative. Due to dyspnea Hydroof R. oryzae. After excluding mucormycosis of other
cortisone is given at 100 mg VEV q6h · 3d. Mycology
organs and systems, the patient was treated with
serology was positive for Paracoccidioides brasiliensis,
itraconazole and gradually recovered. Mucormycosis
after this itraconazole is indicated at 100 mg p.o.
due to R. oryzae is an infection associated with high
o.d. · 6 months. Hydrocortisone was omitted. HIV
mortality, especially in forms with rhinocerebral
ELISA was positive, confirmed by Western-blot. No
involvement and in patients with immune depression
hematological or lymphocytic alterations were obor untreated diabetes (case 1). Subcutaneous forms
served up to this moment. Due to supplies problem in
(case 2) have a better prognosis, especially if the
the institution patient was referred to another one,
patient is immunocompetent and non-diabetic.
where she dies 3 days after due to pulmonary and
cardiovascular complications, postmortem diagnosis as
Reference
death cause was endocarditis.
Conclusions: HIV infection and endemic tropical 19
1 Romano C et al. Case report. Fatal rhinocerebral zygomycosis
diseases interaction has become a major concern, but
due to Rhizopus oryzae. Mycoses 2001; 44: 1–6.
its mechanisms are still poorly understood. PCM, a
South America endemic deep mycosis, may provide
an interesting model to investigate this, as features of
most HIV-PCM-coinfected patients are difficult to
P218
classify into standard acute and chronic PCM forms.
Molecular evidence of recurrent Histoplasmosis
with 9-year latency in a patient with Addison’s
disease
P217
Two cases of rhinocerebral and subcutaneous
zygomycosis
A. Ghilardi, L. Massai and C. Romano
Dermatology, Department of Clinical Medicine and
Immunological Science, Siena, Italy
Case 1: A 50-year-old diabetic farmer developed
rhinocerebral zygomycosis after left zygomaxillary
fracture, treated with systemic antibiotics. A necrotic
erythematous edematous lesion appeared in the same
140
P.R. Hsueh,1 T.C. Tseng,1 S.J. Liaw,1 C.Y. Wang,1
L.N. Lee,1 S. Huang1 and C.H. Hsiao2
1
Laboratory Medicine and Internal Medicine and
2
Pathology, National Taiwan University Hospital,
Taipei, Taiwan
Histoplasmosis has rarely been reported in Taiwan. A
total of five patients with microbiologically documented histoplasmosis have been identified prior to
2005. Among these five patients, three were HIVinfected, one had recurrent infection, and all
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
acquired infection from other countries. We herein
present a case of fatal histoplasmosis in a patient
with Addison’s disease and long-term use of corticosteroid. The patient acquired Histoplasma capsulatum
in China and initially presented with a laryngeal
mass. He developed fulminant pneumonia and
respiratory failure 9 years later. Nucleotide
sequences of internal transcribed spacer (ITS) regions
of rRNA genes of the H. capsulatum isolate recovered
from biopsied lung tissue and the extracted fungal
DNA from the laryngeal lesion were identical indicating the recurrence nature of infection.
P219
A Trichoderma strain repeatedly isolated
from blood serum, skin lesions, sputum and
pharingeal samples of a pediatric patient
with ALL
A.S. Kantarcioglu,1 T. Celkan,2 A. Yucel,1
S. Kurugoglu3 and K. Altas1
1
Cerrahpasa Medical Faculty, Microbiology and
Clinical Microbiology, 2Cerrahpasa Medical Faculty,
Department of Pediatrics Hematology-Oncology and
3
Cerrahpasa Medical Faculty, Department of
Radiology, Istanbul, Turkey,
Trichoderma was isolated from soil, grains, paper and
textiles. Human cases are limited to severely weakened patients or emerge as complication of dialysis.
We report a Trichoderma isolation from a 9-year-old
boy with ALL in neutropenia. Thoracal and abdominal CT scans revealed pulmonary infiltrates suggesting a mold infection, intestinal edema, tyflitis. He
developed necrotic ulcerated skin lesions. Liposomal
AMB was started (5 mg kg–1 d–1).Blood, urine, stool
cultures were negative. Aspergillus GMN antigen
could not be detected twice in the serum. Tissue
sample was not obtained because of thrombocytopenia. A series of blood, pus from skin lesions, sputum
and pharingeal specimens were obtained repeatedly
in three different days with 3–5 day intervals. Blood
samples were collected in anti-coagulant free sterile
vacuum tubes. Deep cough spontaneous sputum
samples (not saliva) were collected early in the
morning and studied freshly. Pus from skin lesions
and throat specimens were obtained using sterile
swabs avoiding contamination with flora organisms.
Aseptically prepared sera were studied by direct
microscopy and culturing. Direct microscopical
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
examination was carried by Ehrlich-Ziehl-Neelsen,
methylene blue, Giemsa stained slides. The specimens
were inoculated onto Sabouraud dextrose agar
(SDA), brain-heart infusion agar, cooked ship’s blood
agar and incubated at 25, 30, 37 C. Direct examination of sera, pus from skin lesions, sputum and
throat samples revealed fungal cells and hyaline
hyphae. Cultures on SDA showed white mycelial
growth after 4 days of incubation at 30 C. The same
fungus was isolated repeatedly as a single microorganism from serum, pus, sputum, pharingeal samples and was examined using classical mycological
techniques based on growth rate, macroscopical and
microscopical characteristics of pure colony on
differential media. The isolate first produced whitish-green floccose colonies, then became green due
20 to abundant condition formed concentric white and
green rings. Colony reverse on PDA plates was
colorless in diffuse day light, but a lemon yellow
diffusable pigment was evident that grew in dark.
Conidiophores pyramidally branched i.e. long repeatedly branched lateral branches below, with short
branches near the apex. Phialides were flask-shaped,
phialoconidia were globose, smooth walled and
accumulated in balls at the apex. Smooth walled
chlamydoconidia either single or in triplets were
abundant. The isolate was phenotypically identified
as Trichoderma sp. In vitro susceptibility of the isolate
was examined by NCCLS M38-A reference broth
macrodilution method. MICs (lg ml–1) were as
follows: AMB>16, ITZ, MCZ, KTZ, TRBTrichoderma.
Positive blood culture is classified as proven disseminated infection by EORTC criteria, in our case
hemoculture remained negative for the fungus, it
was isolated from serum cultures.
P220
Genotypic and phenotypic study of Fusarium
solani species complex
M. Azor, J. Cano, J. Gené and J. Guarro
Universitat Rovira i Virgili, Unitat de Microbiologia,
Reus, Spain
Fusarium solani sensu lato is an important plant
pathogen, also able to invade human tissue, resulting
in a wide diversity of mycoses. It is responsible for
cutaneous as well as subcutaneous infections, but
also causes serious systemic infections, usually fatal
141
xxx
for immunocompromised patients. Due to the high
intraspecific genetic variation demonstrated in recent
studies testing mainly non-clinical isolates, F. solani
has been considered, in fact, an species complex. The
delineation of such species could be of extreme
importance from a clinical point of view. We have,
therefore, performed a morphological and molecular
study involving numerous isolates, morphologically
identified as F. solani, mainly isolated from clinical
sources from different countries. Forty clinical (30
from Brazil, 15 from Spain, three from Qatar and two
from USA) and 10 environmental isolates from Spain
morphologically identified as F. solani, were included
in this study. DNA sequences from three loci [nuclear
ribosomal internal transcribed spacer (ITS) region, a
portion of the translation elongation factor (EF-1)
gene and a portion of b-tubulin gene] were analysed
to investigate phylogenetic relationships and species
limits within the F. solani species complex. As a
result of the parsimonic analyses of the combined
dataset (1719 bp), a total of 5000 most parsimonious trees were produced from a heuristic search. In
the consensus tree we observed three main clades,
which received a bootstrap support of 100%, and a
total of 45 haplotypes. Clustering was similar to that
observed in the particular trees of the different genes
analysed. We have found no correlation between
genetic clades and geographical origin. However, we
have found important variations in the macro- and
microconidia morphology and in growth rate on
potato dextrose agar at 25/37 C, which correlated
with several of the phylogenetic clades.
P221
Chronic maxillary sinusitis due to
Schizophyllum commune – a case report
K. Mühlethaler,1 P. Bosshard,2 M. Arnold,3
A. Baumann4 and S. Zimmerli5
1
Institute for Infectious Diseases, University of Berne,
Berne,2Institute of Medical Microbiology, University
of Zürich, Zürich,3Institute of Pathology, University of
Berne,4University Hospital, Department of ENT, Head
and Neck Surgery, and5University of Berne, Institute
for Infectious Diseases, Berne, Switzerland
Infections caused by Schizophyllum commune, a
basidiomycetous fungus found on rotten wood are
very rare. Inhaled basidiospores may cause recurrent
sinusitis, the best documented clinical manifestation.
142
In addition, cases of allergic bronchopulmonary
mycosis, pulmonary fungus ball, meningitis, brain
abscess and onychomycosis have been described.
Most patients were successfully treated with surgery
followed by amphotericin B or itraconazol, but the
role of antifungal drugs is not known.
Case: We report the case of a 28-year-old woman
who presented with recurrent maxillary sinusitis of
2 months duration. Despite several courses of antibiotic therapy she did not fully recover. She complained of increasing left-sided frontal headaches and
a swollen left upper eyelid. We found a distinct
swelling of the left lid, discreet diplopia upon gaze to
the left, and a deviation of the massively edematous
endonasal septum to the left. On a CT-scan she had
left-sided pansinusitis with a bony erosion between
frontal sinus and orbita. Extended surgery revealed
friable grayish masses in the left maxillary sinus. She
was treated with oral voriconazole for 3 months and
her symptoms improved rapidly.
Diagnostic studies: Histologic examination showed
edematous respiratory mucosa with mild lymphoplasmocytic infiltration and eosinophilia. In the adjacent mucus were sparse septated and branching
hyphae (PAS and silver-methenamine stains). No
tissue invasion was documented. The initial diagnosis
was chronic sinusitis with fungal hyphae compatible
with Aspergillus spp.Culture on Sabouraud dextrose
agar grew molds. The isolate was subcultured on
mycosel agar (0.1% cyclohexamide) and on potato
dextrose agar. DNA was extracted using the InstaGeneTM matrix. The internal transcribed spacer region
was amplified and sequenced.
Results: Growth characteristics (white, spreading,
woolly colony on Sabouraud dextrose agar; hyaline,
wide septated hyphae; absence of conidia; formation
of lateral spicules; growth at 35 C, cyclohexamidetolerance and a pronounced odor) suggested a
basidiomycete. Because typical characteristics such
as clamp connections and fruiting bodies were
missing, molecular identification was performed.
Sequence analysis revealed S. commune.
Conclusions: Schizophyllum commune is an emerging agent of chronic sinusitis. Clinical presentation
and histopathologic findings are similar to sinusitis
caused by Aspergillus and certain other molds. It
should be included in the differential diagnosis of
patients with chronic sinusitis refractory to standard
therapy. Voriconazole appears to be an efficient
antifungal alternative to amphotericin B and itraconazole.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
P222
The ecology of fungi in patients with cystic
fibrosis (CF)
Y. Nagano,1,2 C. Millar,1 S. Elborn,1,2 C. Goldsmith1
and J. Moore1,2
1
Bacteriology, Northern Ireland Public Health
Laboratory, and2Queen’s University of Belfast,
Respiratory Medicine Research Group, Belfast, UK
Patients with cystic fibrosis (CF) suffer from chronic
micrial infections, which are mainly due to bacteria,
where Pseudomonas aeruginosa, Staphylococcus aureus
and members of the Burkholderia cepacia complex
(BCC) account for a high occurrence of infection.
There is less information on the occurrence of fungi
in these agents, including the yeasts as well as the
filamentous moulds, such as Candida spp. and
Aspergillus spp., respectively. The reasons for underreporting of such mycology agents may be due to (i)
inappropriate laboratory techniques to culture such
organisms in a mixed population of rapidly growing
Gram-negative bacteria, and (ii) a lack of clinical
understanding of what the presence of a diverse
variety of fungal agents represent in CF patients. The
aim of the current study was to identify fungi in 30
patients by employing conventional mycological
culture, as well as a ribosomal RNA PCR-direct
automated sequencing approach. Sputum was
obtained following physiotherapy from adult CF
patients (n = 30) attending the Northern Ireland
Regional Adult CF centre. Of these, 26/30 (87%)
were chronically colonised with Gram-negative
organisms. Enhanced mycological culture with a
CF-derived fungal selective culture medium (NM;
Nagano medium) for 2 weeks at 22 C demonstrated
the presence of yeasts in 15 (50%) patients and
filamentous fungi in only three (10%) patients. Of
these, the majority of the yeasts belonged to the
genus Candida, including C. dubliniensis, as well as
the presence of Exophiala (Wangiella) dermatitidis (the
black yeast) and Scedosporium apiospermum (Pseudallescheria boydii), Penicillium olsonii, Penicillium sp. and
Aspergillus sp., as determined by sequence analysis of
the short internal transcribed spacer (ITS2) region.
Conventional laboratory analysis failed to detect the
Scedosporium or Exophilia organisms, due to overgrowth by Gram-negative organisms. In addition,
when the ITS2 region was employed as a primary
diagnostic tool, all 30 patients were positive for the
presence of fungal DNA, with between one and three
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
bands present. In conclusion, we demonstrate that
several potentially important fungi may be missed if
mycological culture methods are not refined in order
to select out genera that require additional workup.
Additionally, without the ability to detect such
agents in sputum, antifungal susceptibilities would
not be performed, thereby compromising antifungal
treatment options, in an already complicated
management area, due to resistance problems.
A polyphasic approach employing both enhanced
mycological culture combined with molecular
(rDNA) detection and identification will serve to help
determine the presence of these in the sputum of
patients with CF. Only when we have the able to
reliably detect and identify such agents, can we
address issues of clinical significance of such agents
in patients at a higher risk of fungal infection, such
as in lung transplantation.
P223
Emerging fungal pathogens in domestic
animals
R.S. Ovchinnikov, M.G. Manoyan and A.N. Panin
The All-Russia State Center for Quality of Animal
Medicines (VGNKI), Laboratory of Mycology,
Moscow, Russia
A number of emerging fungal pathogens were
reported in the field of medical mycology in last
years. On the contrary, in veterinary mycology data
concerning this question is rather restricted. Here,
we describe a few cases of dermatomycoses in
domestic animals caused by emerging fungal pathogens, focusing on their appearance in damaged
tissues. Totally 253 clinical samples were studied in a
3-year period. Animals belonging to different species
(including horses, pets, fur-bearing animals, birds,
reptiles) and presenting skin lesions were mycologically investigated. Clinical significance of isolated
non-dermatophyte fungi was evaluated according to
Walshe–English criteria. Among rasehorses, dematiaceous non-dermatophyte fungi were recurrently
isolated. Doratomyces stemonitis was the most common species. It was presented in skin scrapings in the
form of brown branching mycelia and 1-celled
conidia. Other dark-pigmented fungi were presented
by Alternaria alternata and Cladosporium cladosporoides. Multi-celled brown phragmospores in skin
samples were typical for A. alternata, and elongated
143
xxx
1 or 2-celled conidia – for C. cladosporoides. Keratinophylic fungus Scopulariopsis brevicaulis was isolated
from horses in some cases. Some atypical fungal
pathogens were detected in dogs and cats. In akitainu-breed dog skin lesions were caused by black yeast
Aureobasidium pullulans. Microscopically, affected hair
shafts were covered by rudimentary club-like pseudomycelium. In another case hyphomycete
Wardomyces spp. was yielded from skin lesions in
basset. One-celled spherical brown spores and
branching mycelium could be observed microscopically in skin samples. An atypical causative agent was
diagnosed in a blue fox. Microscopic examination
revealed branching hyaline mycelium and a great
number of clavate conidia in short chains. Isolated
fungus showed the same morphological features and
was identified as Scopulariopsis acremonium. An
outbreak of skin disease among pigeons keeping in
a same pigeonloft was revealed. Affected birds
showed total feather loss in neck area (‘naked neck’).
Mycological examination allowed to isolate causative
agent of infection – Aspergillus niveus. Conidial heads
distinctive for Aspergillus genus were detected in
affected feathers. In reptiles Fusarium moniliforme was
detected as a most common causative agent, followed
by Penicillium chrysogenum, Alternaria alternata, Acremonium spp., Humicola spp., Phoma spp., Chaetomium
globosum, Mucor circinneloides. Lethal deep mycoses
accompanied by superficial lesions caused F. moniliforme and P. chrysogenum were ascertained in some
reptiles in this study. The same fungi were obtained
both from superficial samples and postmortem probes
from internal organs. Thus, mycoses caused by
emerging fungal pathogens were detected in a
variety of animal species. Clinical significance of
such infections in veterinary should not be underestimated.
