Background: The Applied Biosystems HIV Genotyping System facilitates HIV resistance genotyping but is limited to nucleotides between codon 1 of protease and codon 324 of RT. Resistance mutations may exist both 5 (protease cleavage sites in gag) and 3 (RT codon 333) to this region. Therefore, it would be useful to extend the range of this system beyond the standard limits.

Design: The HIV Genotyping System v2.1 was used to sequence HIV isolates from 109 individuals in the WITS. Chromatograms from sequencing reactions using the most 5 and 3 primers (primers F and C, respectively) were examined to reveal the range of useful sequence information and the presence of resistance mutations in regions beyond the standard limits.

Results: Primer F chromatograms could be read over 200 nucleotides 5 into Gag, limited by band spreading far from the primer site. This includes all of p6 and p1 and part of p7, including the frame-shift site and second zinc-finger domain. No gag cleavage site mutations were observed. However, 10 isolates (9%) contained a 9, 18, or 36 nucleotide repeat, in each case ending at codon 10 of P6, where mutations may interfere with virion maturation. Primer C chromatograms read only 50 nucleotides 3' past RT codon 324, limited by a strong stop on the sequencing template.  7 (6%) of isolates had mutation at RT codon 333. Codon 333 mutation was associated with mutation at codon 184 (p<0.001), or 211 (p=0.0013), and there was a trend toward association with mutation at codon 215 (p=0.11). There was a variable site at codon 334, with 24 (22%) isolates mutant. Mutation at this site was not associated with mutation at codons 215 or 184, but was associated with mutation at codon 211 (p<0.001). (All tests Fisher exact.)

Conclusions: The Applied Biosystems HIV Genotyping System can be used to examine HIV sequences in Gag and 3' regions of RT beyond the standard range. Analysis of these regions in isolates from the WITS reveals an insertion site in p6, a variable site at RT codon 334, and codon 333 mutations associated with presence of mutation at codons 184 or 211.