Medical Mycology 2003, 41, 149–161
Received May 2002; Accepted 8 November 2002
Fungal biodiversity – as found in nasal mucus
W. BUZINA, H. BRAUN, K. FREUDENSCHUSS, A. LACKNER, W. HABERMANN & H. STAMMBERGER
ENT Department, Karl-Franzens-University, Graz, Austria
The biodiversity of fungi isolated from the nasal mucus of patients suffering from
chronic rhinosinusitis and from healthy persons was monitored over 28 months.
Mucus samples were obtained by flushing the noses of patients with saline or by
endoscopic sinus surgery. Fungi from mucus were cultivated on agar plates.
Identification was performed microscopically and by polymerase chain reaction with
subsequent sequencing of the ribosomal internal transcribed spacer region.
Altogether, 619 strains of fungi were cultivated from 233 subjects. Eighty-one
species were identified, with a maximum of nine different species per person. The
most prevalent isolates belonged to the genera Penicillium, Aspergillus, Cladosporium, Alternaria and Aureobasidium. Whereas Aspergillus and Penicillium spp.
occurred in more or less the same numbers throughout the year, Cladosporium spp.,
Alternaria spp. and Aureobasidium pullulans showed a significantly higher
occurrence during late summer and early autumn.
Keywords
chronic rhinosinusitis, fungi, nose, sinuses
Introduction
The prevalence of chronic diseases of the airways,
especially asthma and chronic rhinosinusitis (CRS), but
also common allergies, has been increasing every year
for decades in the ‘westernized world’ [1–5]. Involvement of fungi in asthma may have been first suggested by
Moses Maimonides in the 12th century, when he
described the frequent occurrence of wheezing in damp
weather [6]. In 1726, Sir John Floyer noted the
development of violent asthma in a patient who had
just visited a wine cellar [7]. Hansen published a case of
asthma caused by fungi in 1928 [8].
Mycoses or fungus balls of the sinuses were first
reported in 1885 by Schubert [9], and Kecht [10] found
98 cases of Aspergillus-associated sinusitis in the following 90 years. From 1976 to 1990, Stammberger obtained
data from over 400 patients with massive aspergillosis of
the sinus treated at the university Ear, Nose and Throat
Department at Graz, Austria [11].
In 1983, Katzenstein et al. [12] identified Aspergillus
Correspondence: W. Buzina, ENT University Hospital, KarlFranzens-University Graz, Auenbruggerplatz 26-28, A 8036 Graz,
Austria. Tel: þ43 316 385 3606; Fax: þ43 316 385 7643; E-mail:
[email protected]
ª
ª 2003
2003 ISHAM
ISHAM, Medical Mycology, 41, 149–161
species in the thick mucus of the nose and the sinuses in
patients suffering from CRS and nasal polyposis, and
introduced the term ‘allergic Aspergillus sinusitis’. Later
the disease name ‘allergic fungal sinusitis’ (AFS) was
coined, after other fungi were demonstrated to produce
the same symptoms. Although clinicians are able to
clearly recognize this group of patients based on their
symptoms and laboratory findings, there remains ongoing controversy as to whether this truly is an allergic
disease and, if so, whether or not fungi are the causative
allergens [13]. Ponikau et al. [14] recently suggested that
there was a much higher incidence of fungal etiology in
CRS than had previously been suspected. With improved
techniques, they were able to identify fungi, albeit not
always demonstrated as causal, in the sinonasal mucus of
over 90% of patients with CRS and nasal polyposis, as
well as 100% of healthy controls. In related microscopic
studies, they also, in many cases, found clusters of
eosinophils grouped around the fungal elements in the
nasal mucus. From this, they concluded that fungi acted
as triggers for an immune response mediated by
eosinophils. The term ‘eosinophilic fungal rhinosinusitis’
(EFRS) was introduced to replace AFS. The aim of our
study was to independently evaluate the presence of
fungi in the sinonasal tract of healthy controls and
patients with CRS.
150
W. Buzina et al.
More than 100 000 species of fungi have been
described, and the total number is estimated to be more
than 1.5 million species worldwide [15]. Of this large
number, only about 100 are known to be regularly
involved in human mycoses, and a few hundred more
occur as opportunists [16]. Some chronic allergic diseases
of the respiratory tract are so strongly associated with
reactions against particular fungi that they are named
after the source or occupational environment producing
the heavy fungal inoculum involved (for example, cane
cutter’s disease, farmer’s lung, sick building syndrome).
It is not yet known, however, how many fungi are able to
cause allergies and chronic airway inflammations. Allergies are conventionally conceived as arising in response
to cellular proteins and carbohydrates, but in recent
years it has been realized that mycotoxins and other
secondary metabolites produced by fungi, sometimes in
or on body sites, may be involved. The degree to which
this occurs is unclear. All this leads to a growing
necessity to show which fungi are prevalent in patients’
environments at various times, and even more importantly, which fungi can be found at affected sites in
patients suffering from immunologically mediated diseases of the respiratory tract. Because the question of
whether all fungi or only a limited number of species can
trigger significant immune responses is still not solved,
our aim was to elucidate all fungi in the sinonasal cavity
of CRS patients, regardless of quantity (e.g. single spore,
hyphal filament), developmental stage (e.g. spores,
conidia, hyphae) or organismal physiology (e.g. psychrophilic and not potentially able to grow in patients or
thermotolerant and possibly able to establish a growing
inoculum in patient airways).
Cultivation of fungi followed by macro- and microscopic examination of morphological characters is still
the cheapest, and for many species the most reliable,
technique for identification. However, there are many
fungi that fail to produce distinct features allowing
reliable identification in culture [17–19], and many
species are often not identified because they are not
listed in the identification keys or textbooks commonly
possessed by medical mycology laboratories. In the last
Table 1
few years polymerase chain reaction (PCR)-based
methods have been used to identify medically relevant
fungi. These molecular techniques provide novel tools to
type fungi that cannot be identified to the species level
by microscopy alone and therefore often appear as
‘mycelia sterilia’ in medical and ecological publications.
