Antifungal Susceptibility Profile of Candida Albicans Isolated from Vulvovaginal Candidiasis in Xinjiang Province of China

Liang Yan . Xiao-dong Wang . Seyedmojtaba Seyedmousavi . Juan-na Yuan . Palida Abulize . Wei-hua Pan . Nong Yu . Ya-li Yang . Hai-qing Hu .Wan-qing Liao . Shu-wen Deng

Abstract

We investigated the antifungal susceptibility profiles of 207 independent Candida albicans strains isolated from patients with vulvovaginal candidiasis (VVC) in Xinjiang Province of China. Using CLSI M27-A3 and M27-S4 guidelines, anidulafungin and micafungin were the most active drugs against C. albicans showing an MIC50/MIC90 corresponding to ravuconazole (0.063/1 lg/mL), itraconazole (0.125/ 1 lg/mL), amphotericine B (0.5/1 lg/mL), isavuconazole (0.063/2 lg/mL), 5-flucytosine (1/2 lg/ mL), voriconazole (0.125/4 lg/mL), and fluconazole (0.5/4 lg/mL). 96.1% (199)–100.0% (207) isolates were sensitive to the three echinocandins tested, amphotericine B and 5-flucytosine. The in vitro activity of triazoles against all isolates tested was variable; itraconazole and voriconazole had reduced the activity to almost half of the isolates (55.1% (114) and 51.2% (106) susceptible, respectively). Fluconazole was active against 76.3% (158) isolates tested. The new triazoles ravuconazole, isavuconazole and posaconazole showed good in vitro potency against 89.9% (186)–95.2% (197) of isolates with the geometric mean MIC (lg/mL) of 0.10, 0.12 and 0.14 lg/ mL, respectively. In conclusion, our study indicates that for effective management of systemic candidiasis in Xinjiang Province of China, it is important to determine the susceptibility profiles of isolated C. albicans from patients with VVC.

Keywords Antifungal susceptibility Candida albicans Vulvovaginal candidiasis Xinjiang

Introduction

Vulvovaginal candidiasis (VVC) poses a serious challenge to public health. 70–75% of women suffer at least one VVC episode during their lives, and half of them will experience a recurrence [1]. Evidence shows that disease is recurrent in almost 8% of women aged 15–50 years old. Recurrent vulvovaginal candidiasis (RVVC) is defined as four or more episodes of disease per year [2]. Several studies reported that C. albicans remains the most frequently isolated species from VVC (76–89%) [2], followed by C. glabrata (7–16%) [2], C. parapsilosis, C. tropicalis, and C. krusei [3]. An increasing prevalence of fungal resistance is also reported in global and local antifungal surveillance studies [4–23]. The increased RVVC incidence and drug resistances causes an important public health issue and poses significant challenges to implement appropriate and effective management strategies. Antifungal susceptibility testing of isolated Candida strains therefore plays a useful role in managing Candida infections.
Despite previous reports on antifungal susceptibility of C. albicans causing VVC in Beijing, Shanghai, Shenzhen and Zhanjiang [4, 14, 19–22], there are limited data regarding the antifungal resistance of C. albicans isolated from VVC in Xinjiang Province of China, a multi-ethnic area with different climates and under developed economies compared to the rest of country. In the current study, we therefore investigated the antifungal susceptibility profile of a large collection of C. albicans isolates obtained from VVC patients in Xinjiang Province.

Materials and Methods

Isolates and Identification

We investigated a collection of 207 C. albicans strains isolated from 207 adult women between October 2015 and February 2017 at the First Hospital of Xinjiang Medical University, Xinjiang, China. During examination, all patients showed signs and/or symptoms suggestive of vaginitis, including pruritus vulvae, vulvar burning, vaginal soreness and irritation, dyspareunia, pain or discomfort during urination and abnormal vaginal discharge, which could be diagnosed with VVC along with culture-positive vaginal secretion. Approval of the research was acquired from the Research Ethics Committee of the hospital, and written consent was gained from all patients involved.
All samples were collected with sterilized vaginal swabs. Swabs were cultured on CHROM agar Candida and incubated for 48 h at 35–37 C. All isolates were identified to the species level by sequencing 26S ribosomal DNA gene D1/D2 domains with primer pairs NL-1 (50-GCATATCAATAAGCGGAGGAAAAG) and NL-4 (50- GGTCCGTGTTTCAAGACGG), as described previously [24]. The obtained sequences were compared to the NCBI nucleotide database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to verify species-level identity of each isolate. The geographical origin, clinical data and GenBank accession numbers for the generated D1/D2 sequences are listed in Supplementary Table S1.

