Vulvovaginal candidiasis (VVC) is one of the most common clinical forms of candidiasis among 10% of women during childbearing age that can be developed in a recurrent form [ 1 , 2 ]. The VVC is characterized by multiple symptoms, such as itching, dyspareunia, pruritus, and erythema, which may decrease the life quality of women. Diabetes mellitus, overuse of antibiotics, and pregnancy can increase the complications and severity of this infection [ 3 ].
In most studies, Candida albicans has been reported as the most common cause of this infection, compared to other Candida species [ 4 , 5 ]. However, the prevalence of non-albicans Candida (NAC) spp. including C. glabrata, C. krusei, and C. parapsilosis is increasing [ 6 ]. The reason for the increase in non-albicans spp. is the lack of accurate diagnosis of the causes of vaginitis, and improper use of antifungal medicines [ 2 , 6 ]. Due to the emerging resistance of Candida isolates to the limited antifungals available in the clinical setting, it is essential to establish in vitro antifungal susceptibility testing before the treatment of Candida infections, particularly VVC. Therefore, accurate identification of the causative agents of this infection and determination of antifungal susceptibility testing for Candida isolates can be effective in reducing the drug resistance [ 7 ].
Among the antifungal agents with the highest resistance, azoles resistance was the most prevalent [ 8 ]. Although antifungal drug resistance has been frequently reported among NAC spp., the reports of VVC infection caused by fluconazole-resistant C. albicans have been rare [ 6 , 9 , 10 ]. There were only a few Iranian studies available, especially in Northeastern Iran on susceptibility testing for Candida isolates collected from VVC. Hence, the present study aimed to perform antifungal susceptibility tests on Candida isolates obtained from women with VVC by disk diffusion technique.
Materials and Methods
This study was approved by the Ethics Committee (ethics code: IR.MUMS.fm.REC.1394.204).
In this study, 108 Candida isolates were obtained from 189 vaginal discharge specimens of women suspected of VVC (determined by a midwife or gynecologist) and referred to the health centers of Mashhad University of Medical Sciences, Mashhad, Iran to be examined. According to the above, the symptoms were pruritus, vaginal soreness, dyspareunia, external dysuria, and abnormal vaginal discharge. As described previously, the swabs were examined for direct microscopy using potassium hydroxide, cultivated on sabouraud dextrose agar, and the Candida colonies were purified using CHROMagar Candida [ 11 ].
At the first stage of this study, Candida isolates were identified using the polymerase chain reaction-restriction fragment length polymorphism method using Msp I enzyme [ 11 ]. Based on the inclusion criteria, only the purified and identified Candida isolates obtained from women affected with VVC were included in the study. The exclusion criteria were the contamination of Candida isolates with saprobe fungi.
According to the results, 84, 15, 6, 2, and 1 of the isolated spp. of Candida were C. albicans, C. glabrata, C. kefyr, C. parapsilosis, and C. tropicalis, respectively. To perform antifungal susceptibility testing, the disk diffusion method was performed according to the M44-A2 protocol [ 12 ]. Briefly, Müller Hinton agar (Sigma, Germany) base medium was prepared according to the instructions of the manufacturer and distributed on plates. Yeast suspensions prepared with standard McFarland 0.5 turbidity were cultured uniformly using sterile swabs on plates containing Mueller-Hinton agar (with 2% glucose and 0.5 μg/ml methylene blue).
After 15 min, when the culture medium was dried, commercial disks containing voriconazole, itraconazole, fluconazole, and ketoconazole (Liofilchem, Italy) were placed on the plate. The plates were incubated for 24 h at 35 °C, and the diameter of the growth inhibition zone around each disk was measured. Fluconazole-, itraconazole-, ketoconazole-, and voriconazole-impregnated paper disks contained 100, 50, 15, and 1 μg of antifungals, respectively. The zone diameters were measured. According to the table of antifungal discs recommended by the instructions of the manufacturer (Liofilchem, Italy), their antifungal test profile was reported as sensitive, susceptible dose-dependent, and resistant. The results were analyzed in SPSS software (version 16) using Pearson’s chi-squared test.
