Azoles resistance of candida species causing vulvo-vaginitis in reproductive age women at a tertiary care setting

Objectives: To study prevalence and resistance pattern to azoles of candida species causing vulvovaginitis in reproductive age women. Methods: Samples were collected from Hayatabad Medical Complex from November 2018 to May 2019. This cross-sectional study was conducted at the department of Microbiology, Institute of Pathology and Diagnostic Medicine, Khyber Medical University Peshawar, Pakistan. A total of 369 high vaginal swabs were collected. Candida was isolated by vaginal swabs inoculation on Sabouraud’s dextrose agar (SDA). Colonies on SDA were inoculated on Candida CHROM agar to identify candida species. Wet film microscopy and Gram staining were performed. Biochemical identification was done with 20C AUX. Antifungal susceptibility testing was done by disc diffusion and broth micro-dilution methods to find the resistance pattern of azole drugs. Fluconazole, Clotrimazole, Miconazole, Voriconazole and Itraconazole were the azoles drugs used. Results: Among 43%(n=158) positive candida cases, 44%(n=85) were non pregnant women while 41%(n=73) were pregnant. The Candida species distribution of 158 isolates was as follow; Candida albicans (C. albicans) 46.2%(n=73), Candida krusei (C. krusei) 29.1%(n=46), Candida parapsilosis (C. parapsilosis) 19%(n=30) and Candida glabrata (C. glabrata) 5.7%(n=9). Overall Candida isolates were highly resistant 72%(n=113) to Fluconazole while least resistant 21.5 % (n=34) to Itraconazole. Conclusion: C. albicans is the most prevalent specie involved in Vulvovaginal candidiasis. Candida species were found to be least resistant to Itraconazole followed by Voriconazole, Miconazole, Clotrimazole and Fluconazole.


INTRODUCTION
Candida is the most important opportunistic pathogen among yeast. The Candida spectrum of infection is wide, ranging from local mucosal involvement to life-threatening disseminated infections. Vulvovaginal candidiasis (VVC) is the most common infection of female lower genital tract caused by Candida species. It has been reported that 75% of women get at least one episode of vulvovaginal candidiasis during their lives and 50% of them have multiple episodes. 1

Original Article
Azoles resistance of candida species causing vulvo-vaginitis in reproductive age women at a tertiary care setting Candida albicans is the most prevalent specie causing vulvovaginitis. There is a rise in infection rate due to non-albican species like C. glabrata, C. krusei, C. parapsilosis, C. tropicalis, C. dublinensis and some rare species. Non-albican species are involved in recurrent candidiasis and are more resistant to antifungal drugs. Candida krusei is inherently resistant to Fluconazole while C. glabrata is only 3-7% of resistant. 2 Vulvovaginal candidiasis is more common in the reproductive age and pregnancy due to increase in estrogen level, which causes deposition of glycogen that favors growth of Candida. Risk factors are antibiotic use, oral contraceptives, intrauterine contraceptive device, pregnancy, immunocompromised conditions like steroids use, Diabetes Mellitus, and chemotherapy. 3 In the past few decades, there is improvement in the antimicrobial testing and focus remained on cost effective, precise and reproducible testing methods. Clinical Laboratory Standards Institute (CLSI) guidelines are used as reference scheme available for yeast susceptibility testing, where disc diffusion and broth micro dilution methods are considered for antifungal susceptibility testing. 4 Topical and oral azoles are conventionally used to treat vulvovaginal candidiasis. The most common anti-fungal used to treat vulvovaginal candidiasis is Fluconazole due to its easy availability and oral dosage Clotrimazole and Miconazole are the commonly used topical azoles in our set up. There is a rise in Candida strains resistant to one or more of the azoles due to their widespread use. The newer Azoles like Voriconazole and Itraconazole have broad spectrum activity against the Fluconazole resistant Candida species. Cross resistance has been reported in Fluconazole resistant Candida species against the newer Azoles. 5 In our clinical set up, bacterial culture and sensitivity of the specimens are commonly done but fungal cultures are not done. There is no national surveillance program to monitor the prevalence of Candida species and antifungal resistance. Only direct microscopy of the specimens is performed to report Candida species while identification of the Candida at species level and antifungal susceptibility testing are not done. Keeping this present state of dealing with Candida, this project was designed to study the prevalence of different Candida species and their resistance pattern.  6 Sampling technique was nonprobability convenient sampling. All women of childbearing age (married) both pregnant and nonpregnant having signs and symptoms of vulvovaginitis were included in the study. Women taking antifungal drugs were excluded. Written informed consent was obtained from all patients. After enrolment in the study, demographic data were collected from patients using a structured questionnaire and two high vaginal swabs were collected from each patient using aseptic techniques. 7 Samples were then inoculated on Sabouraud's dextrose agar. Candida present in the samples were grown as creamy white colour colonies which were then sub-cultured on Candida CHROMagar for species identification. 8 Wet film microscopy and Gram staining were also performed for identification. The isolates were identified by carbohydrate assimilation method using API 20C AUX system (Bio Merieux). Pure Candida isolates were stored at -80 °C in micro bank. 9 Susceptibility testing to Azoles was conducted according to the Clinical and Laboratory Standards Institute guidelines. For quality control American Type Culture Collection (ATCC) 90028 was used. Fluconazole (25µg), Clotrimazole (10 µg), Miconazole (50µg), Voriconazole (1µg) and Itraconazole (10µg) (Himedia) were tested by using disc diffusion method (DD). Candida colonies were transferred to saline. Turbidity was matched with 0.5 McFarland standards. The suspension was inoculated on Muller Hinton agar with the help of a swab. Filter paper discs containing antifungals were applied on the Muller Hinton media. Interpretation of antifungal was done according to the Clinical and Laboratory Standards Institute guidelines -M44A document.

