Computational protocol: High levels of pre-treatment HIV drug resistance and treatment failure in Nigerian children

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Protocol publication

[…] Before ART initiation and every six months during follow-up, a study blood sample (6 ml EDTA tube) was collected for HIV VL testing using the Roche Cobas AmpliPrep TaqMan® (Cobas Amplicor; Roche Diagnostics, Switzerland) to detect virological failure. For all children with a pre-treatment VL >1000 cps/ml, population-based sequencing of the HIV-1 pol gene was performed by the reference laboratory of the Institute of Human Virology in Abuja, Nigeria. HIV-1 RNA was extracted from 200 µL plasma, amplified and sequenced as previously described [] at the National WHO HIV drug resistance reference laboratory of the Institute of Human Virology in Abuja, Nigeria, using an in-house method and primers designed and optimized for CFR02_AG and subtype G isolates []. Obtained sequences were visually inspected using Sequencher (Gene Codes Corp., Ann Arbor, MI, USA) at two independent laboratories to verify that each nucleotide base was covered at least by three reads, one of which had to be in the opposite direction of the other two. Sequences were first aligned using HIVAlign (www.hiv.lanl.gov/content/sequence/VIRALIGN/viralign.html). Sequence-genetic relatedness was assessed in MEGA version 5.2.2 (http://www.megasoftware.net/). Samples of which sequences were <1.0% different and had been processed on the same day were re-processed and re-sequenced to rule out cross-sample contamination. To ensure quality of the data set, each sequence was checked before they were submitted to ViroScore® []. Major drug resistance mutations were identified based on the 2009 WHO list for surveillance of transmitted resistance [] using the Stanford Calibrated Population Resistance analysis tool version 6.0 []. Susceptibility of the prescribed ART regimen was determined through calculation of the genotypic sensitivity score (GSS) using the Stanford algorithm (Version 7.0) []. Reduced susceptibility to the prescribed regimen was defined as GSS<3, that is, <3 fully susceptible drugs. HIV-1 subtyping was performed using the REGA HIV-1 subtyping tool V3 []. Genotypic resistance testing was conducted retrospectively and, therefore, results could not be used by the treating physicians to make the basis of their treatment decisions.The study has received ethical clearance from the Health Research and Ethics Committee of the Lagos University Teaching Hospital, and was conducted in compliance with Good Clinical Practice guidelines and the principles of the Declaration of Helsinki. All laboratory procedures were conducted according to Good Laboratory Practice guidelines. […]

Pipeline specifications

Software tools Sequencher, MEGA, Viroscore, REGA HIV Subtyping Tool
Application Phylogenetics
Organisms Human immunodeficiency virus 2, Homo sapiens
Diseases HIV Infections
Chemicals Nucleosides