The results of antifungal susceptibility testing of four antifungal agents, fluconazole, ketoconazole, itraconazole, and voriconazole, are summarized in Table 1.
|Susceptibility profile||Antifungal agents|
|Susceptible dose-dependent (%)||20.4||7.4||23.1||5.5|
According to this Table, Candida isolates showed the highest sensitivity to voriconazole and ketoconazole. Furthermore, they showed the highest drug resistance to fluconazole and itraconazole, in that order. The results of the antifungal profile of different Candida spp. are shown separately in Table 2. Based on these results, the highest rates of sensitivity and resistance of C. albicans and C. glabrata to fluconazole were 58.3% and 9.3%, respectively.
|Candida spp. (No.)||Antifungal agents|
|S (%)||SDD (%)||R (%)||S (%)||SDD (%)||R (%)||S (%)||SDD (%)||R (%)||S (%)||SDD (%)||R (%)|
|C. albicans (84)||63 (58.3)||14 (12.9)||7 (6.5)||77 (71.3)||5 (4.7)||2 (1.8)||61 (56.5)||18 (16.7)||5 (4.8)||78 (72.3)||4 (3.7)||2 (1.8)|
|C. glabrata (15)||3 (2.8)||2 (1.8)||10 (9.3)||13 (12.1)||2 (1.8)||0 (0)||4 (3.7)||7 (6.4)||4 (3.6)||15 (14)||0 (0)||0 (0)|
|C. kefyr (6)||2 (1.9)||4 (3.8)||0 (0)||5 (4.6)||1 (0.9)||0 (0)||6 (5.6)||0 (0)||0 (0)||4 (3.7)||2 (1.8)||0 (0)|
|C. parapsilosis (6)||0 (0)||2 (1.8)||0 (0)||2 (1.8)||0 (0)||0 (0)||2 (1.8)||0 (0)||0 (0)||2 (1.8)||0 (0)||0 (0)|
|C. tropicalis (1)||1 (0.9)||0 (0)||0 (0)||1 (0.9)||0 (0)||0 (0)||1 (0.9)||0 (0)||0 (0)||1 (0.9)||0 (0)||0 (0)|
|Total||69 (63.9)||22 (20.4)||17 (15.7)||98 (90.7)||8 (7.4)||2 (1.9)||74 (68.6)||25 (23.1)||9 (8.3)||100 (92.7)||6 (5.5)||2 (1.8)|
The highest rates of sensitivity and resistance of C. albicans to ketoconazole were 71.3% and 1.8%, respectively. Moreover, the highest rates of sensitivity and resistance of C. albicans to itraconazole were 56.5% and 4.8%, respectively. In addition, the highest rates of sensitivity and resistance of C. albicans to voriconazole were 72.3% and 1.8%, respectively. However, with the exception of fluconazole (P=0.001), there was no significant difference between various spp. of Candida and the antifungals of clotrimazole (P=0.994), itraconazole (P=0.146), and voriconazole (P=0.596).
Due to the limited access to some antifungal agents in many parts of the world, the azole antifungals are the most frequent class used to treat Candida infections. Therefore, such antifungals are often preferred for the treatment of various Candida infections as they are available, inexpensive, exhibit limited toxicity, and are administrated orally [ 9 ]. On the other hand, there are an incredibly large number of documents about the intrinsic and acquired resistance to azole antifungals among some Candida spp. [ 5 ]. However, it is essential to minimize such resistance emergence to both preserve and improve the azole class of antifungals for the treatment of Candida infections.
One way to reduce antifungal resistance is to establish in vitro antifungal susceptibility testing prior to antifungal therapy. Therefore, the present study aimed to evaluate the antifungal susceptibility of Candida isolates obtained from women with VVC in Mashhad, Northeastern Iran. Although according to the extensive literature, C. albicans is the most common causative agent of VVC, C. glabrata is reported as the most common NAC spp. [ 4 , 11 ]. However, drug resistance has often been reported in NAC spp., compared to C. albicans [ 9 ].
