Species distribution and susceptibility profiles of Candida species isolated from vulvovaginal candidiasis, emergence of C. lusitaniae

Background and Purpose: The aim of the current study was to investigate the epidemiology of vulvovaginal candidiasis (VVC) and recurrent VVC (RVVC), as well as the antifungal susceptibility patterns of Candida species isolates. Materials and Methods: A cross-sectional study was carried out on 260 women suspected of VVC from February 2017 to January 2018. In order to identify Candida species isolated from the genital tracts, the isolates were subjected to polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) using enzymes Msp I and sequencing. Moreover, antifungal susceptibility testing was performed according to the Clinical and Laboratory Standards Institute guidelines (M27-A3). Results: Out of 250 subjects, 75 (28.8%) patients were affected by VVC, out of whom 15 (20%) cases had RVVC. Among the Candida species, C. albicans was the most common species (42/95; 44.21%), followed by C. lusitaniae (18/95; 18.95%), C. parapsilosis (13/95; 13.69%), C. glabrata (8/95; 8.42%), C. kefyr (6/95; 6.31%), C. famata (5/95; 5.26%), C. africana (2/95; 2.11%), and C. orthopsilosis (1/95; 1.05%), respectively. Multiple Candida species were observed in 28% (21/75) of the patients. Nystatin showed the narrowest range of minimum inhibitory concentration (MIC) (0.25-16 μg/ml) against all Candida strains, whereas fluconazole (0.063-64 μg/ml) demonstrated the widest MIC range. In the current study, C. lusitaniae, as the second most common causative agent of VVC, was susceptible to all antifungal agents. Furthermore, 61.1% of C. lusitaniae isolates were inhibited at a concentration of ≤ 2 μg/ml, while 38.9% (n=7) of them exhibited fluconazole MICs above the epidemiologic cutoff values (ECV). Candida species showed the highest overall resistance against fluconazole (61.3%), followed by itraconazole (45.2%) and caspofungin (23.7%). All of C. albicans strains were resistant to itraconazole with a MIC value of ≥ 1 μg/ml; in addition, 87.5% of them were resistant to fluconazole. Moreover, 100% and 87.5% of C. glabrata strains were resistant to caspofungin and fluconazole, respectively. Conclusion: As the findings revealed, the majority of VVC cases were caused by non-albicans Candida species which were often more resistant to antifungal agents. Candida lusitaniae generally had fluconazole MICs above the ECV. Given the propensity of C. lusitaniae to develop resistance under drug pressure, antifungals should be administered with caution. The emergence of these species justify the epidemiological surveillance surveys to watch out the distribution of yeast species.

