الفهرس 
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Abstract
A hospital-based cross-sectional study was conducted on 300 patients presented with clinical picture suggestive of vulvovaginal candidiasis attended to obstetrics and gynecology outpatient clinics of Al Azhar Assuit University Hospital between August 2018 and June 2019. The patients were investigated for vulvovaginal candidiasis by vulvovaginal swabs cultured on SDA, Candida isolates were detected in 75 patients, 76 Candida isolates were recovered from 75 patients as one patient had a mixed infection by two different isolates. The association between VVC and many risk factors including age, hormonal use, antibiotics, steroid, IUD, DM, history of vaginitis, and frequent intravaginal douches were assessed; statistical analysis showed that women ≤ 40 years old had a threefold higher risk of getting VVC than the elder one. Also, patients with frequent intravaginal douching or with a history of vaginitis were at a higher risk of acquiring VVC. Different Candida species were identified by germ tube test, growth at 45°C, culture on cornmeal tween 80, Hichrom agar, and biochemical tests (KB006 HiCandida Identification Kits). Accordingly, C. albicans was the predominant isolated species (47 isolates, 61.8%) followed by C. glabrata (22 isolates, 29%), C. kruzi (5 isolates 6.6%), and lastly C. parapsilosis (2 isolates 2.6%) The sensitivity of 76 Candida isolates to antifungals were tested for 2 polyenes (Nystatin and Amphotericin B) and 4 azoles (Fluconazole, Voriconazole, Itraconazole, and Miconazole ) by the disc diffusion method, Nystatin exhibited an excellent efficacy against all Candida spp except one C .glabrata isolate was resistant (1.3%). A good activity of Amphotericin B was detected against C.parapsilosis, C.albicans & C.glabrata with percentages of (100%, 89.8 &81.8%), respectively, but less effect was observed against C. krusie (60%).Susceptibility to different azoles drugs was various among different Candida spp. Fluconazole and Voriconazole showed outstanding efficacy against 47 C. albicans isolates with the sensitivity of 100%, less susceptibility of C.albicans was detected to Itraconazole (80.9%) and notably little effect of Miconazole against C.albicans (23.4%) was recorded. Regarding C. glabrata Miconazole was the most effective azole drug with (95.5%) sensitivity. Voriconazole showed (68.1%) sensitivity, it has been observed that fluconazole and Itraconazole had the least effect on C.glabrata with (45.5 % & 36.4%) sensitivity, respectively. Regarding the 12 fluconazole-resistant C. glabrata isolates a detailed analysis of cross-resistance among the four tested azoles showed that one isolate was resistant to all azoles, three isolates were resistant to three azoles (Fluconazole, Itraconazole, and Voriconazole), and 8 isolates were resistant to itraconazole and fluconazole. C. krusie was the most resistant species to azole drugs among different Candida species; all strains were resistant to Fluconazole and Itraconazole. Miconazole and Voriconazole, resistance rates were 60% and 40%, respectively. For 2 isolates of C.parapsilosis complete susceptibility to all tested azoles was found.
The MIC of fluconazole was detected in 22 vaginal C. glabrata isolates by E test. There was a difference between the results of the disc diffusion method and E test for fluconazole, disc diffusion method gave a higher rate of the resistant phenotype. Quantitative real-time PCR was used to evaluate the expression levels of Fluconazole resistance genes (ERG11, CDR1, SNQ2, and PDR1) in C. glabrata. These four genes were quantified and normalized relative to the housekeeping gene, β actin, C.glabrata isolates were tested as they were the most frequent fluconazole species. Quantitative RT–PCR experiments showed that the mean relative gene expression levels of the ERG11 gene were 1.43 ± 0.53 (mean ± standard error of the mean). There was no statistically significant difference in the ERG11 expression levels in resistant isolates compared to susceptible control isolates (P-value 0.53). While, the mean relative gene expression levels of the CDR1 gene were 3.45 ± 0.69, which was significantly greater in resistant isolates relative to susceptible control isolates (P = 0.004). The mean relative gene expression levels of the SNQ2 gene was 3.71±1.32, there was a statistically significant difference in the SNQ2 expression levels between resistant and susceptible control isolates (P = 0.038). As regards, PDR1 gene, the mean relative gene expression levels were 1.56 ± 0.17, there was a statistically significant difference in the PDR1 expression levels between resistant and susceptible control isolates. Ten of the 12 (83.3%) fluconazole-resistant C. glabrata isolates expressed CgCDR1 at higher levels than susceptible control isolate, while EGR11 was overexpressed only in 4 (33.3%) isolates compared to susceptible isolates. 9 (75%) fluconazole-resistant C. glabrata isolates upregulated SNQ2 while PDR1 showed overexpression in 11 (91.7%) of the 12 fluconazole-resistant isolates compared to susceptible isolates Concomitant upregulation of more than one efflux pump genes (CgCDR1, and CgSNQ2) and their regulator CgPDR1 was noticed among the studied fluconazole-resistant C. glabrata isolates. CgCDR1, CgSNQ2, and CgPDR1 were simultaneously overexpressed in six isolates, while CgCDR1 and CgPDR1 were concurrently overexpressed in three isolates, two isolates upregulated CgPDR1 and CgSNQ2, and only one isolate upregulated CgCDR1 and CgSNQ2