Although the sequence database necessary to reliably
identify the majority of fungi based on PCR amplifications does not yet exist, existing databases do contain
most common or economically important species, as well
as a number of other fungi sufficient to allow approximate grouping of many test sequences with related
organisms. In this study, sequences of the ribosomal
internal transcribed spacer (ITS), one of the most
commonly sequenced loci in mycological studies, were
employed to aid in the identification of cultures from
sinonasal materials.
Material and methods
Fungal strains and cultivation
In total, 233 nasal and sinus mucus samples were
examined over 28 months (Table 1). The samples
originated from 210 patients suffering from CRS and
from 23 healthy volunteers (control group). Mucus from
104 patients (aged 12–68 years, mean 45.6) and from the
control group (aged 18–54 years, mean 33.0) was
obtained by flushing each nostril with 10 ml sterile
0.9% NaCl solution as described by Ponikau et al. [14].
One hundred and six mucus samples were collected
during functional endoscopic sinus surgery (FESS) from
the affected sinuses of the patients (aged 14–85 years,
mean 46.8). The mucous material was treated with
0.05 parts of mucolytic dithiothreitol (Sputolysin1,
Calbiochem, La Jolla, CA, USA) to release fungal
elements and these were sedimented by centrifugation
[14]. All samples were incubated at 25 oC on Sabouraud’s glucose agar (SGA; 10 g of peptone, 40 g of
glucose, 15 g of agar per liter of deionized water),
Czapek-Dox agar (CZA; 30 g of sucrose, 3 g of NaNO3,
1 g of K2HPO4, 0.5 g of KCl, 0.5 g of MgSO4 7 H2O,
0.01 g of FeSO4 7 H2O, 15 g of agar per liter of
Types of samples taken and overall results of fungal culturing
Positive
Age
Species/person
Group
Persons
Number
%
Min.
Max.
Mean
Strains
Max.
Mean
Control
Flushing
FESS
23
104
106
21
95
89
91.3
91.3
84.0
18
12
14
54
68
85
33.0
45.6
46.8
75
331
213
9
9
6
4.0
3.2
1.9
Control: healthy control; Flushing: chronic rhinosinusitis (CRS) patient, mucus obtained by flushing; FESS: CRS patient, mucus obtained by FESS (functional
endoscopic sinus surgery).
ª 2003 ISHAM, Medical Mycology, 41, 149–161
Fungal biodiversity
deionized water), malt extract agar (MEA; Merck,
Darmstadt, Germany), and brain–heart agar (BHA;
Merck). One hundred milligrams of chloramphenicol
and 40 mg of gentamicin were added to each medium to
prevent bacterial growth. Whenever more than one
culture grew on a single Petri dish, every isolate was
transferred to new dishes to obtain cultures of every
single strain. Yeasts were transferred to CHROMagarTM
Candida plates (Becton Dickinson, Cockeyville, MD,
USA) for classification of different species of Candida
[20]. The cultures were examined twice a week for up to
6 weeks and identified by microscopy based on their
morphology. Strains without sporulation were incubated
on Corn Meal Agar (CMA; 40 g of corn meal, 10 g of
glucose, 15 g of agar per liter of deionized water) to
enhance the production of conidia. All fungi that failed
to produce distinct features for reliable identification
were examined using PCR-based techniques.
DNA isolation
DNA was extracted from fungal cells using a DNA
extraction kit for fungi (Invitek, Berlin, Germany) with
minor modification. In this procedure, cultivated material (10–50 mg) was homogenized in 1.5 ml Eppendorf
tubes with a small pestle, suspended in 500 ml of lysis
buffer (Invitek) and mixed vigorously for 30 min.
Samples were then centrifuged; the supernatant was
removed, followed by the addition of 200 ml of binding
buffer and 15 ml of carrier suspension (Invitek). After
10 min incubation at room temperature, the samples
were centrifuged for 1 min at 10 000 rpm, the pellet was
washed twice with 800 ml of wash buffer (Invitek) and
dried for 10 min at 65 oC. Thereafter, the pellet was
resuspended in 100 ml of pre-warmed (65 oC) elution
buffer (Invitek) and incubated for 10 min at 65 oC. After
centrifugation for 1 min at maximum speed, the supernatant was removed and stored at 25 oC until further
examination.
PCR
The 5.8S ribosomal DNA (rDNA) and the flanking ITS
regions (ITS1 and ITS2) were amplified using the
primers ITS1f [21] and ITS4 [22], which were prepared
commercially by Metabion (Planegg-Martinsried, Germany). PCR was carried out in a reaction mixture
containing 14.8 ml of sterile bidistilled water, 5.0 ml of
10 PCR Buffer (Amersham Pharmacia, Uppsala,
Sweden), 1.25 ml each of dNTP (10 mm), 1 U of Taq
DNA polymerase (Amersham Pharmacia), 2.5 ml of
each primer (10 mm), and 20 ml of fungal DNA (10–
50 ng ml1). One reaction mixture containing water in
place of DNA template was used as a contamination
ª 2003 ISHAM, Medical Mycology, 41, 149–161
151
control. For PCR in the thermocycler (GeneAmp 2400,
PE Biosystems, Foster City, CA, USA) the following
parameters were chosen: 2 min at 95 oC, followed by 35
cycles of 45 s at 95 oC, 45 s at 48 oC and 90 s at 72 oC,
with a final extension at 72 oC for 10 min. The
amplification products (2 ml each) were visualized after
gel electrophoresis and staining in ethidium bromide
under UV light on a transilluminator.
Cycle sequencing
Excess primers and dNTPs were removed using chromatography columns (Microspin S-300 HR, Amersham
Pharmacia). To sequence the entire ITS region with the
enclosed 5.8S rDNA, we used the primers ITS1f and
ITS4 in a concentration of 1.6 mm. Sequencing was
carried out using the ABI PRISM BigDyeTM Terminator
Cycle Sequencing Kit (PE Biosystems) according to the
manufacturer’s recommendations. The parameters for
cycle sequencing in the thermocycler GeneAmp 2400
(PE Biosystems) were 18 s delay at 96 oC, followed by 25
cycles with 18 s at 96 oC, 5 s at 50 oC and 4 min at 60 oC.
