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

[摘要] It is estimated that 32.8 million people are living with Human ImmunodeficiencyVirus (HIV) globally with the number of people receiving antiretroviral therapy inlow- and middle- income counties increasing to more than 5 million people in 2009.These successes are threatened by treatment failure and the development of resistanceto treatment. With an estimated 3.7% patients failing first line treatment after 2 yearsand 17.9% after 4 years on treatment there is a need for a practical and cheap in-housedrug resistance assay that can be used to provide drug resistance data to clinicians andto use as a research tool to investigate drug resistance. In this study we attempted tooptimize 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 antiretroviralresistance in patients on the Western Cape Mother-To-Child-Transmission (MTCT)regimen.Quality control samples were received from The National Institute of CommunicableDiseases AIDS Virus Research Unit, The Round Robin HIV-1 genotyping assessmentsystem from the University of Würzburg and the QCMD assessment system wereused for the optimization and validation of an in-house drug resistance assay. TheViroSeq™ HIV-1 Genotyping System was used for comparison of sample andmutation detection.It was possible to optimise and validate a genotyping assay for diagnostic testing andresearch use by comparison with the ViroSeq™ HIV-1 Genotyping System andevaluation with external quality assessment systems. This assay could subsequentlybe used to determine the development of genotypic-antiretroviral resistance in patientstreated according to the provincial prevention of mother-to-child-transmission(PMTCT) protocol in the Western Cape (single dose nevirapine (sd-NVP), combinedwith a short course Zidovudine (AZT)). Patient samples were collected from pregnantwomen who took part in the Western Cape PMTCT program and visited theTygerberg Obstetrics Clinic and Delft Community Hospital. EDTA blood wasobtained to measure CD4-cell count, viral load, and to do genotyping for viral subtypeand the presence of resistance mutations. Information on prior exposure toantiretroviral therapy was also collected. A detected resistance rate of 17.1% in this predominantly HIV-1 subtype C population is lower than previously recorded whensd-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 therisk 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 separatelyto obtain PCR products, of 487 and 804 base pairs respectively for sequencing. Thetwo PCR products were sequenced with three and five primers respectively tosequence the complete PR and approximately 250 amino acids of the RT gene. Thesequences generated, thus, are analysed and aligned with the Sequencer V4.7 softwareto obtain a consensus sequence of approximately 1200 base pairs for analysis ofresistance mutations in the protease and reverse transcriptase genes.The developed assay was hence further simplified and improved, by combining thePR and RT assay into one, which was optimised and validated for use in the routinediagnostic setting. The final genotyping assay uses 8 primers for sequencing to obtaina 1200 bp sequence for genotyping that contains the protease and the 5' of the reversetranscriptase genes in which antiretroviral resistance associated mutations are found.The assay was accredited by SANAS in 2008.

[发布机构] Stellenbosch University