1. Background
In healthy individuals, the capacity of Candida, as a commensal, to produce either systemic or superficial infections is depended on the host immune system and different risk factors (1). Bacterial vaginosis, vulvovaginal candidiasis (VVC), and trichomoniasis are the most frequent types of vaginitis, among which VVC is the second most frequent form after bacterial vaginosis (2). This infection is one of the most common gynecological disorder among women who are in their childbearing age. About 75% of sexually active women have at least once experienced symptomatic VVC, which is usually presented as itching, soreness, burning, and abnormal curd-like vaginal discharge (1). According to several studies, non-albicans candida species are increasing as causative agents of RVVS in the world. (3). Candida albicans, followed by C. glabrata, are the most frequent causative agents of VVC, and they are also a part of the indigenous flora of the gastrointestinal tract and skin (1-5). Furthermore, in local and global antifungal surveillance studies, a growing prevalence of fungal resistance has been reported (1-4). The growing incidence of VVC, RVVC, and drug resistance led to an essential public health issue, and it is a challenge for the treatment strategy of clinicians (3). Although there are many effective antifungal drugs, both oral and topical use for treating VVC decreased susceptibility of some vaginal yeast isolates to some antifungal agents (4). One of the main points in guiding appropriate therapy selection for mycoses is the antifungal susceptibility patterns of pathogenic fungi (2-4, 6, 7). An estimation of antifungal effectiveness, monitoring of the development of drug resistance, ensuring beneficial treatment outcome, and therapeutic potential of unproven compounds may also be provided by antifungal susceptibility testing (4, 6, 7).
2. Objectives
The present investigation aimed to define in vitro antifungal susceptibility profile of 108 clinical isolates of C. albicans and C. glabrata against itraconazole (ITC), voriconazole (VRC), fluconazole (FLC), amphotericin B (AMB), nystatin (NYS), and caspofungin (CAS).
3. Methods
3.1. Clinical Isolates
Seventy-seven strains of C. albicans and 31 strains of C. glabrata used in this study were isolated in 2017 in Tehran. The test strains were sub-cultured on potato dextrose agar (PDA, Merck, Germany) with chloramphenicol (Merck, Germany) and incubated at 37°C for 48 h. The species-level identification of clinical isolates was achieved using the previously described PCR- restriction fragment length polymorphism (RFLP) method. Briefly, PCR products of the ITS1-5.8S-ITS2 region were subject to digestion with the restriction enzyme MspI, and the strains were identified by comparing the electrophoretic RFLP patterns with those profiles obtained previously (8).
3.2. In vitro Antifungal Susceptibility Testing
Antifungal susceptibility testing (AFST) was performed according to the recommendations proposed by the Clinical and Laboratory Standards Institute (CLSI) M27-A3 and M27-S4 documents (9, 10). The antifungal agents tested were itraconazole (ITC), voriconazole (VRC), fluconazole (FLC), amphotericin B (AMB), nystatin (NYS) and caspofungin (CAS) (Sigma, St. Louis, MO, USA). The drug dilution ranges tested were 0.063 - 64 mg/L for FLC and NYS, 0.016 – 16mg/L for ITC, VRC and, AMB, and 0.008 - 8 mg/L for CAS. C. parapsilosis (ATCC 22019) were used as quality control strains. Briefly, homogeneous conidial suspensions were spectrophotometrically measured at the 530 nm wavelength and a percent transmission within the range of 75 - 77%. The final inocula suspension adjusted to 0.5 - 2.5 × 103 conidia/mL in RPMI-1640 with L-glutamine and without sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) buffered at pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS, Sigma-Aldrich, St. Louis, MO, USA). The 96-well microplates were incubated at 35°C and examined visually after 24 and 48 h to determine MIC values. Minimum inhibitory concentration (MIC) was determined according to the recommendations proposed by the CLSI M27-A3 and M27-S4 documents. The geometric mean (GM) MICs, MIC50, MIC90, and MIC ranges were calculated. Following the CLSI M27-S4, resistance to FLC for C. albicans was defined as MIC ≥ 8 mg/L and MIC ≥ 64 mg/L for C. glabrata, for VRC, MIC of ≥ 1mg/L was defined as resistant for C. albicans and C. glabrata and CAS, MIC of ≥ 1mg/L was defined as resistant for C. albicans and MIC of ≥ 0.5 mg/L C. glabrata. Isolates with MIC ≥ 2 mg/L for AMB were considered resistant.
3.3. Statistical Analysis
The statistical differences between the mean values were evaluated with SPSS (version 17.0) using student t-test. Statistical significance was set at 0.05.
4. Results
Our results showed 8 (25.8 %) and 6 (7.8 %) C. glabrata and C. albicans isolates resistance to FLU, respectively. Furthermore, resistance to VRC and ITC were observed in 8.4% and 3.7% of all isolates, respectively, and six isolates (5.6%) had intermediate MIC to CAS.