P224
Fungal pathogens from respiratory medicine
patients and their susceptibility to new and old
antifungal drugs
21 M.A. Petrou
With the exception of systemic fungal infections,
fungi recovered from patients referred to Respiratory
Medicine are often ignored on the assumption that
treatment is of little value, thus it is not required.
144
Infections such as fungal balls and ABPA persist for
many years and most clinicians consider the use of
antifungals as unnecessary mainly due to lack of
evidence. As a result, culture and susceptibility of the
fungus is typically neglected. One other factor
dissuading clinicians in pursuing treatment is hospitalisation; patients prefer to be treated at home using
oral agents. Bronchial alveolar lavages, sputa, blood
cultures, tissue biopsies oral swabs, urine and wound
drains specimens from more than 250 Respiratory
Medicine patients, were cultured (spun deposits of
respiratory specimens) and the isolates were identified using standard mycological procedures. The
MICs were determined using the NCCLS method
with minor modifications. Eight different species of
Aspergillus, 13 other species of filamentous fungi and
15 species of yeasts were isolated. A. fumigatus
accounted for almost 50% and Candida albicans near
20% of all isolates. A. terreus and Scedosporium
apiospermum were resistant to Amphotericin B
whereas all other isolates were sensitive. C. krusei
had reduced sensitivity to Flucytosine, all other
yeasts were sensitive and most but not all filamentous fungi were resistant. All the filamentous fungi,
C. glabrata, C. inconspicua and C. krusei were resistant
to Fluconazole, C. pelliculosa, C. rugosa, Saccharomyces cerevisiae and Trichosporon beigelii had reduced
sensitivity and all other isolates were sensitive.
Fusarium solani and Rhizomucor pusillus were resistant to Itraconazole, Acremonium species, C. glabrata
and S. cerevisiae had reduced sensitivity and all other
isolates were sensitive. Rhizopus arrhizus, R. pusillus,
were resistant to Voriconazole, Acremonium species,
Alternaria alternata, F. solani, Paecilomyces variotii,
Penicillium species and C. glabrata had reduced sensitivity and all other isolates were sensitive. Susceptibility data obtained with Caspofungin for these
isolates will also be presented. These data will be
compared to those obtained from haematology/
oncology and other patients. Both Itraconazole and
Voriconazole were shown to be highly active against
most isolates and in particular Aspergillus. Both drugs
are available for oral treatment and now Clinicians
have a choice. The choice of drug depends entirely on
drug bioavailability, distribution in organs such as
the lung, patient’s renal and liver functions, concomitant treatment and compliance. The variety of
species and susceptibility data found in this study
recommend screening of respiratory patients. Furthermore it invites Clinicians to consider treatment,
thus reducing morbidity and in some cases mortality
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
in this group of patients who might also have other
underlining diseases that potentially convert a chronic fungal infection into a systemic fatal infection.
P225
Trends in mortality due to paracoccidioidomycosis in Venezuela
P.M. Rifakis,1 A.J. Rodriguez-Morales,2 G. Provenza,3
J. Benitez,4 C.N. Rodriguez3 and P. Meijomil3
1
Medicina, Pérez de León Hospital,
Caracas,2Universidad de Los Andes, Center for
Research JWT, Trujillo, 3Mycology, Clinical Infectious
Diseases Collaborative Research Group, Caracas and
4
Enviromental Health, Ministry of Health, Maracay,
Venezuela
Introduction: A systemic and endemic emerging
mycosis in Latin America, paracoccidioidomycosis, is
characterized by its chronicity and by the severity of
the disseminated form in healthy individuals, as well
as in immunocompromised individuals co-infected
with HIV, resulting, in the latter, in a mortality rate
in the range of 30–45%. In countries such as Brazil
where this disease is endemic, it carries a high
mortality rate. For this reason in this report, we
analyzed the trends in mortality due to infections
caused by Paracoccidioides brasiliensis in Venezuela in
an 8-year period.
Methods: Therefore, a review of Paracoccidioidomycosis mortality in Venezuela, between 1995 and 2002,
was done, using mortality records from Ministry of
Health, using descriptive and analytical statistics.
Results: For this period, 53 patients died from
Paracoccidioidomycosis (PCM), 84.9% were males
and 15.1% females (P < 0.05). Annual mean number
of deaths from PCM was 6.6 ± 3.7 deaths per year.
From total PCM deaths, 64.2% were >50 years old.
Deaths from PCM in young individuals (<20 years old)
were recorded in just 3.8% patients. From total PCM
death patients, 28.3% (15) were pulmonary Paracoccidioidomycosis and 9.4% (5) were disseminated
Paracoccidioidomycosis. Annual mean number of
deaths from pulmonary PCM was 1.9 deaths per year.
Paracoccidioidomycosis mortality in this period has
been without significant changes (linear regression:
r2 = 0.416, P = 0.0842), although each year has
been tending to decrease the incidence. Mean annual
mortality rate was 0.28 (±0.15) per million inhabitants (range, 0.13–0.58).
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
Discussion: Although in references countries (as
Brazil), the reported mean annual mortality rate was
1.45 per million inhabitants, indicating a downward
long-term trend (reduction of 31.28%) in last years,
in Venezuela this figure is significantly less than that,
but as reported in Brazil is also decreasing in our
country. Paracoccidioidomycosis should be suspected
in patients with an appropriate travel history who
experience weight loss and have pulmonary,
mucosal, and cutaneous lesions, although in immunocompromised patients is possible to seen disseminated diseases, as occurred in almost 10% of fatal
cases reported in this series.
P226
Trends in mortality due to Pneumocystis carinii
pneumonia (PCP) in HIV patients from
Venezuela
A.J. Rodriguez-Morales,1 J. Benitez,2 C.N. Rodriguez3
and P. Meijomil3
1
Center for Research JWT, Universidad de Los Andes,
Trujillo, 2DGSACS, Ministry of Health, Maracay, and
3
Microbiology Lab, JGH West General Hospital,
Caracas, Venezuela
Introduction: The causes of death in AIDS have
evolved since 1988 following the widespread use of
prophylactic and antiretroviral therapies in patients
with HIV infection. Although this, PCP remains as an
important cause of illness and death in HIV-infected
persons.
Methods: Therefore, a review of PCP mortality in
HIV patients from Venezuela, between 1996 and
2002, was done. For this period, 8030 patients died
from HIV, 83.2% were males and 16.8% females
(P < 0.01). Annual mean number of deaths from
HIV was 1123.8 ± 108.3 deaths per year.
Results: From total HIV death patients, 20.7% were
30–34 years old, 19.3% 35–39 years old, 16.8%
25–29 years old (P < 0.01). Deaths from HIV in
children (<5 years old) were recorded in just 1.97%
patients. From total deaths, 7.32% (588) were
attributed to PCP. Annual mean number of deaths
from PCP-HIV was 84.0 ± 25.3 deaths per year.
From total PCP-HIV deaths, 83.8% were males and
16.2% females (P < 0.01); according age: 22.8%
were 30–34 years old, 21.9% 35–39 years old,
16.8% 25–29% (P < 0.01). PCP deaths in <5 years
old occurred in 1.7%. Although treatment
145
xxx
availability HIV and PCP-HIV mortality in this period
has been withouth significant changes (linear
regression: r2 = 0.51, P = 0.071; r2 = 0.136,
P = 0.415).
Conclusions: PCP has not been the leading cause of
death in our country since many years ago, but the
frequency of PCP and its mortality rate should be
continuously reduced; throughout the developing
world, the rate of coinfection with Mycobacterium
tuberculosis and PCP is high, ranging from 25% to
80%, evidencing that PCP remains as significant
problem in HIV-infected patients, although the
HAART availability.
P227
Central nervous system infection due to
Scedosporium apiospermum after near
drowning: a fatal case in a child
1
2
2
3
P228
1
Kotsiou, Dotis, Iosifidis, Velegraki, Tamiolaki and
Roilides2
1
Pediatric Intensive Care Unit, Hippokration Hospital,
and 23rd Department Pediatrics, Aristotle University
of Thessaloniki, Thessaloniki, and 3National University
of Athens, Reference Mycology Laboratory, Athens,
Greece
Background: Scedosporium apiospermum, an anamorph of Pseudallescheria boydii, is a filamentous
fungus resistant to almost all antifungal agents.
Invasive infection due to S. apiospermum, especially
when it involves the central nervous system (CNS), is
associated with very poor prognosis. It usually occurs
in immunocompromised patients.
Case report: A previously healthy 6-year-old boy
was admitted to our pediatric intensive care unit
(PICU) in a coma, with hypoxemia, metabolic
acidosis, dehydration due to osmotic diuresis and
unstable vital signs, after near drowning in a sewage
reservoir. Laboratory tests and imaging studies
revealed lung infection. Pseudomonas aeruginosa and
Staphylococcus aureus were isolated from multiple
bronchial cultures, so broad-spectrum antibiotic
therapy was administered. Approximately 2 weeks
later new bronchial cultures yielded Aspergillus
fumigatus and Aspergillus terreus, thus antifungal
therapy with amphotericin B lipid complex (ABLC,
5 mg kg–1 day–1) was initiated. At the beginning of
the 3rd week, due to protracted seizures and
neurological deterioration, a brain computed tomog-
146
raphy (CT) revealed right frontal and parietal
subarachnoid hemorrhages and diffuse brain edema.
In cerebrospinal fluid cultures, S. apiospermum grew,
and voriconazole (8 mg kg–1 day–1) was added to
ABLC intravenously. Despite the combined treatment, CT performed during the subsequent days
showed progression of the disease. The condition of
the child rapidly deteriorated to the point of clinical
brain death 35 days after admission to the PICU and
18 days after initiation of voriconazole administration. Cultures taken from the sewage reservoir 1 month after the accident did not grow
S. apiospermum.
Conclusion: CNS infection due to S. apiospermum is
a very rare complication of near drowning and is
followed by extremely poor outcome despite antifungal therapy.
Fatal disseminated Trichosporon asahii
infection in a neutropenic patient with acute
myeloid leukemia
F. Sanchez,1,4 M. Lopez,1 N. Cliville,1 C. Rodriguez,2,4
C.V. Larrosa,2 R. Martino3 and P. Coll1,4
1
Microbiology, Hospital de Sant Pau, 2Pathology,
Hospital de Sant Pau, 3Clinical Haematology, Hospital
de Sant Pau, and 4Genetics and Microbiology,
Universitat Autnoma de Barcelona, Barcelona, Spain
Case report: A 43-year-old man presented to the
hospital with a 2–3 weeks history of fever and
progressive fatigue. Initial laboratory tests disclosed a
total leukocyte count of 121 400 mm–3, a hematocrit
level of 29% and a platelet count of 100 000 mm–3.
The bone marrow biopsy revealed acute myeloblastic
leukemia (subtype: M5) and the patient received
induction chemotherapy with idarubicin, cytarabine
and etoposide. The day after induction the patient
developed fever with no clinical response to empirical
use of cefepime and teicoplanin. Five days later,
empirical antifungal therapy with caspofungin was
initiated, but rapidly discontinued when a highresolution CT scan was highly suggestive of viral
infection (numerous pulmonary nodules). Acyclovir
was started. On day 11, the patient initiated a
astrointestinal syndrome with serious liquid depletion,
hypernatremia, hypokalemia and metabolic alkalosis.
Four days later, he developed acute respiratory failure
and transfer to ICU was required for ventilatory and
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
hemodynamic support. Empirical therapy with fluconazol was initiated, but was changed to voriconazole 8 days later in view of the microbiological results
from broncoalveolar lavage. Despite the treatment, the
patient died on day 20 with multiorgan failure.
Microbiological findings: Blood sample cultures obtained on days 1, 2, 3, 4, 6, 8, 13, 14, 15 and 16
yielded negative results, and only two hemoculture
bottles, one taken at admission and another on day 5,
were positive for caogulase-negative staphylococci.
Flu A virus was recovered from a nasopharyngeal
aspirate obtained on day 4. On hospital day 17, a
broncoalveolar lavage was positive for Trichosporon
asahii, and continued to be recovered in concurrent
culture with Acinetobacter baumanii 2 days latter.
Autopsy results: Histologically, the lungs showed
severe bilateral basal bronchopneumonia and
numerous hyphae and arthroconidia were observed
widely dispersed throughout the lungs. T. ashii was
also found in liver, spleen and gastrointestinal tract.
Multiple acute ulcers were observed in the gastrointestinal tract. Acinetobacter baumanii was isolated
from postmortem blood culture and lungs.
in the soil and in organic material. Patients are
infected either by trauma or inhalation. Presented
here are five cases of phaeohyphomycosis, four of
which were in immunocompromised patients.
P229
P230
Five cases of phaehyphomycosis
Gangrenous necrosis of the diabetic foot caused
by Fusarium acutatum
A. Skiada,1 E. Petrikkou,1 F.D. Madopoulou,1
A. Mitrousia-Ziouva,1 M. Aggelopoulou,2
G. Kosmadakis,3 B. Seitanidis,4 G.L. Daikos1 and
G. Petrikkos1
1
Research Laboratory for Infectious Diseases and
Antimicrobial Chemotherapy, 2A’ Dpt. Internal
Medicine, University of Athens, 3Laikon Hospital,
Renal Transplantation, 4Hygeia Hospital,
Hematology, Athens, Greece
The term ‘Phaeohyphomycosis’ includes various
clinical syndromes, which are due to the dematiaceous black fungi. Infections vary from superficial to
systemic, involving several viscera and the brain. All
these syndromes are characterized by infiltration of
the tissues by hyphae and pseudohyphae of a very
dark colour. This colour is due to melanin or other
pigments in the fungal cell walls. The commonest
fungi that produce phaeohyphomycosis are Bipolaris,
Exophiala, Cladosporium, Wangiella, Curvularia and
Alternaria, and they are ubiquitous in nature, found
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
Sex
Age
Fungus
Site of
infection
Underlying
disease
Female
Female
42
50
lternaria alternata
A. alternata
Soft tissue
Sinuses
None
Brain lymphoma
Male
35
A. alternata
Soft tissue
Male
28
Bipolaris spicifera
Soft tissue
Female
67
Cladosporium sp.
Hard palate
Kidney
transplantation
Kidney
transplantation
Acute
myelogenous
leukemia
Therapy
AmBisome
mBisome
caspofungin
Itraconazole
terbinafin
Ambisome
caspofungin
AmBisome
The outcome was favourable in the first three
cases, whereas the last two patients died, one due
to concomitant sepsis and the other due to the
fungal infection. Phaeohyphomycosis is one of the
emerging fungal diseases and must be always
included in the differential diagnosis in susceptible
patients.
S.J. Taj-Aldeen,1 J. Gene,2 I. Al Bozom,1 W. Buzina,3
J.F. Cano2 and J. Guarro2
1
Laboratory Medicine and Pathology, Hamad
Medical Corporation, Doha, Qatar, 2Mycology Unit,
Rovira I Virgili Univesity, Reus, Reus, Spain and
3
Institute of Hygiene, Graz, Austria
Foot infections are common and serious complications of diabetic patients. We report a case of a
68-year-old diabetic foot patient with Fusarium sp.
infection that developed into gangrenous necrosis.
Fusarium sp. was isolated on two successive occasions with no other associated microorganisms.
Histopathology demonstrated tissue invasion these
findings suggesting infection rather than colonization. A detailed morphological study demonstrated
that the isolate belonged to Fusarium acutatum,
which was confirmed by rDNA sequencing. This
fungus is only known in Asia and has not been
previously reported as a human pathogen.
