Background:  Previous studies have reported that drug-exposed persons who had wild type HIV-1 at baseline by bulk sequencing responded poorer to treatment regimens containing a similar drug when compared to drug-naïve persons. The development of sensitive point-mutation assays has allowed the identification of minority HIV-1 variants persisting below the 20% frequency threshold of sequence detection. These methods improve the sensitivity of resistance testing by 10- to 100-fold and can differentiate mutations at frequencies above those that arise naturally. Sensitive real-time polymerase chain reaction (PCR) -based assays with detection cutoffs between 0.3 and 2.0% were used to reassess resistance mutations in drug-naïve persons. These assays revealed that resistance mutations are indeed hidden in wild type virus populations and that these variants may double current estimates of transmitted resistance. Furthermore, in transmitted mutant virus populations, sensitive testing identified ~30% more ≥2-drug class resistance. Sensitive detection assays are currently being applied to assess whether low-frequency mutants impact the efficacy of ART. To evaluate whether minority resistance mutations in drug-naïve persons correlated with poor virologic suppression, PCR-based assays were used to retrospectively examine baseline viral RNA samples from persons who participated in triple-drug treatment studies. Additionally, nevirapine resistance mutations and HIV-1 transmission were evaluated following single-dose nevirapine (SD-NVP) exposure during delivery of the second child in women who were previously exposed or unexposed to SD-NVP. 

Conclusions:  The findings from these studies imply that low-frequency treatment-relevant mutations persisting above naturally occurring levels in drug-naïve patients are associated with virologic failure and provide evidence that minority mutations can have clinical consequences. Additional sensitive testing is necessary to further evaluate the impact of minority proviral variants and of linked low-frequency mutations on antiretroviral responses.