Table 1 lists the geometric mean (GM) MIC, MIC ranges, MIC50, and MIC90 of six antifungal agents against 108 candida strains (C. albicans; n: 77 and C. glabrata; n: 31). CAS (GM: 0.05 mg/L), AMB (GM: 0.12 mg/L), VRC (GM: 0.13 mg/L), and ITC (GM: 0.15 mg/L) had low MIC against all tested strains, whereas FLC (GM: 3.2 mg/L) and NYS (GM: 0.7 mg/L) had higher MIC. The MIC90 for all isolates were CAS: 0.25 mg/L. AMB: 0.5 mg/L, ITC: 0.5 mg/L, VRC: 1 mg/L, NYS: 2 mg/L and FLC: 32 mg/L. The MIC ranges across all isolates were as follows: CAS: 0.016-0.5 mg/L, AMB: 0.032 - 0.5 mg/L, ITC: 0.063 - 1 mg/L, VRC: 0.016 - 2 mg/L, NYS: 0.125 - 2 mg/L and FLC: 0.25 - 64 mg/L. The GM MICs of C. glabrata strains were as follows: CAS (GM: 0.08 mg/L), AMB (GM: 0.16 mg/L), ITC (GM: 0.34 mg/L), VRC (GM: 0.38 mg/L), FLC (GM: 16.53 mg/L) and NYS (GM: 0.82 mg/L), whereas for C. albicans strains the GM MICs were: AMB (GM: 0.11 mg/L), CAS (GM: 0.05 mg/L), ITC (GM: 0.10 mg/L), VRC (GM: 0.08 mg/L), FLC (GM: 1.56 mg/L) and NYS (GM: 0.59 mg/L). Our results indicated that no statistically significant differences existed in the patterns of susceptibility to each compound within species.
Table 1.Geometric Mean of MIC, MIC Ranges, MIC50, and MIC90 Values Obtained by Testing the Susceptibility of 108 Candida isolates Obtained from 108 Vulvovaginal Candidiasis Patients to the Antifungal Agents
| Strains/ Antifungals | GM | Range | MIC50 | MIC90 | R (%) | SSD (%) | I (%) | S (%) |
|---|---|---|---|---|---|---|---|---|
| C. albicans (n:77) | ||||||||
| AMB | 0.11 | 0.032 - 0.5 | 0.125 | 0.5 | 77 (100) | |||
| CAS | 0.05 | 0.016 - 0.5 | 0.063 | 0.25 | 4 (5.2) | 73 (94.8) | ||
| FLC | 1.56 | 0.25 - 16 | 2 | 8 | 6 (7.8) | 11 (14.2) | 60 (78) | |
| VRC | 0.08 | 0.016 - 1 | 0.125 | 0.5 | 4 (5.2) | 8 (10.4) | 65 (84.4) | |
| ITC | 0.10 | 0.063 - 1 | 0.063 | 0.25 | 3 (4) | 7 (9) | 67 (87) | |
| NYS | 0.59 | 0.125 - 2 | 0.5 | 2 | ||||
| C. glabrata (n:31) | ||||||||
| AMB | 0.16 | 0.063 - 0.5 | 0.125 | 0.5 | ||||
| CAS | 0.08 | 0.032 - 0.5 | 0.063 | 0.25 | 2 (6.5) | 29 (93.5) | ||
| FLC | 16.53 | 2 - 64 | 16 | 64 | 8 (25.8) | 5 (16.1) | 18 (58) | |
| VRC | 0.38 | 0.063 - 1 | 0.5 | 1 | 5 (16.1) | 26 (80.6) | ||
| ITC | 0.34 | 0.125 - 1 | 0.5 | 1 | 2 (6.5) | 5 (16.1) | 24 (77.4) | |
| NYS | 0.82 | 0.25 - 2 | 0.5 | 2 | ||||
| Total (n:108) | ||||||||
| AMB | 0.12 | 0.032 - 0.5 | 0.125 | 0.5 | ||||
| CAS | 0.06 | 0.016 - 0.5 | 0.063 | 0.25 | 6 (5.6) | 102 (94.4) | ||
| FLC | 3.2 | 0.25 - 64 | 4 | 32 | 14 (13) | 16 (14.8) | 78 (72.2) | |
| VRC | 0.13 | 0.016 - 2 | 0.125 | 1 | 9 (8.4) | 8 (7.4) | 91 (84.2) | |
| ITC | 0.15 | 0.063 - 1 | 0.125 | 0.5 | 4 (3.7) | 9 (8.3) | 95 (88) | |
| NYS | 0.7 | 0.125 - 2 | 0.5 | 2 |
Abbreviations: MIC, minimum inhibitory concentration; GM, geometric mean; MIC50, minimal concentration that inhibits 50% of isolates; MIC90, minimal concentration that inhibits 90% of isolates; CAS, caspofungin; AMB, amphotericin B; ITC, itraconazole; VRC, voriconazole; FLC, fluconazole; NYS, nystatin; R, resistance; SSD, susceptible dose-dependent; I, intermediate; S, susceptible.
