SPECIES DISTRIBUTION AND ANTIFUNGAL SUSCEPTIBILITY OF VAGINAL CANDIDA ISOLATES

Introduction: Vulvovaginal candidiasis (VVC) is the most common Candida infection in females of reproductive age. Data concerning the species identification and antifungal susceptibility of vaginal Candida isolates are of great importance for the infection management. Aim: The aim of the present study was to determine the species distribution and antifungal susceptibility of Candida isolates obtained from vaginal samples of women with VVC. Material and methods: A total of 125 vaginal Candida isolates were collected and tested. The definitive species identification was performed by VITEC2 Compact ( Bio Merieux, France ) using YST ID cards. The minimum inhibitory concentrations (MICs) of nine antifungal agents were determined by the commercial system Micronaut-AM ( Merlin Diagnostika GmbH, Germany ). Results: Overall, 8 Candida species were established among the vaginal yeast isolates. The most common was C. albicans (77.6%), followed by C. glabrata (12%), C. krusei (4%), C. kefyr (2.4%), C. spherica (1.6%), C. lusitaniae , C. utilis , and C. sake (each one 0.8%). All C. albicans and 20 non-albicans Candida (NAC) were susceptible to nine antifungal agents. In the group of NAC, 8 isolates were resistant to fluconazole – 5 C. krusei with intrinsic resistance, 2 C. spherica , and 1 C. sake . The fluconazole MICs of C. spherica and C. sake


INTRODUCTION
Candida albicans and some other Candida species are common colonizers of the female genital system.The colonization is a predisposing factor for development of vulvovaginal candidiasis (VVC).VVC is a widespread disease in otherwise healthy women, predominantly during the reproductive years.The potential risk factors for VVC include pregnancy, use of antibiotics, cytostatics, corticosteroids, oral contraceptives, and the presence of underlying diseases such as diabetes, AIDS, etc. (1)(2)(3)(4)(5)(6)(7)(8).Although not life-threatening illness, its global rate and negative effect on life quality warrant a thorough study of the problem (9).In the majority of cases, the causative agent of VVC is C. albicans but a share of non-albicans Candida (NAC) species is also increased (5,10,11,12).The treatment of VVC involves application of topical or oral azoles but as is known, some NAC possess natural or acquired resistance to azole antifungals.Because of that, the precise species identification and antifungal susceptibility testing are of great importance for the proper therapy.The aim of the present study was to determine the species distribution and antifungal susceptibility of Candida isolates obtained from vaginal samples of women with VVC.

Yeast isolates
The study covers 125 vaginal Candida isolates collected from hospitalized female patients and outpatients with vaginal candidiasis in Pleven, Bulgaria, for the period 2018 -2022.All yeasts were isolated in the routine laboratory work.The pure cultures of yeast strains were stored in skim-milk

ADDRESS FOR CORRESPONDENCE:
Hristina Yotova Hitkova Faculty of Pharmacy, Medical University -Pleven, 1 St. Kliment Ohridski Str., 5800-Pleven, Bulgaria email: hitkova@abv.bgmedia at -20 o C before testing in vitro.Since personal data were not used, this study was not under ethics approval.

Species identification
The identification of Candida species was carried out by microscopic, cultural and biochemical methods.The preliminary identification was based on the growth with different color on CHROMagar Candida (BD, UK), and the microscopic characteristics on a cornmeal agar (BD, UK).The CHROMagar Candida is used for differentiating Candida albicans from nonalbicans Candida.On the cornmeal agar, the specific chlamydospores of C. albicans were observed.The germ tube test which is not routinely done in laboratories was used for rapid detection of C. dubliniensis and C. albicans.Both yeasts form germ tubes after inoculation in sera and incubation at 37º C for 2-4 hours.The definitive species determination was done by the automatic system for biochemical identification VITEK 2 Compact (Bio Merieux, France) using YST ID cards.The results were generated automatically by the apparatus database and these with a confidence level more than 89% probability were accepted as correct.

Figure 1 Species distribution of 125 vaginal Candida isolates
The results of antifungal susceptibility are shown in Table 1.They reveal high susceptibility of all C. albicans isolates to the nine antifungal agents.The MICs of fluconazole ranged from 0.25 -2 μg/mL and the calculated MIC 50 and MIC 90 were 0.5 and 1 μg/mL, respectively.The fifteen C. glabrata strains were also susceptible to all antifungals with fluconazole MICs between <0.002 and 0.5 μg/mL.Overall, non-albicans Candida kept their antifungal susceptibility with the exception of the naturally resistant to fluconazole species C. krusei (fluconazole MICs: 32 -128 μg/mL).C. spherica and C. sake showed a high level of resistance to fluconazole with MICs: 32 -128 μg/mL and MIC -16 μg/mL, respectively.These isolates also expressed resistance to other azoles (voriconazole, posaconazole, and itraconazole) with MICs 4 -8 μg/mL for C. spherica, and 1 -2 μg/mL for C. sake.All C. albicans and non-albicans species were susceptible to amphotericin B (MICs: 0.031 -1 μg/ mL), 5-fluorocytosine (MICs: 0.0625 -4 μg/mL), and the MICs of echinocandins were within susceptible breakpoints.

DISSCUSION
Herein we presented information concerning the species identification and antifungal susceptibility of Candida spp.isolated from women with vulvovaginal candidiasis.Our results are in line with the concept of the global prevalence of C. albicans as a cause of VVC.It is generally accepted that C. albicans is responsible for 70 to 90% of the fungal vulvovaginitis (14).Some publications pay attention on the molecular analyses to distinguish C. albicans from its closely related species C. dubliniensis and C. africana.Using the hwp1 gene amplification, Klesiewicz et al. (15) detected 94.17% C. albicans and 5.22% C. dubliniensis among vaginal yeasts, phenotypic identified as C. albicans.The same authors revealed 97.85% compliance between the VITEK2 system and the molecular assay, and correct identification of all C. albicans sensu stricto with the VITEK2.In our study, C.albicans strains were also identified by the VITEK2 Compact with excellent and very good confidence levels with more than 93% probability.A report of genital tract candidiasis in Poland showed C. albicans in 78.3% of the cases (16).These data are very close to our findings.Similar rates of C. albicans vulvovaginal candidiasis were published in Iran, Taiwan and Australia -67%, 67.3% and 73%, respectively (17,18,7).Holland et al. (5) detected C. albicans in 89% of a total of 1221 vaginal isolates, and others published an even higher rate -92.3% (19).C. albicans was also found as a dominant species of VVC in China and it was confirmed in 84.71% of overall 543 vaginal isolates (20).Hedayati et al. (10) determined three main species -C.albicans (42.5%), C. glabrata (21.9%) and C. dubliniensis (16.4%) among 66 yeasts obtained from females with VVC.In contrast, C. glabrata (57.4%) and C. albicans (25.9%) were detected as the primary and secondary species among pregnant women in Ghana (12).In our study, C. glabrata was the second leading causal pathogen of VVC.This observation is consistent