Cross-clade simultaneous HIV drug resistance genotyping for reverse transcriptase, protease, and integrase inhibitor mutations by Illumina MiSeq

Abstract

BackgroundViral resistance to antiretroviral therapy threatens our best methods to control and prevent HIV infection. Current drug resistance genotyping methods are costly, optimized for subtype B virus, and primarily detect resistance mutations to protease and reverse transcriptase inhibitors. With the increasing use of integrase inhibitors in first-line therapies, monitoring for integrase inhibitor drug resistance mutations is a priority. We designed a universal primer pair to PCR amplify all major group M HIV-1 viruses for genotyping using Illumina MiSeq to simultaneously detect drug resistance mutations associated with protease, nucleoside reverse transcriptase, non-nucleoside reverse transcriptase, and integrase inhibitors.ResultsA universal primer pair targeting the HIV pol gene was used to successfully PCR amplify HIV isolates representing subtypes A, B, C, D, CRF01_AE and CRF02_AG. The universal primers were then tested on 62 samples from a US cohort of injection drug users failing treatment after release from prison. 94% of the samples were successfully genotyped for known drug resistance mutations in the protease, reverse transcriptase and integrase gene products. Control experiments demonstrate that mutations present at ≥ 2% frequency are reliably detected and above the threshold of error for this method. New drug resistance mutations not found in the baseline sample were identified in 54% of the patient samples after treatment failure. 86% of patients with major drug resistance mutations had 1 or more mutations associated with drug resistance to the treatment regimen at the time point of treatment failure. 59% of the emerging mutations were found at frequencies between 2% and 20% of the total sequences generated, below the estimated limit of detection of current FDA-approved genotyping techniques. Primary plasma samples with viral loads as low as 799 copies/ml were successfully genotyped using this method.ConclusionsHere we present an Illumina MiSeq-based HIV drug resistance genotyping assay. Our data suggests that this universal assay works across all major group M HIV-1 subtypes and identifies all drug resistance mutations in the pol gene known to confer resistance to protease, reverse transcriptase and integrase inhibitors. This high-throughput and sensitive assay could significantly improve access to drug resistance genotyping worldwide.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0122-8) contains supplementary material, which is available to authorized users.

No Substantial Evidence for Sexual Transmission of Minority HIV Drug Resistance Mutations in Men Who Have Sex with Men

“…Overall, our results suggested that minor resistant variants may emerge de novo shortly after transmission, when the small effective population size limits efficient purge by natural selection (30,31). Future studies using recent sequencing technologies that provide longer reads and are less prone to single-base errors (MiSeq or PacBio with or without Primer ID) (32,33) or single-genome amplification/ Sanger sequencing in different settings (i.e., ART-experienced HIV-infected source partners) within a similar Bayesian framework would be informative for detecting clinically relevant minority variants and investigating their propensity for transmission.…”

Diagnosis of Human Immunodeficiency Virus Infection

“…Deep sequencing. The three different types of deep sequencing instruments currently in use include Illumina MiSeq (296,297), the Roche 454/GS Junior pyrosequencer (295,298,299), and the Ion PGM 200 sequencer (300). The operational principles of these methods are similar and can be completed to yield DRM reports in 1 to 2 days.…”

HIV-1 genotypic drug resistance testing: digging deep, reaching wide?

“…For the required template enrichment, the current fullgenome protocols rely on amplification of large genomic regions with subtype-wide primers and therefore perform less well at viral loads below 1000-3000 copies/ml, making these assays currently more appropriate for surveillance efforts than for monitoring of individual patients [28,29]. However, several studies have already shown that experienced laboratories can achieve accurate NGS results at frequencies as low as 1% [30,31 ,32,33 ].…”