Candida nivariensis as a New Emergent Agent of Vulvovaginal Candidiasis: Description of Cases and Review of Published Studies

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1 DOI /s 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 García-Martos. Manuel Rodríguez-Iglesias Received: 12 July 2015 / Accepted: 13 December 2015 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. García-Martos M. Rodríguez-Iglesias (&) Clinical Microbiology Lab, Puerta del Mar University Hospital, University of Cádiz, Cádiz, Spain manuel.rodrigueziglesias@uca.es 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 % 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.

2 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. 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 inco 2 atmosphere). These isolates were not able to form germ tubes, chlamydospores, pseudohyphae or ascospores. 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 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 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 (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. 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 Pregnancy (gestational weeks) No No Pruritus vulvar Yes Yes Yes No Discharge profuse Yes Yes Yes Yes Predisposition and/or therapy Contraceptives Clotrimazole Fosfomycin Diabetes Treatment Fluconazole Clorhexidina/boric Fluconazole Itraconazole/boric

3 Table 2 Minimal inhibitory concentration to antifungal compounds in C. nivariensis Antifungal MIC50 MIC90 Posaconazole Voriconazole Itraconazole Fluconazole 2 4 Micafungin Anidulafungin Caspofungin Amphotericin B 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 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 Table 3 Overview of published reports of C. nivariensis isolates 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 UK Borman et al. [3] fluid, peritoneal fluid, lung biopsy 1 Oral rinse Indonesia Wahyuningsih et al. [13] 1 Pleural fluid Australia Lockhart et al. [14] 2 Blood, sputum India Chowdhary et al. [15] 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, Poland Swoboda-Kopec et al. [20] peritoneal fluids 7 Vagina China Li et al. [21] 1 Toenails China Feng et al. [22] 4 Vagina Spain This study

4 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 MALDI- TOF to obtain a definitive identification. 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