Analysis of sequences
Sequence analysis was performed using an automated
sequence analyzer (ABI PRISM 310, PE Biosystems) in
conjunction with the abi prism auto assemblerTM
software (version 140, Applied Biosystems Division
1995, PE Biosystems) and aligned using bioedit sequence alignment editor, which is available as freeware
at http://jwbrown.mbio.ncsu.edu/BioEdit/bioedit.html.
All sequence data were submitted to GenBank (http://
www2.ncbi.nlm.nih.gov/). Accession numbers are
AF455394–AF455544 and AF461413–14. ITS sequences
were compared with entries in genomic databanks using
Internet freeware from European Bioinformatics Institute (EMBL) at http://www2.ebi.ac.uk/fasta33/ to find
homologies. All cultures were reviewed microscopically
for an eventual discrepancy to the molecular results
obtained.
Histology
Forty-seven mucus samples obtained during endoscopic
sinus surgery were fixed in formalin and embedded in
paraffin. Serial sections were prepared and stained with
Gomori methenamine silver (GMS).
Statistics
The x2 test was used to compare frequencies. To test the
hypothesis that two independent samples have come
from the same population, the Wilcoxon rank sum test
152
W. Buzina et al.
was applied. A significance level of P 5 0.05 was
considered statistically significant. All statistical procedures were carried out using the spss program (SPSS
Inc., Chicago, IL, USA).
Results
From 104 mucus samples obtained by flushing the nose
of the patients with saline, 95 showed fungal growth
(91.3%) (Table 1). Altogether 331 strains of fungi were
identified, the samples contained up to 9 different species
per patient, with an average of 3.2 species. The group of
23 persons without symptoms of CRS (healthy controls)
produced fungi in 21 mucus samples (91.3%), and 75
strains were isolated. The maximum number of species
obtained in a single control sample was 9, the average
4.0. In mucus obtained from 106 CRS patients during
endoscopic sinus surgery, 89 samples were fungus
positive (84.0%). The number of isolates in this group
was 213, the maximum was 6 species per sample, and the
average was 1.9. A total of 619 isolates of fungi were
cultivated from 233 samples investigated. Four hundred
and fifteen were identified at the species level (81
species), 148 to the generic level (7 genera), 22
basidiomycetous fungi to the level of the phylum, (this
means that they were classified as unknown basidiomycetes based on their ITS sequences), and 34 strains that
were not identifiable by microscopy or molecular
techniques were left as unidentified sterile mycelia. The
most prevalent genera were Aspergillus (125 isolates,
20.2%), Penicillium (123, 19.9%), Cladosporium (84,
13.6%), Candida (38, 6.1%), Alternaria (34, 5.5%), and
Aureobasidium (32, 5.2%). Alternaria spp., Cladosporium spp. and Aureobasidium pullulans showed a
significant seasonal fluctuation (P 5 0.001) with maxima
in prevalence in late summer/early autumn, and minima
in late winter/early spring (Fig. 1). The occurrence of the
other taxa did not show a significant prevalence during
the 28 months examined.
From 47 patient mucus samples examined microscopically for fungal content, 33 were fungus positive
(70.2%). The spectrum of fungal elements detected in
nasal mucus varied among single conidia, hyphal fragments, and septate branched hyphae (Table 2).
Fig. 1 Seasonal distribution of A:
Aspergillus and Penicillium, B: Alternaria, Cladosporium and Aureobasidium. Note the different scales in
A and B.
ª 2003 ISHAM, Medical Mycology, 41, 149–161
Fungal biodiversity
Discussion
When fungi from nasal mucus were obtained by flushing
the noses of CRS patients and healthy persons, the
proportion of individuals positive for fungal growth was
exactly the same (91.3% positive). The results of
Ponikau et al. [14], who used almost the same cultivation
technique as used in this study, showed a slightly higher
result (96 and 100% positive for patients and controls,
respectively). Quite contradictory results for the proportion of patients yielding fungal cultures from nasal mucus
are found in the literature: 80.0% (prior to surgery) and
33.3% (one to several month after surgery) [23], 61.5%
[24], 39.3% [25], 48.6% [26], 42.1% [27], 73.3% [28],
80.0% [29], 84.6% [30], and 86.4% [31]. The discrepancies are, in our view, based on differences in how various
researchers obtained and treated nasal mucus for fungal
cultures.