The VVC affects not only mature women but also young girls. The predisposing factors for young girls to develop VVC include the anatomical features of vagina and its closeness to the rectum, lumbar hair, small labia minor, thin vaginal epithelium, deficiency in estrogen hormones, and genetic issues [2,3]. In addition, some of the risk factors associated with the development of VVC include antibacterial agent usage, hormonal fluctuations during pregnancy, immune system weakness, use of intrauterine devices for birth control, poor personal hygiene, metabolic disorders (e.g., diabetes mellitus), and stress [4,5]. The VVC is a highly common problem in diabetic women, owing to a number of factors, such as increase of vaginal mucosa due to the high deposition of glycogen in vaginal tissue [6]. According to the results of the research conducted around the world, the most common causative agent of VVC is Candida albicans (77-95%), followed by non-albicans Candida (NAC) species (20-30%) [7][8][9]. However, the results of the recent studies are indicative of the growing increase of NAC species [10,11]. Among NAC species, C. lusitaniae remains a less common cause of vulvovaginitis worldwide; however, this species has a noticeable role in the recurrence of vaginitis [1,12]. Moreover, based on some reports, this species shows resistance to amphotericin B and cross-resistance to echinocandins and azoles [13,14]. However, there are limited data regarding the antifungal susceptibility of C. lusitaniae causing VVC.
Azole antifungal agents can be used in the treatment of VVC like it is used for other superficial fungal infections [15]. It is difficult to perform an epidemiological investigation about the incidence, diagnosis, and treatment of VVC given the high rate of self-treatment with over-the-counter (OTC) medications and also the treatment of patients without prescribing laboratory examinations by physicians [16]. The incidence of VVC resistant to azole antifungal is on a growing trend due to the excessive administration of fluconazole and other azoles. The VVC self-diagnosis and self-treatment with OTC antifungal product can occasionally lead to the perpetuation of symptoms and RVVC [17].
Susceptibility to azoles in Candida species is highly variable. The treatment failure, as well as the recurrence and relapse of infection due to the emergence of NAC species resistant to conventional azoles is a worrisome problem [18,19]. Regarding this, the present study was conducted to identify the etiologic agents of VVC among the women attending a Gynecology Clinic in Babolsar, northern Iran, using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and evaluation of the in vitro antifungal susceptibility of Candida species isolated from patients with VVC to eight antifungal drugs. These antifungals included fluconazole (FLC), itraconazole (ITC), miconazole, clotrimazole (CLO), nystatin (NYS), ketoconazole (KET), caspofungin (CAS), and tioconazole (TIC). The ultimate goal was to determine the role of multiple Candida species in the incidence of RVVC.

Fungal isolates and patient characterization
A cross-sectional study was carried out on 260 nonpregnant women referring to the Gynecology and Obstetrics Department of Hazrat-e-Zainab Hospital, Babolsar, Iran, from February 2017 to January 2018. All participants enrolled in the study signed consent forms. The women with the clinical evidence of VVC, including burning, itching, cheesy discharge, and pain during intercourse were enrolled in the study. On the other hand, the subjects who had recent vagina douche or any form of antifungal therapy and those unwilling to participate were excluded from the study. The RVVC was defined as four or more episodes of culture-proved VVC in a year.
Two samples of cervical/vaginal discharge were collected from each patient by means of sterile saline wetted cotton-tipped swabs. One swab was used for direct microscopy, and the other one was applied for culture assay. Preliminary diagnoses of specimens were performed using the KOH (10%) mount, gram stain, culture on Sabouraud dextrose agar (SDA) (Merck, Germany), SDA supplemented with 0.5% chloramphenicol, and CHROMagar Candida incubated at 37°C for 48-72 h [20]. The CHROMagar Candida as a differential culture medium can facilitate the identification of mix yeast species in the clinical sample presumptively.
Serial dilutions of mix yeasts were set up on CHROMagar Candida to differentiate and recognition them. The evidence of budding yeast cell with pseudohyphae in direct microscopy and yeast growth was considered VVC. Species identification of grown yeast was performed conventionally using germ tube production in horse serum, chlamydospore test on corn meal agar with Tween 80, and colored colonies. For accurate identification, the yeast isolates were subjected to further investigation, including molecular methods.

DNA Extraction
Genomic DNA was extracted according to our previously described method [20] with some modifications. The isolates were identified by means of ITS1-5.8S-ITS2 gene amplification. Briefly, a loopful of 48-hour grown colonies was suspended in 300 µl of lysis buffer (200 mmol -l Tris-HCl [pH: 7.5], 25 mmol -l EDTA, 0.5% [w/v] SDS, 250 mmol -l NaCl) and then incubated at 100°C for 15 min and centrifuged. The supernatant was added with 200 µl of 3.0 mol -l sodium acetate and incubated at -20°C for an hour and then centrifuged at 12,000 g for 10 min. The supernatants were precipitated with an equal volume of cold isopropanol, centrifuged at 10,000 g for 10 min, washed with 70% of cold ethanol, air-dried, suspended in 50 µl TE buffer (10 mM Tris, 1 mM EDTA pH 8), and finally stored at -20°C until needed.

