Evaluation of a Cost Effective In-House Method for HIV-1 Drug Resistance Genotyping Using Plasma Samples

<div><p>Objectives</p><p>Validation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples.</p><p>Design</p><p>The validation includes the establishment of analytical performance characteristics such as accuracy, reproducibility, precision and sensitivity.</p><p>Methods</p><p>The accuracy was assessed by comparing 26 paired Virological Quality Assessment (VQA) proficiency testing panel sequences generated by in-house and ViroSeq Genotyping System 2.0 (Celera Diagnostics, US) as a gold standard. The reproducibility and precision were carried out on five samples with five replicates representing multiple HIV-1 subtypes (A, B, C) and resistance patterns. The amplification sensitivity was evaluated on HIV-1 positive plasma samples (n = 88) with known viral loads ranges from 1000–1.8 million RNA copies/ml.</p><p>Results</p><p>Comparison of the nucleotide sequences generated by ViroSeq and in-house method showed 99.41±0.46 and 99.68±0.35% mean nucleotide and amino acid identity respectively. Out of 135 Stanford HIVdb listed HIV-1 drug resistance mutations, partial discordance was observed at 15 positions and complete discordance was absent. The reproducibility and precision study showed high nucleotide sequence identities i.e. 99.88±0.10 and 99.82±0.20 respectively. The in-house method showed 100% analytical sensitivity on the samples with HIV-1 viral load >1000 RNA copies/ml. The cost of running the in-house method is only 50% of that for ViroSeq method (112$vs 300$), thus making it cost effective.</p><p>Conclusions</p><p>The validated cost effective in-house method may be used to collect surveillance data on the emergence and transmission of HIV-1 drug resistance in resource limited countries. Moreover, the wide applications of a cost effective and validated in-house method for HIV-1 drug resistance testing will facilitate the decision making for the appropriate management of HIV infected patients.</p></div

Phylogenetic analysis of the sequences generated during the evaluation of an in-house method.

<p>The phylogenetic tree was generated by using the PhyML software to create a maximum likelihood tree <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087441#pone.0087441-Chaturbhuj1" target="_blank">[13]</a>. The reference sequences were obtained from the Los Alamos HIV Database (<a href="http://www.hiv.lanl.gov" target="_blank">www.hiv.lanl.gov</a>). IHDR- sequences generated by an in-house method; VSQ- sequences generated by the ViroSeq method; R1 to R5- sample used in the reproducibility study (hilighted in blue); P1 to P5- sample used in the precision study (hilighted in red); A, B, C, D, E are the replicates of the same sample.</p

Pairwise sequence identity analysis between the in-house and the ViroSeq method.

<p>Pairwise sequence identity analysis between the in-house and the ViroSeq method.</p

Primers used in the In-house method for HIV-1 drug resistance genotyping.

<p>Primers used in the In-house method for HIV-1 drug resistance genotyping.</p

Drug resistance-associated amino acid positions in protease and reverse transcriptase from 26 proficiency testing panel plasma samples genotyped by the in-house and the ViroSeq methods.

<p>Note: Partial discordant positions are shown in bold.</p

Reproducibility and Precision of an in-house method.

<p>Reproducibility and Precision of an in-house method.</p