Abstract
BackgroundIt is largely unknown how frequently low-abundance HIV drug-resistant variants at levels under limit of detection of conventional genotyping (<20% of quasi-species) are present in antiretroviral-experienced persons experiencing virologic failure. Further, the clinical implications of low-abundance drug-resistant variants at time of virologic failure are unknown.Methodology/Principal FindingsPlasma samples from 22 antiretroviral-experienced subjects collected at time of virologic failure (viral load 1380 to 304,000 copies/mL) were obtained from a specimen bank (from 2004–2007). The prevalence and profile of drug-resistant mutations were determined using Sanger sequencing and ultra-deep pyrosequencing. Genotypes were interpreted using Stanford HIV database algorithm. Antiretroviral treatment histories were obtained by chart review and correlated with drug-resistant mutations. Low-abundance drug-resistant mutations were detected in all 22 subjects by deep sequencing and only in 3 subjects by Sanger sequencing. In total they accounted for 90 of 247 mutations (36%) detected by deep sequencing; the majority of these (95%) were not detected by standard genotyping. A mean of 4 additional mutations per subject were detected by deep sequencing (p<0.0001, 95%CI: 2.85–5.53). The additional low-abundance drug-resistant mutations increased a subject's genotypic resistance to one or more antiretrovirals in 17 of 22 subjects (77%). When correlated with subjects' antiretroviral treatment histories, the additional low-abundance drug-resistant mutations correlated with the failing antiretroviral drugs in 21% subjects and correlated with historical antiretroviral use in 79% subjects (OR, 13.73; 95% CI, 2.5–74.3, p = 0.0016).Conclusions/SignificanceLow-abundance HIV drug-resistant mutations in antiretroviral-experienced subjects at time of virologic failure can increase a subject's overall burden of resistance, yet commonly go unrecognized by conventional genotyping. The majority of unrecognized resistant mutations correlate with historical antiretroviral use. Ultra-deep sequencing can provide important historical resistance information for clinicians when planning subsequent antiretroviral regimens for highly treatment-experienced patients, particularly when their prior treatment histories and longitudinal genotypes are not available.
Highlights
HIV genotyping technologies other than the conventional HIV genotyping have been used to show that viral variants in an HIVinfected person, whether acutely or chronically infected, are more genetically diverse than previously appreciated by conventional HIV genotyping assays [1,2,3,4,5,6,7,8,9]
The differences in the numbers of drug-resistant mutations (DRMs) detected by the two methods remained statistically significant when mutations were classified into nucleoside reverse transcriptase inhibitor (NRTI), nucleoside reverse transcriptase inhibitor (NNRTI), and protease inhibitor (PI) (Figure 1)
DRMs at levels,20% of viral quasispecies made up 36% of the total number of mutations detected by ultra-deep sequencing; the vast majority were undetected by standard HIV genotyping
Summary
HIV genotyping technologies other than the conventional HIV genotyping have been used to show that viral variants in an HIVinfected person, whether acutely or chronically infected, are more genetically diverse than previously appreciated by conventional HIV genotyping assays [1,2,3,4,5,6,7,8,9]. Current genotyping assays are based on population sequencing of reverse transcriptase polymerase chain reaction (RT-PCR) generated products of HIV protease (PR) and reverse transcriptase (RT) genes This technology has been a major advancement in the understanding and management of HIV drug resistance in clinical practice, a major limitation is the inability to detect lowabundance drug-resistant mutations (DRMs) at levels ,20% of the viral quasi-species [10,11]. A growing number of studies have shown that low-abundance DRMs can be detected in chronically-infected antiretroviral-naıve individuals using ultra-sensitive allele-specific PCR assays or by ultra-deep sequencing methods [8,14,15,16,17] These studies show that baseline low-abundance DRMs undetected by conventional sequencing, in particular non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations, are associated with poor treatment response in persons initiating antiretroviral therapy (ART). The clinical implications of low-abundance drug-resistant variants at time of virologic failure are unknown
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