Background:  Sensitive point-mutation assays can improve identification of resistance mutations by uncovering minority mutants persisting below bulk sequence detection. We applied validated, sensitive tests to re-evaluate transmitted drug resistance and to assess whether minority mutants at baseline correlated with poor virologic suppression under ART.

Methods:  We tested for minority resistance mutations in viral RNA from 2o groups of newly diagnosed individuals, those with wild type virus (n = 211) and those with drug-resistance mutations (n = 303) by conventional sequencing. We used real-time polymerase chain reaction (PCR) -based assays for L90M in protease, and M41L, K70R, K103N, Y181C, and M184V in reverse transcriptase. We also performed retrospective sensitive testing on baseline samples from drug-naïve persons who participated in treatment studies with efavirenz/lamivudine (EFV/3TC) + abacavir (ABC) or zidovudine (ZDV). Blinded plasma-virus samples from 101 virologic failures (>50 RNA copies/mL by 48 weeks) and 158 treatment successes were tested for 3 treatment-relevant resistance mutations, K103N, Y181C, and M184V.

Results:  Sensitive testing identified drug-resistance mutations in 29 of 211 (14%) wild type samples, including 10 M41L, 10 K70R, 8 K103N, 3 Y181C, 3 M184V, and 3 L90M. Of the wild type samples, 4 had minority 2-class resistance mutations. The assays also identified an additional 2 to 9% of each mutation in samples known to have other resistance mutations. This resulted in a cumulative 30% increase in the 6 mutations tested and a 40% increase in ≥2 drug-class resistance. Of the 259 trial participants, the PCR assays detected 16 individuals with resistance mutations at baseline as compared to 7 persons by bulk sequencing. Of 7 persons, 6 (86%) had sequence-detectable mutations and of 9 persons, 7 (78%) with low-frequency mutants at baseline experienced virologic, failure. The association between resistance mutations and virologic failure increased substantially (from p = 0.015 to p = 0.00076) when low-frequency mutants were included. In a logistic model, people with minority resistance mutations had 8.1-times the odds of experiencing treatment failure than those without mutations, independent of virus load and CD4 counts.

Conclusions:  Minority mutations nearly double the prevalence of drug resistance in transmitted HIV-1 and associate with virologic failure. The data suggest that sensitive testing can benefit both detection of drug-resistant HIV and ART management of infected persons.