METHODS
Broth micro-dilution (BMD) was performed by micro-titer plate method following M27A CLSI guidelines. Inoculum was prepared by picking Candida colonies from 24 hours culture on Sabouraud's dextrose agar. The colonies were suspended in 5ml of 0.85% saline and adjusted to 0.5 McFarland standards. The suspension was diluted 1:50 followed by a dilution of 1:20 in Roswell Park Memorial Institute Medium RPMI 1640 (M1972 HI media part A, D glucose was added). Stock solutions of Clotrimazole, Miconazole, Itraconazole and Voriconazole (Sigma, USA) were prepared in dimethyl sulfoxide (DMSO) at concentration of 1600 µg/ ml. Fluconazole was prepared in distilled water in concentration of 1280µg/ml. Final concentration of Fluconazole (64-0.125 µg/ml) and Clotrimazole, Miconazole, Itraconazole and Voriconazole (16-0.03µg/ml) were made in RMPI 1640 media. Drug dilutions inoculum of 100µl was transferred to the column 1-10 of the 96-well plates (Nest, China). Column 11 and 12 were growth control and sterility control, respectively. Inoculum of 100µl was added to columns 1-11. Reading of MIC were taken after 24-48 hours. Minimum inhibitory concentration (MIC) break-points for Fluconazole, Itraconazole and Voriconazole were determined according to M27A document. 10 For Miconazole and Clotrimazole, there are no interpretive breakpoints available in CLSI document. For Clotrimazole resistant breakpoint of ≥1µg/ml reported was used. 11 For Miconazole resistant breakpoint of ≥16µg/ml was used. 12 Statistical Analysis: All data were transferred to excel sheet and analyzed by using SPSS version 20. Frequencies and percentages were calculated for different categorical variables. Mean and standard deviation was calculated for numerical variables like age and zone of inhibition. Percent agreement was calculated for disc diffusion and broth microdilution methods. Data was presented in the form of tables. Candida species distribution found among 158 of isolates was as follow; Candida albicans 73(46.2%), Candida krusei were 46 (29.1%), Candida parapsilosis were 30 (19%) and Candida glabrata were nine isolates (5.7%).

RESULTS
By disc diffusion method candida species showed susceptibility pattern as follows; Fluconazole was resistant in 75.9%, susceptible dose dependent in 1.9% and sensitive in 22.2% of isolates. Clotrimazole was sensitive in 20.3%, SDD in 15.2% and resistant in 64.6% of isolates. Miconazole was sensitive in 18.4%, SDD in 16.5 and resistant in 65.2% of isolates. Voriconazole was sensitive in 36.7, SDD in 2.5% and resistant in 60.8% of isolates. Itraconazole was sensitive in 64.6%, SDD 11.4% in and resistant in 24.1% of isolates as shown in Table-I Table-II. The non-albican Candida species showed higher resistance as compared to Candida albicans . Table-II Table-III. A total of 46 resistant Candida krusei isolates had MIC of 64µg/ml (fluconazole), 30 had MIC in range of 1-16 µg/ml (clotrimazole), 31 had MIC of 16µg/ml (miconazole), 28 had MIC in range of 4-16µg/ml (voriconazole), 2 isolates had MIC in range of 1-8µg/ml (itraconazole) Table-III. A total of 24 resistant candida parapsilosis isolates had MIC of 64µg/ml (fluconazole), 20 had MIC in range of 1-16 µg/ml (clotrimazole), 20 had MIC of 16µg/ml (miconazole), 5 had MIC in range of 4-16µg/ml (voriconazole), 10 had MIC in range of 1-16µg/ml (itraconazole) as shown in Table-III. Of the resistant Candida glabrata isolates eight had MIC of 64µg/ml (fluconazole), seven had MIC in range of 1-16µg/ml (clotrimazole), five had MIC of 16µg/ml (miconazole), five had MIC in range of 4-16µg/ml (voriconazole), and two isolates had MIC of 1µg/ml (itraconazole) as shown in Table-   There was a good agreement between DD and BMD methods in identifying the percentage of resistant Candida species for Fluconazole, Clotrimazole, Miconazole, Voriconazole and Itraconazole. However, some isolates showed resistance to azoles antifungals by DD method were found to be sensitive by BMD method. Percent agreement between the two antifungal susceptibility methods for Fluconazole, Clotrimazole, Miconazole, Voriconazole and Itraconazole was found to be 94% ,82%, 95%, 90% and 86% respectively.

