Mycopathologia
DOI 10.1007/s11046-015-9978-y
Candida nivariensis as a New Emergent Agent
of Vulvovaginal Candidiasis: Description of Cases
and Review of Published Studies
Pilar Aznar-Marin . Fátima Galan-Sanchez . Pilar Marin-Casanova .
Pedro Garcı́a-Martos . Manuel Rodrı́guez-Iglesias
Received: 12 July 2015 / Accepted: 13 December 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract Candida nivariensis is a new emergent
agent related to human infections in the vaginal tract
and other localizations, but the phenotypic characteristics are very similar to Candida glabrata and can be
misidentified and underdiagnosed. We described four
cases of vulvovaginitis identified by matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry and confirmed the results with PCR amplification and sequencing of the entire ITS genomic
region (ITS1, ITS2 and 5.8 rRNA). We reinforce the
need for new diagnostic tools for the correct identification of yeast infections.
Keywords Candida nivariensis MALDI-TOF Vaginitis Emerging yeasts
Introduction
While Candida albicans remains the most common
yeast species implicated in symptomatic vulvovaginitis, recent reports have described a relative decrease in
P. Aznar-Marin F. Galan-Sanchez P. Marin-Casanova P. Garcı́a-Martos M. Rodrı́guez-Iglesias (&)
Clinical Microbiology Lab, Puerta del Mar University
Hospital, University of Cádiz, Cádiz, Spain
e-mail: [email protected]
its proportional impact compared to non-Candida
albicans species, with C. glabrata, C. parapsilosis, C.
tropicalis, C. lusitaniae and C. krusei as emerging
significant opportunistic pathogens. These species of
Candida are readily identified by conventional phenotypic-based methods, which rely upon a combination of morphological features coupled with the
abilities of the organisms to ferment several sugars
or assimilate a variety of carbon and nitrogen sources.
However, conventional methods for yeast identification are slow, and chromogenic media have been
formulated to detect different Candida species on the
basis of the color of the colonies, but they are only
useful in a limited number of species [1–3]. Matrixassisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF MS) has emerged
as a powerful and rapid tool for the identification of
bacterial and yeast pathogens, and several studies have
reported species identification rates of 92–99 %
among collection of yeasts and yeast-like organisms
and clinical samples [4–8]. Molecular approaches
have also been developed and were designed mostly
for the ribosomal RNA (rRNA) genes [9, 10]. Due to
the expansion of Candida non-albicans infection, and
their different antifungal susceptibility pattern, accurate identification is essential for clinical management.
C. glabrata represent a species complex. A correct
identification of the species is important for appropriate treatment and may be important for the management of the patients infected by the various C.
glabrata look-alike species.
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Mycopathologia
In 2005, Candida nivariensis, a yeast species
genetically related to C. glabrata, was described in a
Spanish hospital [3] from 3 patients over a 3-year
period. Subsequently, it was described as a causative
agent of candidemia and oropharyngeal candidiasis.
Its further isolation from deep, usually sterile body
fluids indicates that this pathogen is widely distributed
in clinical specimens and relevant in human infections
[1]. C. nivariensis exhibit frequently multidrug resistance to azole antifungal agents and have MICs similar
to or even more elevated than those for C. glabrata.
We describe four cases of C. nivariensis identified by
MALDI-TOF and confirmed by sequencing as an
etiologic agent of vulvovaginal candidiasis.
The identification based on colony morphology and
carbohydrate assimilation (ID32C, bioMerieux,
Marcy-l’Êtoile, France) was uncertain. Biochemically,
only the fermentation of trehalose can distinguish C.
nivariensis from C. glabrata; however, C. glabrata
developed pink-colored colonies in CHROMagar. The
isolates were analyzed by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF). Mass
spectra acquisition and analysis were performed on a
Bruker Microflex platform using MALDI Biotyper
(Bruker Daltonik, Bremen, Germany) following our
protocol as previously described [11]. All isolates were
identified as C.nivariensis with score ranging between
1.802 and 2.086, indicating secure genus and probable
species identification. To confirm MALDI-TOF
results, PCR amplification and sequencing of the entire
ITS genomic region (ITS1, ITS2 and 5.8 rRNA) using
primers ITS-1 and ITS-4 as previously described [11]
were done. Sequence was edited using Chromas v. 2.23
software (Technelysium Pty. Ltd, Brisbane, Australia)
and entered into BLAST search provided by Genbank
for species identification. A percent similarity score of
100 % between the query sequence and the C.nivariensis NRRL Y-48269 (access no. JN882341) was
obtained. Susceptibility testing was performed using
Sensititre Yeast One 10 (Trek Diagnostic Systems,
Cleveland, USA) according to CLSI guidelines, and
the results are summarized in Table 2, showing
sensitivity to all antifungals tested.
Patients and Methods
We described four cases in women presented at
hospital with vulvar pruritus and profuse vaginal
discharge. The age range was between 20 and 33 years
old. Two patients were pregnant and another has
diabetes. All were treated with fluconazole or itraconazole and boric acid responding well to treatment
(Table 1). To obtain the sample, two sterile cottontipped commercial swabs were used to collect discharge from high vagina and transported to the
Laboratory of Microbiology for culture. One of the
swabs was used for Trichomonas culture using Roiron
medium, while the other was plated on CHROMagar
Candida, blood agar, MacConkey agar and chocolate
agar. Gram stain shows the presence of yeasts cells.
After 48 h of incubation at 30 °C, white colonies grew
on CHROMagar Candida, as well as in blood and
chocolate agar (incubation at 37 °C in CO2 atmosphere). These isolates were not able to form germ
tubes, chlamydospores, pseudohyphae or ascospores.
