HIV Drug Resistance Testing by High-Multiplex “Wide” Sequencing on the MiSeq Instrument

Abstract

f Limited access to HIV drug resistance testing in low-and middle-income countries impedes clinical decision-making at the individual patient level. An efficient protocol to address this issue must be established to minimize negative therapeutic outcomes for HIV-1-infected individuals in such settings. This is an observational study to ascertain the potential of newer genomic sequencing platforms, such as the Illumina MiSeq instrument, to provide accurate HIV drug resistance genotypes for hundreds of samples simultaneously. Plasma samples were collected from Canadian patients during routine drug resistance testing (n ‫؍‬ 759) and from a Ugandan study cohort (n ‫؍‬ 349). Amplicons spanning HIV reverse transcriptase codons 90 to 234 were sequenced with both MiSeq sequencing and conventional Sanger sequencing methods. Sequences were evaluated for nucleotide concordance between methods, using coverage and mixture parameters for quality control. Consensus sequences were also analyzed for disparities in the identification of drug resistance mutations. Sanger and MiSeq sequencing was successful for 881 samples (80%) and 892 samples (81%), respectively, with 832 samples having results from both methods. Most failures were for samples with viral loads of <3.0 log 10 HIV RNA copies/ml. Overall, 99.3% nucleotide concordance between methods was observed. MiSeq sequencing achieved 97.4% sensitivity and 99.3% specificity in detecting resistance mutations identified by Sanger sequencing. Findings suggest that the Illumina MiSeq platform can yield high-quality data with a high-multiplex "wide" sequencing approach. This strategy can be used for multiple HIV subtypes, demonstrating the potential for widespread individual testing and annual population surveillance in resource-limited settings.A dvances in highly active antiretroviral therapy (HAART) in recent decades have resulted in sustained decreases in HIVrelated morbidity and mortality rates. HIV-infected individuals who receive treatment now have nearly normal life expectancies, such that HIV is now considered a manageable chronic disease (1, 2); antiretroviral therapy (ART) not only provides benefits at the individual patient level but also results in a population-level advantage through HAART-induced suppression of HIV replication and the inherent prevention of onward transmission of the virus, termed "treatment as prevention" (3-6).Drug resistance testing is an essential complement to HAART, enabling clinicians to identify patients infected with drug-resistant HIV and to prescribe the appropriate antiretroviral regimens (7). Lack of access to HIV drug resistance testing acts as a major barrier to long-term treatment success, either through prescription of ineffective regimens in the case of transmitted resistance or through decreased ability of physicians to identify causes of treatment failure (7-9). Cases of unsuppressed viremia allow continued transmission and can compromise the management of HIV on both the patient and population levels. These challenges are particula...

Prevalence and clinical impacts of HIV-1 intersubtype recombinants in Uganda revealed by near-full-genome population and deep sequencing approaches

“…However, our results also suggest that an average MiSeq coverage depth of 10–100 can still very accurately predict Sanger subtyping results across all genomic regions in these Ugandan non-subtype B HIV samples. This agrees with previous observations that MiSeq can accurately predict Sanger-derived rt sequences at coverage depth below 100 [23]. Furthermore, we showed that MiSeq was more successful in yielding full-length GP120 sequences compared to Sanger sequencing, supporting the move to deep sequencing in HIV env genetic studies.…”

HIV-1 drug resistance and resistance testing

“…In NGS deep sequencing the depth of coverage refers to the number of viral variants sequenced, typically several hundred. Researchers have shown that harnessing the cost savings of NGS using a “wide sequencing” strategy, wherein many samples are processed in a single run with a lesser depth of coverage, could be a viable method of genotypic testing in LMICs (Dudley et al, 2012; Lapointe et al, 2015). Cost-effectiveness models using NGS platforms have not been published, but the estimated cost per sample is much lower than SGRT at 1 to 20 USD (Dudley et al, 2012; Lapointe et al, 2015).…”

Low-Frequency Drug Resistance in HIV-Infected Ugandans on Antiretroviral Treatment Is Associated with Regimen Failure

“…These variants are usually present as minority members of the virus population, which can be selected to dominate over drugsusceptible variants under drug pressure (25)(26)(27). For this reason, a series of ultrasensitive HIV-1-genotyping assays, based on deep sequencing (next-generation sequencing [NGS]), have been developed to detect drug-resistant HIV-1 variants at levels below 20% of the viral population in an infected individual (24,(28)(29)(30)(31). Several studies have associated early detection of these minority HIV-1 drug-resistant variants with subsequent treatment failure (32)(33)(34)(35)(36)(37); however, with the advent of single-pill once-a-day (QD) cART regimens, treatment failures in HICs are rare, and the relevance of minority members of the viral population to the ART outcome is still under debate (32,(38)(39)(40)(41).…”