147
xxx
P231
Preliminary study on the yeast flora in reptiles
digestive tract
A. Moretti,1 V. Vidotto,2 L. Boncio,3 J. Moretta,4
F. Lisi,4 C. Marini,5 C. Panzieri5 and F. Agnetti5
1
Department of Biopathological Science and
Hygiene, University of Perugia, Perugia, 2Department
of Medical and Surgical Disciplines, University of
Turin, Turin, 3Department of Medical and Surgical
Specialties and Public Health, University of Perugia,
Terni, 4Veterinary Clinician and 5State Veterinary
Institute, Perugia, Italy
The relationship between reptiles and pathogenic
fungi was studied to establish the potential antifungal activity of some snakes venom: for example, it
was demonstrated that the venom of Crotalus durissus
cumanensis is a strong antifungine substance. Little
data are available on mycotic diseases in reptiles and
the correlation between these animals and pathogenic fungi. It is known that reptiles are susceptible
for skin diseases, especially those farmed in captivity;
alimentary deficiencies may cause cutaneous diseases, visible by desquamation or colour alterations;
lights, thermostats and other heating systems of the
reptile house can contribute to induce burns, visible
as ulcers, abrasions, or other skin lesions. According
to some authors cutaneous lesions in reptiles may be
also caused by pathogenic fungi. Very few data are
available about reptiles mycotic diseases caused by
yeasts; in literature, it is reported only a case of
systemic mycosis by Trichosporon beigelii in a rock
rattlesnake (Crotalus lepidus klauberi). In this context,
Authors performed a preliminary study in order to
investigate the digestive tract yeast flora of some
healthy reptiles. Twenty animals, five adult pythons
(Python molurus bivattatus), five adult ball pythons
(Python regius), and 10 iguanas (Iguana iguana) were
examined. The pythons belong to a zoological garden
in Central Italy (Perugia), while the iguanas to some
pet-shops in the city of Terni; all the reptiles
considered are farmed in vivarium with an inside
temperature of 25 C. Age, sex and healthy status of
each animal were recorded. From each iguana,
mucosal specimens from the oropharinx and the
cloaca were obtained, using sterile cotton swabs; on
the contrary, the pythons were investigated only
with swabs from the cloaca, since the difficulty to
open their mouth. For fungal isolations, Sabouraud
dextrose agar supplemented with 20 U.I. penicillin
148
and 40 lg ml–1 streptomycin was used; the plates
were incubated at 26 C for 10–12 days. Yeasts were
identified by microscopic examination, Germ-tube
test and on the basis of carbohydrate assimilation
patterns. The Isolated yeasts were: Candida albicansm
(form six iguanas), Candida tropicalis and C. albicans
(from two iguanas), Rhodotorula minuta and C. albicans (from two iguanas), C. tropicalis and C. albicans
(form four ball pythons) and Candida guillermondii
(form four pythons). Two ball pythons (10%) were
negative for yeasts presence. The authors retain that
further epidemiological investigations are necessary
to have a clear picture of the mycological status in
snakes in other Italian areas, to understand the
interactions of the yeast-cell with the reptile mucosal
surfaces (critical step in the pathogenesis of invasive
process) and to evaluate the potential risk of human
transmission, since most of reptiles are farmed in
domestic habitat.
P232
On the presence of yeasts in the external ear
canal of healthy pigs
A. Moretti,1 L. Boncio,2 V. Vidotto,3 E. Bollo,4
R. Salvatori,5 F. Agnetti,6 M. Sensi6 and V. Grelloni6
1
Department of Pathobiological Sciences and
Hygiene, University of Perugia, Perugia, 2Department
of Medical and Surgical Specialties and Public Health,
University of Perugia, Terni, 3Department of Medical
and Surgical Disciplines, 4Department of Animal
Pathology, University of Turin, Turin, 5Veterinary
Clinician and 6State Veterinary Institute, Perugia, Italy
Clinical cases of otitis externa, media and interna in
pigs were observed and different etiological agents
were isolated and their role was evaluated. Some
Authors already reported clinical cases of mycotic
external otitis in fattened pigs and in their mothers
associated to yeasts, Malassezia pachydermatis (86.1%
of cases) and Candida genus particularly. Considering
the poor literary data, the Authors, in this preliminary study, examined samples of auricular swabs
collected from healthy swine, belonging to different
management breeding, in order to establish the
occurrence of the yeast flora. Samples of ear wax
from external ear canals of 100 pigs were collected,
using sterile cotton swabs, inoculated onto Sabouraud glucose agar and m-Dixon agar media, and
incubated at 26 C for 2 weeks. For the identification
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
of the yeast species, morpho-physiological characteristics were considered. Twenty-seven percent of the
animals examined were positive for yeasts. Within
positive swine, the following yeasts were isolated:
M. pachydermatis (14.8% in sows), Candida albicans
(37%), C. tropicalis (29.6%), C. guillermondii and
C. krusei (18.5%); C. albicans was singularly isolated
in the 50% of cases and in association with
C. tropicalis, C. guillermondii and C. krusei in the
remaining 50%. Yeasts of the genus Malassezia are
known to be components of the microflora of human
skin and of many warm blooded animals. From
1959, when it was first reported the presence of the
genus Pityrosporum in swine, the values of prevalence observed in most recent studies are variable and
the species isolated are different (M. furfur, M. sympodialis, M. slooffiae) since the taxonomic revision in
1996. The carriage of M. pachydermatis to pig was
also reported in normal skin. Further studies on a
greater number of pigs will be necessary to establish
the prevalence of these lipophilic yeasts, both in ear
canal and skin, with the aim of understand their
spread capability and their responsibility for clinical
cases of otitis and dermatitis. The presence of a
fungal species in the auricular canal may be justified
considering the biological relations that the
microorganism develops with the host and the
environment where animals live, as a source of
contamination; this environment, together with the
geographical and seasonal variations and the lipidic
composition of the ear wax are important factors,
influencing the ear mycotic flora ecosystem.
controls. A total of 8.3% of diabetics had clinical
evidence of oral candidosis, of which, 36% wore their
dentures overnight and were tobacco smokers. None
of the controls had any clinical evidence of oral
candidosis. Positive yeast was detected in 58.3% of
diabetics compared with 30% in healthy controls
(P < 0.001). C. albicans was the most prevalent species in both diabetics and controls (81.8% and 76.9%,
respectively) followed by C. tropicalis, C. parapsilosis
and C. glabrata. Candida kefyr and C. krusei were
isolated from diabetics but not from controls at a
combined rate of 1.3%. All C. albicans recovered from
diabetics and controls were susceptible to amphotericin B, ketoconazole, itraconazole and fluconazole.
Non-albicans Candida isolates were shown to have
higher azole MIC values than C. albicans isolates.
Candida was detected more frequently in diabetic
denture wearers than in control counterparts in all
anatomic sites sampled (P < 0.05). The prevalence of
Candida was significantly higher in diabetics (both
denture wearers and dentate patients) compared with
that in the respective control group (P < 0.05).The
frequency of Candida isolation was significantly higher
in smokers than in the non-smokers both in diabetics
and controls (P < 0.001).Our findings are in line with
previously published data regarding oral candidosis in
diabetic patients.
P241
1
Prevalence of oral Candida infection in diabetic
patients
K.H. Abu-Elteen and M.A. Hamad
Department of Biological Sciences, Hashemite
University, Zarqa, Jordan
The prevalence, species distribution and antifungal
susceptibility profile among Candida species isolates
from the oral cavity of Jordanian diabetic patients was
determined using the imprint culture method. The
contribution of smoking and dental status to the
prevalence and distribution of Candida species was also
evaluated. A total of 262 individuals were enrolled in
the study, 132 were diabetics (47 males and 85
females) and 130 (44 males and 86 females) healthy
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
P242
Frequency of otomycosis in ENT clinic of
Baqiyatallah (A.S) Hospital
M.A. Afshari,1 R. Kachooii2 and M. Ajalooieyan2
Microbiology and 2E.N.T, Baqiyatallah, Tehran, Iran
Otomycosis is one of the fungal infections of the
external ear canal seen in the subtropical area of
the world. The most common fungus which affects
the external ear tropical canal is Aspergillus and
Candida albicans. The early symptom in such patients
is pruritus. This infection is treated by intensive
cleaning of the external ear and administrating of
suitable drugs. This research which lasted 2 years
(2002, 2004), is about patients who referred to ENT
clinic (23 cases were male and 12 cases were female),
Otomycosis was diagnosed in these patients by
clinical assessment and confirmed by fungus laboratory tests. Clinical diagnosis was based on clinical
symptom and observing the fungus component in
external ear canal by Otoscopy. The lab specimen
149
xxx
was taken by wet soap with strile physiologic serum
or washed with strile physiologic serum and cultured
on the two types of cultured field. The results shows
that 20 patients out of 35 cases (57/1%) suffered
Otomycosis which were 12 person (34/3) male and
eight person (22/8) female. Most of the cases were
between 30 and 40 years old. The most common
fungus component showed as following. Candida
albicans five cases (15/7%). In recent study the
percent of patients which suffered from Otomycosis
was 57/1. It shows that this disease can not
diagnosed entirely by clinical symptom, thus libratory diagnosis’ is essential.
Keywords: otomycosis, Aspergillus, C. albicans, Baqiyatallah hospital.
P243
Deep-seated fungal diseases in immunocompromised patients in Iran
S.H. Bassiri Jahromi and A.A. Khaksar
Pasteur Institute of Iran, Medical mycology, Tehran,
Iran
During the last two decades or so, the incidence of
fungal infections has increased dramatically. Deepseated mycoses are creating serious problems for
clinicians working with certain populations of
patients, such as those with cancer, the immunocompromised, and physiologically compromised. A
study of fungal isolated for identification of deep
fungal infections, risk factors and etiologic agents in
immunocompromised patients was carried out in the
section of Medical Mycology, Pasteur Institute of Iran
from 1994 to 2001. Eighty two immunosupress
patients with deep fungal infection were retrospectively analyzed for etiology and risk factors. They had
one or more predisposing factors to disseminated
fungal infections. Diagnosis was established by
demonstration of fungus in direct and cultural
examinations. Candida spp. were isolated in 67%
(36.5% C. albicans and 30.5% non-albincans), and
Aspergillus spp. were isolated in 15% of cases. The
most frequent risk factors were hematologic malignancy (ALL, lymphoma, hodjekin, multiple myloma)
and diabetes mellitus. This study suggest that in
immunocompromised patients, fungal infections
especially in saprophytic infections, back ground
evaluation and clinical future, correspondence of
150
clinical symptoms and laboratory examinations
should be considered and investigation of other
factors which created the infection will lead us to a
clear picture of patient situation.
P244
Epidemiologic surveillance of cutaneous fungal
infection in Tehran, Iran from 2000 to 2004
S.H. Bassiri Jahromi,1 A.A. Khaksar,1 G. Sadeghi1 and
A. Amir Khani2
1
Medical Mycology and 2Epidemiology, Pasteur
Institute of Iran, Tehran, Iran
Background: Cutaneous fungal infections are common in Tehran, Iran, and causative organisms
include dermatophytes, yeasts, and non-dermatophyte
molds. These organisms are in constant competition
for their particular environmental niche, often
resulting in the emergence of one or more predominant pathogens and displacement of other less
competitive species. Changes in the incidence of
fungal pathogens can be followed from laboratory
culture results of infected cutaneous tissues over
time. These data can be used to ascertain past and
present trends in incidence, predict increases in
antifungal resistance and the adequacy of our
current pharmacologic repertoire, and provide
insight into future developments.
Objective: This study identifies epidemiologic trends
and the predominant organisms causing superficial
fungal infections in Tehran, Iran.
Methods: A total of 13502 specimens were collected from clinically suspected tinea corporis, tinea
cruris, tinea capitis, tinea faciei, tinea pedis, tinea
manuum, and finger and toe onychomycosis from
2000 through 2004. Specimens were submitted to
the Medical Mycology section, Pasteur Institute of
Iran in Tehran, for fungal culture and identification,
and the incidence of each species was calculated.
Results: Dermatophytes remain the most commonly
isolated fungal organisms except from clinically
suspected finger onychomycosis, in which case
Candida species comprise >70% of isolates. Epidermophyton floccosum remains the most prevalent fungal
pathogen, and increased incidence of this species was
observed in tinea cruris. As the casual agent of tinea
pedis and toenails. T. rubrum continues to increase in
incidence.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
Conclusion: Consideration of the current epidemiologic trends in the incidence of cutaneous fungal
pathogens is of key importance to investigational
effort, diagnosis, and treatment.
P245
Fungal infections in Germany: results of the
epidemiological study of the National Reference
22 Centre of Systemic Mycoses
M. Borg-von Zepelin, R. Rüchel, M. Weig, U. Reichard
and U. Groß
National Reference Centre for Systemic Mycoses,
University Clinics, Institute of Medical Microbiology,
Göttingen
Fungal infections have increased during the last
decades. The epidemiological data mostly have been
evaluated in the USA, only few European studies are
available and no German study has been published
so far. Since July 2004, the National Reference Centre
of Systemic Mycoses (NRZ) has started a survey on
fungal infections in Germany to get insight in the
epidemiological situation of systemic fungal infections. Therefore clinical laboratories of the whole
part of the country have been requested to send
fungal isolates coming out of primary sterile materials to the NRZ together with information on the
patients. All fungal isolates have been differentiated
according to standard procedures and testing for
antifungal drug susceptibilities were performed by Etest according to the manufacturer and have been
tested according to the NCCLS procedures for
confirmation.
23 Results and conclusions: More than 400 fungal
isolates have been included in this study until now.
Fifty-seven percent of the isolates were identified as
C. albicans, followed by C. glabrata (21%), C. parapsilosis (6.1%), C. tropicalis (5.5%) and C. krusei (1.3%).
However, the percentage of non-albicans species with
43% is high. Thirteen percent of all fungal isolates in
this survey showed decreased susceptibility to fluconazole (MIC > 32 mg l–1). The study additionally
provides further data on the situation on antifungal
drug susceptibilities in Germany. With selected
strains the results of the E-test will be compared to
data generated by the NCCLS procedures. These
results underline the importance of fungal infections
in Germany, and confirm the high percentage of nonalbicans species.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
P246
Candidemia trends in an adult intensive care
unit
J. Garbino,1 P. Rohner,1 J.A. Romand,2
K. Bouchuigui-waf3 and D. Lew1
1
Division of Infectious Diseases, 2Surgical Intensive
Care and 3Microbiology Laboratory, University of
Geneva Hospitals, Geneva, Switzerland
Background: A large proportion of nosocomial
infections are acquired in intensive care units (ICUs).
Candida spp. are the most important non-bacterial
nosocomial pathogens, and candidemia carries a
high risk of mortality in critically ill patients. Candida
spp. are responsible for the majority of nosocomial
fungal infections.
Methods: Hospital medical records of adult patients with candidemia were reviewed from January
1989 to December 2004. Demographic information, overall mortality, and predisposing factors
for the development of fungal infections were
retrieved.
Results: A total of 404 episodes of candidemia
were reviewed. 140 (35%) developed in critical
care: 87 (62%) in surgical ICU and 53 (38%) in
medical ICU. The median age of patients with
candidemia was 58 years (range 17–92); 87 (62%)
were males. Eighty-five patients (60%) died within
30 days. Candida albicans was responsible for 72%
(n = 101) of infections acquired in ICUs. Nonalbicans Candida species (28%) were equally distributed throughout the study period. C. glabrata was
identified in 14 episodes, followed by C. parapsilosis
(n = 7), C. tropicalis (n = 5), C. krusei (n = 2),
C. guilliermondii (n = 1), C. lusitaniae (n = 1),
C. pseudotropicalis (n = 1), and unspecified Candida
species (n = 9). No change in the distribution of
C. albicans versus non-albicans Candida species
identified over years was noted. We observed a
trend toward a decrease in candidemia from 1989
to 1994 (n = 77) to 1995–2004 (n = 62). Most
strains of C. albicans (94%) remained highly sensitive to fluconazole.
Conclusion: Fungal infection remains a severe
disease associated with high crude mortality. We
observed a trend toward a decrease in candidemia
during the study period. C. albicans was the most
commonly isolated species, and no shift was observed
towards non-albicans Candida species.
151
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P247
Phenotypic and genotypic analysis of Candida
sp. responsible for infections in liver transplant
recipients in Children’s Memorial Health
Institute
that some infections were caused by hospital
isolates.
