Background: The transmission of drug-resistant HIV in newly identified infected populations has become an underlying epidemic which can be better assessed with sensitive resistance testing. Since minority drug resistant variants cannot be detected by bulk sequencing, methods with improved sensitivity are required. Thus, the goal of this study was to evaluate if transmitted drug resistance mutations at minority levels in Japanese patients could be identified using highly sensitive allele-specific PCR (AS-PCR).
Materials and methods: Samples were taken from newly diagnosed HIV/AIDS cases at the National Nagoya Hospital from January 2008 to December 2009. All samples were bulk sequenced for HIV protease and reverse transcriptase. To detect minority populations with drug resistance, we used AS-PCR with mutation-specific primers designed for seven reverse transcriptase inhibitor resistance mutations, M41L, K65R, K70R, K103N, Y181C, M184V, and T215F/Y, and for three protease inhibitor resistance mutations, M46I/L and L90M.
Results: We studied 149 newly identified HIV cases. Bulk sequencing detected 8 cases with NRTI resistance mutations (one with A62V, one D67E, one T215D, one T215E, two with T215L and two T215S) and 15 with PI resistance mutations (one with N88D and 14 with M46I). Results obtained by AS-PCR and bulk sequencing demonstrated good concordance but the AS-PCR enabled the detection of seven additional drug-resistant cases (one M41L, two with K65R, two with K70R, and one M184V) in the RT region. Additionally, AS-PCR assays identified 15 additional cases with M46I, five with M46L and four cases with L90M in the protease region.
Conclusions: Using AS-PCR substantially increased the detection of transmitted drug resistance in this population from 15.4% to 26.8%, further supporting the benefit of sensitive testing among drug-naïve populations. Since the clinical impact of minority drug-resistant populations is not fully comprehended for all mutations, follow-up studies are needed to understand their significance for treatment.