Detailed comparisons of different HIV drug resistance assays are needed to identify the most useful assay(s) for different clinical settings and to facilitate comparison of results from studies that use different methods. In our cohort of NVP-exposed infants analyzed at 6–8 weeks of age, population sequencing-based assays (ViroSeq and GeneSeq HIV) identified the majority of infants who had NVP-resistant HIV, and the results obtained with these two assays were similar for 95% of the samples tested (NVP resistance: yes/no). GeneSeq HIV did detect more NVP resistance mutations than ViroSeq. However, this changed the resistance interpretation for only two samples. Assay discordance between ViroSeq and GeneSeq could reflect test-to-test variability. In two previous studies, high variability was observed among laboratories using population sequencing-based methods for HIV genotyping.28,29
In most perinatal studies, the volume of infant plasma available for analysis is limited, and assay procedures must often be adapted for low-volume samples. The viral load for one discordant sample was low (3977
copies/ml, sample 214), and sampling bias could have caused assay discordance between ViroSeq and GeneSeq HIV for detection of K101E (). All of the other 29 samples that had viral load data available had viral loads >20,000
c/ml. However, even in samples with high viral loads, discordant results may be obtained because of assay variability or stochastic sampling of resistant viruses in samples with minority resistant populations.
In three samples (343, 362, and 441) K103N was detected at very low levels by LigAmp (2.6–5.4% of the viral population), and was also detected by ViroSeq and GeneSeq HIV. In all three cases, the ViroSeq and GeneSeq electropherograms were consistent with very low levels of the mutant strains. In validation studies, ViroSeq reliably detected mixtures present at 40% or more of the viral population, when the viral load was between 2000 and 5000
and GeneSeq HIV has been reported to reliably detect mixtures present at 10–20%.23
However, these assays clearly detect some mutations at lower levels in some samples. In a previous study, we analyzed samples with both ViroSeq and LigAmp for K103N detection. ViroSeq detected K103N in 71.4% of samples with a LigAmp result of 5–10% and in 16.9% of samples with a LigAmp result of 1–5%.18
In this report, LigAmp detected low-level NVP resistance mutations that were not detected by ViroSeq (12 mutations in 12 infants; only one of those mutations was detected by GeneSeq HIV). However, in 11 of those 12 infants, at least one other NVP mutation was detected by ViroSeq, so the LigAmp results did not change overall the resistance interpretation (yes/no for presence of NVP resistance). In other samples, LigAmp did not detect resistance mutations that were detected by ViroSeq (e.g., due to alternate codon use). Minority variants assays, such as LigAmp, are most useful in settings in which drug resistance mutations are likely to be present at low levels. For example, when we analyzed infants in the same cohort who were HIV infected after 6 weeks of age by breast-feeding (late-infected infants),3
LigAmp identified NVP resistance in six of eight infants tested; ViroSeq did not detect resistance in any of those infants, reflecting the low levels of the NVP-resistant HIV variants in those samples.3
LigAmp was also useful for analysis of the persistence of NVP resistance mutations in women and infants after sdNVP, since NVP-resistant variants often fade to levels that cannot be detected by ViroSeq within months of sdNVP exposure.16,30
Unlike population sequencing-based genotyping assays, LigAmp also provides information on the level of mutations in the viral population. For example, we were able to use LigAmp to show that the level of NVP-resistant variants in women after sdNVP exposure is influenced by HIV subtype31
and that repeated use of sdNVP is not associated with selection of higher levels of resistant variants.3
Unlike genotypic assays, the PhenoSense HIV assay provides a direct measure of drug susceptibility. We identified 12 samples with discordant results from GeneSeq HIV and PhenoSense HIV testing. In all but one case, GeneSeq HIV predicted resistance that was not detected by PhenoSense HIV. In 9 of 12 cases, the relevant mutations were present as mixtures. Previous studies have found genotypic assays to be more sensitive than phenotypic assays when resistant variants are present as mixtures.23
Variations in phenotypic susceptibility are also seen with some drug resistance mutations,23
which could explain detection of mutations in some samples that retain susceptibility in the PhenoSense HIV assay. Finally, some cases of genotypic–phenotypic discordance in non-subtype B samples may reflect limits in our understanding of resistance in diverse HIV strains.
This report provides a detailed comparison of four different resistance assays in a well-defined clinical cohort. In our cohort of 6-week-old infants exposed to NVP-based regimens for pMTCT, population sequencing-based methods (ViroSeq and GeneSeq HIV) were the most informative and had concordant results for 95% of the samples. LigAmp was useful for the detection and quantification of minority variants, but identified only one additional infant with NNRTI resistance. PhenoSense HIV provided a direct and quantitative measure of NNRTI susceptibility, but did not identify any additional infants with NNRTI resistance. In other settings, use of minority variants and phenotyping assays should be considered for analysis of resistance, depending on the study cohort, study design, and objectives.