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Title: Genotypic heterogeneity and antifungal susceptibility profile of clinical Candida glabrata isolates in Kuwait
Authors: Ahlam Fayadh Alanazi 
Supervisor: Prof. Suhail Ahmad
Keywords: candidemia;Genotype heterogeneity;Kuwait;antifungal Susceptibility
Issue Date: 2012
Publisher:  Kuwait university - college of graduate studies
Abstract: The incidence of invasive candidiasis in critically ill immunocompromised patients is increasing and is associated with high mortality. Early detection of candidemia and accurate identification of Candida species improve prognosis. Candida glabrata is among the four most common Candida species causing mucosal and blood stream infections. Phenotypically identified C. glabrata isolates now represent a complex of three species, i.e., C. glabrata, C. nivariensis and C. bracarensis which exhibit varying susceptibility to azoles warranting species-specific identification. This study determined the presence of C. nivariensis and C. bracarensis among phenotypically identified C. glabrata isolates (n=216) in Kuwait for the first time. Drug susceptibility testing was performed to determine rates of resistance to four antifungal agents and molecular fingerprinting of C. glabrata isolates was performed to determine genotypic relatedness for better control and prevention of these infections. All isolates were tested by Chromogenic Candida agar, a multiplex (m) PCR developed to yield species-specific amplicons from C. glabrata (360 bp), C. nivariensis (250 bp) and C. bracarensis (180 bp) and by uniplex species-specific PCR assays. The results were confirmed by DNA sequencing of ITS region of rDNA from 30 selected isolates. Antifungal susceptibility testing was performed by Etest on RPMI 1640 medium against amphotericin B, fluconazole, voriconazole and 5-flucytosine. Molecular fingerprinting of 80 C. glabrata isolates was done by PCR sequencing of intergenic spacer (IGS)-2 region of rDNA and genotypic relationship was deduced by constructing neighbor-joining phylogenetic tree by MEGA4 software. None of 216 C. glabrata-complex isolates was identified as C. nivariensis or C. bracarensis by mPCR and uniplex species-specific PCR assays indicating that C. nivariensis and C. bracarensis are not clinically important species in Kuwait. DNA sequencing of ITS region confirmed the identification of 30 selected isolates as C. glabrata and also showed limited genotypic heterogeneity. Only 1 of 204 and 9 of 196 C. glabrata isolates were resistant to amphotericin B and voriconazole, respectively while 16 and 42 of 206 isolates were resistant and dose-dependent susceptible, respectively, to fluconazole. Ten of 16 fluconazole-resistant isolates were susceptible to voriconazole suggesting that voriconazole is likely to be more effective against C. glabrata infections. Fingerprinting of 80 C. glabrata isolates identified 66 isolates as unique strains while 14 isolates were grouped in 6 clusters with two or three isolates in each cluster. The data show that majority of C. glabrata isolates in Kuwait are not clonally related implying that transmission of these infections among patients is a rare event.
Appears in Programs:0520 Microbiology (M.Sc.)

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