In the present study, although C. albicans (78%) was the predominant causative agent of VVC, C. glabrata (14%) was the most common NAC spp. In other parts of this region, the spp. distribution has a similar pattern [ 4 , 11 , 13 ]. Therefore, most patterns of antifungal results have been reported for these dominant spp. In the present study, 92% and 90% of Candida isolates were sensitive to voriconazole and ketoconazole, respectively.
Among the studied spp., C. albicans and C. glabrata had the highest rates of sensitivity to voriconazole that were 72.3% and 14%, respectively. Furthermore, the same spp. showed similar sensitivity rates to ketoconazole that were 71.3% and 12.1%, respectively. However, the resistance rate of C. albicans isolates against ketoconazole and voriconazole was 1.8%.
In a study conducted by Khan et al. in Lebanon, voriconazole was more effective (97.5%) against C. albicans spp., compared to fluconazole (90%) and itraconazole (87.5%) [ 14 ]. In a case reported by Adampour et al., ketoconazole was more effective against Pichia fermentans (C. firmentaria, a variety of firmentaria) isolated from a vaginitis, compared to fluconazole, itraconazole, and voriconazole. Therefore, it is better to examine and evaluate every Candida isolates via an in vitro antifungal susceptibility test before treatment [ 10 ].
In the current study, Candida isolates also showed the highest rates of antifungal resistance to fluconazole (15.7%) and itraconazole (8.3%). Moreover, C. glabrata and C. albicans had the highest resistance rates against fluconazole that were 9.3% and 6.5%, respectively. Furthermore, the resistance rates of C. albicans and C. glabrata against itraconazole were 4.8% and 3.6%, respectively. In contrast, Wang et al. in China found that the resistance rates of C. albicans against fluconazole and itraconazole were 16.6% and 51.5%, respectively. They also found that the resistance rates of C. glabrata to fluconazole and itraconazole were 73.3% and 61.9%, respectively [ 8 ].
In a study carried out by Wang et al., nystatin demonstrated the highest impact against Candida spp. However, in the present study nystatin was not available to be examined. The increase in resistance against fluconazole and itraconazole in the aforementioned study can be attributed to the frequent empiric prescription for sporadic VVC, which may result in resistant C. albicans and C. glabrata causing recurrent VVC infection to emerge.
Furthermore, it should be mentioned that C. glabrata has an inherent resistance to fluconazole too. However, an increase in resistance against antifungal agents may have major consequences resulting in poor outcomes and more chronic forms among women with VVC. Similar results were confirmed by Yassin et al., who reported that C. glabrata showed resistance to most used antifungals (i.e., fluconazole, itraconazole, clotrimazole, nystatin, and terbinafine) [ 15 ]. It should be noted that the pattern of antifungal susceptibility in members of the genus Candida is different, with the wide use of antifungals in clinical practice.
One of the limitations of this study was the lack of sufficient same antifungal powders to perform the microdilution broth method simultaneously, and to check the accuracy of the obtained results. Moreover, we did not have any Candida strain as quality control and carried out the procedures according to the instructions of the manufacturer of disks.
Hence, the development of high-level and multidrug resistance among Candida spp., especially C. glabrata has become a global public health concern. Therefore, it is important to know the spp. distribution of Candida prior to treatment.
The results indicated that among the studied Candida spp., C. glabrata, and C. albicans showed the highest resistance to antifungal drugs. Furthermore, the Candida spp. isolated from VVC showed the highest rate of sensitivity to voriconazole and ketoconazole and the lowest rate of sensitivity to fluconazole. Further studies on greater sample sizes and more isolates and spp. of Candida are recommended.
The authors would like to appreciate the staff of Medical Mycology and Parasitology Laboratory in Gha’em Hospital, Mashhad, Iran. This paper was extracted from a student thesis submitted in partial fulfillment of the requirement for the Master’s degree. It was approved by the Research Committee of the Faculty of Medicine and financially supported by the Research Deputy (No. 931194) of Mashhad University of Medical Sciences, Mashhad, Iran.
H. Z. interpreted the data and prepared the manuscript. Z. K. collected specimens and performed the project. A. F. designed and planned the study. All authors read and approved the final manuscript.
Conflicts of interest
The authors declare that there is no conflict of interest in this study.
The authors declare no financial interests related to the materials of the study.
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