P248
Isolation and identification of yeast species in
B. Garczewska, W. Kaminska and D. Dzierzanowska 24 throuth cultures of Iranian soldiers
The Children’s Memorial Health Institute, Clinical
Microbiology and Immunology, Warsaw, Poland
M. Ghahri
Tarbiat Modarres University, Medical Mycology,
Yeasts are the most frequent etiological factors of
Tehran, Iran
fungal infections in hospitalized patients. Due to
many risk factors, such as immunosupression,
A total of 568 Iranian soldiers went under study for
prolonged antibiotic therapy, presence of central
isolation and identification of yeast flora of oral
venous catheters or long hospital stay, liver
cavity. A sterile swab was used for each individual
transplant recipients are especially susceptible to
and specimens collected from nasopharynx area,
opportunistic infections caused by Candida sp.
then inoculated to Petri dishes containing Sabouraud
Although Candida albicans is still the most common
Dextrose Agar and incubated for 48 h at 37 C. All
fungal pathogen in transplantation settings,
colonies counted and stocked in distilled water until
increasing rates of infections caused by other
further analysis. Yeasts identified by disk diffusion
species, such as Candida tropicalis, Candida parapsitest as a technique published in Journal of Medical
losis, Candida glabrata and Candida krusei have been
Microbiology, Vol. 20 (1985) by Sobczak. The results
observed in recent years. This change in etiology is
were compatible with Api 20C AUX as much as
accompanied by an increasing resistance to anti95.3%. A number of standard strains collected from
fungal agents, especially to azole drugs. The aim of
Iranian type culture collection analyzed as a conthis work was to analyse phenotypes and genotypes
firmatory tests. 51.4% of Petri dishes were positive
of Candida isolates responsible for colonization and
for yeast species and 318 isolated Yeast colonies
infections in liver transplant recipients. A total of
identified. C. albicans, Candida pseudotropicalis, Candida
85 strains of Candida sp. isolated from various
tropicalis and Candida guillermondi were the frequent
clinical materials (blood, urine, BAL, CSF, bile,
yeast species isolated from throuth of soldiers.
venous catheters, wound and mucosal swabs) were
cultured from seven children. Isolates were identified by biochemical reactions (API ZYM and ID 32
P249
C tests, bioMerieux). Antifungal susceptibility testThe study of dermatophytosis in wrestlers and
ing was performed according to NCCLS recommenits relationship with wrestling
dations. All isolates were subjected to genotyping
by RAPD method. The most frequent cause of
J. Hashemi, A. Nasrollahi and P. Hossainjani
systemic fungal infections in our patients was
Department of Mycology, Islamic Azad University,
C. albicans. In two patients during fluconazole
treatment resistance to this agent and other azole 25 Tehran, Iran
drugs emerge (fluconazole MIC 32–64 mg l–1).
Mats were carried on 500 wrestles in 37 wrestling
Genetic analysis revealed clonal and endogenous
salons during 6 months in Tehran. There were 144
character of fungal infections. In three patients
doubtful cases to dermatophytosis and 188 samples
fungal infections were confirmed by mucosal colwere collected from different regions of their bodies.
onization of the pharynx and/or anus. Comparison
Classic method (scraping) was used for sampling of
of RAPD patterns of Candida parapsilosis cultured
dermatophytic lesions. Seventy-four samples were
from patients and hospital environment showed
152
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
collected from 37 mats include 37 samples by
mouquette (a kind of carpet) and 37 samples by an
initiatively method (direct culture). Direct culture has
been done by a cordless vacuum cleaner. The
laboratory examination showed that 171 skin scrap
out of 188 specimen were positive for dermatophyte
agent (90.96%). Out of them 133 wrestlers (26.6%)
had Tinea corporis gladiatorum. Four cases of mats
(10.81%) had contamination to Dermatophytes.
Trichophyton tonsurans was the only dermtophyte
that was isolated from wrestling and wrestling mats.
Highest contamination rate on the basis of weight in
the group of 76–80 kg was (34.15%) and on the
basis of age in the group of 16–17 was (31.50%).
Chi-squared test revealed a statistical difference
between the two groups of wrestlers with regarding
to continual contact lesion location with body of
contestant or surface of mat, similar lesion in exercise
or competitive constant, continual contact with
water and treatment and relapse history
(P < 0.01). In the other wise, chi-squared and
Fisher’s exact test did not reveal a statistical
difference between the two groups (positive and
negative cases) with regarding to using of sauna,
external and internal travel and contact with
26 animals (P < 0.05) In this survey for the first time
cordless mini vacuum cleaner method for sampling of
Mat surface was used and isolated colony count were
compared mouqette (a kind of Carpet). The student
test showed that there was a significant difference
between the two method. Therefore it means the
sampling by this instrument is better than classic
method.
various fungal diseases in cases admitted to Yazd
central laboratory from 2002 to the end of 2004.
Sampling had been performed from the patients, who
referred to this laboratory by the physicians. After
clearing the specimen in 10–20% KOH and lactophenol, microscopic exam of direct smears, and if requested by the physician, culture on Sabouraud’s dextrose
agar and Mycosel agar (SC and SCC) had been done.
The demographic data and results of tests on 3744
such cases were evaluated and analyzed by SPSS Win
software. Of total 3744 cases, 1508 cases (40%) were
positive for superficial and epidermal fungal agents,
including 855 males and 653 females. There was a
significant (P = 0.0) difference between two sexes
regarding the relative frequencies of infections. Dermatophytosis (43.6%) and tinea versicolor (33.4%)
were the most common infections, and erythrasma,
candidiasis, other yeasts, and saprophytes comprised
other infections in decreasing order of frequencies. The
most commonly afflicted age groups were
20–29 years (26.7%) and 10–19 years (26%).The
least commonly infected persons belonged to
‡60 years old group. Upper extremities (17.8%) and
axilla (1.6%) were the most and least commonly
infected areas, respectively. Only 169 cases (17%) of
966 requested cultures were positive, and the zoophilic
fungus trichophyton verrucosum (26.6%) was the most
frequently isolated organism. Trichophyton schoenleinii
(1.2%) had the least frequency.
Keywords: fungal infections, frequency, Yazd.
P251
Study of the airborne fungi in Yazd
P250
A 3-year fungal infections in cases admitted to
Yazd central laboratory
A.A. Jafari,1 M.H. Hekmati1 and M. Ahmadiah2
1
Department of Paramedicine and 2Health
Department, Yazd Medical University, YAZD, Iran
Cutaneous fungal infections are among the relatively
common mycoses, comprising a considerable percent
of admissions to physicians, especially dermatologists.
Knowing the frequency, distribution, and geographic
status of this disease can be helpful for planning of
control and prevention of them by the health system
managers. The objective of this descriptive, retrospective study was to determine the relative frequencies of
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
A.A. Jafari nodoushan1 and M.H. Ahmadiah2
1
Paramedicine Department and 2Public Health
Department, Yazd Medical University, YAZD, Iran
The general purpose of this study was to evaluate the
type and frequency of fungal spores in different areas
of Yazd, one of the industrial cities of Iran. The
stratified sampling method was used for collecting of
air samples from different areas (urban, rural and
industrial areas) of Yazd using the Anderson single
stage Impactor. From 216 collected samples in
different areas and in different seasons, totally
8320 colonies from 21 genuses of fungi were isolated
that Aspergillus (18.2%), Penicillium (15%) and
alternaria (12%) were the most prevalent isolated
153
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fungi. Cladosporium, yeasts, Rhizopus, Mucor, Fusarium, scopolariopsis and other fungi were another
isolates. Maximum and minimum contamination
rates were seen at noon and the early morning
respectively. Also the highest rate of allergen fungi
was seen in the industrial areas in compare with the
rural and urban areas (t = 6.9, and P £ 0.001). Our
results showed that the average of colony forming
units (CFU) from each sample in autumn was more
than the other seasons that there was seen a
statistical differences between autumn and winter
(t = 2.6, P £ 0.001). In stormy days especially in
summer the rate of fungal spores and thermophilic
fungi was dramatically higher than normal days.
Keywords: airborne, fungal spores.
P252
Occurrence and genotype analysis of Candida
strains repeatedly isolated from the same
patients
B. Krasnode˛bska-Szponder, L. Szymaniak,
I. Wojciechowska-Koszko, M. Mnichowska and
S. Giedrys-Kalemba
Department of Microbiology and Immunology,
Pomeranian Medical University, Szczecin, Poland
fistula, endotracheal tube and bedsores. For species
identification biochemical tests ID32C were used.
Genetic relatedness of isolates was examined with
PCR-RAPD method using various primers for each
examined species.
Results: The presence of nine species of Candida has
been found: Candida parapsilosis – 38 strains, Candida
glabrata – 33 strains, Candida albicans – 28 strains,
Candida tropicalis – six strains and Candida norvegensis, Candida lusitaniae, Candida kefyr, Candida dubliniensis and Candida sake totally making eight strains.
The largest number of genotypes was observed
among C. albicans species: 11 genotypes and three
unique strains. A diversity of genetical profiles
among the other species was smaller: Candida
parapsilosis – 8 genotypes, four unique strains,
Candida glabrata – five genotypes, six unique strains,
Candida tropicalis – two genotypes. A large number of
genetical types were common for strains from
different patients.
Conclusion: A great species and genetic diversity of
isolated Candida spp. strains was affirmed. Strains of
the same genetic type remained for some time at the
unit; they colonized the same patients and caused
exogenous infections of endemic or even epidemic
character.
P253
Introduction: More frequent infections, especially
with Candida genus fungus, have been observed last
years by both clinicians and mycologists. It is
connected not only with development of mycological
diagnostics but also closely related to wide use of
antibiotics, immunosuppressive drugs and development of such fields as intensive care, cardiac surgery
or transplantology.
Objective:
The aim of this study has been an analysis of
species and genotype diversity of Candida spp. strains
isolated from repeated collections from the same
hospitalised patients at the intensive care unit.
Material and methods: One hundred and thirteen
Candida strains spp. have been analysed. The
strains were taken at an intensive care unit of
Independent Clinical Hospital No 2 in Szczecin,
Poland. They were isolated in the years 2000–
2001 from physiological sites of their colonization
– oral cavity, anus, navel, interdigital space
between the toes, vagina, from various clinical
samples – urine, BAL, or obtained from insertion,
154
Prevalence and genotypes characterization of
Pneumocystis jiroveci colonization among
Spanish cystic fibrosis patients with and without lung transplantation
F.J. Medrano,1 C. de la Horra,1 M.A. Montes-Cano,1
J. Dapena,2 N. Respaldiza,1 I. Mateos,3 J.M. Varela1
and E.J. Calderon1
1
Internal Medicine, 2Paediatrics and 3Microbiology,
Virgen del Rocio University Hospital, Seville, Spain
Background: Cystic fibrosis (CF) is the most
common serious hereditary disorder in the Caucasians population. The chronic bronchopulmonary
damage related with infection by opportunistic
pathogens is the most serious clinical problem
among these patients. The chemoprophylaxis of
these infections and, recently, the lung transplant
has improved the prognosis and the survival of the
CF patients. In recent studies Pneumocystis jiroveci
has been identified by nested-PCR in the respiratory
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
tract of CF patients. However, there are many
aspects unknown about the epidemiology and
clinical relevance of P. jiroveci infection in CF and
the utility of chemoprophylaxis against P. jiroveci in
these patients. The aim of this study was to provide
information about the prevalence and genotype
distribution of P. jiroveci colonization, among CF
patients from Spain.
Methods: A cross-sectional study was performed to
identify by nested PCR the mt LSU rRNA gene of
P. jiroveci in respiratory samples from CF patients;
the genotype was analyzed by direct sequencing.
Results: The overall prevalence of P. jiroveci colonization among 100 consecutive CF patients was 24%.
The rate of colonization was higher in patients with
lung transplant (LT) when compared without LT
recipients (41.5% vs. 21.5%). The polymorphisms
85C/248C (42.8%), 85T/248C (28.5%) and 85A/
248C (21.4%) were identified in our CF population;
in one case a mix of genotypes was founded.
P. jiroveci colonization was founded in five out of
14 (37.7%) patients receiving chemoprophylaxis
with trimetroprim-sulfamethoxazol and in five out
of 29 (17.2%) patients treated with azithromycin.
Interesting, none of them developed Pneumocystis
pneumonia during 2 years of follow-up.
Conclusions: There is a high prevalence of P. jiroveci carriers among CF patients in Spain. The result of
this study suggests that chemoprophylaxis with
cotrimoxazol and azithromycin may prevent Pneumocystis pneumonia but not avoid colonization.
microbiology culture, with special stains and conventional cultures, then identifying the organisms by
morphology and with automated systems. In this
period, 893 patients with fungal infections were
evaluated, 56.7% female and 43.3% female. Mean
age was 39.7 ± 24.9 years old, 11.5% belong to the
group <10 years old. Most common clinical mycotic
infection was urinary tract infection (34.2%)
followed by vascular catheter-related infection
(11.4%), vaginal infections (10.8%), lower respiratory tract infections (10.8%), fungemia (7.6%),
among others. In these patients were identified 17
species of fungi, for a total of 1002 strains: Candida
albicans (47.3%), Candida sp. (45.0%), Torulopsis
glabrata (2.0%), C. parapsilosis (1.1%), T. candida
(1.1%), C. tropicalis (1.0%), C. intermedia (0.6%),
Cryptococcus neoformans (0.6%), Aspergillus sp.
(0.4%), C. guillermondii (0.2%), C. catenulata (0.1%),
C. krusei (0.1%), C. lusitaniae (0.1%), Mucor sp.
(0.1%), Sporothrix schenckii (0.1%), Trichophyton
mentagrophytes (0.1%), Trichosporon sp. (0.1%). The
presence of Candida species in the urine is frequent
among hospitalized patients. It represents a major
challenge to the physician because it is unclear
whether candiduria represents colonization or infection, whether the bladder or the kidney is involved in
infection, or whether it represents a surrogate
marker for systemic infection. This picture is more
complicated because there are few prospective studies
addressing the issue of when and how to treat a
patient with candiduria, possibly with fluconazole as
the drug of choice, provided the agent is not a
resistant species.
P254
Epidemiology of fungal infections in a general
hospital of Caracas, Venezuela
C.N. Rodriguez,1 A.J. Rodriguez-Morales,2
A. Garcia,1 B. Pastran1 and P. Meijomil1
1
Microbiology, West General Hospital, Caracas and
2
Los Andes University, Center for Research JWT,
Trujillo, Venezuela
Fungal infections are emergent diseases in hospital
institutions. Increase on immunosuppressive diseases
and conditions have been influencing the epidemiological pattern of mycoses in hospitalized patients.
For these reasons we studied the epidemiology of
fungal infections in a general hospital of Caracas,
Venezuela, in a 10-year period, between 1992 and
2003. All clinical samples were processed at the
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
P255
Superficial and cutaneous mycoses among referal patients in Kermanshah Medical Mycology
Lab 2002
27 Nazari
Microbiology, Ali Mikaeili, Kermanshah, Iran
Dematophytosis and pityriasis versicolor are frequent mycoses in human. In this study, 1 year
positive superficial and cutaneous mycoses were
collected and following result were obtained: all of
referal persons are 1800, dermatophytosis with
36% had frequent diseases and pityriasis versicolor
12%, erytherasma 7%, trichomycosis 2%. In this
study dematophytosis more frequent among chil-
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dren 0–10 age and pityriasis in 30–40 years old
group.
P256
Dermatophytoses in children: 5 years experience
H. Papavasileiou, I. Varzakakos, A. Makri,
L. Papavasileiou, K. Avgerinakou and A. Vogiatzi
Clinical Microbiology, Penteli Children’s Hospital,
Athens, Greece
Introduction: Dermatophytoses are superficial
mycoses caused by fungi that can invade stratum
corneum and keratinized tissues. The incidence is
higher in children living in closed communities
(nursing homes, schools) and those who frequently
come in close contact with soil and animals.
Purpose: The aim of this study was to determine the
epidemiological profile of dermatophytoses in children who attended the outpatient clinic of our
Hospital.
Material and Methods: During a 5-year period
(2000–2004) 830 samples were obtained from
suspicious lesions of the skin, hair and nails in
children 6–15 years old. The material was taken
from the periphery of the skin lesion by scraping it
off. The samples were examined by direct microscopy, using 20% potassium hydroxide solution and
staining with methylene blue. At the same time,
the specimens were cultured in Sabouraud-Chloramphenicol agar and Dermatophyte-agar (Dermato-T, BioMerieux). Identification of the fungi was
based on their microscopic characteristics (structure,
micro-macroconidia)
and
macroscopic
morphology of the colonies (texture and surface
color).
Results: Out of 830 children, fungi isolated in 220
(26.5%). The isolated fungi were, 194 (88%) dermatophytes, 15 Candida spp. (6.9%), 10 Malassezia
furfur (4.6%) and 1 Fusarium spp. (0.5%). The most
frequently isolated dermatophytes were, Microsporum
canis (48.5%), Trychophyton mentagrophytes
(11.3%), Trychophyton tonsurans (10.3%) and Trychophyton violaceum (7.7%). Girls suffer more
frequently (59.8%). Skin was more frequently affected by fungal infections (77.3%), than scalp (16.5%)
or nails (6.2%). The higher incidence of these
infections was during the summer and autumn
months.