Contamination of isolation plates by airborne spores
was excluded by performing all dilution and incubation
steps under a clean bench. Several negative controls
inoculated only with sterile saline showed no fungal
growth (data not shown). Therefore, it can be assumed
that the fungi isolated all derived from nasal mucus or
Table 2
Results of fungal cultures and microscopy for chronic rhinosinusitis patients and controls
Patient
code
Sample Microscopic
type* resulty
Taxa isolatedz
Patient
code
Sample Microscopic
type* resulty
Taxa isolatedz
125
132
135
204
106
153
199
4
7
12
25
58
94
S
S
S
S
S
S
S
F
F
F
F
F
F
SBH
SBH
SBH
SBHC
SH
HC
H
H
H
H
H
H
H
87
116
126
151
159
202
115, 146
1
2
3
6
8
9
F
S
S
S
S
S
S
F
F
F
F
F
F
N
N
N
N
N
N
N
X
X
X
X
X
X
37
36
65
68
70
77
F
F
F
F
F
F
H
D
D
D
D
D
10
11
14
F
F
F
X
X
X
79
104
61
161
148
127
170
175
184
107, 137
121
140
112
5
13
15
17
F
F
F
S
S
S
S
S
S
S
S
S
S
F
F
F
F
D
D
D
DC
D
D
D
D
D
D
C
C
C
N
N
N
N
16
18
19
20
21
22
23
24, 42,
95
26
27
28
29
30
31
32
33
34
35
38
F
F
F
F
F
F
F
F
X
X
X
X
X
X
X
X
F
F
F
F
F
F
F
F
F
F
F
X
X
X
X
X
X
X
X
X
X
X
50
74
F
F
N
N
40
F
X
pros
afum
nide
ccla, calb, cber, nide
bhaw
ccla, apor
pspe
afum, avit, ccla, apul, pspe, nide
calb, ccla, hdem, pspe, umay
calb, ccla
pspe, ccla, bcin, hsch
scom
aalt, ccla, apul, aspe, calb, ucha,
bbas, enig, nide
aspe, pcom, aalt
afum, aspe
pdig
afum, trob
anid, pspe, afum, avit, pexp, sscl,
bbas, basi
ccla, basi
basi
calb, pspe, bbas
calb
pcom, tcuc
calb, basi, pdig
afla
ccla, pspe, aalt, aspe, bbas, enig
pspe
elat
afum, apul, pcom, pisl, ccla
hdem, ccla, anid, pspe, aalt
afum, ccla, aalt, basi, anig, pspe
ccla, aspe, pspe, pvar, aalt, trob,
apul
nide
pspe
ª 2003 ISHAM, Medical Mycology, 41, 149–161
153
ccla, aalt, sscl, enig, nide
ccla, basi
palb
calb, pspe, ares, ccla
hdem
badu
afum, aalt
afum, basi
afum, aspe, apul, ccla, basi
atet, ccla, pcom, hdem
afum, pver, ccla, aalt, afis
aalt, ccla, pspe, sscl, pglo, cpal,
basi, cpar
aalt, ccla, apul, basi
pspe, apul, fspe
afum, pspe, apul, ccla, nide,
badu
ccla, sscl, avit, ahum
pcom, ccla, afum, apul, aalt
afum, aspe, pspe, sscl
ccla, aalt, aspe, pspe, ahum, sscl
aalt, cgla
afum, aspe, ccla, fspe, aalt, lmic
ccla, aalt, cher
pspe
pcom, afum, pspe, apul, cglo
pspe, pcom, aalt
ccla, pspe, aspe, apul, avit, sscl
ahum, hdem, ccla, sscl
ccla, pspe, afum, pglo, aalt
pspe, ccla, apul, aspe, acla
pspe, ccla, afum, nide
aspe, scom
aspe, afum, pspe, aalt
aspe, afum, tfus
ccla, pspe, tvir, tcuc, aust, ahum,
aalt
pspe, aspe, afum, ccla, pisl,
hdem, ahum, ppap
154
W. Buzina et al.
Table 2
Results of fungal cultures and microscopy for chronic rhinosinusitis patients and controls—cont.
Patient
code
Sample Microscopic
type* resulty
Taxa isolatedz
Patient
code
41
43
45
46, 62
47
48
49
51, 64,
73
52
53
54
55
56
F
F
F
F
F
F
F
F
X
X
X
X
X
X
X
X
basi, pspe, cten
aspe, basi
afum, ccla, pspe, tvir
aspe
aspe, nide
pcom, arub, sscl
aspe, pspe, tinh
afum
F
F
F
F
F
X
X
X
X
X
57
59
63
66
67, 100
71
72
75
76
80
81
82
83
84
85
86
88
89
90
91
92
93
96
97
98
99
101
39, 44,
60, 69,
78, 102,
103
105, 200
108
109
110
111
113
114
117
118
119
120
122
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
pspe, anid, arub, ahum, basi
pspe, aspe, anid
afum, anig, pspe
pspe, afum
afum, bbas, hann, aspe, cten,
cpar
anid
csph
afum, anid, pspe, tmuc
hann
calb
cher
pcom, sscl, arsp, calb
pcom, pexp, cten
avit, hsch, afum, nide
anid, tcuc
anid, enig
calb, scom
anid, pisl, ccla, basi, nide
nide
afum, anid, aspe, pspe, ccla
etax, basi, nide
ppap, basi
afla
calb, cpar, ccla
aspe, pspe, ccla, badu
afum, aalt, pspe, ccla, cfam
aalt, ccla, pspe, apul, nide
ccla, pspe, nide, scom, fspe, nide
aspe, anig, enig, foxy, pspe
anig, pspe
coxy, pspe, aspe, afla, sscl, enig
calb, anig, acla, ccla, pspe
S
S
S
S
S
S
S
S
S
S
S
S
X
X
X
X
X
X
X
X
X
X
X
X
128,
130
133
136
139
142
144
145
149
150,
156
157
158
162
163
164
165
167
169
171
172
173
174
176
177
178
179
180
181
182
183
185
123,
129,
155,
187,
189,
192
190
193
195
196
197
201
203
206
207
208
209
141,
138,
147,
154,
168,
198,
211
212
213
basi
aspe, anig, pspe, ucha, fsol
calb, aalt, enig, ccla, pspe
aalt, apul, anid, pspe, ctri, anig
ccla, apul, basi
apul, ccla, calb, nide
avit
ccla, apul, aspe, tcuc, nide
anid, aspe, pspe, ccla, aalt
calb, aalt, ucha, apul, ccla, pspe
cgla, aspe, pspe, apul
pspe, sscl
Sample Microscopic
type* resulty
Taxa isolatedz
210
S
S
S
S
S
S
S
S
S
166 S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
124, S
134,
186,
188,
191,
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
afum
aalt, sscl, baus
cgla, apul, pspe, afum
scom
tcuc
anig, pspe, nide
ckru
rory, astr, pspe
suni, pign, pspe
calb, badu
calb, basi, pspe, aspe, bbas
pspe, fspe
calb
pspe, basi
ctro, scom
aalt
aspe, nide, gcan
enig
badu
afum, pspe
apul, enig, pspe
aspe, sscl, arsp, pgla
bhaw
ccla, pspe, apul, plil, nide
ccla, anig, aalt, pmva, lbic
cher, sfla
ctro, pspe
hdem, ppul, pdig, ccla, nide
cfam, ucha, gspe, apul
aalt, cher, nide
calb, apul
anig, pspe
pspe
S
S
S
S
S
S
S
S
S
S
S
131, S
143,
152,
160,
194,
205
C
C
C
X
X
X
X
X
X
X
X
X
X
X
X
apul, ccla, pspe, nide
pspe, nide
aspe
pspe, coxy
ccla, enig
apul, ccla, basi
calb, pspe, nide
nide
ccla, bcin, pglo, nide
calb, pspe, ccla, misp
pspe, aspe, calb, aara, nide
–
X
X
X
aalt, apul, ccla
ccla, aspe, cgui, sbre
apul, hsch
ª 2003 ISHAM, Medical Mycology, 41, 149–161
Fungal biodiversity
Table 2
Results of fungal cultures and microscopy for chronic rhinosinusitis patients and controls—cont.
