Background: We and others have shown that subtype C HIV-1 isolates from patients failing on a regimen containing stavudine (d4T) or zidovudine (AZT) exhibit thymidine-associated mutations (TAMs) and K65R which can impair the efficacy of Tenofovir (TDF) at second line. Depending on the various studies, the prevalence of K65R substitution as determined by the Sanger method ranges from 4 to 30%. Our aim was to determine whether ultra-deep pyrosequencing (UDPS) could provide more information than the Sanger method about selection of K65R in this population of patients.
Methods: 27 subtype C HIV-1 isolates from treated patients failing on a regimen with d4T or AZT plus lamivudine (3TC) plus nevirapine (NVP) or efavirenz (EFV) and who had been sequenced by Sanger were investigated by UDPS at codon 65 of the reverse transcriptase (RT). 18 isolates from naïve patients and dilutions of a control K65R plasmid were analysed by Sanger plus UDPS.
Results: Analysis of Sanger sequences of subtype C HIV-1 isolates from naïve patients exhibited expected polymorphic substitutions compared to subtype B but no drug resistance mutations (DRMs). Quantitation of K65R variants by UDPS ranged from <0.4% to 3.08%. Sanger sequences of viral isolates from patients at failure of d4T or AZT plus 3TC plus NVP or EFV showed numerous DRMs to nucleoside reverse transcriptase inhibitors (NRTIs) including M184V, thymidine-associated mutations (TAMs) plus DRMs to non- nucleoside reverse transcriptase inhibitors (NNRTIs). Two K65R were observed by Sanger in this series of 27 samples with UDPS percentages of 27 and 87%. Other samples without K65R by Sanger exhibited quantities of K65R variants ranging from <0.4% to 0.80%, which were below the values observed in isolates from naïve patients.
Conclusions: While Sanger sequencing of subtype C isolates from treated patients at failure of d4T or AZT plus 3TC plus NVP or EFV exhibited numerous mutations including TAMs and 8% K65R, UDPS quantitation of K65R variants in the same series did not provide any more information than Sanger.