Discussion
The first report of C. nivariensis was done in Canary
Islands, Spain [1]. It was isolated from blood, bronchoalveolar lavage and urine samples, from three
patients of the same hospital. C. nivariensis has isolated
Table 1 Characteristics of the patients studied
Characteristic
Case 1
Case 2
Case 3
Case 4
Age
20
33
25
32
Pregnancy (gestational weeks)
Pruritus vulvar
31
Yes
27
Yes
No
Yes
No
No
Discharge profuse
Yes
Yes
Yes
Yes
Predisposition and/or therapy
Contraceptives
Clotrimazole
Fosfomycin
Diabetes
Treatment
Fluconazole
Clorhexidina/boric
Fluconazole
Itraconazole/boric
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Mycopathologia
Table 2 Minimal inhibitory concentration to antifungal compounds in C. nivariensis
Antifungal
MIC50
MIC90
Posaconazole
0.03
0.125
Voriconazole
0.03
0.125
Itraconazole
0.25
0.5
Fluconazole
2
4
Micafungin
0.015
0.015
Anidulafungin
0.015
0.015
Caspofungin
0.06
0.125
Amphotericin B
0.25
0.25
from in clinical specimens and relevant in human
infections as blood, peritoneal fluid, pleural fluid,
catheter, respiratory samples, urine, toenails and vulvovaginal infection [12–22] (Table 3). Predisposing risk
factors include diabetes mellitus and immunosuppression as VIH coinfection and renal transplantation [6, 11].
Vulvovaginitis is the most frequent clinical presentation
in women [17, 22], appearing as risk factors for
pregnancy, premenstrual phase, diabetics, oral contraceptives, using soap with pH acid and previous antibiotic
therapy. The natural ecological niche of C. nivariensis is
garden soils or potted plant but is evident in the
adaptation to human environment. The infection is
Table 3 Overview of
published reports of C.
nivariensis isolates
acquired through sexual transmission or from fecal
reservoir, which explains the isolation from urine or
vaginal exudates and the recurrence of episodes.
C. nivariensis and C. bracarensis have shown to be a
close genetic relative of C. glabrata [23]. Although it
seems to have a global distribution around the five
continents, nowadays, the total number of clinical cases
reported is small, possibly due to the impossibility to
distinguish phenotypically this species from Candida
glabrata [3]. Biochemically, only the fermentation of
trehalose can distinguish both species, and a rapid test
(GLABRATA RTT) has been developed, but it needs
the careful choice of primary culture media and they are
needed alternative systems [24]. In CHROMagar, C.
nivariensis grow as white colonies, while C. glabrata
shows pink-colored colonies, but does not permit its
morphological differentiation from other related species as C. bracarensis, C. norvergensis and C. inconspicua [2]. A finding was the ability of MALDI-TOF
MS to distinguish within C. glabrata clade, which
presently rely on molecular methods for species
discrimination [6–8]. Despite its several advantages,
MALDI-TOF MS requires substantial initial setup cost
and is yet to develop a global database of all common
and uncommon pathogens.
There is a controversial issue with the azole
resistance in C.nivariensis. The strains isolated from
Number of
isolates
Source
Country
References
1
Blood
Spain
Alcoba-Florez et al. [1]
1
Blood
Japan
Fujita et al. [12]
16
Blood, oral cavity, pelvic, abscess, ascitic
fluid, peritoneal fluid, lung biopsy
UK
Borman et al. [3]
1
Oral rinse
Indonesia
Wahyuningsih et al. [13]
1
Pleural fluid
Australia
Lockhart et al. [14]
Chowdhary et al. [15]
2
Blood, sputum
India
1
Blood
Spain
López-Soria et al. [16]
5
Vagina, bronchoalveolar lavage
India
Sharma et al. [17]
1
Blood
France
Parmeland et al. [18]
1
Urine
UK
Gorton et al. [7]
2
Blood, vagina
Malaysia
Tay et al. [19]
13
Urine, tracheal aspirate, abscess,
peritoneal fluids
Poland
Swoboda-Kopec et al. [20]
7
Vagina
China
Li et al. [21]
1
Toenails
China
Feng et al. [22]
4
Vagina
Spain
This study
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Mycopathologia
Japan and UK were found to be resistant to azoles, who
report high fluconazole MICs ([64 mg/L) [3, 12],
while other authors have reported MICs of fluconazole
ranging from 0.5 to 16 mg/L [13–17]. Our isolates did
not showed a high level of resistance to this antifungal
agent, but the treatment with fluconazole was not
effective in a patient. Other failures have been reported
by other authors. The MICs of the azole antifungal
agents for C. nivariensis are significantly higher than
those for standard C. glabrata isolates tested with the
same antifungal agents and were equivalent to the
MICs observed for a subset of the most azole-resistant
C. glabrata strains. Moreover, C. nivariensis isolates
are at least as resistant as C. glabrata isolates to
itraconazole and are more resistant than C. glabrata to
fluconazole and voriconazole. Considering that C.
nivariensis has been reported from Europe, Asia and
Australia, it has a propensity to antifungal resistance,
and further studies focusing on its prevalence and
antifungal susceptibility profile are warranted [25].
In conclusion, although C. nivariensis is rare,
correct identification is clinically important as this is
newly described species. The limitations of the current
routine diagnostic methods have highlighted the
requirement for alternative diagnostic approaches
such as MALDI-TOF MS. This method can save time
and costs compared to molecular methods. In our
laboratory, we have implemented a new algorithm
identifying white colonies on CHROMagar as isolates
that require further investigation for correct identification in clinically relevant samples, and have introduced MALDI-TOF for the routine identification of
yeasts. The identification of novel pathogenic Candida
spp requires the use of new alternatives as MALDITOF to obtain a definitive identification.
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