Patient
code
Sample Microscopic
type* resulty
Taxa isolatedz
Patient
code
Sample Microscopic
type* resulty
Taxa isolatedz
214
215
216
217
218
C
C
C
C
C
X
X
X
X
X
222
C
X
219
220
221
C
C
C
X
X
X
223, 230
224
225
226, 228
227
229
231
232, 233
C
C
C
C
C
C
C
C
X
X
X
X
X
X
X
X
acla, pspe
ccla, ainf
ccla, gspe, pros
pspe, apul, ccla, aspe, umay
pspe, aspe, afum, anig, ccla,
pver, umay, tvir, bbas
ccla, pspe, aspe, astr, cten, nide
ccla, apul, nide
afum, ccla, pspe, apul, lmic, vlec,
mcib, anid
155
ccla, pspe, aalt, aspe, anid, avit,
pchr
ccla, pspe
ccla, pspe, nide
apul, nide
pspe
ccla
afum, pspe, bbas
pspe, aspe, anid, ccla, aalt, nide
*Sample type: C, healthy control; F, chronic rhinosinusitis (CRS) patient, mucus obtained by flushing; S, CRS patient, mucus obtained by surgery.
yMicroscopic fungal structures: B, branched; C, conidia; D, hyphal debris; H, hyphae; N, Negative; S, septate; X, not determined.
zTaxa: aalt, Alternaria alternata; aara, Aphanocladium araneareum (recently renamed Lecanicillium aphanocladii); acla, Aspergillus clavatus; afis, Aspergillus
fischerianus (Neosartorya fischeri); afla, Aspergillus flavus; afum, Aspergillus fumigatus; ahum, Ampelomyces humuli; ainf, Alternaria infectoria; anid, Aspergillus
(Emericella) nidulans; anig, Aspergillus niger; apor, Aporospora sp.; apul, Aureobasidium pullulans; ares, Aspergillus restrictus; arsp, Arthrinium sp.; arub,
Aspergillus rubrobrunneus (Eurotium rubrum); aspe, Aspergillus sp; astr, Acremonium strictum; atet, Aspergillus tetrazonus; aust, Aspergillus ustus; avit,
Aspergillus vitis (Eurotium amstelodami); badu, Bjerkandera adusta; basi, Basidiomycetes; baus, Bipolaris (Cochliobolus) australiensis; bbas, Beauveria bassiana;
bcin, Botrytis cinerea; bhaw, Bipolaris (Cochliobolus) hawaiiensis; calb, Candida albicans; cber, Cunninghamella bertholletiae; ccla, Cladosporium
cladosporioides; cfam, Candida famata (Debaryomyces hansenii); cgla, Candida glabrata; cglo, Chaetomium globosum; cgui, Candida (Pichia) guilliermondii;
cher, Cladosporium herbarum; ckru, Candida krusei; coxy, Cladosporium oxysporum; cpal, Curvularia pallescens; cpar, Candida parapsilosis; csph, Cladosporium
sphaerospermum; cten, Cladosporium tenuissimum; ctri, Curvularia trifolii; ctro, Candida tropicalis; elat, Eutypa lata; enig, Epicoccum nigrum; etax,
Echinodontium taxodii; foxy, Fusarium oxysporum; fsol, Fusarium solani (Nectria haematococca); fspe, Fusarium sp.; gcan, Geotrichum candidum; gspe,
Ganoderma sp.; hann, Heterobasidion annosum; hdem, Hormonema dematioides; hsch, Hypocrea schweinitzii; lbic, Leptosphaeria bicolor; lmic, Leptosphaeria
microscopica,; mcib, Myriosclerotinia ciborium; misp, Microdochium sp.; nide, not identified; palb, Penicillium alberechii ined.; pchr, Penicillium chrysogenum;
pcom, Penicillium commune ; pdig, Penicillium digitatum; pexp, Penicillium expansum; pgla, Penicillium glabrum; pglo, Phoma glomerata; pign, Phellinus
igniarius; pisl, Penicillium islandicum; plil, Paecilomyces lilacinus; pmva, Paecilomyces variotii; ppap, Pleospora papaveracea; ppul, Pleurotus pulmonarius; pros,
Penicillium roseopurpureum; pspe, Penicillium sp.; pvar, Penicillium variabile; pver, Penicillium vermiculatum (Talaromyces flavus); rory, Rhizopus oryzae; sbre,
Scopulariopsis brevicaulis; scom, Schizophyllum commune; sfla, Schizopora flavipora; sscl, Sclerotinia sclerotiorum; suni, Saccharomyces unisporus; tcuc,
Thanatephorus cucumeris; tfus, Trichaptum fusco-violaceum; tinh, Trichoderma inhamatum; tmuc, Trichosporon mucoides; trob, Tricholoma robustum; tvir,
Trichoderma viride; ucha, Ulocladium chartarum; umay, Ustilago maydis; vlec, Lecanicillium (Verticillium) lecanii.
from sinus materials obtained by FESS, and thus either
represented organisms colonizing the sinus or nasal
surfaces or organisms belonging to the normal incident
spores impacting into nasal mucus from indoor or
outdoor air. The latter category was considered relevant
for evaluation because there might be a number of
dormant fungal propagules whose power as triggering
agents for immunological responses is significant. When
Table 3
mucus samples were collected endoscopically from the
affected areas of the sinus, the quantity of fungal cultures
obtained was lower than that obtained in samples from
nasal lavage. Eighty-nine of 106 (84.0%) patients
evaluated using FESS, compared with 95 of 104
(91.3%) flushing patients had fungi in their sinuses. A
x2 test showed a P-value 5 0.001. Also the number of
different species per patient was lower (P ¼ 0.037), with
Synopsis of numbers of chronic rhinosinusitis patients and controls yielding predominant fungal types.