Antifungal Susceptibility Testing

All isolates were tested for in vitro antifungal susceptibility to 11 antifungal agents according to the CLSI reference guideline M27-A3 and M27-S4 [25, 26]. Antifungal drugs tested were anidulafungin (ANF), caspofungin (CAS), micafungin (MFG), amphotericin B (AmB), 5-flucytosine (5-FC), fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), posaconazole (POS), isavuconazole (ISA), and ravuconazole (RAV). Anidulafungin, voriconazole, isavuconazole purchased from Toronto Research Chemicals Inc., micafungin provided by Astellas Pharma, and remaining antifungal were obtained from Sigma-Aldrich). C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were used as control strains for all experiments. All isolates were sub-cultured onto Sabouraud Dextrose Agar at 35 C for almost 24 h for viability and purity. Colonies were suspended in sterile saline, and the final inoculum concentration of the suspension was adjusted to 0.5–2.5 9 103 CFU mL-1 with RPMI 1640 broth medium. The 96-well plates were incubated for 24 or 48 h at 35 C, and minimum inhibitory concentrations (MIC) were determined visually.
Drug concentration ranges, time of MIC reading and interpretive breakpoints used for 11 antifungal agents are listed in Table 1. Although interpretive criteria for the susceptibility to amphotericin B remain elusive, we here classified MIC ^ 1 lg/mL as susceptible and MIC ] 2 lg/mL as resistant referring to previous studies [8–10, 13, 16–18]. There are no interpretive breakpoints for posaconazole, isavuconazole and ravuconazole yet.

Results

MIC distribution, MIC50/MIC90, geometric mean values (GM), susceptibility rate (S%), susceptibledose dependent rate (SDD%) and resistant rate (R%) of 207 C. albicans isolates tested to 11 antifungal agents are summarized in Table 2.
The values of MIC50/MIC90 for all isolates used in this study are as follows (in increasing order): anidulafungin and micafungin were the most active drugs against C. albicans as they had the lowest MIC50/MIC90 (0.016/0.0313 lg/mL), followed by caspofungin (0.25/0.25 lg/mL), posaconazole (0.125/0.5 lg/mL), ravuconazole (0.063/1 lg/mL), itraconazole (0.125/1 lg/mL), amphotericine B (0.5/ 1 lg/mL), isavuconazole (0.063/2 lg/mL), 5-flucytosine (1/2 lg/mL), voriconazole (0.125/4 lg/mL), fluconazole (0.5/4 lg/mL).
According to the CLSI M27-S4 guideline, of the 207 C. albicans isolates, 96.1% (199)–100.0% (207) isolates were susceptible to the three echinocandins tested, and the MIC50/MIC90 of both micafungin and anidulafungin were 0.016/0.0313 lg/mL, which were tenfolds less than that of caspofungin (0.25/0.25 lg/ mL) (Table 2).
The in vitro activity of triazoles against 207 isolates of C. albicans was variable; itraconazole and voriconazole had reduced activity to approximately half of the isolates (susceptibility rate 55.1% (114) and 51.2% (106), respectively). Furthermore, 34.8% (72) isolates were susceptible-dose-dependent (SDD) and 10.1% (21) isolates were resistant to itraconazole; for voriconazole, 16.9% (35) isolates were SDD and 31.9% (66) were resistant. Fluconazole was active against 76.3% (158) isolates tested. The triazoles, posaconazole, isavuconazole and ravuconazole showed good in vitro potency with MICs less than 1 lg/mL against 89.9% (186)–95.2% (197) C. albicans isolates tested.
As expected, all C. albicans isolates tested were susceptible to amphotericin B. Six isolates showed resistance to 5-flucytosine, but remained sensitive to the remaining list of antifungals tested.

