title: Concordance between allele-specific PCR and ultra-deep pyrosequencing for the detection of HIV-1 non-nucleoside reverse transcriptase inhibitor resistance mutations.
abstract: Recent advances in genotyping technologies have allowed for detection of HIV-1 drug resistance mutations present at low levels. The presence and percentage of Y181C and K103N drug-resistant variants in the blood of 105 subtype C HIV-infected infants who failed single-dose nevirapine prophylaxis for HIV transmission were compared using two highly sensitive genotyping methods, allele-specific PCR (AS-PCR) and ultra-deep pyrosequencing. Significant correlations in detection between both methods were found for both Y181C (correlation coefficients of 0.94 [95% CI 0.91-0.96]) and K103N (0.89 [95% CI 0.84-0.92]) mutations. The majority of discordant specimens (3/5 Y181C and 8/11 K103N) had wild-type variants when population sequencing was used, but mutant variants were detectable at very low levels (≤5%) with either assay. This difference is most likely due to stochastic variations in the appearance of mutant variants. Overall, both AS-PCR and ultra-deep pyrosequencing methods have proven to be sensitive and accurate, and may confidently be used where feasible.
authors.affiliation: Centre for HIV and STIs, National Institute for Communicable Diseases of the National Health Laboratory Service, Johannesburg, South Africa; Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa. Electronic address: gillianh@nicd.ac.za.
authors.forename: Gillian M
authors.initials: GM
authors.lastname: Hunt
authors.affiliation: Centre for HIV and STIs, National Institute for Communicable Diseases of the National Health Laboratory Service, Johannesburg, South Africa; Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa. Electronic address: lynnm@nicd.ac.za.
authors.forename: Lynn
authors.initials: L
authors.lastname: Morris
authors.affiliation: Johns Hopkins University School of Medicine, Baltimore, MD, United States. Electronic address: akmoorthy@gmail.com.
authors.forename: Anitha
authors.initials: A
authors.lastname: Moorthy
authors.affiliation: Empilweni Services and Research Unit, Rahima Moosa Mother and Child Hospital, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa. Electronic address: ashraf.coovadia@wits.ac.za.
authors.forename: Ashraf
authors.initials: A
authors.lastname: Coovadia
authors.affiliation: ICAP, Columbia University Mailman School of Public Health, New York, NY, United States. Electronic address: eja1@cumb.columbia.edu.
authors.forename: Elaine J
authors.initials: EJ
authors.lastname: Abrams
authors.affiliation: Empilweni Services and Research Unit, Rahima Moosa Mother and Child Hospital, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa. Electronic address: renate.strehlau@wits.ac.za.
authors.forename: Renate
authors.initials: R
authors.lastname: Strehlau
authors.affiliation: Gertrude H. Sergievsky Center, College of Physicians and Surgeons and Department of Epidemiology, Mailman School of Public Health, Columbia University, New York, NY, United States. Electronic address: lk24@cumc.columbia.edu.
authors.forename: Louise
authors.initials: L
authors.lastname: Kuhn
authors.affiliation: Johns Hopkins University School of Medicine, Baltimore, MD, United States. Electronic address: dpers@jhmi.edu.
authors.forename: Deborah
authors.initials: D
authors.lastname: Persaud
mesh_terms: D000483:Alleles; D024882:Drug Resistance, Viral; D005260:Female; D060005:Genotyping Techniques / Q000379:methods; D015658:HIV Infections / Q000821:virology; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000145:classification / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D008826:Microbial Sensitivity Tests / Q000379:methods*; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase
keywords: Allele-specific PCR; HIV-1 drug resistance; Low-frequency NNRTI variants; Ultra-deep sequencing