156
Summary: Microsporum canis was the most frequently dermatophyte isolated. Girls are more frequently affected than boys. It was found that 70% of
the children infected had come in contact with
animals (cats, dogs). We believe that is essential to
treat the affected children as well as the animals
which come in close contact with them, in order to
minimize these infections.
P257
Fusarium solani keratitis as cause of a double
corneal transplant – report of a case
M.D. Pinheiro,1 P. Alves-Faria,2 A. Gonçalves,2
R.J. Moreira2 and F. Falcão-Reis2
1
Laboratory of Microbiology and 2Department of
Ophtalmology, Hospital de S. João, Porto, Portugal
Fusarium solani and other fungi such as Penicillum
spp. and Aspergilluss spp., are agents of hyalohyphomycosis, a fungal infection that presents with
hyaline, light-coloured, hyphal elements, branched
or unbranched and without pigmented walls.
Fusarium spp. grow quickly in different culture
media, producing colonies displaying a variety of
colour and texture. Microscopically, septate hyaline
filaments, 3–8 lm in diameter are observed; the
production of fusoid macroconidia and microconidia are characteristics of the Fusarium genus. It is a
common soil saprophyte and causes a broad
spectrum of diseases in humans, from minimal
local invasion to disseminated disease. It is frequently involved in eye pathology when ocular
trauma is a predisposing factor. In fact, Fusarium
spp. are the most common cause of fungal keratitis
worldwide. Here, we report a case of Fusarium
keratitis in the setting of a double corneal transplant. In May 2004, a 14-year-old girl complained
of decreased vision, pain and photophobia of the
right eye. A necrotizing keratitis with deep stroma
involvement was diagnosed; no ocular trauma was
referred. A topical treatment with ofloxacin and
dexamethasone 0.1% started, four times a day. Six
days later, she returned to hospital with similar
complains; there was evidence for an increase of
the stroma infiltrate, a biopsy for cultural exams
was made and topical trifluridin and clotrimazole
were added. While the culture revealed negative,
despite intensive treatment, vision deterioration
continued. The patient was then operated to
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
implant a penetrating corneal graft. The cornea
was put in liquid culture media and sent to the lab
for cultural exams. After 24 h at 35 C, the liquid
was subcultured to Sabouraud-chloramphenicolgentamicin at 25 C; 2 days later colonies were
observed with morphologic features of Fusarium
spp., later identified as Fusarium solani. Susceptibility testing to amphotericin, itraconazole, caspofungin and voriconazole (VO) resulted in minimal
inhibitory concentrations of 0.5 lg ml–1, 16 lg ml–
1
, 16 lg ml–1 and 8 lg ml–1, respectively. In spite
of the therapy, re-growth of the fungal infiltrate in
the corneal interface and anterior chamber occurred, and 2 weeks later a second penetrating
corneal graft was transplanted. The anterior chamber was previously irrigated with VO and the
corneal graft submersed in VO for 20 min, before
transplantation. Therapy continued with topical
and systemic VO for 3 months. One year after, the
final vision was reduced to 20/30. It was not
possible to establish a predisposing factor to the
onset of the infection but the initial antibiotic and
steroid treatment is likely to have contributed for
the adverse evolution. Nevertheless, the case
stresses the importance of submitting suspicious
samples for thorough microbiological evaluation
in order to plan a well grounded therapeutic
strategy.
P258
Epidemiology of fungaemia in a general hospital in spain
V. Pulian Morais,1 C. Caro Narrillos,2 A. Figueira
Vazquez,3 P. Alvarez Garcia,1 M. Hernandez Blanco,1
A. Pascual Duran1 and M. Garcia Campello1
1
Microbiology, C.H. Pontevedra, 2Internal Medicine,
C.H. Pontevedra and 3C.H. Pontevedra, Laboratory,
Pontevedra, Spain
Objectives: To describe the characteristics of candidemia episodes, the distribution rate and susceptibility pattern of Candida spp. isolated in blood
cultures during 2004 at a tertiary care hospital in
Spain.
Material: A retrospective study of those blood
samples processed for fungal culture from patients
with suspected invasive fungal infection during
2004 was performed. Fungaemia was defined as
isolation of fungus at least from one blood culture
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
with in a 21 day period. Blood cultures were
developed in Bactec System 9240 and when a 7-day
period was finished a subculture in Candida ID2
agar plates at 35 C was realized. The isolated were
identified by colony morphology and by ID32C.Antifungal MIC was carried out by Fungitest for
Amphotericin B, 5-Fluorocytocin, Fluconazole and
Itraconazole. Medical charts were reviewed to
record clinical and demographic characteristics
presented before collection of the first blood sample
positive for Candida spp.
Results: A total of 1009 bloodstream cultures from
192 patients were studied (114 males, 78 females).
All patients were adults except for one preterm
newborn. 18 (9.3%) funguemia episodes were
observed in 17 patients; in two patients a multiple-species candidemia was detected (Candida albicans and Candida parapsilosis); one patient had two
different episodes with Candida parapsilosis and
Candida krusei in each one. The species distribution
was: Candida parapsilosis 10 (50%), C. albicans six
(30%), Candida glabrata two (10%), Candida tropicalis
one (5%) and Candida krusei one (5%). Underlying
disease was haematological malignancy in seven
(41 %) patients, solid tumour in three (17%),
traumatism in two (11%), respiratory insufficiency
in one, diabetes in one, Behcet Disease in one, HIV
infection in one and preterm newborn in one.
Associated risk factors for candidemia included
intravascular catheter (10 central and two peripheric devices) in 15 patients, broad spectrum
antibiotics in 13, admission in ICU in five and
previous surgery in three. The rate of mortality was
53% (9/17).
Conclusions: Candidemia is still a nosocomial
infection associated with high mortality rate. Nonalbicans Candida spp. predominated as etiologic
agents of candidemia. Candida parapsilosis was the
main etiology of candidemia in this study. C. krusei
and C. glabrata were isolated when previous antifungal therapy was undertaken. The use of cromogenic
media was useful to detect mixed candidemia. No
reduced susceptibility to azoles was detected among
non-C. glabrata/krusei isolated as it is already wellknown. The risk factors more frequently associated to
candidemia in our study (intravascular catheter,
antibiotherapy, ICU stay, prior surgery) are in
agreement with published data from other European
countries. Haematological malignancy was the most
frequent underlying disease in our patients. We
reinforce the necessity of continuous epidemiologic
157
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surveillance to follow dynamics of candidemia which
will help to develop effective, preventive and therapeutic strategies.
the causal agent, the anthropophilic species were the
most common dermatophyte of tinea.
P260
P259
Epidemiology of dermatophytosis in Khouzestan province, southwest of Iran
A. Rafiei,1 M. Radmanesh,2 R. Yaghobi,2 M. Mapar,2
M. Omidian,2 S. Rasaei,2 R. Taddion2 and
M.B. Mousavi2
1
Ahwaz Jundishapour University of Medical Sciences,
Infectious and Tropical Diseases Research Center and
2
Ahwaz Jundishapour University of Medical Sciences,
Dematology, Ahwaz, Iran
Cutaneous fungal infection especially dermatophytosis are common in different parts of Iran. Dermatophyte organisms are in constant competition for their
particular environmental niche. These data can be
used for ascertain past and present trends in
incidence and the emergence of more predominant
dermatophytes. The aims of this study were to
identify the epidemiologic trends and causative
organisms of dermatophytosis in Khuzestan province. A total of 2953 specimen were collected from
patients clinically suspected to have fungal infection
from 2002 through 2005. Materials collected from
hair, nail and skin and were investigated by direct
examination and cultured in Sabouraud dextrose
agar and mycobiotic Agar (Difco). Fungal colonies
were identified by macroscopic and microscopic
examination and supplementary tests. The prevalence of dermatophytosis among the suspected
patients was 24.1%. The most predominant types of
infection of dermatophytosis were as follows: tinea
cruris (40.1%), tinea corporis (14.5%), tinea pedis
(14.5%), and tinea capitis (10%) tinea pedis (6.7%),
tinea faciei (6.5%), tinea ungium (6.0%) and tinea
barbae (1.2%). The most frequent dermatophyte
organisms
were:
Epidermophyton
floccosum,
Trichophyton Verrucosum, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton Viollaceum,
Microsporum canis and Trichophyton schoenleinii. The
frequency rate of dermatophytosis was higher in
males than females. The duration of infection had a
wide range from 70 days to 20 years. Age patients
were from 4 months to 85 years old, but
21–40 years age group was found to be the most
common infected group (45.2 %). In conclusion as
158
Prevalence of fungal infections in Tonb isiand
M. Riazipour,1 A. Karimi2 and M.A. Afshari1
1
Mircobiology, Faculty of Medicine, Baqiyatallah
Medical Sciences University and 2Biological Science,
Imam Hosein University, Tehran, Iran
Various factors may affect distribution of fungal
infections including presence of disease sources,
susceptible hosts, and predisposing environmental
conditions. The objective of the present study was to
determine the prevalence of superficial, cutaneous
and subcutaneous fungal infections amongst the
residents of Big Tonb, one of the several islands in
southern Iran. Randomly selected individuals were
clinically examined for presence of fungal lesions.
Suspected lesions evaluated by current mycological
methods namely sampling, direct microscopy and
culturing on appropriate medium. The results
showed that about 40% of the examined individuals
had lesions resembling to superficial and cutaneous
fungal lesions and there was no lesion suspected to
subcutaneous mycoses. Tinea versicolor (4.6%),
pytirosporosis (4.6%), trichomycosis axilaris (4.6%),
and tinea pedis (2.3%) were the only clinically found
mycoses confirmed by mycological methods. It is
concluded that despite the tropical climate and low
sanitary conditions, the prevalence of superficial
mycoses in Tonb Island is not higher than the other
parts of Iran.
Keywords: fungal infections, prevalence, Tonb.
P261
Skin dermatophytosis outbreak at a school
M.L. Rosado,1 C. Verı́ssimo,1 R. Sabino,1 H. Ponte2
and J.C. Brandão1
1
Mycology, National Institute of Health Dr. Ricardo
Jorge, Lisbon and 2Centro de Saúde da Azambuja,
Azambuja, Portugal
The Mycology Unit of the Portuguese National Institute of Health Dr. Ricardo Jorge is a leading public
health institution in Medical Mycology and has an on-
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
going Dermatohphytes Surveillance program running
since 2001. The program aims to determine and track
changes in prevalence and species distribution within
the national territory and includes reported episodes of
academic dermatophytoses of children. During 2004,
an outbreak of dermatophytoses in a school of the
region Lisbon and Tagus was reported. The outbreak
involved 20 students and two teachers with skin
lesions in various body parts. Twelve cases of dermatophytoses were confirmed where one species only was
involved: Trichophyton mentagrophytes. rDNA 26S
(D2 region) sequence analysis confirmed identification
and outbreak agent.
P262
Dermatophytosis in Denmark in year 1993 and
2003
D.M. Saunte,1,2 E.L. Svejgaard,2 M. Haedersdal2 and
M.C. Arendrup1
1
Mycology and Parasitology, Statens Serum Institut
and 2Dermatology and Venerology, Bispebjerg
Hospital, Copenhagen, Denmark
Introduction: Trichophyton (T) violaceum and
T. tonsurans used to represent approximately 45%
of the dermatophyte infections in Denmark in the
beginning of 1900. These dermatophytes disappeared in 1930 but are now isolated again from
skin and hair specimens. In other Nordic countries a
similar change in epidemiology has been observed,
and especially among immigrants with tinea capitis,
T. violaceum, Microsporum (M.) audouinii, T. soudanense and T. tonsurans are raising in numbers. This
study focuses on the species distribution of dermatophytes infections in Denmark during a 10-year
period to investigate if there is a change in aetiology.
Methods: A total of 47.873 samples received for
analysis for dermatophytes at the Statens Serum
Institute, Copenhagen, Denmark in the years 1993
and 2003 were included in the study. The samples
included skin, nail and hair samples from patients
from all parts of Denmark.
Results: Year 1993: all together 20.772 skin specimens were received and 3809 (18%) revealed a
dermatophyte positive culture. The species distribution was as follows: T. rubrum 63% (n = 2391),
T. mentagrophytes 24% (n = 921), M. canis 9%
(n = 346), Epidermophyton (E) floccosum 3%
(n = 96), T. violaceum 1% (n = 38) and T. verrucosum
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
<1% (n = 17). Year 2003: 27.101 skin specimens
were analysed and 5623 (21%) revealed a dermatophyte positive culture. T. rubrum represented 76%
(n = 4260), T. mentagrophytes 15% (n = 816), M. canis 4% (n = 224), E. floccosum <1% (n = 8), T. violaceum 2% (n = 99), T. tonsurans <1% (n = 46), M. audouinii <1% (n = 9), T. verrucosum <1% (n = 8),
T. terrestre <1% (n = 36) and M. gypseum <1%
(n = 5). During this period the frequency of nail
specimens has raised from 18% to 54% of all specimens
analysed. The hair specimen frequency is about the
same.
Conclusion: The epidemiology of dermatophytosis
in Denmark is changing. Notable is an overall increase
of 48% of dermatophyte culture positive samples over
the last 10 years. T. rubrum, which is still the most
frequent species, is increasing, which correlates with
the three fold rise in nail specimens. Infections caused
by T. mentagrophytes, M. canis and E. floccosum are
decreasing and infections with more ‘exotic’ antrophophilic infections such as T. violaceum, T. tonsurans
and M. audouinii are raising in numbers.
P263
African histoplasmosis treated with
itraconazole: a 2-year follow-up
H. Sequeira,1 C. Garcia,2 L. Soares Almeida,3
J.N. Maia e Silva,2 J. Pignatelli,2 A. Pereira4 and
M.A. Marques Gomes1
1
Laboratório de Micologia, 2Serviço de Dermatologia,
3
Laboratório de Histopatologia and 4Serviço de
Infectocontagiosas, Hospital de Santa Maria,
Clinica Universitária de Dermatologia, Lisboa, Portugal
Introduction: Skin lesions are more frequent in
African histoplasmosis than in its American form;
the course of the disease is usually chronic, although
some patients can developed a more rapidly progressive and disseminated infection.
Case report: We describe a case of a 63-year-old
black male born in Guiné Bissau, resident in Portugal
since 1994. The patient had a 6-month history of
granulomatous lesions in the skin of the left eye, nose
and trunk. Some of these lesions were ulcerated with
serous exudates. Blood analyses did not reveal
significant alterations including serology for HIV 1
and 2. Thoracic CT scan revealed pulmonary fibrosis
and axillar, mediastinic and hilar lymphadenopathies. Cutaneous histopathology was suggestive of
159
xxx
cryptococcosis or histoplasmosis. Mycological study –
the direct examination on KOH 20% of scales, crusts,
serous fluid and cutaneous biopsy – showed the
presence of oval and sometimes budding large yeast
cells. Cultures on Sabouraud Dextrose Agar and
Mycobiotic Agar incubated at 24ordm; and 37ordm;,
developed Histoplasma capsulatum var. duboisii, thus
leading us to the conclusion that the patient was
affected by African histoplasmosis. He was treated
with itraconazol 200 mg day–1 P.O. during 1 year,
with total remission of cutaneous lesions and
improvement of the radiologic parameters.
Comments: In African histoplasmosis the relapses
are more frequent than in American form. A long
follow-up period is therefore mandatory before a
patient is considered cured. Our patient was observed
during the last 2 years and there were no clinical,
laboratorial or radiological alterations.
P264
Rhodothorula mulcilaginosa peritonitis in a
patient on ambulatory peritoneal dialysis
H. Sequeira,1 S. Silva,2 P. Silva,2 E. Almeida,2
F. Abreu,2 J. Barbas,2 M. Martins Prata2 and
M. Marques Gomes1
1
Laboratório de Micologia da Clı´nica Dermatológica,
Hospital de Santa Maria and 2Serviço de Nefrologia e
Transplantação Renal, Hospital de Santa Maria,
Lisboa, Portugal
Aim: To report a case of fungal peritonitis by Rhodotorula mucilaginosa (syn. R. rubra) in a patient on
continuous ambulatory peritoneal dialysis (CAPD).
