Genotypic resistance to nucleoside and non-nucleoside reverse transcriptase inhibitors in HIV-1 infected patients by duplex selective polymerase chain reaction

Abstract

Drug resistance is a major contributing factor to the failure of antiretroviral therapy. The development of drug resistance further complicates clinical management because of the high level of cross-resistance within the drug classes. In Thailand, most individuals are infected with HIV-1 subtype A/E. Much have been reported for genotypic resistance of HIV-1 in North America and Europe where HIV-1 subtype B predominates. However, little is known about the HIV-1 genotypic resistance in other part of the world, particularly in regards to other subtypes. Resistance testing has recently been recommended as a helpful monitoring assay for management of HIV-1 infection. Sequencing analysis is a standard genotypic resistance assay. However, it is expensive and is not widely accessible. Simpler and cheaper assays are needed. The amplification refractory mutation system (ARMS) have been applied for development of the K103N/Y181C and Q151M/T215Y/F duplex selective PCR. These assays were used for comparison of genotypic resistance between antiretroviral-naive (group I; n=20) and antiretroviral-experienced (> 6 months) (group II; n=25) HIV-1 infected Thais. To develop a successful duplex selective PCR, several factors need to be considered. The most important factor is the good primer design including the strength and position of nucleotide mismatches in the primer. Moreover, subtype specific sequences and mutations should also be considered in primer design. By using duplex selective PCR assay for genotypic resistance evaluation in HIV-1 infected Thais, more than 94% showed concordant results with the sequencing analysis. There was a significant difference of genotypic resistance between ART-naive patients (group I) and ART-experienced (> 6 months) HIV-1 infected patients (group II) (p< 0.01, Chi-square test). No mutations were observed in the ART-na.ve, while 23 of the 25 (92%) ART-experienced patients harbored drug-resistant mutant viruses. T215Y/F mutation is the most common genotypic resistance found in this study (80%). The second most common mutation found was NNRTI associated mutations (K103N, Y181C) (overall=24%, however in NNRTI failure was 67% (6/9)). The MDR Q151M was found only 4%. These findings suggest that HIV resistance occurs under selective pressure of antiretroviral drugs and also emphasize that resistance testing is helpful to guide switching regimens in patients who fail therapy. With the high sensitivity (96%) and specificity (98%), as well as lower cost, these duplex selective PCR assays are thus valid for further cost-effective HIV drug resistance surveillance study in resource limited countries. For clinical uses in detection of multi-nucleoside and NNRTIs resistances however needs further investigation in a larger scale study.