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Prevalence of Candida nivariensis and Candida bracarensis in vulvovaginal candidiasis
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Jianling Lia*, Yingying Shana*, Shangrong Fana**, Xiaoping Liub
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a Department of Obstetrics and Gynecology, Peking University Shenzhen Hospital, Shenzhen, 518036 China;
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b Department of Laboratory Science, Peking University Shenzhen Hospital, Shenzhen, 518036 China;
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*Jianling Li and Yingying Shan contributed equally to this study.
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**Correspondence: Shangrong Fan. Department of Obstetrics and Gynecology, Peking University Shenzhen
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Hospital, Shenzhen 518036, China. Tel.:+86 755 83923333-5505; Fax: +86 755 83923333-8208
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E-mail address:
[email protected] (SR Fan)
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Abstract
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Candida nivariensis and Candida bracarensis were isolated from patients with vulvovaginal candidiasis (VVC).
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Candida nivariensis and Candida bracarensis were found in presumptive Candida glabrata isolates, which
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were identified using the API Candida system. We retrospectively re-examined vaginal presumptive Candida
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glabrata isolates for Candida nivariensis and Candida bracarensis from January 1, 2003, through December 31,
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2012, via detection of the ITS1 region and the 5.8S ribosomal RNA gene. Among 301 presumptive Candida
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glabrata isolates, 293 isolates were confirmed as C. glabrata (97.34%), 7 isolates were identified as C.
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nivariensis (2.33%) and 1 isolate was identified as C. bracarensis (0.33%). The C. nivariensis and C.
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bracarensis isolates were confirmed by sequencing. All C. nivariensis isolates were susceptible to, nystatin and
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susceptible or susceptible dose-dependent, to fluconazole, itraconazole, miconazole, and clotrimazole. The C.
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bracarensis isolate was susceptible to nystatin and the tested azoles. Among the seven patients with VVC
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caused by C. nivariensis and who were treated with various antifungal agents, only one patient achieved
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mycological eradication at both the day 7-14 and day 30-35 follow-ups. The C. bracarensis isolate was isolated
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from a symptomatic pregnant woman; additional data for this patient were unavailable. We conclude that C.
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nivariensis and C. bracarensis existed in the vaginal samples of patients with VVC.Therapeutic efficacy in the
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patients with C. nivariensis was poor and inconsistent with the observed in vitro antifungal susceptibility, which
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requires further study.
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Keywords
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Vulvovaginal candidiasis; Candida glabrata; Candida nivariensis; Candida bracarensis
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Introduction
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Vulvovaginal candidiasis (VVC), affects up to 75% of women of child-bearing age at least once in their lifetime
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and is predominantly caused by Candida albicans [1-4]. An increase in the prevalence of non-albicans species,
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mainly C. glabrata, in VVC has been reported [1]. Two new species, Candida nivariensis and Candida
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bracarensis, have recently been reported as emerging pathogens that are phenotypically indistinguishable from
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Candida glabrata based on conventional chromogenic media or biochemical panels such as the API Candida
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system [5-13]. Several studies have shown that C. nivariensis and C. bracarensis are rare in clinical isolates
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[14-16] and less susceptible than Candida glabrata to azole antifungals [10, 17]. Few data of clinical
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significance regarding C. nivariensis and C. bracarensis in VVC are currently available [16, 18]. The
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differentiation of these species from C. glabrata may be of major importance in understanding their clinical and
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epidemiologic role in VVC. The purpose of this study was to determine the occurrence of C. nivariensis and C.
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bracarensis in VVC in southern China and to analyze the clinical characteristics, drug susceptibility and
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therapeutic efficacy of these patients.
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Methods
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Clinical isolates and reference strain
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Vaginal swabs were obtained from patients with VVC attended the Gynecology Department Clinic, Peking
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University Shenzhen Hospital from January 1, 2003 through December 31, 2012. The specimens were plated on
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CHROMagar (Biocell Laboratory Ltd, Zhengzhou, China) for 24 to 48 hours at 37°C in ambient air. The isolates
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were identified using the standard API Candida system (Biomerieux, France) and stored in medium containing 2%
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glucose, 2% peptone and 20% glycerol at –70°C.
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Molecular identification
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The presumptive C. glabrata isolates were removed from the –70 °C freezer and revived on a Sabouraud agar
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plate for 24 to 48 hours at 37°C in ambient air. One single yeast colony from the isolates was suspended in a
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microcentrifuge tube containing 50 µL of lysis buffer for direct polymerase chain reaction (PCR) to detect the
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microorganisms (Takara Biotechnology Co., Ltd,Dalian,China). The contents were heated at 80°C for 15 min
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and then centrifuged. One microliter of the supernatant was used for the PCR. The four primers used (universal
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reverse primer: UNI-5.8S 5’-ACCAGAGGGCGCAATGTG-3’ and species-specific forward primers: GLA-f
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5’-CGGTTGGTGGGTGTTCTGC-3’, NIV-f 5’-AGGGAGGAGTTTGTATCTTTCAAC-3’ and BRA-f
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5’-GGGACGGTAAGTCTCCCG-3’), the composition of the PCR mixture, and the PCR conditions were in
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accordance with the methods described by Romeo et al [19]. The PCR products were electrophoresed on a 2%
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(wt/vol) agarose gel in TBE buffer and then visualized using a Unitec system (Cambridge, USA) following
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ethidium bromide staining (0.5 mg/ml). The PCR products obtained from the C. nivariensis and C.bracarensis
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isolates were sequenced（Applied Biosystems 3730xl DNA Sequencer）using primers identical to those used for
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the PCR.
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Germ tube test
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The isolates were incubated in sterile test tubes containing 0.5 ml of serum at 35°C for 2.5 to 3 hours. A drop of
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the yeast/serum mixture was placed on a clean microscope slide, covered with a cover slip, and microscopically
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examined. The appearance of small filaments projecting from the cell surface indicated germ tube test positivity.
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The reference strain used was C. albicans (ATCC 90028).
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Chlamydospore test
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The isolates were plated on corn meal agar plates supplemented with 1 % Tween 80 for 24 to 72 hours at 25 °C
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in ambient air. The wet mount was examined microscopically. The appearance of chlamydospore indicated
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chlamydospore test positivity. The reference strain used was C. albicans (ATCC 90028).
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Antifungal susceptibility testing
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In vitro susceptibility of Candida for fluconazole, itraconazole, miconazole, clotrimazole,and nystatin was tested
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using a commercial agar diffusion test obtained from Rosco Laboratory (A/S Rosco, Taastrup, Denmark). The
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Neo-Sensitabs tablet assay was performed according to the manufacturer's instructions [20]. The quality control
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isolate Candida albicans ATCC 90028 was tested in the same manner. The endpoints for the antifungal agents
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were interpreted according to the manufacturer's instructions.
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Treatment regimens
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The isolates used in this study were collected from several previous clinical trials. The patients were treated with
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the following antifungal agent regimens: including: oral fluconazole 150 mg one dose; fluconazole 150 mg two
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doses and nystatin vaginal suppository 20 mIU daily for 14 days; miconazole nitrate vaginal suppository 1200
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mg one dose; and miconazole nitrate vaginal suppository 1200 mg
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refered Candida negative or positive on Candida culture at 14(7-14) days and 35(30-35) days after last
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antifungal agent dosing.
two doses. Eradication or failure was
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Statistical methods
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Values of the results are expressed as the means unless otherwise indicated. Each variable was tested for
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between-groups differences by Student’s t test or the chi-squared test where appropriate. Statistical significance
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was set at P
