Transmitted HIV-1 drug resistance can compromise initial antiretroviral therapy (ART); therefore, its detection is important for patient management. The absence of drug-associated selection pressure in treatment-naïve persons can cause drug-resistant viruses to decline to levels undetectable by conventional bulk sequencing (minority drug-resistant variants). We used sensitive and simple tests to investigate evidence of transmitted drug resistance in antiretroviral drug-naïve persons and assess the clinical implications of minority drug-resistant variants. We performed a cross-sectional analysis of transmitted HIV-1 drug resistance and a case-control study of the impact of minority drug resistance on treatment response. For the cross-sectional analysis, we examined viral RNA from newly diagnosed ART-naïve persons in the US and Canada who had no detectable (wild type, n = 205) or one or more resistance-related mutations (n = 303) by conventional sequencing. Eight validated real-time PCR-based assays were used to test for minority drug resistance mutations (protease L90M and reverse transcriptase M41L, K70R, K103N, Y181C, M184V, and T215F/Y) above naturally occurring frequencies. The sensitive real-time PCR testing identified one to three minority drug resistance mutation(s) in 34/205 (17%) newly diagnosed persons who had wild-type virus by conventional genotyping; four (2%) individuals had mutations associated with resistance to two drug classes. Among 30/303 (10%) samples with bulk genotype resistance mutations we found at least one minority variant with a different drug resistance mutation. For the case-control study, we assessed the impact of three treatment-relevant drug resistance mutations at baseline from a separate group of 316 previously ART-naïve persons with no evidence of drug resistance on bulk genotype testing who were placed on efavirenz-based regimens. We found that 7/95 (7%) persons who experienced virologic failure had minority drug resistance mutations at baseline; however, minority resistance was found in only 2/221 (0.9%) treatment successes (Fisher exact test, p = 0.0038). These data suggest that a considerable proportion of transmitted HIV-1 drug resistance is undetected by conventional genotyping and that minority mutations can have clinical consequences. With no treatment history to help guide therapies for drug-naïve persons, the findings suggest an important role for sensitive baseline drug resistance testing.

Conventional sequence analysis detects human immunodeficiency virus (HIV)–1 drug resistance mutations in ∼40% of women shortly after they receive int

The 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. We 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. Combined, 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.

With drug-resistant HIV-1 present in at least 10%–20% of new infections in Western countries and in >60% of patients failing antiretroviral therapy (ART), monitoring HIV-1 drug resistance is becoming increasingly important for assessing its impact on therapeutic measures of virus control and for guiding treatment. The sensitivity limitations of conventional bulk genotyping often lead to an underestimation of the total burden of drug resistance in a patient, as resistant variants escape detection when present at low frequency within the viral quasispecies. Using sensitive resistance testing methods, a few investigators have linked low-frequency mutations to poor treatment outcomes, while other studies have shown no correlation. Understanding the technical limitations of sensitive testing methods and the relevance of the amount of a particular resistance mutation in the context of different ART regimens will help to define the clinical benefit of low-frequency resistance testing. Paramount to interpreting the clinical utility of sensitive testing is evaluating resistance mutations selectively, at biologically significant frequencies, and using methods that have been broadly validated on clinical specimens.

We examined CD4⁺ cell count and plasma viral load patterns among Botswana TDF/FTC Oral HIV Prophylaxis Trial (TDF2 study) participants who seroconverted, comparing participants assigned to receive tenofovir/emtricitabine with participants assigned to receive placebo. We also evaluated for antiretroviral drug resistance among the breakthrough HIV infections. Among nine seroconverters assigned to tenofovir/emtricitabine and 24 to placebo, there were no significant differences in their CD4⁺ cell count or viral load profiles over time. Of the four participants who seroconverted on-study while receiving tenofovir/emtricitabine, none became infected as a result of drug-resistant HIV; moreover, no resistance mutations emerged following seroconversion.

To substantiate reports of greater emergence of the K65R nucleoside reverse transcriptase inhibitor (NRTI) mutation in human immunodeficiency virus type 1 (HIV-1) subtype C, we examined natural low-level K65R expression in subtype C relative to subtypes B and AE. We used allele-specific polymerase chain reaction to screen HIV-1 amplified by reverse-transcription high-fidelity polymerase chain reaction from subtype C–infected South African women and infants and CRF01(subtype AE) from Thailand; all subjects were NRTI naive. We found low-level K65R of unknown clinical significance in NRTI-naive subtype C–infected women and infants at frequencies above the natural occurrence in subtypes B and AE. The frequent appearance of subtype C frameshift deletions at codon 65 supports a propensity for transcription error in this region.