Background:  Drug resistance mutations selected by combination nevirapine (NVP) + lamivudine (3TC) + zidovudine (AZT) may identify low adherence and potential second-line treatment strategies. Patterns of drug resistance were identified in treated subtype C-infected AIDS patients from Chitungwiza, Zimbabwe, using a sensitive allele-specific polymerase chain reaction (AS-PCR) for the K103N mutation compared with consensus pol sequencing.

Methods:  Plasma samples with viral load >400 copies/ mL were extracted and reverse transcribed and amplified by PCR. The first-round amplicon was tested with a quantitative AS-PCR for K103N. A positive K103N AS-PCR was defined as >0.2% prevalence of asparagine (AAC or AAT). Di-deoxy consensus sequencing was performed by homebrew nested sequencing with primers optimized for subtype C pol. 

Results:  Of 87 subjects on treatment, 25 (29%) had a viral load >400 copies/mL at 16, 24, or 48 weeks. We obtained 29 sequences from 20 of 25 subjects. Of these 20, 4 (20%) achieved viral suppression by 48 weeks, while 16 were viremic (median viral load = 4.075, range 3.01 to 5.2 log₁₀ copies/ mL).  Of the 20 subjects with sequences, 11 had an nucleoside reverse transcriptase inhibitor (NRTI) -associated mutation:  10 an M184V, 1 D67DN, and all 11 had concurrent non-NRTI (NNRTI) -resistance mutations. In total, 15 subjects had NNRTI-resistance mutations:  8 had the K103N mutation alone, 7 had other NNRTI-resistance mutations—including A98G, K101E, K103S, V108I, Y181C, Y188C, G190A, and K238N—in various combinations, and 1 had only a Y181C mutation. AS-PCR was performed on amplicon from the 29 sequenced samples; 12 subjects had K103N detected by AS-PCR. In 29 paired sequence/AS-PCR samples, 9 pairs were both AS-PCR and sequence positive for K103N, 5 samples were positive by AS-PCR but negative by sequence, and 1 sample positive by sequence but not by AS-PCR. Of 29 pairs, 8 had an NNRTI-resistance mutation by sequence, Y181C or G190A/S, but were designated wild type by a K103N AS-PCR.  Only 1 of the 29 paired samples with wild type sequence was identified to have K103N by AS-PCR.  

Conclusions:  In subtype C HIV infection, NVP+3TC+AZT predominantly selects NVP (K103N) and lamivudine (M184V) mutations. Thymidine analog mutations are rare within the first year of treatment. NVP resistance includes K103N in 53% and G190A/S or Y181C in the other 47%. In 75% of subtype C samples with virologic failure and consensus mutation(s) associated with NNRTI resistance, AS-PCR for the K103N mutation identifies >0.2% AAC or T, although AS-PCR for K103N may be negative in early failure with G190A/S or Y181C by population sequence.