Fungal type
No. [%] of
positive surgical
patients
No. [%] of positive
patients sampled by
flushing
Total no. [%] of
positive patients
No. [% ] of
positive controls
Aspergillus fumigatus
Aspergillus (all species)
Alternaria (all)
Candida albicans
Candida (all)
Cladosporium (all)
Penicillium (all)
Aureobasidium
Basidiomycetes (all)
5
25
9
16
22
23
48
13
20
31
85
21
10
15
45
60
13
30
36
110
30
26
37
68
108
26
50
3
15
4
–
1
16
16
6
3
[4.7]
[23.6]
[8.5]
[15.1]
[20.8]
[21.7]
[45.3]
[12.3]
[18.9]
ª 2003 ISHAM, Medical Mycology, 41, 149–161
[29.8]
[81.7]
[20.1]
[9.6]
[14.4]
[43.3]
[57.7]
[12.5]
[28.8]
[17.1]
[52.4]
[14.3]
[12.4]
[17.6]
[32.4]
[51.4]
[12.4]
[23.8]
[13.0]
[65.2]
[17.4]
[4.3]
[69.6]
[69.6]
[26.1]
[13.0]
156
W. Buzina et al.
an average of 1.9, compared with 3.2 (patients) and 4.0
(control) species per person in mucus obtained by
flushing. This difference may be explained by the fact
that, with irrigation, fungi will be flushed out of not only
an affected sinus, but also the entire nasal area consisting
of the inferior, middle and superior as well as the
common nasal meatus, the anterior ethmoidal sinus
clefts, and portions of the nasal vestibulum and the
vibrissae. Mucus sampling during endoscopic sinus
surgery does not collect fungi from the anterior portions
of the nose, but from the diseased sinus area only. Not
only did the number of fungi isolated show differences
between surgical and flushing patients, but the occurrence of some taxa differed also. Whereas the total
percentage of controls and flushing patients growing
Aspergillus is not significantly different (57.7 vs. 73.9%),
there was a significant lower occurrence of Aspergillus in
the surgery group (20.8%, P 5 0.001) than in either of
these groups. Regarding the commonly detected Aspergillus fumigatus, the differences between the three
groups examined are even more prominent. In the
controls 13.0% grew this fungus, compared with 29.8%
in the flushing group and only 4.7% of the surgery
patients (P 5 0.001). Perhaps the heavier mucus of the
patients traps more propagules in the anterior portions
of the nose than are trapped by controls, but these
propagules are found less frequently in the affected
sinuses of the surgery group. Cladosporium appeared to
be found most commonly in the control group at 65.2%,
but the difference from other groups was found not to be
statistically significant, perhaps as an artifact of the low
sample number. However, the difference between
flushing (42.3%) and surgery (20.8%, P 5 0.001) patients was significant for this genus. This leads to the
conclusion that some classically pathogenic fungi such as
Aspergillus are actually establishing to some degree in
nasal mucus of patients, and this is best seen by flushing
studies, whereas Cladosporium and perhaps other classic
seldom- or never-pathogenic fungi are present in mucus
more or less entirely as impacted, nongrowing propagules and are rapidly eliminated by mucociliary transport
in the healthy subject.
The genera Aspergillus, Penicillium, Cladosporium
and Alternaria were the most prevalent taxa in all three
groups of persons investigated. The same genera appear
as the most frequent fungi in the environment in a
variety of publications [32–34]. In Aspergillus, A. fumigatus was the most common species. This correlates with
results from Köck et al. [35] obtained 1997 in the region
(Graz, capital of Styria in south-eastern Austria) where
this study was also performed. A. fumigatus is the main
agent of aspergillosis in immunocompromised patients
and is also responsible for a variety of other diseases (see
Ref. 16 for a list of the literature). Apart from
A. fumigatus, 10 species of Aspergillus were identified
in our samples (Table 2). Penicillium was present as 12
different identified species, but many isolates were not
identified to the species level because of the high level of
variety found in this genus, and are listed in Table 2 as
Penicillium sp. Rainer et al. [34] reported a spectrum of
more than 38 species of Penicillium from an Innsbruck,
Austria hospital that was environmentally sampled over
just a six-month period. In the genus Cladosporium we
identified five species, the most common of which was
C. cladosporioides. This species was also found to be one
of the prevalent fungi in the environment in several
studies [32,36,37], although Rainer et al. [34] found
C. herbarum to be the most common species in
Cladosporium in air samples from the hospital studied
in Innsbruck. Problems concerning the identification of
different species of Alternaria and Ulocladium were
discussed recently by de Hoog & Horré [18]. We
differentiated A. infectoria and A. alternata, with a high
majority of the latter (94%). Four isolates with an
appearance strongly suggesting nonsporulating Alternaria were identified as Ulocladium chartarum based on
ITS sequences. The related Pleospora papaveracea, a
pathogen of poppy plants, was isolated from CRS
patients twice. Differences between CRS patients and
healthy subjects were found in the occurrence of
Candida yeasts, which were present as seven species. In
mucus samples obtained endoscopically from the sinus,
10.3% of all isolates were Candida, mostly C. albicans
(72.7%). In mucus samples obtained by flushing the nose
with saline from CRS patients, 4.5% of isolates were
Candida (66.7% C. albicans) and in the healthy control
group one strain of C. guilliermondii (Pichia guilliermondii) was detected (1.3%). In terms of patient
numbers, 17.6% of all patients, including those sampled
by endoscopy and those sampled by flushing, grew at
least one Candida isolate, compared with 4.3% of
controls; 12.4% of patients grew C. albicans, whereas
no control patient did so. However, these differences in
the occurrence of Candida are not significant (P ¼ 0.176
for Candida, P ¼ 0.091 for C. albicans). Apart from
Candida spp., we have isolated a related representative
of the family Saccharomycetaceae, Saccharomyces unisporus, from a patient with CRS.
