In-house genotypic antiretroviral resistance test : optimisation and validation for use in research and diagnostics
Date
2011-03
Authors
Claassen, Mathilda
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : University of Stellenbosch
Abstract
It 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.
Description
Thesis (MScMedSc)--University of Stellenbosch, 2011.
Keywords
Resistance, HIV infections -- Effect of drugs on, Molecular, Genotyping, HIV infections -- Antiretroviral treatment, Theses -- Medical virology, Dissertations -- Medical virology