Case report: A 69-year-old man, farmer with endstage chronic renal failure secondary to diabetic
nephropathy, initiated CAPD in February 2004. Two
months after starting this renal replacement therapy,
the patient was admitted to our hospital with complain
of diffuse abdominal pain and cloudy dialysate,
without fever. Mycological study of several samples
of dialysate liquid was studied. Direct examination on
Lactophenol blue showed globose, multilateral budding yeast cells, and cultures on Sabouraud Dextrose
Agar and Mycobitic Agar incubated at 24ordm;,
37ordm; and 42ordm; for 1 week, developed yeast
pink colonies. Auxanografic methods and biological
tests led to its identification as R. mulcilaginosa. In vitro
susceptibility tests were performed and the strain was
sensitive to amphotericin B and 5-fluorocytosine and
160
resistant to itraconazol, ketoconazol and fluconazol.
Initial treatment was started with fluconazol
(200 mg day–1 IP), until mycological results were
available, and then the first antifungus was stopped
and began amphotericin B (1 mg kg–1day–1 E.V.). The
patient became asymtomatic, but after 1 month of
treatment, the cell count in the effluent liquid diminished but didn’t return to normal values. Two dialysate
samples revealed R. mulcilaginosa. The catheter was
removed, the patient initiated hemodialysis without
evidence of peritoneal infection.
Comments: Fungal peritonitis is rare. R. mulcilaginosa is yeast with worldwide distribution and isolated
from skin and other biological products as a saprophyte. However, cases of infection have been reported,
most of them associated with the presence of catheters.
The patient received first fluconazol, and then amphotericin B with some improvement; but removal of the
catheter was probably fundamental for the resolution
of the infection. The origin of the present peritoneal
infection was unknown. Perhaps our patient acquired
the infection through his work as a pharmer.
P265
Fungal colonization and infection in the intensive care unit (ICU): preliminary results of an
epidemiology study
A. Skiada,1 I. Pavleas,2 A. Mitrousia-Ziouva,1
A. Mega,2 K. Rigas,2 P. Zioga,3 D. Madopoulou,1
M. Kaloudioti,2 G. Thomopoulos2 and G. Petrikkos1
1
Research Laboratory for Infectious Diseases and
Antimicrobial Chemotherapy, University of Athens,
2
Laikon Hospital, Intensive Care Unit and
3
Department of Microbiology, Laikon Hospital,
Athens, Greece
Introduction: Fungal infections have been increasing during the last 20 years and Candida is an
important pathogen in the hospital setting and
especially in the ICU. Hyphomycetes may also cause
infections in the critically ill, although not as often.
Aim: To record the species of fungi colonizing
patients in the ICU and producing infections and to
identify risk factors for systemic mycoses.
Material and Methods: Ongoing prospective epidemiological study of the incidence of colonization and
infection by fungi in an ICU, of the predisposing factors
and of the use of antifungal drugs, either as prophylaxis or as treatment. The demographic characteristics
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
of all patients admitted in the ICU are recorded, as well
as the reason of admission, the underlying disease,
disease severity as estimated by the APACHE II score
and possible predisposing factors such as central
venous catheters, antibiotics, corticosteroids and chemotherapy, radiotherapy, diabetes mellitus, neutropenia, renal failure, mechanical ventilation of more than
7 days duration, surgery, trauma and burn. Bronchial
secretions are cultured every week and blood cultures
are drawn whenever there are clinical indications.
Results: The study started on August 2004 and it is
in progress. We report the results of the first 9 months.
Eighty-three patients were admitted to the ICU, of
whom forty had colonization and/or infection by fungi
(48%). One hundred and eighteen cultures of bronchial secretions cultures grew fungi (63 C. albicans, 16
C. tropicalis, two C. krusei, two C. glabrata, four
C. parapsilosis, one C. kefyr, 19 Candida sp., one
Saccharomyces cerevisae, three Aspergillus flavus, seven
A. fumigatus). The hyphomyces were isolated in four
patients and represented colonization. Five patients
developed candidemia (12% of colonized patients),
four by C. albicans and one initially by C. parapsilosis
and subsequently C. krusei. From these patients two
died, but their death was not attributed to candidemia.
Conclusions: Colonization by Candida in the ICU is
approximately 50%, whereas candidemia is much
rarer (6%). From the non-albicans species tropicalis is
the commonest and among the hyphomycetes A. fumigatus. Regarding the predisposing factors no statistically significant results can be drawn at present.
P266
Genotyping of Madurella mycetomatis by
selective amplification of restriction fragments
(AFLP) and subtype-correlation with geographical origin and lesion size
W.W.J. van de Sande,1 R. Gorkink,2 G. Simons,2,3
A. Ott,1 A.O.A. Ahmed,4 H. Verbrugh1 and A. van
Belkum1
1
Medical Microbiology and Infectious Diseases,
Erasmus MC, Rotterdam, 2Department of Microbial
Genomics, Keygene NV, Wageningen, 3PathoFinder
BV, Nijmegen, Netherlands and 4University of
Khartoum, Mycetoma Research Group, Khartoum,
Sudan
molecular typing studies identified Sudanese isolates
of this fungus as clonal, and polymorphic genetic
markers have not yet been identified. Here, we report
on the selective amplification of restriction fragments
(AFLP) analysis of 37 Sudanese clinical isolates of
M. mycetomatis. Out of 93 AFLP fragments generated, 25 were polymorphic of which 12 were found
in a large fraction of the strains. These 12 fragments
were present in at least more than 15% of the strains.
Comparative analysis of the markers resulted into a
genetic tree, composed of two main (clusters I and II)
and one minor cluster (cluster III). Seventy-five
percent of the strains found in cluster I originated
from Central Sudan while in cluster II the origin of
the strains was more heterogeneous. Furthermore,
strains found in cluster I were generally obtained
from larger lesions than the strains found in cluster II
(chi-squared test for trend, P = 0.03). The size of the
lesion appeared strongly associated with duration of
the disease. However, the association between lesion
size and cluster did not decrease after adjustment for
disease duration. No linkage with either cluster was
found for sex or age of the patients, with duration of
disease or with antifungal susceptibility for itraconazole, ketoconazole, fluconazole, voriconazole, amphotericin B or 5-flucytosine. AFLP could also be used
to discriminate two isolates from one patient. This
patient suffered from two independent lesions which
became apparent on different time points. With AFLP
it was possible to show that the isolates were indeed
not identical. Two of the 12 polymorphisms found in
a large fraction of the strains showed sequence
homology with putative virulence genes. Marker A7
was homologous to an endo-1,4-beta-glucanase gene
and marker B4 was homologous to a casein kinase I
gene. Markers A12 and B3 were 97% identical to
each other and were homologous to a protein with
an unknown function. Marker B4 (homologous to a
casein kinase I gene), seemed to be associated with
geographic origin (Fisher’s exact, P = 0.001) and
increased MIC to amphotericin B (Mann–Whitney,
P = 0.02). Unidentified marker A4 seemed to be
associated with lower MIC’s to amphotericin B
(Mann–Whitney, P = 0.02). No associations for the
other markers with geographic origin, lesion size, sex
or age of the patients, the duration of the disease or
the antifungal susceptibilities were found. This is the
first report on genetic markers which may be used to
study pathogenicity in M. mycetomatis.
One of the causative organisms of mycetoma is the
fungus Madurella mycetomatis. Previously, extensive
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
161
xxx
P267
P268
Epidemiology of human cutaneous fungal
infections in Umbria region (central Italy) from
1994 to 2004
Interactive study to evaluate clinical features
and epidemiology of candida vulvovaginitis
L. Boncio,1 F. Agnetti,2 A. Moretti,3 V. Vidotto,4
M. Diamanti1 and M. Papini1
1
Department of Medical and Surgical Specialties and
Public Health, University of Perugia 2State Veterinary
Institute, 3Department of Biopathological Science
and Hygiene, University of Perugia and 4Department
of Medical and Surgical Disciplines, University of
Turin, Torino, Italy
Cutaneous fungal infections are common in Italy,
and etiological agents include dermatophytes and
yeasts. These organisms are in constant competition
for their particular environmental niche, often
resulting in the emergence of one or more predominant pathogens and displacement of other less
competitive species. Changes in the incidence of
fungal pathogens can be obtained from laboratory
culture results of infected cutaneous tissues occurred
over time. Our data can be used to ascertain past and
present trends in fungal incidence in Central Italy.
This study identifies epidemiologic trends and the
predominant organisms causing superficial fungal
infections in Umbria region. A total of 3510 specimens were collected from clinically suspected tinea
corporis, tinea cruris, tinea capitis, tinea faciei, tinea
pedis, tinea manuum and finger and toe onychomycosis from 1994 to 2004. Specimens were submitted
to the Mycology Laboratory of Dermatological Section, University of Perugia, (Terni, Umbria), for
fungal culture and identification, and the incidence
of each species was counted. Dermatophytes remain
the most commonly isolated fungal organisms except
from clinically suspected finger onychomycosis,
where Candida species comprise most of the 80% of
isolates. As the causal agent of tinea capitis, Microsporum canis continues to increase in incidence,
achieving exclusionary proportions in Terni’s area.
An increased incidence of Trichophyton rubrum was
observed in toe onychomycosis and tinea pedis.
Epidermophyton floccosum reduced its incidence
through the years. On the contrary, the incidence
of Trichophyton mentagrophytes didn’t change
through the decennium considered. The evaluation
of the current epidemiologic trends in the incidence
of cutaneous fungal pathogens is a key of importance
to investigational efforts, diagnosis and treatment.
162
T.A. Romanovskaya,1 A.Y. Sergeev,2 V.Y. Sergeev2
and B.I. Shpigel1
1
Institute of Allergology and Clinical Immunology and
2
All-Russian National Academy of Mycology,
Moscow, Russia
Vaginal candidosis (VVC) affects significant proportion of women reaching child-bearing age. To study
epidemiology of VVC, anonymous questionnaires
were used several times with varying success, this
experience was limited by several factors. Knowing
the time of first onset of VVC and a rapidly growing
Internet audience in Russia, we used special anonymous questionnaire to examine the features of VVC
at WWW site at http://www.iaci.ru/candida. Main
traffic to this resource was generated by search
engines with queries for trush and candidosis. During
20 months of the resource activity 3784 women
have participated in 18-question online survey.
Special system was used to avoid duplicate answers
or participation of non-target audience. Among
responders, 2108 records were found evaluable and
were further analysed. Of these, 34% were the
citizens of Moscow, 52.3% – citizens from other
regions of Russia and CIS, and 13.7% – of other
regions. Sixty-eight per cent of responders were
employees, 20.2% – students and 11.1% – housewives. Mean age of the patients was
26.5 ± 6.03 years (allowed range 15–60). Among
them, 36.2% reported VVC duration of less than
1 year, 44.2% – from 1 to 5 years, and 19.6% –
more than 5 years. Duration of the disease positively
correlated with symptoms frequency (36.1% – less
than three episodes per year, 15.1% – more than
three, 28.4% – each month and 20.4% – persistent
VVC). Among prominent associated factors, 5.8%
noted pregnancy, 27.7% – treatment with antibiotics, 6.6% – hormonal contraception, 32.4% – other
cause and 27.6% – no cause. About 20% of women
reported Candida balanitis in a partner. Forty-one per
cent of VVC responders reported history of one or
more genitourinary infections. Labial herpes episodes
through year were reported by 44.9%. About 19%
noted the presence of atopic condition and 41.2% –
history of allergic reactions. Thirty-eight per cent of
respondents reported history of VVC management
only as recommended/administered by medical spe-
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
cialist, 15.3% were used to self-treatment and 35.1%
had history of either experience. The responding
audience displayed preference for topical treatment of
candida vulvovaginitis with antifungal formulations
for gynecological use (about 25%) and 30.5%
preferred combination of topical and systemic antifungals. Self-assessment of topical therapy effectiveness was in favor of topical antifungals, yet more
chronic patients reported less confidence towards
topical therapy. Online Internet questionnaires
appear to be useful in examining diseases with onset
in young adulthood and especially superficial fungal
infections in which researchers frequently lack social
and epidemiological data.
P271
Analysis of risk factors for Candida infection in
non-neutropenic patients with lung cancer and
with chronic obstructive pulmonary disease
H. Batura-Gabryel1 and B. Brajer2
1
Department of Pulmonary Diseases and
2
Department of Pulmonary Diseases, University of
Medical Sciences, Poznań, Poland
Neutropenia belongs to the most important risk
factors of Candida infection and candidosis. Sometimes Candida infection and candidosis may be
diagnosed in patients without neutropenia. Study
aim: The analysis of Candida infection risk factors and
oral candidosis in patients with lung cancer (LC) and
in patients with chronic obstructive pulmonary
disease (COPD) without neutropenia. Material and
methods: LC group – 82 patients aged between 35
and 85 (the average of 62 years) before anticancer
treatment and COPD group – 55 patients aged
between 40 and 79 (the average of 60 years). The
stage of the disease in COPD and LC and performance
status in LC were assessed. The identification of fungi
isolated from both groups sputum was made using ID
C 32 test (bioMerieux). There were established the
risk factors with statistically significant influence on
the positive result of mycological examination of
sputum and on the occurrence of the oral candidosis
symptoms (relative risk and 95% confidential interval were calculated).
28 Results: 1. The fungi belonging only to the Candida
species (especially Candida albicans) were isolated and
identified from the sputum 2. Oral candidosis was
present in 22% of LC and in 25.4% of COPD patients
(P < 0.05) 3. To the most important risk factors for
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
Candida infection in all the examined patients dental
prosthesis, antibiotics treatment, COPD diagnosis
(P < 0.05), hospitalisation in ICU (P < 0.06), and
to the risk factors of oral candidosis antibiotics and
intravenous catheters were included (P < 0.05). In
COPD group the most important risk factors for
Candida infection were the antibiotics treatment,
glycocorticosteroids, using intravenous catheters,
and smoking for oral candidosis (P < 0.05). In LC
the significant risk factors associated with Candida
infection belong to dental prosthesis, and the previous exposure to antibiotics in patients with oral
candidosis. In both groups oral candidosis was
observed, more often in patients in late stages of
the diseases (stage III and IV in LC, severe and very
severe COPD).
Conclusions: 1. Risk factors statistically influenced
on the course of Candida infection are varied in
COPD and LC group. 2. COPD and LC patients
without neutropenia belong to Candida infection and
candidosis risk groups. 3. Normal number of polymorphonuclear granulocytes in blood can not be
exclusion criteria for mycological examinations.
Keywords: lung cancer, COPD, Candida, risk factors.
P272
The follow-up of the vaginal candidiasis in
diabetic rats by evaluating the levels of the
oxidative biomarkers in plasma and tissue
samples
S. Kustimur,1 A. Kalkanci,1 G. Akbulut,2 B. Gonul,2
E. Bulduk2 and N. Aksakal3
1
Microbiology, 2Physiyology, 3Public Health, Gazi
University Faculty of Medicine, Ankara, Turkey
Diabetes is a proven predisposing factor for vulvovaginal candidiasis. A multitude of in vivo studies have
been performed utilizing antioxidants in experimental
diabetic models. The effects of antioxidants on oxidative stress are measured through certain observable
biomarkers. The aim of this study is to determine the
relation between diabetes and vaginal candidiasis in
terms of oxidative biomarker levels in a vaginal
candidiasis model of the diabetic rats. All of the
animals were tested for malondialdehyde (MDA),
sulphydrile groups or glutathione (RSH) and ascorbic
acid (C Vitamine-C vit) levels in their plasma and
vaginal tissue samples. Wistar albino rats, weighting
initially 200–250 g and 4 weeks of age, were obtained
163
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from Medical School of Gazi University. All rats were
randomly divided into five groups: (1) Untreated
diabetic control group (STZ, n = 15). Streptozotocine
induced to suffer diabetes by intraperitoneal injection
of 2% STZ (45 mg kg–1). (2) Vaginal candidiasis in
diabetic group (STZ + Can, n = 15). Rat model of
vaginitis was performed as described previously by
using the inoculation of yeast suspensions into the
vagina. (3) Benflourex-treated diabetic and vaginal
candidiasis group (STZ + Bnf + Can, n = 15) treated
with intragastric benflourex (50 mg kg–1) for 21 days.
(4) Benflourex-treated diabetic and fluconazole treated
vaginitis group (STZ + Bnf + Can + Flz, n = 15) was
additionally treated with fluconazole (150 mg kg–1)
intragastrically. (5) Untreated diabetic and treated
vaginitis group (STZ + Can + Flz, n = 15). All the
groups were observed 21 days. The glucose test was
29 performed using Glucotrend (Roche, USA ). More than
200 mg dl–1 glucose levels are accepted as ‘positive’
for diabetes. MDA, RSH and C Vit levels were measured
spectrophotometrically. The results were evaluated
statistically using Mann–Whitney U-test. In the
vaginal candidiasis group, 500 CFU ml–1 C. albicans
was isolated from vaginal cultures. In the group of
treated diabetes; 19 CFU ml–1 C. albicans was isolated.