Discussion

Candida species contribute to a significant percentage of women’s healthcare-related fungal infections worldwide [3]. In order to find a way to prevent and control such infections, it is important to select as early as possible the antifungal treatment of choice, and understand the resistance profile of causative agents to various antifungals.
We investigated the antifungal susceptibility of 207 C. albicans isolates obtained from the patients with VVC in Xinjiang province, which is the largest and most westerly province in China with the population consisting of Han Chinese and Muslims, and separated from the densely populated areas of the country. Table 3 was summarized data on susceptibility of VVC C. albicans isolates to five common antifungal drugs in different studies from 2003 to 2017 [4–22]. To the best of our knowledge, our report is the first study addressing the antifungal susceptibility of C. albicans isolates from VVC patients to 11 antifungals in Xinjiang province. Our findings would be helpful in guiding effective clinical therapy regimes.
The echinocandins have been widely used clinically for candidemia and invasive candidiasis due to their negligible toxicities, generally fungicidal activities and lack of cross-resistance with azoles [27, 28]. Our results showed good activity of the three echinocandins against the majority of C. albicans isolates tested. Of note, micafungin and anidulafungin (MIC50/MIC90: 0.016/0.0313 lg/mL) showed higher potency than caspofungin (MIC50/MIC90: 0.25/
in vitro efficacy of echinocandins against C. albicans obtained from VVC. Shi et al. [22] reported that caspofungin showed good activity to the vaginal C. albicans isolates in southern China. Kalkanci et al. [10] reported MIC values of caspofungin against 46 C. albicans isolates from acute VVC in Turkey (MIC50/ MIC90: 0.25/0.5 lg/mL). Boikov et al. [29] also showed that caspofungin, micafungin and anidulafungin showed good activity against 60 C. albicans strains isolated from VVC with a MIC50/MIC90 of 0.25/0.5 lg/mL, 0.008/0.008 lg/mL and 0.008/ 0.008 lg/mL, respectively. More recently, Sharifynia et al. [30] also reported that caspofungin (geometric means: 0.27 lg/mL) was the most active antifungal against 26 C. albicans strains isolated from RVVC patients in Iran.
Furthermore, in our study all vaginal C. albicans isolates were susceptible to micafungin, which was in agreement with that reported on C. albicans isolates causing systemic Candida infections [31]. Activity of anidulafungin to C. albicans isolates from VVC was similar to that of micafungin except one isolate showing resistance. Similarly, Pfaller et al. presented similar results on anidulafungin against C. albicans causing invasive infections [32].
As shown in Table 3, fluconazole (the recommended drug of choice for VVC) was active against most C. albicans isolates from VVC in the majority of previous reports [4–22] (Table 3). Multiple studies in Argentina, Brazil, Turkey, America, Italy, showed the susceptibility rate of 90–100% on fluconazole against C. albicans isolates from VVC [5, 8–10, 12, 13, 16]. In China, except two reports from Shenzhen with similar susceptibility rate (98.1% S and 88.3% S) (Ge et al. [14], Zhang JY et al. [15]) to western countries, most reports presented lower susceptibility rate (70–83% S, Table 3) in Shenzhen [20], Shanghai [4] and Beijing [21]. The data shown in our study (76.3% S) were in agreement with previously published reports from China.
Studies in Turkey [7, 9, 10], Brazil [13], and America [12], also indicated good in vitro activity of itraconazole (70.7–99.5% S, Table 3) against C. albicans isolates from VVC. However, in China, itraconazole was active against only half isolates in the most reports (62.1% S, 46.5% S, 48.8% S, Table 3) [14, 21, 22], except two studies in Shenzhen (85.0% S and 90.6% S, Table 3) [19, 20]. In our results, susceptibility of C. albicans isolates from VVC to itraconazole (55.1% S) in Xinjiang province was the same as those at other areas of China. Of note, 34.8% isolates were SDD and 10.1% were resistant to itraconazole. The less antifungal activity of itraconazole was likely associated with relatively highfrequency use in clinical setting in China [33], requiring the attention of clinicians in this situation in China.
