ANALYSIS OF CANDIDA SPECIES DISTRIBUTION ISOLATED FROM WOMEN WITH VULVOVAGINAL CANDIDIASIS AND THEIR ANTIFUNGAL SUSCEPTIBILITY PROFILE

Background. Candida species is usually a commensal fungus residing in the vagina, in about 30 %–50 % of healthy adults and remains the most common agent isolated from clinical samples of patients diagnosed with vulvovaginal candidiasis (VVC). Culture is not recommended for all patients on a routine basis but is informative for the exclusion of non-albicans infections resistant to azoles, or recurrent VVC. Identification of Candida species and antifungal susceptibility testing are key elements in the correct management of recurrent VVC. The authors of this paper aimed to study the spectrum of pathogenic Candida species. The antifungal sensitivity patterns of the isolated strains during the years 2017-2019 are also presented. Material and methods. A descriptive study of Candida species distribution and antifungal susceptibility profile was performed. Identification of isolated Candida species was performed by the MALDI-TOF MS, bacterial strains by Vitek 2 automated system (BioMérieux) and antifungal susceptibility profiles by Fungitest (Bio-Rad). Results. Of the 1030 recovered strains, C. albicans species predominated 83.8% and Candida non albicans -16.2%. The most common species of the Candida non-albicans, were C.glabrata (7.5%). Also, it was observed that in 56.6% of the cases C. albicans presented bacterial associations. The most common microbial association was C. albicans and S.aureus (26.6%), followed by C. albicans and E.coli (20.8%). Candida spp. showed moderate antifungal resistance. The resistance rate of C. albicans isolates to miconazole, fluconazole and itraconazole was 25.5%, 17.0% and 11.4% compared to non-albicans Candida strains which showed a resistance rate of 10,8%, 14.4% and 10%, respectively. Conclusions. C. albicans is the most frequently isolated species among patients with VVC, but there is also an increase share of non-albicans Candida species such as C. glabrata. C. albicans isolates showed higher indices of resistance, than Candida non-albicans.

The complex research conducted by a group of authors has been estimated that 75 % of all women will experience at least one episode of VVC in their lives. Between 40 % and 50 % of initially infected women will experience a second episode [5,6].
The study focused on the analysis of the articles that approached mainly factors predisposing to VVC highlighted the following factors: contraceptives usage, and also poor personal hygiene and some sexual and clothing habits [5].
Literature described VVC reports on studies based on women's questionnaires on their self-reported history of vulvovaginal candidiasis, but without laboratory diagnoses that confirm infection with Candida [7].
Other studies show that frequently the women practice self-diagnosis and treatment across the counter, while others merely endure the infection which leads to the development of antifungal resistance thus the problem of antifungal resistance [8,9].
According to research data, the clinical diagnosis of vaginal candidiasis is unreliable, and laboratory confirmation is needed which will identify the etiological agents and their susceptibility to antifungals for providing effective therapy [7,9].
The scientific research published during the last two decades presents evidence that, the most common Candida species associated with VVC are C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei. The authors highlighted that commonly a single species, but two or more species have been found in some women with VVC (1-10 %) [10].
Also, other studies demonstrated that in more than 70 % of cases, VVC is caused by C.albicans, followed by C. glabrata, C. tropicalis, C. parapsilosis and C. krusei [11,12].
In recent times, however, there has been a notable shift in the etiology of candidiasis with non-albicans Candida (NAC) species gaining prominence. An increasing trend of VVC caused by non-albicans Candida species (NAC) has been reported due to the widespread use of antifungal agents that are frequently available over-the-counter [13,14].
Since none of the clinical signs of vulvovaginal candidiasis is pathognomonic, clinical the diagnosis must always be confirmed by laboratory methods [15].
There is evidence from few studies which emphasizes the importance of laboratory diagnosis in management of VVC and which have diagnosed the vulvovaginal candidiasis through laboratory confirmation of infection in symptomatic women [16].
Currently, some studies and guidelines recommend laboratory culture of the vulvovaginal specimen for the effective management of VVC, accordingly clinical evaluation and laboratory culture should be the standard diagnostic method [17].
Culture is not recommended for all patients on a routine basis, as colonization can be detected rather than infection. This diagnostic method is informative for the exclusion of non-albicans infections resistant to azoles, or in the case of a recurrent VVC [18,19,20].
The purpose of this study was undertaken to evaluate the Candida species distribution isolated from vaginal swab and their antifungal susceptibility patterns.

