We identified 145 samples from individuals in the Rakai cohort collected at the time of HIV seroconversion (see Methods). Previous analysis of HIV subtype in the gag
, and gp41 regions using a multiregion hybridization assay [6
] identified these samples as subtype A (n
= 37), subtype D (n
= 61) or intersubtype recombinant (n
= 47). HIV resistance testing (GeneSeq HIV and PhenoSense HIV assays) was successful for 104 (71.7%) of the 145 samples. Failure to obtain results for the remaining 41 samples most likely reflected the low volume of plasma available for testing (0.2 ml versus 3 ml typically requested). In these low-volume samples, low viral load also contributed to assay failure; the 41 samples that failed testing had significantly lower viral loads than the 104 samples with test results (P
= 0.003, ). There were no significant differences in the age, gender, year of seroconversion/sample collection, or CD4 cell count of individuals with vs. without resistance test results (). Individuals who had samples collected closer to their last study visit with a negative HIV test result were more likely to have a resistance test result; however, this association was not statistically significant (P
= 0.063, ).
Characteristics of study participants.
For each of the 104 samples with resistance test results, we determined the pol
region subtype. The subtypes were: A (n
= 26, includes subtypes A and A1), C (1), D (n
= 66), A/D (n
= 9), C/D (n
= 1), and complex intersubtype recombinant (n
= 1). Genotypic testing (GeneSeq HIV assay) was performed to identify mutations associated with reduced susceptibility to nucleoside reverse transcriptase inhibitors (NRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors. Seven of the 104 samples (6.7%) had a mutation associated with NRTI resistance (one mutation in each sample: M41L (n
= 2), E44D (n
= 3), V118V/I (n
= 1), K219K/R (n
= 1)), and one sample had a mutation associated with NNRTI resistance (E138A). However, none of these mutations was sufficient to predict reduced NRTI or NNRTI susceptibility in the GeneSeq HIV assay. The mutations M41L (detected in two subtype D samples) and K219K/R (detected in one subtype A1 sample) were the only mutations identified that are among those used for genotypic surveillance of transmitted NRTI drug resistance [11
]. We did not detect any of the mutations used for genotypic surveillance of transmitted NNRTI drug resistance [11
]. All of the 104 samples had at least one mutation detected in HIV protease associated with reduced protease inhibitor susceptibility (e.g. K20R, M36I). However, none of the 104 samples had a mutation pattern predictive of reduced protease inhibitor susceptibility, and only three samples had a mutation used for genotypic surveillance of transmitted protease inhibitor drug resistance [11
]: one subtype A sample had I47V, one subtype D sample had F53L, and one subtype A1 sample had N88D. It should be noted that the presence of single mutations from the list used for genotypic surveillance of transmitted drug resistance is not expected to always be reflected by phenotypic changes [11
The PhenoSense HIV assay measures the susceptibility of HIV in the test sample to a panel of antiretroviral drugs. Ten (9.6%) of the 104 samples had evidence of reduced susceptibility to one or more antiretroviral drugs in the PhenoSense HIV assay (two subtype A and eight subtype D samples, ), including one individual with partial susceptibility to didanosine (ddI), one individual with resistance to nevirapine (NVP), and eight individuals with resistance and/or partial susceptibility to one or more of the protease inhibitors. Some of the samples with discordant resistance results (GeneSeq HIV = susceptible, susceptible, PhenoSense HIV = resistant or partially susceptible) had genotypic polymorphisms identified; complex interactions of these polymorphisms may have resulted in reduced drug susceptibility to NVP [12
] and some of the protease inhibitors. We found no genotypic explanation for reduced phenotypic susceptibility to ddI, saquinavir/ritonavir, or tipranavir/ritonavir (see ).
PhenoSense HIV and GeneSeq HIV results for samples with reduced antiretroviral drug susceptibility.
In the PhenoSense HIV assay, hypersusceptibility is defined as a fold change IC50
less than 0.4 (indicating that replication of the test virus is inhibited by 50% at a drug level 2.5-fold or less compared with the reference strain). Hypersusceptibility to one or more antiretroviral drugs was detected in 53 (51.0%) of the 104 samples, including 19 (73.1%) of 26 samples with subtype A, 28 (42.4%) of 66 samples with subtype D, four (44.4%) of nine samples with A–D recombinant strains, and two (66.6%) of the three samples with other strains (one each: C, C–D, and complex; P
value for A vs. D = 0.012 [19/26 vs. 28/66]; P
value for A vs. non-A = 0.013 [19/26 vs. 33/78], z-test). Seven (6.7%) of the 104 individuals had hypersusceptibility to the NRTI, zidovudine; 28 (26.9%) of the 104 individuals had hypersusceptibility to one or more of the NNRTIs; 34 (32.7%) of the 104 individuals had hypersusceptibility to one or more of the protease inhibitors. The proportion of individuals who had hypersusceptibility to the NNRTI, delavirdine (DLV), was significantly higher for subtype A than D (P
<0.001, ). In a previous study, subtype A samples were found to have a lower replication capacity than subtype D samples [13
]. However, in our data set, there was no significant difference between the median replication capacity of samples with vs. without DLV hypersusceptibility for subtype A (median: 28.23 vs. 27.49, P
= 0.526.) or subtype D (median: 37.99 vs. 71.96 P
Hypersusceptibility of subtype A vs. subtype D strains.
We next analyzed the Rakai sequence set to see whether we could identify mutations associated with hypersusceptibility to any of the antiretroviral drugs. In subtype D, we found that EFV hypersusceptibility was associated with amino acid substitutions at codon 11 in HIV reverse transcriptase. Among the 66 subtype D samples, five of 12 samples with EFV hypersusceptibility had substitutions at codon 11 (3Q, 1T, and 1H); in contrast, only two of 54 samples without EFV hypersusceptibility had a substitution at codon 11 (1Q, 1N; P value = 0.001, Fisher Exact test). As this association was observed within a single subtype (D), it should not be influenced by subtype-based difference in compatibility with the resistance test vector. The association of EFV hypersusceptibility with codon 11 substitutions was also observed in a separate data set of 182 subtype D samples previously submitted to Monogram Biosciences for testing (unpublished data).