5. Discussion
Several previous studies were reported that C. albicans (76 - 89%) was the most common causative agent of infection in women with VVC, followed by C. glabrata (7 - 16%) from different countries (1, 2, 11-15). The rise of VVC incidence and drug resistance leads to an important public health issue and challenges clinician’s treatment strategies (1). Antifungal susceptibility surveillance investigation was exerting an increasingly essential role in pursuing the progress of antifungal resistance and beginning primary antifungal therapy. With the growing application of antimicrobial agents, the susceptibilities of the candida might be altering now and then (1-4). In the present study, the drug susceptibility profile of 108 C. albicans and C. glabrata isolates, as the two most frequent causative agents of VVC, was tested against six antifungal drugs. Based on the results in the present study, CAS (GM: 0.06 mg/L), AMB (GM: 0.12 mg/L), VRC (GM: 0.13 mg/L) and ITC (GM: 0.15 mg/L) had low MICs against all tested species, while NYS (GM: 0.7 mg/L) and FLC (GM: 3.2 mg/L) had higher MICs. As Table 1 shows, CAS had the lowest GM MIC with 0.05 mg/L, 0.08 mg/L against C. albicans and C. glabrata clinical strains, respectively. Azole drugs, especially FLC, are a major group of antifungals drugs that have been used for the treatment of VVC and RVVC (16). Azole drugs disrupt the biosynthesis of the fungal-specific membrane ergosterol by inhibiting the 14α-sterol demethylase enzyme encoded by the ERG11 gene (17). Fluconazole is the most commonly prescribed azole drug that is used for C. albicans infections (18). Our results showed 7.8% of C. albicans strains were resistant to FLC. According to our results, resistance to FLC among C. albicans strains was reported in several studies (19-25). However, in several reports, all C. albicans strains were sensitive to FLC (26-28). It was shown that almost all women diagnosed with FLC-resistant C. albicans had experienced previous exposure to FLC (29). The rates of azole drug resistance are variable, and they can be affected by the prescribing patterns of clinicians for both prophylaxis and treatment (16). Our results showed that 25.8% of C. glabrata strains were resistant to FLC. In line with our findings, resistance to FLC in C. glabrata isolates was reported in several studies (1, 21, 30-32). One important reason for the development of azole resistance in clinical strains of C. glabrata is the presence of mutations in some genes, especially the PDR1 gene (33). In the present study, 4.6% of all Candida strains (C. albicans: 4% and C. glabrata: 6.5%) were resistant to ITC. Resistance to ITR was detected in the previous studies (20, 32, 34, 35), and in contrast, some researchers did not observe any resistance to ITC (36). In the study conducted by Gross et al. in Costa Rica, 100% of C. albicans isolates were susceptible to ITC (37). Voriconazole has a wide range of activity against filamentous, dimorphic fungi and yeasts (including fluconazole-resistant Candida spp.) (38). Our results were shown 16.1% of C. glabrata and 5.2% of C. albicans strains were resistant to VRC. Although VRC was approved for the treatment of invasive aspergillosis in 2002 by the US Food and Drug Administration (FDA), this antifungal drug was prescribed for the treatment of esophageal candidiasis, candidemia, disseminated Candida infections in viscera and skin (39). Mukasa et al. and El-Feky et al. reported that 6.6% and 10.5% of C. albicans isolates were resistant to VRC (4, 22). Nystatin is a member of polyene drugs, and in the form of an ointment or vaginal pessary, it has been used for four decades (40). In the previous study, Carrillo- Munoz et al. reported the NYS MIC90 values for 11 C. glabrata and 55 C. albicans clinical isolates as 4 mg/L and 2 mg/L, respectively (41). Furthermore, in South Africa, in patients with human immunodeficiency virus (HIV) and healthy individuals, NYS MICs for the 589 oral isolates ranged from 2 to 16 mg/L with reported MIC90 of 8 mg/L for the 466 C. albicans species (42). Our results showed that NYS had lower MIC in comparison to Carrillo-Munoz et al. (41) and Blignaut et al. (42) findings. Caspofungin, as a member of echinocandins, acts by inhibiting 1,3-ß-D-glucan synthase has good activity against candida species (43). As exhibited in Table 1, CAS had the lowest MIC with GM of 0.05 mg/L and 0.08 mg/L for C. albicans and C. glabrata, respectively. Our findings showed that 5.6% of Candida strains had an intermediate MIC range to CAS. Several other investigations also confirmed that CAS was excellent antifungal activity against Candida spp. (2, 43, 44). In the study conducted by Castanheira et al., the MIC90 value for AMB was reported as 1 mg/L in all Candida species, except for C. krusei that had a MIC of 2 mg/L (21). Furthermore, in another study, the MIC 90 value for AMB in the 1,310 isolated C. albicans was detected 1 mg/L that is higher than the present study (0.5 mg/L) (45).
5.1. Conclusions
Based on the results of the present study, CAS, AMB, and VRC had the lowest MIC against all Candida isolates, respectively. However, recently, resistance to azole among the Candida isolates is constantly increasing worldwide. Therefore, antifungal susceptibility testing can be used to predict clinical responses to malfunctions in the management.
