Allele-specific polymerase chain reaction (ASPCR) to detect resistance mutations in minor variants of HIV-1 subtype C in patients failing highly active antiretroviral therapy (HAART).
The World Health Organization (WHO) has recommended Tenofovir disoproxil fumarate (TDF) as one of the preferred first-line antiretrovirals (ARVs). TDF and Abacavir (ABC) were introduced into the South African National Antiretroviral Treatment Guidelines in 2010. However, exposure to TDF and ABC can result in the development of the K65R and L74V resistance mutations, respectively. The K65R mutation occurs preferably in subtype C viruses, due to the unique polymorphisms found at codons 64 and 65 (which are not present in subtype B). This is a cause for concern in South Africa, where subtype C is the most common HIV-1 subtype. In addition, these mutations may be present in the minor viral population (i.e <20% of the viral population) and it has been shown that the presence of a resistance mutation in a frequency as low as <0.5% may be associated with an increase in the risk of virological failure. This study investigated the prevalence of K65R and L74V in the minor viral population, using Allele-specific PCR (ASPCR), in a cohort of subtype C infected patients that failed their first-line treatment regimen that did not include TDF or ABC. RNA was extracted from stored plasma samples from a subset of the South African Resistance Cohort Study (SARCS) and the pol region was reverse transcribed and amplified using a one-step RT-PCR kit (Invitrogen; California, USA). For both the K65R and L74V mutations, ASPCR was performed using specific and non-specific primers. A specific and non-specific standard curve was optimised for each mutation (using a mutant plasmid control) and these standard curves were used to perform an absolute quantification. Subsequently, the percentage of each mutation (in each sample) was calculated by dividing the quantity of mutant sequences in the sample by the quantity of total viral sequences in the sample and multiplying this ratio by 100. The Limit of Detection (LOD) of the K65R ASPCR was 0.72%. Of the 84 patients that were assayed, the K65R mutation was detected in 7 (8.33%) of the patients. Five of the 7 samples were detected above 1% (i.e 3 were approximately 2%, 1 was 9.48% and 1 was 100%) and 2 were detected below 1% (i.e 1 was 0.88% and the other was 0.93%). The limit of detection for the L74V ASPCR was 0.013%.We found the L74V mutation to be prevalent in 9 (10.7%) of 84 patients. In 4 of the 9 patients, the L74V mutation was found in ≥1% of the viral population (viz. 2.82%, 10.10%, 12.02% and 18.22%) and in the other 5 patients, the L74V mutation was detected in <1% of the viral population (2 were between 0.5% and1%, while 3 were detected between 0.013% and0.5%). In this study, ASPCR detected additional K65R and L74V mutations in the minor viral population of TDF and ABC-inexperienced patients that were missed by standard genotyping. These minorityK65R mutations could contribute to treatment failure in these patients when switched to TDF or ABC-containing ARV regimens. ASPCR is a useful tool for screening for minority mutations before starting or switching regimens.