In the group of treated diabetes and treated vaginal
candidiasis, colony numbers were 12 CFU ml–1. In the
group of untreated diabetes and treated vaginal
candidiasis; 551 CFU ml–1 C. albicans were isolated.
In the treated diabetes groups, MDA (0.902,
0.684 nmol ml–1) and RSH (227, 171 nmol ml–1)
levels were found to be decreased while the levels of C
vit were found to be increased (0.495, 0.371 mm
aa 100 ml–1) (P < 0.05). In the groups of untreated
diabetes vaginal candidiasis were found to be more
serious and oxidative biomarkers were found to be
increased (MDA 1.305, 1.261 nmol ml–1 and RSH
258, 231 nmol ml–1) while the antioxidant C vit levels
were
found
to
be
decreased
(0.170,
0.244 mm aa 100 ml–1) (P < 0.05). Vaginal candidiasis was evaluated as an augmentative factor on
oxidative stress in diabetic cases. Systemic oxidative
stres biomarkes were affected from vaginal candidiasis
although it was a local mucosal infection.
164
P273
Candida albicans induces IL-8 and IL-18 in THP-1
cells
N. Kondori1 and I. Mattsby-Baltzer2
1
Clinical Bacteriology and 2Clinical Bacteriology,
Laboratory Medicine, Gothenburg, Sweden
Candida albicans which is present in humans as a part of
the commensal microbial biota may cause invasive
infections especially in immunocompromised patients.
Protection against Candida infection involves both
innate and acquired immune responses. Monocytes
and macrophages are not only important components
of the cellular defense mechanism by producing
cytokines that enhance chemotaxis and phagocytosis,
but may also contribute by the production of IL-12 and
IL-18, or IL-10 to the Th-cell differentiation (Th1/
Th2). Here we evaluate the pro- and anti-inflammatory responses (IL-1??, TNF-??, IL-6, IL-8, IL-10, IL-12
and IL-18) of a human monocytic cell line, THP-1,
against live C. albicans. C. albicans strain ATCC 64549,
the highly virulent C. albicans strain CA-6, and the live
vaccine strain PCA-2 were added to THP-1 cells
(5 · 105 per well) in yeast cell to monocyte ratios of
0.1, 1, and 10. The THP-1 cells were also stimulated by
a component extracted from the cell wall of C. albicans
termed phosphopeptidomannan (PPM), and LPS.
Secreted cytokines in the cell supernatants were
quantitated by ELISA. THP-1 cells released IL-8 and
IL-18, but not IL-1??, TNF-??, IL-6, IL-10, and IL-12,
when stimulated by live C. albicans. High concentrations of IL-8 were found after 8 and 20 h of incubation.
IL-18 was also found after 8 h of incubation with
C. albicans but only at the yeast cell to monocyte ratio
of 10. After 20 h of incubation IL-18 was seen at all
concentrations tested. Higher concentrations of IL-8
and IL-18 were found after stimulation of THP-1 cells
with the virulent C. albicans strain CA.-6 compared
with the two other strains. Killed (formaldehydetreated) C. albicans or PPM did not induce any cytokine
release. LPS induced, however, production of IL-1??,
IL-6, IL-8, and TNF-??, but not IL-18. Only live
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
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C. albicans is able to induce secretion of a few cytokines
(IL-8 and IL-18) in THP-1 cells. PPM, the major
component of the cell wall is not able to trigger this
cytokine release from the cells. Live C. albicans seems
30 to induce cytokines which promote Th1 immunity.
P274
Nail susceptibility to fungal infection in patients
with type 1 and 2 diabetes under long term
poor glycaemia control
A.B. Macura,1 T. Gasinska,2 B. Pawlik1 and
A. Obloza1
1
Mycology, Microbiology, Krakow and 2Clinic of Int.
Dis. and Oncology, Silesian Medical Academy,
Katowice, Poland
Onychomycosis is a frequent disorder in adults. Its
prevalence increases in diabetics. The objective of the
study was (i) evaluation of finger and toe nail
susceptibility to infection with the fungi Candida
albicans and Trichophyton mentagrophytes in patients
with type 1 and type 2 diabetes under long term poor
control of glycaemia, (ii) checking whether or not
various aetiology of diabetes (autoimmunological in
type 1 but not in type 2) may influence the intensity of
fungal nail infection in a setting of long term poor
control of glycaemia. The materials comprised finger
and toe nail scrapings collected from 26 patients with
type 1 diabetes, 25 with type 2 diabetes, and 22
healthy volunteers who served as controls. All of the
diabetics had increased glycaemia during a long-term
follow-up because the level of glycated haemoglobin
HbA1c exceeded 7.5%. The nail susceptibility to
fungal infection was evaluated using an ex vivo
method i.e. the nail scrapings were incubated with
C. albicans and T. mentagrophytes suspensions in
saline. The extent of infection was expressed in terms
of the abundance of hyphe penetrating the nail
fragments as follows: mild infection (+) - single hyphe;
intermediate infection (++) - numerous hyphe; intensive infection (+++) - abundant hyphe. Intensive
fingernail infection with C. albicans was found in
38.4% of patients with type 1 diabetes, in 28% of
those with type 2 diabetes, and in 22.7% of the
controls. Intensive infection of toenails with C. albicans
was found in 34.6%, 20%, and 22.7% respectively.
Intensive fingernails infection with T. mentagrophytes
was found in 30.7% of patients with type 1 diabetes, in
48% of patients with type 2 diabetes and 4.5% of the
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
controls, while toenail infection in 15.3%, 20%, and
18% respectively. Statistical analysis of the findings
gave evidence that both finger and toe nails were more
frequently and more intensively infected with T. mentagrophytes in patients with type 1 and 2 diabetes than
in controls. It was also established that enhanced
susceptibility of finger and toe nails to T. mentagrophytes infection in patients with long term poor control
of glycaemia did not depend on autoimmunological
aetiology of diabetes: it was similar in patients with
both types 1 and 2 diabetes. However, the susceptibility of finger and toe nails to C. albicans infection in the
same patients did not differ significantly from that in
the controls. No correlation was found between the
intensity of infection and the age of the patients or the
duration of diabetes.
P275
How do macrophages ‘express themselves
when they find Candida albicans?
G. Molero Martı́n-Portugués, L. Martı́nez-Solano,
C. Nombela and C. Gil
Departamento de Microbiologı´a II, Facultad de
Farmacia, Universidad Complutense de Madrid,
Madrid, Spain
Macrophages have a central role in the defense against
systemic candidiasis (1) and Candida albicans induces a
specific response in these cells. To deal with this role,
we are analysing the differential protein expression of
the murine macrophage cell line RAW 264.7 after
confrontation to C. albicans SC5314 strain and
making an expression protein map of this cell line.
Experiments were done with the following samples:
macrophages without activation, macrophages +
C. albicans SC5314 alive (1:1) and macrophages +
C. albicans SC5314 heat inactivated (1:100), because
alive or dead cells exert different effects on the
activation of macrophages (2). Heat inactivated wild
type C. albicans is able to induce a level of stimulation
of macrophages that is similar to that produced by LPS.
Macrophages protein extractions were made at 15, 30,
45 min, 3 and 24 h of incubation. Samples were
immunoprecipitated with anti-phosphotyrosine antibodies (clone PY-20, BD Transduction Lab.) and/or
analysed by western blotting with the same antibodies
to examine the differential protein expression of
phosphoproteins. We are analysing the differential
protein expression of our samples after 45 min of
165
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interaction by 2D-PAGE. At this point, we have
identified 35 proteins of macrophages by MALDI-TOF
and MALDI-TOF-TOF. In order to confirm the phosphorylation of the proteins and to isolate the phosphopeptides, we are using affinity chromatography
columns, for enriching the sample in phosphopeptides.
At this point, two samples (macrophage and macrophage + C. albicans alive) have been subjected to a
comparative analysis using ImageMaster 2D-Platinum computer software. Fifteen spots were upregulated after exposure to C. albicans, whereas 32
spots were down-regulated. One of the most significant
events that happen after encountering between host
and pathogen is that more than 60 spots appear and
49 disappear after this interaction. On the other hand,
preliminar results of in vitro experiments show a
differential location of the tyr-phosphorylated proteins
in the macrophages when confronted to live or HICandida cells using immunofluorescence + confocal
microscopy.
Acknowledgement This work was supported by
grants BIO2003-0030 from the Comisión Interministerial de Ciencia y tecnologı́a (CICYT), Spain and
from the Fundación Ramón Areces.
Reference
1 Romani L. Curr Opi Microbiol 1999; 2: 363–7.
2 Chinen T, Qureshi MH, Koguchi Y, Kawakami K. Clin Exp
Immunol 1999; 115: 491–7.
P276
In vitro fungistatic activity of recombinant
human surfactant protein, SP-D, against clinical
isolates of Aspergillus species
P. Warn,1 A. Sharp,1 P. Waters,2 D.W. Denning1
and U. Kishore2,3
1
Faculty of Medicine, The University of Manchester,
Manchester, 2Institute of Molecular Medicine,
University of Oxford, Oxford, UK and 3Institute of
Medical Microbiology, Justus-Liebig-University,
Giessen, Germany
Background: Aspergillus spp. are the major fungal
pathogens in immunocompromised and neutropenic
patients with mortality rates >50% in treated
patients. It has been shown that lung surfactant
protein D (SP-D) is involved in protective immunity
against opportunistic fungal infection by a combination of agglutinating conidia and enhanced uptake
166
and killing by macrophages and neutrophils. Murine
models of invasive pulmonary aspergillosis treated
with intranasal SP-D demonstrate improved survival
after an otherwise fatal challenge with A. fumigatus
conidia, concomitant with lower CFU counts,
reduced hyphal growth, and raised levels of IFNgamma and TNF-alpha in the lungs.
Methods: Two recombinant variants of human SP-D
(rhSP-D Gly-X-Y and Delta) composed of homotrimeric
neck and carbohydrate recognition domains were
expressed in E. coli and purified by a procedure
involving denaturation-renaturation, ion-exchange,
affinity and gel-filtration chromatography. The Gly-XY variant contains additional 8 Gly-X-Y triplets from
the collagen region of SP-D whereas the Delta variant
lacks any additional residue at its N-terminus. 48
clinical isolates of Aspergillus being 18 strains of
fumigatus (AF), 10 strains each of niger (AN), flavus
(AFL) and terreus (AT) were used in the study.
Susceptibilities were determined using the microdilution plate modification of the CLSI (formerly NCCLS)
M27A2 standard. Culture medium was RPMI
1640 + 2% glucose, inoculum 1 · 105 ml–1, plates
incubated for 24 h at 37 C; the plates were read both
visually and on a spectrophotometer at 490 nm
(rhSP-D range 0.9273–15 mg l and 12.5–200 mg l–
1
for Gly-X-Y and Delta rhSP-D, respectively). In a
second set of experiments involving three isolates of AF
and one each of AN, AFL and AT, growth curves were
determined in similar conditions with readings taken
every 5 min for 24 h (1.5–24 mg l–1).
Results: With Gly-X-Y rhSP-D, all isolates showed
reduced growth in a dose dependent fashion. A total
of 40/48 strains demonstrated >80% reduction in
growth at 15 mg ml–1 with MIC50s of 15, 1.875, 15
and 7.5 for AF, AN, AFL and AT, respectively. With
Delta rhSP-D, less inhibition occurred with 35/38
strains showing reduced growth over the dose range.
A total of 80% reduction in growth was seen in few
isolates (5/48 strains). In the growth rate assays, all
strains demonstrated both an overall reduction in
growth rate (as measured by increasing OD) in a dose
dependent fashion. Reduction in growth rate was
most clearly seen by increased lag time (time until
the OD increased) which increased by 2 h 20 min
and 1 h 30 min in the Gly-X-Y and Delta rhSP-D
assays, respectively.
Conclusions: SP-D substantially reduces the
growth rate of Aspergillus in vitro and this fungistatic
activity can be localized to the lectin domain. This is
an additional mechanism by which SP-D provides
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
protective immunity without involving phagocytes.
Thus, the rhSP-D may be used as a potent barrier to
fungal infection.
P281
Toxicogenic fungal isolated from animal feeds
in Yazd dairy centers
A.A. Jafari nodoushan and M. Hosseinzadah
Paramedicine, Yazd Medical University, Yazd, Iran
Fungal toxins especially aflatoxin, receiving increasing attention worldwide because of the hazard it poses
in animal and human. Several Aspergillus and Pecnicillium species, which are predominant in nature,
contaminate animal feeds. Aflatoxin B1 is a type I
human carsinogen, which contaminated animal food,
extracted in milk and meat from animals eating
mouldy feeds. The general purpose of this study was
to determine the most prevalent fungal species that
contaminate the animal feeds in Yazd dairy centers. 67
animal food samples were cultured in plates containing Sabourauds dextrose agar with chloramphenicol
(SC). A total of 204 colonies from 14 different
saprophyte fungi were isolated that Penicillium
(19.6%), and Aspergillus flavus (17.6%) were the most
prevalence isolated species. Rhizopus species (12%),
Cladosporium (11%), Mucor species (8%), and Aspergillus niger (7%) were the other isolated species.
However Aspergillus flavus showed a higher prevalence
in traditional dairy centers but there was not seen any
statistical significant differences between the traditional and industrial dairy centers (2 £ 2.34,
P £ 0.31). There was seen a statistical significant
differences between isolation of Penicillium puberulum
and the type of dairy centers 2 = 11.3, P £ 0.004).
Keywords: aflatoxin, fungi, animal food.
P282
Effect of gliotoxin on the function of Listeria
monocytogenes-specific effector cells in vitro
C. Kupfahl, G. Geginat and H. Hof
Institut für Hygiene und Mikrobiologie,
Universitätsklinikum Mannheim der Universität
Heidelberg, Mannheim, Germany
Gliotoxin is a mycotoxin produced by several fungi,
including Aspergillus fumigatus. Since a possible
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
immunosuppressive activity of gliotoxin is being
discussed in the literature, we analysed the effect of
gliotoxin on bone marrow derived macrophages,
dendritic cells and T-cells after infection with the
facultative intracellular bacterium Listeria monocytogenes in vitro. In these three cell types gliotoxin
induced apoptosis in a dose dependent manner.
Additionally we found gliotoxin to exert an effect
on cellular function in sub apoptotic concentrations.
These low concentrations of gliotoxin only had a
small effect on NO-production of Listeria-infected
bone marrow derived macrophages. Similarly cytotoxic cell lysis by Listeria-specific CD8 T-cells was not
markedly influenced. In contrast, low dose gliotoxin
treatment of dendritic cells and bone marrow derived
macrophages resulted in a dramatically reduced
production of TNF-alpha and IL-12 after infection
with L. monocytogenes. Furthermore, gliotoxin treatment resulted in a strong reduction of antigen
presentation of infected bone marrow derived macrophages as well as in an almost complete anergy of
specific CD8 T-cells. Taken together, these results
demonstrate that gliotoxin exerts immunosuppressive effects on different immune mechanisms. Therefore it can be speculated that gliotoxin acts as a
virulence factor for fungi during invasive mycoses.
P283
Cryptococcus neoformans: in vitro resistance to
three azoles
R.M. Claro,1 A.I. Aller,1 F. Colom,2 M. Ramı́rez,1
C. Castro,1 E. López,1 A. Romero1 and
E. Martı́n-Mazuelos1
1
Microbiology, H.U.Valme, Sevilla and 2Universidad
Miguel Hernández, Facultad de Medicina, Alicante,
Spain
Objective: Although some fluconazole resistant isolates of Cryptococcus neoformans have been reported
in the last years, there are not so much in the case of
voriconazole because of its more recent introduction.
The aim of this study is to know the rates of
resistance to fluconazole, itraconazole and voriconazole of C. neoformans and to evaluate the possible
existence of cross-resistance mechanisms.
Methods: A total of 80 C. neoformans isolates from
HIV patients were studied. 29 of them were
obtained from patients without HAART from 1994
to 1996; the other 51 were obtained from patients
167
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with HAART from 1997 to 2003. The susceptibility
to fluconazole, itraconazole and voriconazole of the
first group and the susceptibility to fluconazole and
voriconazole of the second one were performed by
the broth microdilution method according to the
NCCLS guidelines (M27-A2 document). Fluconazole
and voriconazole were provided by Pfizer and
itraconazole by Janssen. Fluconazole was dissolved
in sterile distilled water, whilst itraconazole and
voriconazole were dissolved in dimethylsulphoxide.
