Background:  Multi-drug-resistant HIV-1 comprise a minority of the transmitted drug-resistant viruses among individuals with newly diagnosed infections. However, declining resistance mutation levels in the absence of drug pressure and the sensitivity limitations of conventional population sequencing may result in underestimations of multi-drug-resistant virus prevalence. To examine this possibility, we used sensitive detection assays for HIV-1 drug-resistance mutations to reassess the frequency of transmitted multi-drug-resistant viruses.

Methods:  We developed and validated real-time polymerase chain reaction (PCR) point-mutation assays for the detection of the protease L90M, and the reverse transcriptase K70R, K103N, and M184V drug-resistance mutations. We used these assays to evaluate plasma-derived viral RNA collected between 1997 and 2004 from 209 drug-naïve persons in the United States and Canada documented to be infected with drug-resistant viruses.

Results:  The real-time PCR assays detected as little as 0.05% mutant sequences in wild type backgrounds; however, assay cutoffs of 0.2 to 0.6% mutant virus were used for screening purposes. The sensitivities and specificities of the assays on genotyped clinical samples carrying the mutations of interest were found to range between 95 and 99%. Real-time PCR screening of the 209 transmitted HIV-1-carrying resistance-related mutations detected additional mutations that expanded the number of drugs to which the viruses were resistant. The added mutants increased the prevalence of L90M from 8% to 10% (+25%), of K103N from 9% to 10% (+11%), of M184V from 10% to 11% (+10%), and of K70R from 7% to 13% (+80%). Clonal sequencing of representative samples containing newly detected mutants verified the mutations and confirmed their frequencies at ≤13% of the virus sample population.

Conclusions:  Our analysis of transmitted viruses carrying resistance-related mutations found that all 4 resistance mutations examined were underestimated by conventional sequencing. Interestingly, the increase in K70R was disproportionately greater, suggesting that this mutation is present in a higher percentage of drug-naïve persons than previously reported. The newly detected mutations indicated that at least 20% more multi-drug-resistant viruses were present than previously estimated in the population tested and may predict poorer than expected antiretroviral treatment responses in persons appearing to lack particular resistance mutations.