Materials and methods
During the 2017-2019, after microbiological analyzes 1030 isolates were recovered from the clinical samples. Specimens were taken from the posterior fornix of the vagina with sterile swabs and transported to the microbiology laboratory for further processing.
Culture isolation and identification For the isolation of Candida spp., specimens were inoculated on Sabouraud dextrose agar supplemented with chloramphenicol (Oxoid) and incubated at 36°C for 72 hours. Simultaneously, with the fungal strains, other associated bacterial strains were isolated. Identification to the species level was performed by MALDI-TOF MS (Bruker Dalto nics) and bacterial strains by Vitek 2 automated system (BioMérieux).
Antifungal susceptibility testing The in vitro activity of the antifungal agents against each isolate was determined by using Fungitest galleries (Bio-Rad SDP Paris, France).
Inoculum preparation From the isolated colonies an inoculum was prepared by suspending them in 3 ml of sterile saline (aqueous solution of 0.50 % NaCl, pH -7.0). To obtain a turbidity equivalent to that of the McFarland 0.50 standard, the measurements were performed with the PhoenixSpec nephelometer (Becton Dikinson). Subsequently, the antifungal susceptibility test was performed by inoculating the Fungitest microplates and incubating at 37° C for 24-48 hours.
Quality control of the investigation. For quality control, C. albicans (ATCC 10231) was used as reference strain and tested simultaneously with the clinical isolates.

Results and discussions
After microbiological analyzes 1030 isolates were recovered from the clinical samples. Information regarding the prevalence of VVC in Moldova is not well known and varies from one study to another. Literature evidence regarding VVC reports highlighted that this disease it is the second most common infection of the vulvovaginal area of symptomatic women accounting for about 17 % to 42 % [21,22,23].
Regrettably, this disease is routinely diagnosed by sign and symptom and is not confirmed with laboratory investigation when necessary. As a result, the spectrum of yeasts implicated in causing the disease, their drug susceptibility profile is not known in the country.

Медицина / Медицина / Medicine
Studies demonstrate the necessity of identification the Candida species responsible for infections in all patients presenting with VVC especially those with recurrent infections. Therefore, identification will influence selection of antifungals and duration of therapy [23,24].
Simultaneously, compared to previous years, a trend has been observedand that is an increase in the rate of isolation of non-albicans Candida species. This phenomenon could be due to the improvement of laboratory diagnostic practices regarding the identification of fungi or a real higher prevalence of these species [25,26].
The most common microbial association was C. albicans and S.aureus -127 combinations (26.6 %), followed by C. albicans and E.coli -99 combinations (20.8 %). Bacterial associations with non-albicans Candida species or recorded less frequently.
The clinical significance of the identification of the Candida to the species level is definite and completely recognized by the majority of studies on the reason for the diversity of expression of virulence factors and their susceptibility to antifungal agents. The support of the laboratory is crucial in order to fight vaginal Candida infection with targeted therapy [27].
Antifungal susceptibility test results show that most isolates were sensitive to 5-fluorocytosine followed by amphotericin B and ketoconazole. Candida species have shown moderate indices of resistance to miconazole, fluconazole and itraconazole.
Isolates of C. albicans have been shown to be more resistant to these medications than non-albicans Candida. The resistance rate of C. albicans isolates to miconazole, fluconazole and itraconazole was 25.5 %, 17.0 % and 11.4 %, compared to non-albicans Candida strains with a resistance rate of 10 %, 14.4 % and 10 %.
Finally, the variety of non-albicans Candida species involved in human pathology, their rising contribution to fungal infections and the antifungal susceptibility profiles makes their identification at the species level essential for epidemiological investigations, optimizing therapy and patient management.

Conclusions
Candida albicans was the most common isolated fungal species among patients with vulvovaginitis, and there is an increasing tendency of isolation of Candida non-albicans species, a phenomenon found by other similar research.
Analyzing the clinical isolates in this research, associations of Candida spp. with other microorganisms were also observed. Candida albicans with S. aureus was the most common, followed by Candida albicans and E.coli.
Based on the antifungal susceptibility test results, it was found that most isolates were susceptible to 5-fluorocytosine and amphotericin B, ketoconazole and moderately resistant to miconazole, fluconazole and itraconazole. C. albicans have been shown to be more resistant than non-albicans Candida.
Additional studies on a larger sample and evalua ting results of antifungal susceptibility testing are of great importance for optimizing therapy and patient management to fighting vaginal Candida infections with targeted therapy.

Conflicts of interests.
Authors declare the absence of any conflicts of interests and their own financial interest that might be construed to influence the results or interpretation of their manuscript.