An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

Abstract

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.

Long-Range HIV Genotyping Using Viral RNA and Proviral DNA for Analysis of HIV Drug Resistance and HIV Clustering

“…The CDC supplies WHO-designated and CDC-supported President's Emergency Plan for AIDS Relief (PEPFAR) Genotyping Laboratories with the ATCC HIV-1 Drug Resistance Genotyping kit (46) for drug resistance testing. Many experienced genotyping laboratories have developed their own in-house amplification and sequencing protocols (11,(47)(48)(49)(50)(51)(52)(53)(54)(55)(56), including identification of minor viral variants that are normally missed by commercial genotyping kits (57-61). All of these approaches generally include smaller and more restricted regions for testing HIV-1 drug resistance.…”

“…Routine antiretroviral (ARV) drug resistance testing is useful in choosing an optimal treatment regimen and monitoring its efficiency in clinical practice (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). HIV genotyping has been used successfully in research on HIV transmission clusters and HIV transmission dynamics .…”

Drug Resistance Among Adolescents and Young Adults with Virologic Failure of First-Line Antiretroviral Therapy and Response to Second-Line Treatment

“…The SATuRN protocol was designed to be an affordable protocol implementing mostly open source and open access bioinformatics resources for the interpretation of HIV drug resistance. 17 The amplicons generated were sequenced by Sanger sequencing at Molecular Cloning Laboratories, San Francisco, CA. The quality of the consensus sequences generated was verified by phylogenetic tree reconstruction in Geneious software, version 8 (http://geneious .com).…”

HIV drug resistance testing among patients failing second line antiretroviral therapy. Comparison of in-house and commercial sequencing

“…Reagent volumes and PCR conditions for the 1 st and 2 nd rounds were as follows; 94°C for 2 minutes, followed by 30 cycles of 95°C for 30 seconds, 58°C for 20 seconds, 72°C for 2 minutes, and then 72°C for 10 minutes, before a final holding step at 4°C. Second round amplicon was 1.32kb as verified by gel electrophoresis on a 1% agarose gel [10]. …”

“…We sought to develop local HIV drug resistance capacity by adopting a published affordable genotyping method [10]. We performed GRT on plasma from patients failing second-line therapy accessing clinical care at a specialist HIV clinical care center (Newlands Clinic, Harare, Zimbabwe).…”