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In-house genotypic antiretroviral resistance test : optimisation and validation for use in research and diagnostics

dc.contributor.advisorVan Zyl, Gert Uvesen_ZA
dc.contributor.advisorEngelbrecht, Susanen_ZA
dc.contributor.authorClaassen, Mathildaen_ZA
dc.contributor.otherUniversity of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.en_ZA
dc.date.accessioned2011-03-01T14:39:07Zen_ZA
dc.date.accessioned2011-03-14T08:15:17Z
dc.date.available2011-03-01T14:39:07Zen_ZA
dc.date.available2011-03-14T08:15:17Z
dc.date.issued2011-03en_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/6520
dc.descriptionThesis (MScMedSc)--University of Stellenbosch, 2011.en_ZA
dc.description.abstractIt is estimated that 32.8 million people are living with Human Immunodeficiency Virus (HIV) globally with the number of people receiving antiretroviral therapy in low- and middle- income counties increasing to more than 5 million people in 2009. These successes are threatened by treatment failure and the development of resistance to treatment. With an estimated 3.7% patients failing first line treatment after 2 years and 17.9% after 4 years on treatment there is a need for a practical and cheap in-house drug resistance assay that can be used to provide drug resistance data to clinicians and to use as a research tool to investigate drug resistance. In this study we attempted to optimize and validate an in-house drug resistance assay, adapted from Jacobs et al, 2008, to be used as a diagnostic tool and to study the presence of antiretroviral resistance in patients on the Western Cape Mother-To-Child-Transmission (MTCT) regimen. Quality control samples were received from The National Institute of Communicable Diseases AIDS Virus Research Unit, The Round Robin HIV-1 genotyping assessment system from the University of Würzburg and the QCMD assessment system were used for the optimization and validation of an in-house drug resistance assay. The ViroSeq™ HIV-1 Genotyping System was used for comparison of sample and mutation detection. It was possible to optimise and validate a genotyping assay for diagnostic testing and research use by comparison with the ViroSeq™ HIV-1 Genotyping System and evaluation with external quality assessment systems. This assay could subsequently be used to determine the development of genotypic-antiretroviral resistance in patients treated according to the provincial prevention of mother-to-child-transmission (PMTCT) protocol in the Western Cape (single dose nevirapine (sd-NVP), combined with a short course Zidovudine (AZT)). Patient samples were collected from pregnant women who took part in the Western Cape PMTCT program and visited the Tygerberg Obstetrics Clinic and Delft Community Hospital. EDTA blood was obtained to measure CD4-cell count, viral load, and to do genotyping for viral subtype and the presence of resistance mutations. Information on prior exposure to antiretroviral therapy was also collected. A detected resistance rate of 17.1% in this predominantly HIV-1 subtype C population is lower than previously recorded when sd-NVP was administered to HIV-1 subtype C positive patients in PMTCT programs. This could indicate that a dual PMTCT regimen including AZT and NVP reduces the risk of resistance to NVP relative to a regimen that uses sd-NVP. The genotyping assay uses four primers to amplify the PR and the RT gene separately to obtain PCR products, of 487 and 804 base pairs respectively for sequencing. The two PCR products were sequenced with three and five primers respectively to sequence the complete PR and approximately 250 amino acids of the RT gene. The sequences generated, thus, are analysed and aligned with the Sequencer V4.7 software to obtain a consensus sequence of approximately 1200 base pairs for analysis of resistance mutations in the protease and reverse transcriptase genes. The developed assay was hence further simplified and improved, by combining the PR and RT assay into one, which was optimised and validated for use in the routine diagnostic setting. The final genotyping assay uses 8 primers for sequencing to obtain a 1200 bp sequence for genotyping that contains the protease and the 5’ of the reverse transcriptase genes in which antiretroviral resistance associated mutations are found. The assay was accredited by SANAS in 2008.en_ZA
dc.format.extent188 p. : ill.
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : University of Stellenboschen_ZA
dc.subjectResistanceen_ZA
dc.subjectHIV infections -- Effect of drugs onen_ZA
dc.subjectMolecularen_ZA
dc.subjectGenotypingen_ZA
dc.subjectHIV infections -- Antiretroviral treatmenten_ZA
dc.subjectTheses -- Medical virologyen_ZA
dc.subjectDissertations -- Medical virologyen_ZA
dc.subject.lcshMicrobiological assay
dc.subject.otherMedical Virologyen_ZA
dc.titleIn-house genotypic antiretroviral resistance test : optimisation and validation for use in research and diagnosticsen_ZA
dc.typeThesisen_ZA
dc.rights.holderUniversity of Stellenbosch


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