Benjamin Chimukangara1, Bhavini Varyani2, Tinei Shamu3, Junior Mutsvangwa4, Justen Manasa5, Elizabeth White6, Cleophas Chimbetete7, Ruedi Luethy8, David Katzenstein9. 1. Department of Molecular Biology, Biomedical Research and Training Institute, Harare, Zimbabwe; Department of Virology, National Health Laboratory Service, University of KwaZulu-Natal, Durban, South Africa. Electronic address: benjiechim@yahoo.com. 2. Department of Molecular Biology, Biomedical Research and Training Institute, Harare, Zimbabwe. Electronic address: bhavinivaryani@gmail.com. 3. Newlands Clinic, Newlands, Harare, Zimbabwe. Electronic address: TineiS@newlandsclinic.org.zw. 4. Department of Molecular Biology, Biomedical Research and Training Institute, Harare, Zimbabwe. Electronic address: Jnr.mutsvangwa@gmail.com. 5. Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA. Electronic address: jmanasa@gmail.com. 6. Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA. Electronic address: betsyjohn@aol.com. 7. Newlands Clinic, Newlands, Harare, Zimbabwe; Institute of Social and Preventive Medicine, University of Bern, Switzerland. Electronic address: cleophasc@newlandsclinic.org.zw. 8. Newlands Clinic, Newlands, Harare, Zimbabwe. Electronic address: RuediL@newlandsclinic.org.zw. 9. Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA. Electronic address: davidkk@stanford.edu.
Abstract
INTRODUCTION: HIV genotyping is often unavailable in low and middle-income countries due to infrastructure requirements and cost. We compared genotype resistance testing in patients with virologic failure, by amplification of HIV pol gene, followed by "in-house" sequencing and commercial sequencing. METHODS: Remnant plasma samples from adults and children failing second-line ART were amplified and sequenced using in-house and commercial di-deoxysequencing, and analyzed in Harare, Zimbabwe and at Stanford, U.S.A, respectively. HIV drug resistance mutations were determined using the Stanford HIV drug resistance database. RESULTS: Twenty-six of 28 samples were amplified and 25 were successfully genotyped. Comparison of average percent nucleotide and amino acid identities between 23 pairs sequenced in both laboratories were 99.51 (±0.56) and 99.11 (±0.95), respectively. All pairs clustered together in phylogenetic analysis. Sequencing analysis identified 6/23 pairs with mutation discordances resulting in differences in phenotype, but these did not impact future regimens. CONCLUSIONS: The results demonstrate our ability to produce good quality drug resistance data in-house. Despite discordant mutations in some sequence pairs, the phenotypic predictions were not clinically significant.
INTRODUCTION: HIV genotyping is often unavailable in low and middle-income countries due to infrastructure requirements and cost. We compared genotype resistance testing in patients with virologic failure, by amplification of HIV pol gene, followed by "in-house" sequencing and commercial sequencing. METHODS: Remnant plasma samples from adults and children failing second-line ART were amplified and sequenced using in-house and commercial di-deoxysequencing, and analyzed in Harare, Zimbabwe and at Stanford, U.S.A, respectively. HIV drug resistance mutations were determined using the Stanford HIV drug resistance database. RESULTS: Twenty-six of 28 samples were amplified and 25 were successfully genotyped. Comparison of average percent nucleotide and amino acid identities between 23 pairs sequenced in both laboratories were 99.51 (±0.56) and 99.11 (±0.95), respectively. All pairs clustered together in phylogenetic analysis. Sequencing analysis identified 6/23 pairs with mutation discordances resulting in differences in phenotype, but these did not impact future regimens. CONCLUSIONS: The results demonstrate our ability to produce good quality drug resistance data in-house. Despite discordant mutations in some sequence pairs, the phenotypic predictions were not clinically significant.
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