Aureobasidium pullulans was frequently isolated. A
variety of diseases caused by this fungus have been
reported in medical literature (see Ref. 16 for a review).
Because of the high abundance of this black yeast in the
environment [38], the finding of A. pullulans in nasal
secretions, from both patients and healthy persons, was
not surprising. Closely related to A. pullulans is Hormonema dematioides, which was found as seven isolates
ª 2003 ISHAM, Medical Mycology, 41, 149–161
Fungal biodiversity
from patients, but was not obtained from healthy
subjects. The anamorph genus Fusarium was found in
surgical samples from the nasal sinuses as well as in
mucus obtained by flushing the nose. Fusarium spp.,
important plant pathogens and producers of mycotoxins,
are reported repeatedly as causing sinusitis [39–41]. We
have identified the species F. oxysporum, F. solani
(Nectria haematococca) and one isolate identified only
as Fusarium sp.
The causative agent of ‘snow mould’ on grasses is the
fungus Microdochium nivale, formerly known as Fusarium nivale. Cases of infections in humans caused by this
psychrophilic fungus (not growing at or near body
temperature) are not known from the literature. We
isolated one strain of Microdochium sp. from a patient
suffering from sinobronchial syndrome. This finding
should not be interpreted as indicating establishment in
situ or sole causality, but at the same time, the
contribution of such fungi to the development of
eosinophilic inflammation may be significant in some
patients. The genus Leptosphaeria presented with the
species L. bicolor and L. microscopica in one sample
each of nasal mucus from patients suffering from CRS.
Leptosphaeria-like ascospores are one of the elements
commonly recognized in nonviable air spora samples
worldwide. Ampelomyces humuli, which we identified in
six samples obtained by flushing but not in surgical
specimens, is not listed in textbooks as being a fungus
with clinical significance. Epicoccum nigrum, quoted
repeatedly as producer of allergens affecting patients
suffering from asthma and CRS [42–47], was isolated
from 10 patients, but not from healthy persons. The low
numbers involved prevented statistical analysis. Three
species of the Trichoderma–Hypocrea anamorph–teleomorph complex could be identified: Trichoderma
inhamatum, T. viride (Hypocrea rufa) and Hypocrea schweinitzii. One case of an invasive sinusitis caused
by Trichoderma longibrachiatum was reported by Furukawa et al. [48]; however, none of our patients showed
signs of a fungal invasion of the tissue. Arthrinium
phaeospermum has been described as cause of cutaneous
infections [49,50]; we isolated two strains of Arthrinium
sp. from nasal mucus. The entomopathogenic (parasitic
to insects) fungus Beauveria bassiana is rarely found as a
cause of diseases in humans [51]. We isolated this fungus
from nasal mucus in six patients and two healthy
controls. The genera Bipolaris and Curvularia, closely
related to each other (teleomorphs Cochliobolus in both
cases), typically produce dark pigmented hyphae. Two
surgical isolates were identified as Bipolaris hawaiiensis
(teleomorph Cochliobolus hawaiiensis), a known cause
of CRS and sinus mycoses [52–55], and another was
identified as B. australiensis. Curvularia species are also
ª 2003 ISHAM, Medical Mycology, 41, 149–161
157
listed as causing CRS and invasive sinusitis [56–60]. Our
surgical samples from a sinus contained a strain of
Curvularia trifolii, and one strain was identified as
C. pallescens. One isolate each of Paecilomyces variotii
and P. lilacinus, fungi reported repeatedly to cause
sinusitis [61–69], were cultivated from mucus samples
of patients suffering from CRS. The mycoparasitic
fungus Lecanicillium aphanocladii (formerly Aphanocladium aranearum), which we have isolated once, is,
among other fungi, thought to be responsible for
respiratory distress in workers harvesting grain [70]. No
medical case was found for Eutypa lata, the causal agent
of Eutypa dieback, a serious necrotizing disease of
grapevine [71]. Sclerotinia sclerotiorum is known for the
production of ‘Sclerotinia sclerotiorum (1!3)-beta-dglucan’ (SSG), which possesses anti-tumor activity, and
which has effects on the function of human alveolar
macrophages, lysosomal enzyme activity and the secretion of nitric oxide [72,73]. It also acts as an inflammatory
agent and causes mucous membrane irritations [74]. A
case of a phototoxic dermatitis caused by celery infected
by Sclerotinia sclerotiorum is reported in the literature
[75]. In our samples from nasal secretions we found this
species in 14 cases. It was not associated with any case in
which well-formed filaments were seen in direct microscopy.
Acremonium strictum, a fungus reported to be a cause
of a pulmonary infection [76] and invasive infections in
neutropenic patients [77,78], was isolated from one
patient and one control. A case of brain infection
attributed to Chaetomium globosum was reported by
Anandi et al. [79], but the fungus involved was later
reidentified as Chaetomium atrobrunneum by Abbot
et al. [80]. We found the environmentally ubiquitous
C. globosum in the nasal mucus of one patient. The
ascomycetous yeast Geotrichum candidum was isolated
in one case from the maxillary sinus of a patient. Phoma
glomerata has been suspected to be involved in several
mycotic diseases [81]. In our patients we isolated this
fungus in three cases from nasal mucus. Scopulariopsis
brevicaulis, a frequent cause of onychomycoses, was
detected in nasal mucus from one healthy control. Also
able to cause onychomycoses and white piedra, Trichosporon mucoides was obtained by nasal lavage in one
case. The plant pathogenic Botrytis cinerea, causing the
‘noble rot’ of grapes, was reported as the agent of ‘berry
sorter’s lung’ or ‘wine grower’s lung’ [82]. We found this
fungus in nasal lavage from two patients, one living in
the wine region of south-eastern Styria, the other living
next to a wine- and fruit-growing agricultural college.