Molecular identification
For the purpose of molecular identification, the samples were subjected to ITS1-5.8S-ITS2 rDNA amplification and restriction enzyme analysis. The restriction enzyme analysis was performed as previously described [21]. Briefly, for each restriction digestion reaction, 10 μl of the amplified PCR product was digested with 1.5 μl of restriction enzyme buffer, 1 μl of restriction enzyme Msp I (Fisher Scientific, Leicestershire, UK), and 2.5 μl of high-performance liquid chromatography grade water. The reaction mixture (15 μl) was incubated at 37°C for 2 h. Restriction fragments were separated by 2% agarose gel in TBE buffer for 1 h at 100 V.
The identification of C. albicans species complex (i.e., C. africana, C. albicans, and C. dubliniensis) was accomplished using the partial amplification of hyphal wall proteins (HWP1) gene according to the primers designed by Romeo and Criseo (forward, 5′-GCTACCACTTCAGAATCATCATC-3′ and reverse 5′-GCACCTTCAGTCGTAGAGACG-3′) that generate the fragments of 940 and 740 bp for C. albicans and C. africana [22]. The discrimination of C. parapsilosis complex, including C. parapsilosis and C. orthopsilosis, was conducted as previously described [23].

In vitro susceptibility testing
Susceptibility of the grown yeasts to FLC, ITC, MIC, CLO, NYS, KET, CAS, and TIC was evaluated using broth microdilution method as recommended by the Clinical and Laboratory Standards Institute (CLSI) M27-A3 and M27-S4 document guidelines [24,25]. Briefly, the antifungal agents were diluted in the standard RPMI-1640 medium (Sigma Chemical Co. Germany) buffered to pH 7.0 with 0.165 Mmorpholinepropanesulfonic acid (Sigma, Germany) and L-glutamine without bicarbonate to yield two times their concentrations.
The buffer medium was dispensed into 96-well microdilution trays at the concentrations of 0.016-16, 0.063-64, and 0.008-8 μg/ml for ITC/KET/NYS/TIC, FLC, and CAS, respectively. The MIC endpoint was defined as 100% and 80% inhibition for NYS and other drugs, respectively. Yeast inoculum onto Sabouraud dextrose in sterile saline (0.85%) was prepared after 24 h of incubation, resulting in a final concentration of 0.5-2.5×10 3 cells/ml. The plates were incubated at 35°C for 48 h for all antifungals, except for CAS and FLC (for 24 h). For each isolate, drug-free (growth control) and yeast-free (drug control) wells were included, and all isolates were tested in duplicate.
Candida parapsilosis (ATCC 22019) was used as a quality control for each series of MIC plate.

Ethical approval
Ethical approval was obtained from the Research and Ethics Committee of Mazandaran University of Medical Sciences, Mazandaran, Iran, with a reference number of IR.MAZUMS.REC.95.2313 and dated 22 September 2016.

Polymerase chain reaction restriction fragment length polymorphism and HWP1 amplification
Enzymatic digestion with MspI revealed different patterns for yeast isolates ( Figure 1). Furthermore, the partial amplification of HWP1 gene for C. albicans and C. africana strains yielded a single band with sizes of ̴ 1000 and 750 bp, respectively ( Figure 2). A total of 95 Candida strains were isolated from 75 infected patients ( Table 2). The most prevalent species was C. albicans (42/ (Table  3). Two C. africana isolates were mixed with other Candida species; therefore, it was not possible to determine their antifungal susceptibility due to the difficulty of separating them.        (Table 4). Moreover, all C. albicans strains were resistant to ITC with a MIC value of ≥ 1 μg/ml.
In the current study, C. lusitaniae, as the second most common causative agent of VVC, showed susceptibility to all antifungals. In addition, the growth of the majority of these species was inhibited at a concentration of ≤ 2 μg/ml (Table 4). Seven C. lusitaniae isolates exhibited FLC MICs above the epidemiologic cutoff values (ECV; 4-64 μg/ml). Six isolates of C. kefyr showed the highest susceptibility to CAS and TIC with the GM MIC values of 1.41 and 1.12 μg/ml, respectively (Table 4). Furthermore, CAS and TIC also inhibited the growth of 5 C. famata isolates at a concentration of ≤ 0.5 μg/ml. All of C. glabrata strains were resistant to CAS, and 87.5% of them were resistant to FLC (Table 4).

