Sensitivity and Specificity of the ViroSeq Human Immunodeficiency Virus Type 1 (HIV-1) Genotyping System for Detection of HIV-1 Drug Resistance Mutations by Use of an ABI PRISM 3100 Genetic Analyzer

Abstract

The ViroSeq human immunodeficiency virus type 1 (HIV-1) genotyping system is an integrated system for identification of drug resistance mutations in HIV-1 protease and reverse transcriptase (RT). Reagents are included for sample preparation, reverse transcription, PCR amplification, and sequencing. Software is provided to assemble and edit sequence data and to generate a drug resistance report. We determined the sensitivity and specificity of the ViroSeq system for mutation detection using an ABI PRISM 3100 genetic analyzer with a set of clinical samples and recombinant viruses. Twenty clinical plasma samples (viral loads, 1,800 to 10,500 copies/ml) were characterized by cloning and sequencing individual viral variants. Twelve recombinant-virus samples (viral loads, approximately 2,000 to 5,000 copies/ml) were also prepared. Eleven recombinant-virus samples contained drug resistance mutations as 40% mixtures. One recombinant-virus sample contained an insertion at codon 69 in RT (100% mutant). Plasma and recombinant-virus samples were analyzed using the ViroSeq system. Each sample was analyzed on three consecutive days at each of three testing laboratories. The sensitivity of mutation detection was 99.65% for the clinical plasma samples and 99.7% for the recombinant-virus preparations. The specificity of mutation detection was 99.95% for the clinical samples and 100% for the recombinant-virus mixtures. The base calling accuracy of the 3100 instrument was 99.91%. Mutations in clinical plasma samples and recombinant-virus samples were detected with high sensitivity and specificity, including mutations present as mixtures. This report supports the use of the ViroSeq system for identification of drug resistance mutations in HIV-1 protease and RT genes.

Sensitivity of the ViroSeq HIV-1 Genotyping System for Detection of the K103N Resistance Mutation in HIV-1 Subtypes A, C, and D

“…Previous studies have documented high sensitivity and specificity of the ViroSeq system for detection of HIV-1 drug resistance mutations, 1 and excellent performance of ViroSeq for analysis of diverse HIV-1 strains. 10 This report demonstrates that ViroSeq consistently detects the K103N mutation in plasma samples with subtypes A, C, and D at levels over 20% of the viral population, and often detects the K103N mutation at lower levels.…”

“…These assays also detect some mutations that are present at lower levels. Using recombinant viral stocks, we previously demonstrated that ViroSeq reliably detects drug resistance mutations present in 40% of the viral population in samples with viral loads from 2000 to 5000 copies/ml 1 , lower level mixtures were not evaluated in that study. In another study, ViroSeq detected the K103N mutation in a recombinant human immunodeficiency virus type 1 (HIV-1) strain at a level of 10%.…”

Field Evaluation of a Broadly Sensitive HIV-1 In-House Genotyping Assay for Use with both Plasma and Dried Blood Spot Specimens in a Resource-Limited Country

“…The FDA-approved ViroSeq assay amplifies an 1.8-kb fragment covering the entire protease region and codons 1 to 335 of the reverse transcriptase (RT) region. This assay has an amplification detection sensitivity of 2,000 RNA copies/ml of plasma and a DR-associated mutation (DRM) detection sensitivity and specificity of 99.65% and 99.95%, respectively (35). Drug resistance genotyping was performed according to the manufacturer's instructions, and DR interpretations were conducted using both the ViroSeq HIV-1 genotyping system software, V2.6, and the Stanford genotyping resistance interpretation algorithm (http://sierra2.stanford.edu/sierra/servlet/JSierra) to allow for comparison with the in-house assay.…”

High Failure Rate of the ViroSeq HIV-1 Genotyping System for Drug Resistance Testing in Cameroon, a Country with Broad HIV-1 Genetic Diversity

“…The ViroSeq HIV-1 genotyping system was optimized for use on subtype B strains (3) and is currently widely commercialized and used in African settings where non-B HIV-1 strains predominate. The present observations in Cameroon, a country where almost all cocirculating strains are non-B HIV-1 variants, showed the impact of non-B HIV-1 genetic divergence on the performance of this assay.…”