Several isolated species that have never been described
in context of human diseases included Aporospora sp.,
Myriosclerotinia ciborium, and the entomopathogenic
158
W. Buzina et al.
Lecanicillium (Verticillium) lecanii. Every species was
present once in isolations from nasal mucus.
Compared with ascomycetes, filamentous basidiomycetes play a minor role as human pathogenic agents,
although recent investigations have suggested a greater
importance for this group [17]. Basidiomycetous yeasts
of the genus Trichosporon are widespread in the
environment. The species we identified, T. mucoides, is
one of the two species with the highest clinical
significance [16]. The dimorphic plant pathogenic (corn
smut) fungus Ustilago maydis (Ustilaginales) appears in
older medical literature as a causal agent of meningitis,
asthma, and dermatomycosis [83–86]. This fungus was
found in nasal secretions in our study, as was Schizophyllum commune (Stereales), the common split gill
mushroom. A review of cases caused by this fungus was
published by Buzina et al. [17]. There were additional
basidiomycetous fungi found in samples of mucus from
the nose and the sinuses, none of which have been listed
in medical literature to date: the root rot fungus
Heterobasidion annosum (Stereales); the Aphyllophoralean fungi Bjerkandera adusta (white rot fungus),
Ganoderma sp. and Trichaptum fusco-violaceum; the
hardwood trunk rot Phellinus igniarius (Hymenochaetales); the Agaricalean ectomycorrhizal symbiont Tricholoma robustum and the wood-decaying Pleurotus
pulmonarius; and the plant pathogens Thanatephorus
cucumeris (Ceratobasidiales) and Echinodontium taxodii.
Also, we isolated 22 nonsporulating strains for which
analysis of the ribosomal ITS region resulted in
classification as ‘unidentified basidiomycetes’.
The zygomycete Rhizopus oryzae is one of the most
important agents of human mucormycosis, and is most
commonly involved in rhinocerebral infections [87–91].
The patient from whom we isolated the strain suffered
from CRS without any symptoms suggestive of an
invasive infection (microscopic examination was not
carried out).
In total, 34 strains were completely unidentifiable, as
they produced no morphological features allowing
identification, and their ITS sequences showed no
homology with known fungi in international genomic
databanks.
The two most common genera, Aspergillus and
Penicillium, were present in mucus samples throughout
the year, without showing statistical significant seasonal
maxima and minima (Fig. 1). Ebner et al. [32] reported
peaks of Aspergillus in August and November–December, and maxima for Penicillium in June and in the
autumn months, both genera were present throughout
the year. A highly significant (P 5 0.001) different
seasonal distribution was observed for Cladosporium,
Alternaria and Aureobasidium pullulans, fungi found in
nature on decaying plant material and thus having their
main occurrence in late summer and autumn in the
northern hemisphere. Although Cladosporium was
present in mucus samples at every time of the year,
there was a significant accumulation in the months July
to October. In the study of Ebner et al. [32], Cladosporium was most prevalent from the end of May to
September. In a year-round observation performed in
1994 in Istanbul, Turkey, Cladosporium was predominant in July and August, and from September to the
end of January [92]. Alternaria was not found in our
patients and controls from January to March, and had
its maximum in August and September. Ebner et al.
[32] showed almost the same seasonal distribution for
Alternaria with a peak from July to October and
virtually no appearance from January to the end of
April.
In general, the finding and identification of these 84
different species of fungi from sinonasal mucus does not
necessarily imply that all the fungi obtained are causal
agents of the diseases affecting the patients examined. It
could also be that a proportion of these fungi are present
in the respiratory tract in detectable amounts without
being connected to the etiology of the disease. However,
to differentiate those cases from ones in which fungi are
responsible for diseases, it is important to determine all
occurring fungi. Within the spectrum of fungi found in
sinonasal mucus there are also some species of thermointolerant fungi, for example, Microdochium sp. and
Cladosporium cladosporioides. This means that they are
not capable of growing at body temperature, but the role
of their spores in inducing or exacerbating inflammatory
reactions is not yet determined. Much cross-reactivity
may be elicited by fungal allergens, especially within
relatively closely related fungal groups [6], so in cases of
CRS where incident airborne spora may contribute to
the condition, it is not necessary that each individual
fungal species contributing to symptoms be associated
with specific patient sensitization or with heavy or
constant exposure.
Our findings strongly support the data published by
Ponikau et al. [14], that fungi can be found in (almost)
any sample taken from anybody’s nose and sinuses. This
finding seems not to be surprising for mycologists, as a
sticky surface, i.e. sinonasal mucus, when exposed to
ambient air is expected to become covered with
omnipresent airborne fungal spores. But this common
knowledge appears to be contradicted by discrepant
results of fungal cultures from nasal mucus in some
studies (e.g. Refs 23–31, and there are many more!). The
mechanism by which, in some people, those airborne
fungi turn from ‘normal flora’ or ‘normal spora’ into
triggers of inflammatory and immunological reactions,
ª 2003 ISHAM, Medical Mycology, 41, 149–161
Fungal biodiversity
resulting in CRS and polyposis, is currently under
investigation at several research centres.
18
Acknowledgements
19
The authors would like to thank Richard Summerbell for
very helpful comments on the manuscript. We are
grateful to Helmut Mayrhofer and Paul Blanz from the
Institute of Botany, Graz, for giving us the opportunity
to use the sequencer. We also wish to thank Martin
Grube for many fruitful discussions, and Kerstin Schimpl
for her invaluable technical assistance.
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