Abstract
BackgroundMinority drug resistance mutations (DRMs) that are often missed by Sanger sequencing are clinically significant, as they can cause virologic failure in individuals treated with antiretroviral therapy (ART) drugs.ObjectiveThis study aimed to estimate the prevalence of minor DRMs among patients enrolled in a Malawi HIV drug resistance monitoring survey at baseline and at one year after initiation of ART.MethodsForty-one plasma specimens collected from HIV-1 subtype C-positive patients and seven clonal control samples were analysed using ultra-deep sequencing technology.ResultsDeep sequencing identified all 72 DRMs detected by Sanger sequencing at the level of ≥20% and 79 additional minority DRMs at the level of < 20% from the 41 Malawian clinical specimens. Overall, DRMs were detected in 85% of pre-ART and 90.5% of virologic failure patients by deep sequencing. Among pre-ART patients, deep sequencing identified a statistically significant higher prevalence of DRMs to nucleoside reverse transcriptase inhibitors (NRTIs) compared with Sanger sequencing. The difference was mainly due to the high prevalence of minority K65R and M184I mutations. Most virologic failure patients harboured DRMs against both NRTIs and non-nucleoside reverse transcriptase inhibitors (NNRTIs). These minority DRMs contributed to the increased or enhanced virologic failures in these patients.ConclusionThe results revealed the presence of minority DRMs to NRTIs and NNRTIs in specimens collected at baseline and virologic failure time points. These minority DRMs not only increased resistance levels to NRTIs and NNRTIs for the prescribed ART, but also expanded resistance to additional major first-line ART drugs. This study suggested that drug resistance testing that uses more sensitive technologies, is needed in this setting.
Highlights
Rapid scale-up of antiretroviral therapy (ART) over the past decade has remarkably reduced the mortality and morbidity of HIV-positive patients and decreased HIV transmission
The quasi-species nature of HIV-1 makes the detection of drug resistant mutations (DRMs) more difficult, because the commonly-used Sanger sequencing for drug resistance testing is incapable of detecting these drug resistant HIV variants at a level of less than 20% of the viral population.[2,3,4,5]
From the 1% mixed clone, K103N was not detected within the 520 sequence reads; Y181C and M184V were detected at the level of 1.03% with 1335 reads, while G190A was detected at 0.97% with 785 reads
Summary
Rapid scale-up of antiretroviral therapy (ART) over the past decade has remarkably reduced the mortality and morbidity of HIV-positive patients and decreased HIV transmission. Minority drug resistant variants ( known as low-frequency mutants) that are not detected by Sanger sequencing are clinically important, as they can cause virologic failure in patients treated with ART for the first time.[6,7,8,9] Recent studies have demonstrated that particular drug resistant HIV mutant viruses are clinically significant at a level of 1% of the viral population, as the minority variants can replicate quickly and become the predominant viral population through the selective pressure of ART drugs, leading to treatment failure.[9,10] in the absence of drug pressure in treatment-naïve patients, the stability of transmitted DRMs is different.[11] For instance, a transmitted M184V mutation can quickly revert to wild-type due to diminished viral fitness.[12]. Minority drug resistance mutations (DRMs) that are often missed by Sanger sequencing are clinically significant, as they can cause virologic failure in individuals treated with antiretroviral therapy (ART) drugs
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