31 RPMI medium (Sigma ) was used for all drugs. The
final concentrations were 0.12–64 mg l–1 for fluconazole and 0.015–8 mg l–1 for itraconazole and
voriconazole. For all drugs, MIC values were the
lowest drug concentration which inhibited growth
by the 50% (prominent inhibition of growth)
compared with the control. The chosen breakpoints
were ??8 mg l–1 (S) and ??16 mg l–1 (SDD/R) for
fluconazole according to Aller et al. (Antimicrob
Agents Chemother 2000; 44: 1544), and
??0.125 mg l–1 (S) and ??0.25 mg l–1 (SDD/R) for
itraconazole and ??1 mg l–1 (S) and ??2 mg l–1
(SDD/R) for voriconazole according to the last
NCCLS guidelines.
Results: The next table shows the MICs obtained for
the tested drugs.
Number (%) of isolates
Drug
Fluconazole
–1
MIC (mg l )
16 (SDD/R)??
5
8
Itraconazole 0.25 (SDD/R)??
28
0.125
Voriconazole ??2 (SDD/R)
3
??1
Pre-HAART
HAART
Total
(17.24%) 0 (0%)
5 (6.25%)
(S)??
24 (82.76%) 51 (100%) 75 (93.75%)
(96.5%)
–
28 (96.5%)
(S)??
1 (3.5%)
–
1 (3.5%)
(10.34%) 0 (0%)
3 (3.75%)
(S)
26 (89.66%) 51 (100%) 77 (96.25%)
All of the five isolates in the pre-HAART era with
fluconazole MIC ??16 mg l–1 had a itraconazole MIC
??0.25 mg l–1 and three of them had a voriconazole
MIC ??2 mg l–1.
Conclusions: 1. All the C. neoformans isolates with
voriconazole MIC ??2 mg l–1 had high MICs for
fluconazole and itraconazole too. All of them were
isolated in the pre-HAART era, when voriconazole
was not used yet, so a cross-resistance mechanism
could be implicated. 2. There wasn’t found any
susceptible dose dependent or resistance fluconazole
or voriconazole C. neoformans isolate in the HAART
era.
168
P284
Cryptococcus neoformans proteome and cell
cycle regulation
S. Kawamoto,1 M. Ohkusu,1 Y. Yamanaka,2
K. Watanabe,2 T. Sonoda,2 H. Hirano2 and K. Okuda2
1
Research Center for Pathogenic Fungi and Microbial
Toxicoses, Molecular Function, Chiba University,
Chiba and 2Yokohama City University, Yokohama,
Japan
Cryptococcus neoformans has emerged as a major
pathogenic microbe in patients with impaired immunity such as AIDS patients. Like the model yeast
Saccharomyces cerevisiae, Cryptococcus neoformans is
also a budding yeast but it exhibits some cell cycle
characteristics different from S. cerevisiae. As a first
step to elucidate the mechanisms for Cryptococcus
neoformans cell cycle molecularly, we isolated the
C. neoformans homologue of CDC28/Cdc2 (cyclindependent kinase-1, Cdk-1) molecule, the main cell
cycle gene which regulates the major processes in
eukaryotic cell cycle. Genome project of the pathogenic yeast C. neoformans has just completed very
recently (Science 2005; 307: 1321). Proteomics has
become a major focus in the postgenomic researches.
In order to use the molecular data for the research
fields such as phylogenic analysis, pathogenic factor
analysis, and proteomics-based drug discovery/development, we have been performing the proteome
analysis of C. neoformans. In order to further analyze
the control mechanism of C. neoformans cell cycle
molecularly at the protein level, we have been
performing the proteomic analysis of the yeast cells.
We have also been exploring to carry out the
differential proteomic analysis at the different cell
cycle stages under the various cultivation conditions.
Yeast cells cultured in yeast-extract/polypeptone/
glucose medium were disrupted mechanically and
centrifuged to yield the cytosolic soluble fraction. In
this study, the soluble extract was used for 2D
(isoelectric focusing/SDS-PAGE) electrophoresis analysis, and designated protein spot was subjected to
in-gel digestion using trypsin, and the paptides were
further analyzed by amino acid suquencing. We
compared the protein expression profile to each other
and the protein spots remarkably different in expression level were subjected to amino acid sequencing
analysis. The sequence data were referred to SGTC
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
database of the C. neoformans genome project and
some protein spots were identified.
ole offers acceptable coverage of uncommon Candida
species bloodstream infections.
P285
P286
Candida famata sepsis in a child with
non-Hodgkin lymphoma
Kodamaea (Pichia) ohmeri fungemia in a
premature neonate
P.E. Papakonstantinou Eugenia,1
R.E. Roilides Emmauel,2 K.A. Karakoli Anna,1
S.F.V. Sidi-Fragandrea Vasiliki,1
H.E. Hatzipantelis Emmauel,1 B.E. Bibashi Evangelia,3
S.D. Sofianou Danai,3 K.E.D. Koliouskas and
E. Dimitrios1
1
Paediatric Oncology, 23rd Department of Paediatrics
and 3Department of Microbiology, Hippokration
Hospital, Thessaloniki, Greece
S.J. Taj-Aldeen,1 S.H. Doiphode1 and X.Y. Han2
1
Laboratory Medicine and Pathology, Hamad
Medical Corporation, Doha, Qatar and 2Department
of Laboratory Medicine, The University of Texas M.D.
Anderson Cancer Center, Houston, USA
Background and Aims: Patients with haematologic malignancies are highly susceptibly to potentially fatal fungal infections. Candida famata infections
are uncommon, so the clinical presentation and
successful management of a patient treated in our
clinic should be instructive. C. famata infections
account for <1% of fungaemias in a man. Mortality
due to non-albicans Candida species is similar to
Candida albicans varying from 15% to 35%. Several
reports indicate this rare species to be less susceptible
to the currently antifungal agents with the exception
of voriconazole.
Methods: Case report. A 4-year-old girl was diagnosed as suffering from B-cell non-Hodgkin lymphoma and enrolled on the MRC 903 protocol. Four
months later, during severe neutropenia (ANC
0.1 · 109 l–1) she presented with fever and was
started on ceftazidime and amikacin. Liposomal
amphotericin B 5 mg kg–1 day–1 was also added.
C. famata was isolated from multiple blood cultures
taken from both her Hickman catheter and peripheral veins. Voriconazole 8 mg kg–1 day–1 was added
6 days later. Nine days after the initiation of
amphotericin and 3 days after voriconazole, blood
cultures became repeatedly negative for fungi. The
fever subsided 2 days after voriconazole was started
while the patient recovered from myelosuppression.
The catheter was removed a month later in view of
subsequent megatherapy.
Conclusions: Successful treatment in our patient
was probably related to the synergistic action of
antifungal agents combined with rapid recovery from
neutropenia. This case also suggests that voriconaz-
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
Kodamaea ohmeri is an yeast that rarely causes human
infections. We report the first case of K. ohmeri
fungemia in a premature neonate that was successfully treated by liposomal amphotericin B. Biochemical
identification of the yeast was performed by Vitek II
and API and was confirmed by rDNA sequencing. This
organism may represent an emerging challenge to the
hospitalized neonates.
P287
Intracranial abcess due to Cryptococcus
neoformans: an unusual clinical feature in an
immunocompetent patients
O. Töre,1 S. Akçağlar,1 E. Kazak,1 Y. Heper,1
H. Akalın,1 B. Hakyemez2 and B. Ener1
1
Department of Microbiology and Infectious
diseases, and 2Department of Radiology, University
of Uludağ, Bursa, Turkey
Cryptococcus neoformans is the most common fungus
affecting the Central nervous System (CNS) and
usually it causes mennigoencephalitis. Although the
incidence of cryptococcal infection is high when
associated with HIV infection or AIDS, this infection
can occur in an immunocompetent healthy host as
well. In this report we present an unusual case of
intracranial abscess in an immunocompetent patient.
Case report: A previously healthy 54-year-old
woman with complaint of unpleasant smelling and
snoring for 5 months and tinnitus, headache for
2 months. She was apathetic and had superior
oblique paralysis of right eye. The CT imaging
examination revealed multiple cystic lesions in brain.
169
xxx
stereotactic biopsi carried out from the lesion near the
mesencephalon and after evaluation of frozen examination as abscess, ceftriaxon and metronidazol were
immediately started. After isolation and identification
of C neoformans from the biopsi material, therapy
changed into Amphotericin B 1 mg kg–1 day–1. At
day 3, extraventricular drainage system was applied
by neurosurgeons for draining CNS, and 5 cc cerebrospinal fluid (CSF) were drained in every 3 h.
Laboratory examination of ventricular CSF showed
170 mm–3 WBC (with granulocyte predominance),
5 000 000 mm–3 RBC, 41 mg dl–1 glucose. AntiHIV antibodies were found negative twice, and serum
glucose values were at normal levels. There was no
history of contact with pigeons. The examinations of
immune status showed no pathological findings. At
day 11, CSF examination showed 90 mm–3 WBC.
Although, there were budding yeasts on fresh microscopy no mycological and bacteriological growth
were observed on the cultures. The antifungal
therapy was changed with liposomal amphotericin
B 5 mg kg–1 day–1 because of the renal failure. At
day 17, EVDS was drowning back, and therapy was
carried out with fluconazole 2 · 400 mg IV. At day
25, since she has a high fever and pyuri, cefepim was
added. Then cefepim was changed with Meropenem,
regarding the antibiogram of positive urine culture.
High fever regressed until day 38, but then since she
experienced another fever attack, teicoplanin added
to the therapy. At day 39, her condition was
worsened and 40 day after the beginning of cryptococcal abscess treatment patient died.
P291
Variability of laccase activity in Cryptococcus
neoformans and Cryptococcus gattii
E. Alvarado, J.M. Torres-Rodrı́guez, Y. Morera and
T. Jiménez
IMIM. Autonomous University of Barcelona,
Experimental Mycology Research Unit (URMEC),
Barcelona, Spain
Cryptococcus neoformans and C. gattii are the agents of
human and animal cryptococcosis. The virulence of
these species is related with several factors, apparently not regulated co-ordinately. Among the virulence factors, laccase (phenoloxidase) activity has
been considered as one of the most important. The
main goal of this study was determinate the intensity
and speed for L-Dopa oxidation due to laccase
170
production of C. gattii serotype B isolated in Spain
from six goats death of disseminated cryptococcosis.
These strains were compared with reference isolates
of all serotypes of Cryptococcus (n = 8). All the
14 isolates were disrupted with glass pearls and
supernatant was mixed with a solution of L-dopa
2 mol l–1. Each cryptococcal supernatant was
observed visually and measured spectrophotometrically at 450 nm every 10 min until 120, for
evaluation of laccase activity. Supernatant of the
same isolates killed at 56 C for 1 h were used as
negative controls. One unit of enzyme was defined as
the amount that caused a colour change in A
450 nm of 0.03 at 30 C pH 6.8. A formula was
applied for calculate the enzymatic units (EU):
(A 450120 min – A 4500 min)/108 cells ml–1
=(U 10–8 cells ml–1)
In the first 10 min, two isolates produced colour
change (pink pigment development), and nine from 13
(69%) showed laccase activity in 60 min. Only one
strain of C. gattii serotype B showed low laccase
activity after 120 min of incubation. None of the
controls killed yeasts supernatants showed change
colour or spectrophotometer variations. The highest
Laccase activity was observed in three out six C. gattii
serotype B isolated in Spain, on the contrary the lowest
activity was found in C. gattii serotype C. Results of this
study showed that at the same conditions, laccase
activity is variable. Some isolates were fast enzyme
producers but after 2 h of incubation practically all the
strains were actives. Most of C. gattii serotype B
isolated in non-immunocompromised goats presented
increased laccase activity showing high levels of this
pathogenic factor. The technique used for measure the
Laccase activity could be considered as an accurate
method demonstrating this enzyme is expressed fast
and in high amounts in the serotype B of C. gattii
isolated from animal cryptococcosis.
Laccase activity of supernatants of 14 isolates of
Cryptococcus neoformans (n = 4, A and D serotypes)
and C. gattii (n = 10, B and C serotypes) after
120 min of incubation with 2 mM L-dopa solution.
Absorbance 450 nm and corresponding enzymatic
units (EU:U 10–8 cells ml–1).
Strain
A146 A74 D297 D12 B432 B161 B52* B59* B48* B50* B53* B56* C160 C417 Control
T=0
0.06 0.05 0.09 0.10 0.06 0.06 0.04 0.08 0.05 0.06 0.05 0.07 0.05 0.06 0.04
T = 120 0.11 0.24 0.18 0.24 0.14 0.23 0.07 0.16 0.19 0.43 0.58 0.70 0.07 0.12 0.04
EU
1.7
6.3
3.0
4.7
2.7
5.7
1.0
2.7
4.7
12.3 17.7 21.0 0.7
2.0
0
*Isolates from death goats.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
xxx
P292
Abstract unavailable.
Spanish strains is similar to results obtained in other
southern Mediterranean countries.
P293
P294
Extracellular enzymatic activities in Cryptococcus neoformans strains isolated from Spanish
AIDS patients
Candida albicans phospholipase activity in
different part of the body in immunodepressed
patients and healthy subjects
V. Vidotto,1 J. Ponton,2 G. Quindos,2 S. Aoki,3
S. Ito-Kuwa,3 K. Nakamura,3 and E. Bollo4
1
Dipartimento Discipline Medico-Chirurgiche,
Sezione Malattie Infettive, Università di Torino,
Torino, Italy, 2Departamento Inmunologia,
Microbiologia y Parasitologia, Inmunologia,
Microbiologia y Parasitologia, Universidad Pais Vasco,
Bilbao, Spain, 3Molecular Biological Department,
Advanced Research Center, Nippon Dental
University, Niigata, Japan and 4Dipartimento
Patologia Animale, Sezione Anatomia Patologica,
Università di Torino, Torino, Italy
V. Vidotto,1 A. Goglio,2 A. Grisis,2 K. Fukushima,3
S. Aoki,4 F.T. Cannizzo5 and B. Fianchino6
1
Sezione Malattie Infettive, Discipline MedicoChirurgiche, Torino, 2Laboratorio Microbiologia,
Ospedali Riuniti Bergamo, Bergamo, Italy, 3Research
Center for Pathogenic Fungi and Microbial Toxicoses,
Chiba University, Chiba, 4Advanced Research Center,
Molecular Laboratory, Nippon Dental University,
Niigata, Japan, 5Sezione Anatomia Patologica,
Patologia Animale, Torino and 6Laboratorio
Microbiologia e Virologia, Ospedale Amedeo di
Savoia, Torino, Italy
Twenty-eight Cryptococcus neoformans strains isolated
from Spanish AIDS patients and stocked at the
culture collection of the Universidad del Pais Vasco
(Bilbao, Spain) were tested for their extracellular
enzymatic activity by the API-ZYM kit (Biomérieux,
France). Serotype determination of the strains was
performed according to the Crypto-check method
(Iatron Lab., Japan). The results obtained showed the
high positive percentage (more than 90%) among the
28 C. neoformans strains for no. 3 (esterase), no. 4
(esterase-lipase-C8), no. 6 (leucine-arylamidase),
no. 11 (phosphatase acid), no. 12 (phosphoamidase)
and no. 17 (b-glucosidase). These results reveal that
there are several new extracellular enzymatic activities in C. neoformans which can be involved in tissue
invasion and probably in C. neoformans virulence.
Interesting it is also the high values of the colour
scale 4.9 nmol in the enzyme no. 11 (phosphatase
acid). The predominant serotype A (17 among 28)
One hundred and thirteen Candida albicans strains
isolated in different part of the body in
immunocompromised patients and in healthy subjects were tested for their phospholipase activity.
Samples were from vaginal and pharyngeal swabs,
escreate, bronco aspirate lavage, skin and nails
scrapings and emoculture isolated in immunocompromised patients and in healthy subjects from the
Ospedali Riuniti Bergamo, Italy and from the Ospedale Amedeo di Savoia, Torino, Italy. The C. albicans
isolated strains in the different part of the body were
at first identified by the API Candida 20 CAUX
(Biomérieux, France) and then were tested for their
phospholipase activity at 37 C after 3 and 6 days.
This activity was expressed as Pz. The Pz decreased
from from the 3rd to the 6th day in all the samples.
Samples from skin and nails and emoculture showed
lower phospholipase activity than the other samples
after 3 and 6 days.
2005 Blackwell Publishing Ltd • Mycoses, 48, (Suppl. 2) 1–173
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