Discussion
Vulvovaginal candidiasis is a common lower genital tract infection in pregnant and child bearing age women. The majority of patients with VVC are diagnosed by signs and symptoms without using laboratory findings, and infection is not confirmed. The present study targeted non-pregnant women in order to identify the distribution of Candida species in VVC and RVVC cases and determine their antifungal susceptibility patterns. In this study, the prevalence of VVC and RVVC was estimated by laboratory and clinical criteria. Although the prevalence rate of VVC (28.8%) in our study was within the reported range, it was slightly higher than the rates reported by Abbasi Nejat et al. [26], Diba et al. [27], and Hedayati et al. [28]. However, our obtained rate was lower than the prevalence rates presented by Mukasa et al. [29] and Bitew et al. [30].
Some of the potential factors for differences in the occurrence and/or recurrence of VVC among studies are sociodemographic characteristics, diabetes mellitus, dietary habits, personal hygiene, sexual activity, immunological status, and use of antibiotics, immunosuppressant, or oral contraceptives, which are various and conflicting [30]. In the current study, age was investigated as a possible risk factor for the occurrence of VVC and RVVC. Our finding regarding age as an important risk factor was consistent with those of similar earlier studies [27,28] (Table 1). In the current study, the mean ages of the patients with VVC and RVVC were 31±7.5 and 28±4.4 years, respectively, showing a significant difference (P=0.005). Our finding is consistent with those obtained by Bitew et al. [30].
Furthermore, in the current study, out of 75 patients with VVC, 15 (20%) patients were diagnosed with RVVC. The prevalence rates of RVVC were reported as 24.2% and 12.2% in previous studies [26,27]. In the current investigation, a total of six Candida species were detected. The prevalence rate of C. albicans as the most prevalent species associated with vulvovaginitis was obtained as 44.22%. Furthermore, the overall prevalence of NAC species was 55.78% with C. lusitaniae as the most predominant species.
In a study, infection with C. lusitaniae showed a high rate of intrinsic resistance to amphotericin so that susceptibility testing was not required [47]. Although C. lusitaniae clinical isolates show no reduced susceptibility [44], in some research, it elevated the MICs for echinocandins [48], which are used as the first-line therapy of candidemia. Recently, an unusual emerging resistance to echinocandins has been shown due to mutation in FKS genes in clinical cases and experimental animal model [49]. In addition, there is evidence regarding the development of cross-resistance to azoles and echinocandins following combination antifungal treatment [14].
In our study, caspofungin values were below the ECVs of 0.5-1 μg/ml that would be considered nonwild using the ECVs reported by Lockhart et al. [48]. There is no valid CLSI susceptibility breakpoint for less prevalent Candida species like C. lusitaniae. In this case, the ECVs defined as the highest susceptibility endpoint of the wild-type MIC population. The ECVs can facilitate the detection of the emergence of in vitro resistance and help physicians in managing fungal infection where breakpoints are not available [50]. However, these values will not categorize a fungal isolate as susceptible or resistant and do not predict clinical response as breakpoints do [51] The recovery rate of C. albicans as the most common species isolated from patients with VVC was similar with those of numerous studies. Nonetheless, the recovery percentage of NAC vaginitis (55.78%) was higher than the rates reported in two previous studies presenting lower rates of 28.7% and 41.4%, respectively [25,29]. Similar to other studies that reported a high recovery percentage (65.0% and 57.5%) for NAC species in Egypt and Iran [28,52], it seems that there has been a growing shift towards NAC species.
Documented information regarding the prevalence of NAC isolated from Iranian patients causing vulvovaginitis revealed C. glabrata as the most common yeast among the NAC species causing vaginitis [26,28,53]. It should be noted that C. lusitaniae has been rarely reported as the causative agent of VVC in the studies conducted in Iran. It seems that the high prevalence of C. lusitaniae in Babolsar, Iran, is related to some factors, such as the accurate identification of this agent from other Candida species using PCR-RFLP and sequencing, quite good sampling, genetic adaptability of this species to this geographic area, and different populations. In addition, there is no report regarding the distribution pattern of Candida species in this region.
Similar to our study, in a study carried out by Bitew et al. [30], different recovery rates were reported for NAC species. The results of the mentioned study demonstrated C. krusei as the dominant NAC species, followed by C. dubliniensis, C. glabrata, C. tropicalis, C. kefyr, C. parapsilosis, C. guilliermondii, C. lusitaniae, and C. inconspicua. It was suggested that the increase of NAC species, isolated from patients complaining of genital tract infection naturally resistant