Higher susceptibility rate (98.3% S) of C. albicans isolates from VVC to voriconazole had been reported by Dota et al. [13]. In China, two studies from Shanghai and Zhanjiang reported the susceptibility rate of C. albicans isolates from VVC to voriconazole were 81.0 and 81.2%, respectively [4, 22]. The susceptibility rate shown in our study (51.2% S) was however relatively lower, and notably, 31.9% isolates were resistant and 16.9% isolates were SDD in Xinjiang province which were much higher than those in previous reports from Ying C et al., Dota KFD et al. and Shi et al. (14.0% and 5.0%; 0.0% and 1.7%; 7.6% and 11.2%; respectively, Table 3) [4, 13, 22]. It is not known exactly what lead to the regional disparity, but different ethnic compositions, climates and clinical uses of antifungal drugs between Shanghai and Xinjiang possibly contribute to the distinction in susceptibility. Nevertheless, it will remind of clinicians of caution when choosing voriconazole as the therapy for VVC in Xinjiang province.
The Candida surveillance study demonstrated that resistance to fluconazole was highly predictive for resistance to voriconazole [34]. Our results also showed that 14 of 17 fluconazole-resistant isolates were resistant to voriconazole. In addition, isolates with reduced susceptibility to fluconazole showed cross-resistance to itraconazole and voriconazole. Seven isolates were resistant to both of fluconazole and itraconazole, and 14 isolates were resistant to both of fluconazole and voriconazole. Moreover, 5 isolates were resistant to fluconazole, itraconazole and voriconazole.
The good in vitro activity of members of new triazoles (posaconazole, isavuconazole and ravuconazole) against C. albicans isolates resulting in invasive infections has been documented by many studies [35–39]. For VVC, however, there are limited data on vaginal C. albicans isolates. In the present study, the three new triazoles showed good in vitro activity against all C. albicans isolates from VVC patients in Xinjiang province. Approximately, 90% isolates were inhibited by within 1 lg/mL of posaconazole, isavuconazole and ravuconazole (1 lg/mL regarded as the breakpoint of susceptibility for the new triazoles [40–45]). Our finding is in agreement with reports from Northern America [46] and Kuwait [47] which showed good activity of posaconazole against C. albicans isolates from VVC (MIC90: 0.03 lg/mL and 0.064 lg/mL, respectively). However, there are no data available on isavuconazole and ravuconazole about the in vitro susceptibility of C. albicans isolates from VVC currently. For isavuconazole, compared with the results reported in studies on Candida bloodstream isolates (MIC50/MIC90: 0.002/0.004 lg/ mL [35] and 0.015/0.03 lg/mL [36], respectively), our results (MIC50/MIC90: 0.063/2 lg/mL) were significantly higher. Ravuconazole showed good effectiveness against C. albicans isolated from VVC (MIC50/MIC90: 0.063/1 lg/mL), although it had higher MIC values compared to that in the studies against Candida bloodstream isolates (MIC50/MIC90: 0.007/0.03 lg/mL [40] and 0.016/0.125 lg/mL [48]). And the differences of MIC values for isavuconazole and ravuconazole were both above 2 log2 dilution steps, suggesting that isolates from VVC are less susceptible than isolates from invasive infections to isavuconazole and ravuconazole.
Our study also confirmed the good in vitro activity of amphotericin B and 5-flucytosine against C. albicans isolates from VVC again (Table 3) [8–10, 12, 13]. Hundred percentage of the isolates tested were susceptible to amphotericin B which agreed with previous studies [9, 10]. Over 95% of isolates were susceptible to 5-flucytosine, which was consistent with the previous studies [10, 12]. The isolates resistant to 5-flucytosine did not have crossresistance to 10 other antifungal drugs, which was in agreement with previous study by Ribeiro et al. [49].
In conclusion, our finding suggests that antifungal susceptibility testing in Xinjiang province should be performed routinely to help clinicians to develop appropriate therapies with high probability of successfully treating VVC.

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