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

Abstract

The US Food and Drug Administration-cleared ViroSeq HIV-1 Genotyping System (ViroSeq) and other population sequencing-based human immunodeficiency virus type 1 (HIV-1) genotyping methods detect antiretroviral drug resistance mutations present in the major viral population of a test sample. These assays also detect some mutations in viral variants that are present as mixtures. We compared detection of the K103N nevirapine resistance mutation using The US Food and Drug Administration-cleared ViroSeq HIV-1 Genotyping System (ViroSeq) and other population sequencing-based genotyping methods detect mutations present in the major viral population in a test sample. 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%. 2HIV-1 variants with the K103N mutation are often selected in women who receive a single dose of the antiretroviral drug nevirapine for prevention of HIV-1 motherto-child transmission. 3,4 We evaluated the sensitivity of ViroSeq for detection of K103N in 305 clinical plasma samples collected from African women 6 to 8 weeks after single dose nevirapine administration. Samples were collected from women in the HIV Network for Prevention Trial (HIVNET) 012 trial 5,6 (Ugandan women, 146 subtype A samples and 95 subtype D samples) and the Nevirapine-

Sensitive Deep-Sequencing-Based HIV-1 Genotyping Assay To Simultaneously Determine Susceptibility to Protease, Reverse Transcriptase, Integrase, and Maturation Inhibitors, as Well as HIV-1 Coreceptor Tropism

“…Additional studies using a larger number of clinical samples will be needed to verify this limit of detection. Nevertheless, our novel assay was able to reproducibly detect HIV-1 minority variants at frequencies at least 4-fold lower than the 20% reported for the standard Sanger-based sequencing (16,(44)(45)(46)(47)(48).…”

“…Most of these HIV-1 drug resistance or tropism tests correspond to methods developed and used in research settings; however, a few selected assays are commercially available. All current commercial genotypic HIV-1 drug resistance assays are based on population (Sanger) sequencing (10,43,44), limiting the detection of minority variants to those in Ͼ20% of the viral population (44)(45)(46)(47)(48). In the case of HIV-1 tropism determination, only a couple of ultrasensitive deep-sequencing-based genotypic tests are currently used in the clinical setting (63,66).…”

“…All variants with a frequency of Ͼ1 within the population were aligned using ClustalW (78) and the phylogeny reconstructed using the neighbor-joining statistical method as implemented within MEGA 5.05 (79). In this study, a minority variant was defined as a variation detected at Ͼ1% (based on the intrinsic error rate of the system as described below) and Ͻ20% of the virus population, corresponding to those mutations that cannot be determined using population sequencing (44)(45)(46)(47)(48).…”

“…To date, all current commercial genotypic HIV-1 drug resistance assays are based on population sequencing (10,43,44), which can detect only minority variants that are present in Ͼ20% of the viral population (44)(45)(46)(47)(48). However, and although this is still uncertain, drug-resistant HIV-1 minority variants (i.e., those present in as low as 1% of the viral population) have been suggested to be clinically relevant, as they have a high chance of selection under antiretroviral drug pressure conditions (49)(50)(51)(52)(53)(54)(55)(56)(57).…”

Improvement in allele-specific PCR assay with the use of polymorphism-specific primers for the analysis of minor variant drug resistance in HIV-1 subtype C

“…Commercial genotyping assays are limited to the detection of HIV-1 drug resistance when resistant viruses comprise at least 20% of the viruses in an infected individual (Brun-Vezinet et al, 2004;Church et al, 2006;Grant et al, 2003;Palmer et al, 2005). There is widespread interest in understanding the clinical relevance of minor variant drug resistance, that is, when drug resistant viruses are present at less than 20%.…”