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Curr Med Mycol, 2019, 5(4): 26-34 to antifungal agents, is probably related to the widespread and inappropriate use of azole antifungals [1,54]. Therefore, the most logical cause of NAC species emergence is low sensitivity to azole antifungal agents, compared with Candida species isolated frequently from patients [55]. Similar to other studies, mixed infection was seen in our patients [1]. Laboratories should be able to detect mixed cultures in primary cultures because it is an important issue for the management of VVC patients. In addition, the determination of in vitro antifungal susceptibility pattern is highly important before deciding on a specific treatment and introducing new antifungal agents in order to predict the outcome of treatment for the routine surveillance of fungal infections.
Fluconazole is first-line therapy for the treatment and prevention of candidiasis; however, the prolonged use of this antifungal agent has contributed to the development of antifungal drug resistance in Candida isolates. In the present study, C. kefyr, C. glabrata/C. albicans, and C. parapsilosis showed the resistance rates of 100%, 87%, and 57% to FLC, respectively, which were higher, compared to the values reported in other studies [1,26,29].
Regarding ITC, C. glabrata was absolutely resistant to this medication, which is similar to the results obtained by Mukasa et al. [29].
Unlike other reports, in the current study, C. albicans and C. parapsilosis were found to be absolutely resistant to ITC. Abbasi Nejat et al. [26] found that all isolates were highly susceptible to amphotericin B. With regard to CAS, the resistance rates were obtained as 100% and 42.8% for C. glabrata and C. albicans/C. parapsilosis, respectively. In contrast, Bitew et al. [30] found that all of the yeast isolates were 100% susceptible to this medication. In the present study, the isolates showed reduced MICs to caspofungin with an MIC90 of 8 μg/ml and resistance rate of 23.7%.
Overall, in terms of GM MICs, CAS demonstrated potent activity against almost all yeast isolates (n=63) in comparison with FLC, ITC, MIC, CLO, NYS, KET, and TIC. In this study, C. famata was susceptible to all medications. While in a study performed in Uganda, 100% of C. famata showed resistance to ITC, and 50% of them were resistant to CLO [29]. The high prevalence rate of NAC species with reduced susceptibility to azole antifungal agent in the current study is in line with some recent reports that have indicated that resistance to antifungal drugs may also be an important factor for VVC.

Conclusion
As the findings indicated, NAC species were the most common yeast isolates obtained from patients with VVC infection and C. lusitaniae being the generally predominant species. Given the propensity of C. lusitaniae to develop resistance under drug pressure, antifungals should be administered with caution. The emergence of these species justifies the implementation of epidemiological surveillance surveys to watch out the distribution of yeast species. Overall, in vitro antifungal susceptibility testing is an essential measure for choosing the correct antifungal agents for appropriate therapy. However, it is required to perform prospective studies in our region to track the changing trend in antifungal susceptibility and development of mutation under the widespread use and abuse of OTC antifungals, especially in RVVC cases.