Detection of minority drug resistant mutations in Malawian HIV-1 subtype C-positive patients initiating and on first-line antiretroviral therapy

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.

Next Generation Sequencing Reveals a High Prevalence of HIV-1 Minority Variants and an Expanded Drug Resistance Profile Among Individuals Initiated to Antiretroviral Therapy in a Resource-constrained Setting

“…While the actual clinical relevance of minority DRMS remains contested [38][39][40], other studies [19,21,25,31,35] have reported association between minority variants with virologic failure. This concurs with our observation that the higher the frequency of baseline minority mutations the more the viral load count at end point (Kruskal Wallis p < 0.005) when we compared cases and controls.…”

“…The NGS assays have a high sensitivity and offer a platform for high-throughput sequencing, enabling NGS to detect HIV minority variants that constitute as low as 0.05-20% of the HIV viral population [13][14][15]. Minority drug-resistance mutations (DRMs) are of clinical relevance since they can cause treatment failure in individuals initiated on HIV antiretroviral drugs [16][17][18][19]. Minority variants can reduce the e cacy of HIV drugs, for example, etravirine [20] and other NNRTI-based rst-line regimens [21].…”

Building resources to meet evolving laboratory medicine challenges in Africa

“…Thus, new knowledge and disease evolution will continue to press us to change. In this issue, Zhou et al’s 7 response to the need for drug resistance information was to evaluate the application of viral population deep sequencing for people living with HIV in Malawi. They demonstrate that deep sequencing can identify drug-resistant minority variants, making informed drug choices that eliminate rather than select resistant HIV lineages possible.…”

“…Some of the technologies that will aid laboratory contributions to better care are relatively new ones, operating on new platforms and taking advantage of what we now know about the molecular basis of disease. Zhou et al’s 7 next generation sequencing approach is a case in point. But still others will be extensions of tried and true technologies that may not require new skills and infrastructure.…”

“…Both the Zhang 8 and Zhou 7 articles showcase techniques that have already been deployed in other parts of the world but had not previously been piloted in African healthcare settings. In the end, an evidence-based selected suite of essential phenotypic and genotypic tests, including sequence-based ones, must be available to all patients and the more rapidly we can assess test utility in local settings, the sooner African patients will have access to faster and more precise diagnoses.…”