Abstract
BackgroundThe success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing.MethodologyWe developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing.SignificanceCombined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.
Highlights
Active antiretroviral therapy (HAART) can provide sustained clinical benefit for HIV-1 infected persons, but treatment success is jeopardized by drug resistance
We focused on developing and validating nine assays for key drug resistance mutations in subtype B HIV-1 clinical specimens as a basis for later expansion to other virus mutations and subtypes
To ensure sufficient template for repeat testing, virus sequences were first amplified from 5 mL HIV-1 RNA by reverse transcriptase-polymerase chain reaction (RTPCR) using the reverse primer RTP-REV2 [59-CTT CTG TAT GTC ATT GAC AGT CC], and forward primer RTP-F1 [59CCT CAG ATC ACT CTT TGG CAA CG], which span from n.t. 1 in protease to n.t. 777 in RT
Summary
The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. There is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, requires simple and practical methods for routine testing. We developed highlysensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations
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