Phenotypic and molecular antifungal susceptibility testing of yeast isolates from Pretoria

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dc.contributor.advisor Lekalakala, M. Ruth
dc.contributor.coadvisor Dangor, Yusuf
dc.contributor.postgraduate Hnaya, Maren
dc.date.accessioned 2014-08-18T07:06:40Z
dc.date.available 2014-08-18T07:06:40Z
dc.date.created 2014-04-25
dc.date.issued 2014 en_US
dc.description Dissertation (MSc)--University of Pretoria, 2014. en_US
dc.description.abstract Antifungal drug resistance is a growing problem. Several mechanisms contribute to the development of resistance to antifungal agents. In South Africa, little is known about the antifungal susceptibility of local yeast isolates. The aim of this study was therefore to determine the antifungal susceptibility profile of local Candida species and Cryptococcus neoformans isolates and the molecular mechanisms of resistance to different antifungal agents in Pretoria. A total of 250 yeast isolates were collected from the diagnostic laboratory in the Department of Medical Microbiology at the University of Pretoria-National Health Laboratory Services. The isolates were subcultured on Sabouraud dextrose agar media for purity of yeast colonies. Identification to species level was performed using biochemical techniques. The antifungal susceptibility of 87 isolates was determined by the Etest for three azole antifungals (fluconazole, posaconazole and voriconazole), amphotericin B and caspofungin. Clinical breakpoint susceptibility was determined according to the CLSI clinical breakpoint reference methods. Polymerase chain reaction was performed on C. albicans isolates to amplify the ERG11 gene and the FKS1 gene. Sequencing was done for the amplification products and the sequence data were analysed by the CLC genome workbench software. Among the 250 isolates collected, Candida species accounted for 82.8% and C. neoformans accounted for 17.2% of the isolates. C. albicans was the most commonly isolated (76.8% of Candida species), of which 30% were resistant to caspofungin. Fluconazole resistance was detected in 56.7% of C. parapsilosis isolates, the highest fluconazole resistance among Candida species. Cross-resistance was found between fluconazole and voriconazole. Resistance to posaconazole was detected in 66.7% of C. glabrata isolates, whilst all other Candida species and C. neoformans isolates were fully susceptible. Furthermore, C. neoformans var. gattii isolates were less susceptible to azole antifungal agents than C. neoformans var. neoformans isolates. Molecular alterations are one of the mechanisms of resistance to azole and caspofungin antifungal agents. The amino acid substitutions D116E, K128T and V437I were detected in the ERG11 gene of two azole susceptible isolates. The new amino acid substitution E517Q was detected in the ERG11 gene of a resistant isolate. The S642L substitution was detected in the FKS1 gene of all the isolates that were caspofungin resistant and caspofungin susceptible. C. albicans was the most commonly isolated yeast species in Pretoria. Cross-resistance was detected between fluconazole and voriconazole. Therefore, these two agents are not a good alternative to use in the treatment of resistant isolates. With Candida species and C. neoformans isolates, there was less resistance to posaconazole than to fluconazole and voriconazole. The identification of the two varieties of C. neoformans is important in order to establish the differences in their antifungal susceptibility. Resistance to azole and caspofungin antifungal agents existed without the previously described molecular alterations in the ERG11 and FKS1 genes of resistant isolates. Further studies are required to explain the role of new amino acid substitutions, as well as the involvement of other mechanisms in resistance to antifungal drugs. en_US
dc.description.availability unrestricted en_US
dc.description.department Medical Microbiology en_US
dc.description.librarian gm2014 en_US
dc.identifier.citation Hnaya, M 2014, Phenotypic and molecular antifungal susceptibility testing of yeast isolates from Pretoria, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd <http://hdl.handle.net/2263/41376> en_US
dc.identifier.other E14/4/345/gm en_US
dc.identifier.uri http://hdl.handle.net/2263/41376
dc.language.iso en en_US
dc.publisher University of Pretoria en_ZA
dc.rights © 2014 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. en_US
dc.subject Antifungal en_US
dc.subject Yeast en_US
dc.subject Susceptibility en_US
dc.subject Resistance en_US
dc.subject Mechanism of resistance en_US
dc.subject Candida en_US
dc.subject Cryptococcus neoformans en_US
dc.subject Etest en_US
dc.subject Pretoria en_US
dc.subject UCTD en_US
dc.title Phenotypic and molecular antifungal susceptibility testing of yeast isolates from Pretoria en_US
dc.type Dissertation en_US


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