DISCUSSION
Among opportunistic fungi candida species are the most important. Vulvovaginal candidiasis is one of the common infections in females of reproductive age. It is estimated that 75% of child bearing age females have at least one episode of VVC during their lifetime. 13 Numerous studies have been performed to establish the prevalence of vulvovaginal candidiasis. The prevalence found in the present study is compatible to other studies for example 40% prevalence of VVC is reported in Pakistan. 14 Prevalence rates of 45% and 44.8% are reported in Uganda and Lebanon respectively, which are similar to the current study. 7,15 Higher rate (48.4%) of Vulvovaginal candidiasis is reported in India. 16 Lower infection rate i.e., 28.3%, and 22.71% are reported by other researchers. 17,18 This may be related to cultural, geographical, climatic and socioeconomic conditions.
In the present study, the age range of 18-46 years was observed, which is in agreement to studies conducted by various researchers who reported higher incidence in the age group of 15-45 years. This may be due to the high levels of estrogen during reproductive age and use of contraceptives. 19 Our findings are not in agreement with the findings of a study conducted in Nigeria which reported a high incidence of candida albicans during 36-40 years of age while low prevalence (20.42%) in 20-25 years age group. 20 The current study revealed high prevalence of VVC among second trimester of pregnancy of 54.79% followed by 27.40% in third trimester. Study published in Nepal reported 61% in second trimester. 21 Prevalence of 54% in 2nd trimester, 30% in 3 rd trimester and 16% in 1 st trimester was reported in India. 22 These studies are in agreement to the current study. A study done in Brazil, it was found that candida albicans is the most prevalent specie Causing VVC, While C.glabrata is reported second common specie in that study which is not similar to present study which found C.krusei as the second common specie. 23 The results of susceptibility pattern regarding Itraconazole are similar to a study done in Kenya, which revealed Itraconazole was sensitive in 88.33% of isolates. This is in agreement with our study while resistance to Fluconazole and Clotrimazole found is lower than our study. 24 Resistance to Fluconazole, Voriconazole and Itraconazole was in agreement with those found in a study in Turkey. 5 Candida species were most sensitive to voriconazole in another study done in Pakistan study while in our study isolates were more sensitive to Itraconazole drug. 4 In a study conducted in China Candida species revealed infrequent resistance. 25 In another study conducted in Iran results showed 8 (25.8 %) and 6 (7.8 %) of resistance in C. glabrata and C. albican isolates to Fluconazole, respectively. Furthermore, resistance to Voriconazole and Itraconazole were observed in 8.4%, and 3.7% of stains. 26 The resistance rates of Fluconazole, Clotrimazole and Miconazole of the current study are higher than other population because of the irrational and excessive use of these drugs. A good agreement between DD method and BMD was reported in India which is similar to that found in this study. 27

Limitations:
This study was done only in one hospital of Peshawar. It might increase our knowledge about the causative species and susceptibility pattern of azoles antifungal drugs if samples are collected from different hospitals.

CONCLUSION
This study showed that C.albicans is the main causative agent followed by C.krusei, C.parapsilosis and C.glabrata. Antifungal sensitivity testing of Candida species against azoles showed higher sensitivity to Itraconazole followed by Voriconazole, Miconazole, Clotrimazole and Fluconazole. Therefore, Itraconazole is a good therapeutic choice in vulvovaginal candidiasis management. A good agreement was found between DD and BMD methods of susceptibility testing. Disc diffusion method is less laborious and is more convenient to perform.