الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Candida species are important opportunistic pathogens that are responsible for both mucosal and systemic infections, specifically in immunocompromised patients.
Candida infections are commonly treated with azole antifungal drugs, of which fluconazole is the most commonly prescribed and widely used for most candida infections, due to fewer side effects and low cost.
Azole antifungal drugs function by inhibiting the cytochrome P450-dependent enzyme lanosterol 14α-demethylase (Erg11p) encoded by the gene ERG11, which is the most important enzyme involved in the ergosterol biosynthesis pathway. The prolonged and repetitive use of fungistatic azole drugs may lead to the development of resistant isolates. Resistance to azole poses a serious clinical problem in the management of candida infections. Several underlying mechanisms are associated with azole resistance in Candida species; one of the fundamental mechanisms for azole resistance is the overexpression of the drug efflux-pumps genes CDR1, CDR2 and MDR1. Other resistance mechanisms include upregulation of the ERG11 gene, alteration in the ERG11 gene, upregulation of various transcription factors and alterations in other genes involved in the ergosterol biosynthesis pathway.
The present study aimed to characterize the molecular determinants of azole antifungal drugs resistance in Candida species isolated from different types of clinical specimens.
To achieve this aim, clinical Candida isolates were cultured on Sabouraud dextrose agar and stained with the Gram stain for microscopic examination. chromogenic agar (BCA), germ-tube production test, and BD Phoenix instrument were used for species identification. Then, isolates were tested for fluconazole susceptibility using the disk
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diffusion method and the broth microdilution technique. RNA was extracted from fluconazole-non-susceptible isolates and fluconazole-susceptible isolates from C.albicans and C.tropicalis strains using RNAzol® RT and the cells were broken by bead beating. And then, the reverse transcription of the extracted RNA was achieved using Thermo scientific RevertAid First-strand cDNA Synthesis Kit. Quantitative Syber Green Real-Time PCR was performed on cDNA to evaluate the relative gene expression level of azole efflux-pump encoding genes CDR1 and MDR1 in C.tropicalis isolates and CDR1 and CDR2 in C.albicans isolates.
The current study included 111 isolates of Candida species collected from the microbiology laboratory of the Medical Research Institute, Alexandria University, over the period from 2019 to 2021. Isolates were preliminary identified using phenotypic chromogenic agar (BCA) and the most predominant species was C.albicans (n=36), (32%), while C.tropicalis (n=32) ranked second and accounted for 29%, and the remaining were non-albicans candida. A germ tube test was performed as a second step to confirm the identification of C. albicans (n=36) and C.tropicalis (n=32), which provisionally identified by chromogenic agar (BCA). The results showed that 6 of 36 isolates with green colonies on BCA were germ tube negative, while 3 of 32 isolates with dark-blue colonies on chromogenic agar (BCA) were germ tube positive.
Thirty-two isolates out of our 111 Candida isolates were randomly selected for automated identification by BD Phoenix as our reference method. These selected isolates included 10 isolates with green colonies on BCA (C. albicans), 21 isolates with dark-blue colonies on BCA (C. tropicalis), and 1 isolate with beige colonies on BCA (C.parapsilosis, C.glabrata , Ckefyr or C.lusitaniae). In 3/10 C. albicans isolates and 2/21 C. tropicalis isolates, there was disagreement between chromogenic agar (BCA) and the germ-tube production test in the results of the identification of Candida isolates.
The result of BD phoenix identification agreed with that of BCA and germ-tube test in 24/32 isolates by 75%. The strength of agreement between the identification of C. albicans by germ-tube test and Phoenix BD system was very good (p <0.001), whereas a lower value for the strength of agreement was observed with BCA identification.
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Clinical isolates of Candida species (C. albicans, n = 9, and C. tropicalis, n = 16) were identified by BD Phoenix were tested for their susceptibility to fluconazole using the disk diffusion method, and the results were further confirmed using the broth microdilution method. The disk diffusion technique revealed non- susceptibility to fluconazole in 13 out of 16 C. tropicalis isolates (81.25%) compared to 3/9 C. albicans (33.33%), were susceptible dose-dependent of the C. tropicalis isolates 7/13 (53.8%), while for C. albicans, a susceptible dose-dependent result was observed for 1/3 of the isolates.
The results of the Broth microdilution test showed perfect agreement (p= 0.0027) with the results of the disk diffusion method for the 3 isolates of C. albicans that displayed non-susceptibility to fluconazole. While no statistically significant agreement was detected between the results of the 2 methods for the fluconazole non-susceptible C. tropicalis (p =0.374).
The results showed that the accuracy of the disk diffusion method for identifying fluconazole non-susceptibility was low in C. tropicalis (62%), compared with an accuracy of 100% for C. albicans.
The relative gene expression of the CDR2 efflux pump among our C. albicans isolates (n= 3) showed upregulation in the fluconazole non-susceptible isolates (1.99 ± 1.04).On the other hand, the relative gene expression of CDR1 in non-susceptible isolates was at a basal level (0.73 ± 0.24) which did not differ from the fluconazole-susceptible isolates (0.9 ± 0.25). Overall, we did not observe any statistically significant difference in the expression of CDR1 and CDR2 efflux pumps among fluconazole-susceptible and non-susceptible isolates of C. albicans.
Regarding C. tropicalis, our results also showed no statistically significant difference in CDR1 gene expression between fluconazole non-susceptible isolates and fluconazole-susceptible isolates (p= 0.199). CDR1 expression was found to be at a basal level of (0.69 ± 0.76) in the former group, which did not much differ from the expression of this efflux pump among the latter (1.08 ± 0.5). Meanwhile, the MDR1 efflux pump was found to be downregulated among C.tropicalis fluconazole non-susceptible isolates (0.30 ± 0.63) with a basal level of expression among the fluconazole-susceptible isolates (1.16 ± 0.77), and this was statistically significant (p= 0.025).