Our previous studies using an earlier version of the ViroSeq system demonstrated excellent performance for the analysis of subtype B plasma samples (5
) as well as non-subtype B plasma samples from Uganda (primarily subtypes A and D) (16
). In the present report, we demonstrated that the FDA-cleared ViroSeq system (version 2.0) performs well for the analysis of genetically diverse non-subtype B and intersubtype recombinant strains. In the present study, genotypes were successfully obtained for 124 (98%) of 126 samples infected with genetically diverse HIV-1. The ViroSeq system has seven sequencing primers, which provide overlapping, bidirectional sequence data over the entire region analyzed. Bidirectional sequence data are useful for resolving sequence ambiguities and for confirming the presence of nucleotide mixtures. Nucleotide mixtures detected in genotyping assays reflect the presence of genetically diverse viral variants present in plasma samples from most HIV-1-infected individuals. The ViroSeq system is able to accurately detect specific drug resistance mutations representing 40% or more of the viral population in HIV-1 subtype B plasma samples (ViroSeq product insert). However, the detection of minority variants as mixtures may provide additional information about the presence of drug-resistant variants.
The most consistent difference in performance observed in the present study compared to previously reported results for subtype B strains (5
) was in the performance of the A and D alternate primers, which bind to the highly heterogeneous gag
region. For subtype B strains, sequencing was successful with the A and D primers for 92 and 88% of reactions, respectively, and at least one of the primers (either primer A or primer D) was successful with 98.4% of the samples. In contrast, for the genetically diverse strains in this study, the A and D primers were successful for 86 and 30% of the reactions, respectively. Both primers (A and D) failed for 9% of the genetically diverse samples, resulting in some regions of unidirectional sequence data.
The genetic heterogeneity of HIV-1 viruses presents a challenge for molecular assays that depend on oligonucleotide primer hybridization. Nucleotide mismatches between the primer(s) and template have the potential to reduce the efficiency of hybridization, leading to inefficient amplification or failed sequencing runs. The genetically diverse panel of samples used in the present study provided an opportunity to examine the level of nucleotide conservation within primer sites used for sequence analysis across multiple subtypes and CRFs. Consistent with the performance (>95% success rate) of the primary set of ViroSeq sequencing primers (primers A to C and F to H), a relatively high degree of nucleotide conservation was observed at the primer binding sites (Table ). One or no mismatches were present in primer A, B, C, F, and G sites in 90% or more of the samples. For primer A, B, C, F, and G sites, two or fewer mismatches were present in 97, 99, 100, 99, and 99% of the panel members, respectively. The primer H site was less conserved, with two or fewer mismatches for 77% of this genetically diverse sample panel. Interestingly, although the level of conservation was somewhat lower in primer H, it accounted for only 20% of the failed sequencing reactions in our study. Primers A and F accounted for, respectively, 46 and 26% of the failed sequencing reactions, although their overall nucleotide conservation was higher than that for the primer H binding site. The overall level of sequence conservation within the target binding site of a primer is just one of many factors that can influence the efficiency of primer hybridization. The position and nature of the mismatch, primer length, and stringency (temperature and buffer conditions) are additional factors that contribute to mismatch tolerance. For primer F, all eight sequencing failures (reproducible at both laboratories) could be attributed to a mismatch at the 3′ terminus of the primer. The samples with primer failures included a subset (6 to 11%) of subtype A, C, D, and F and CRF02_AG specimens. In all cases, primer A provided sequence coverage on the opposite strand. Two primer G sequencing failures, both due to 3′-terminal mismatches on subtype C strains, were observed. Thus, the position of the mismatch was critical for all sequencing failures with primers F and G. Sequencing failures with primers A, C, and H were generally associated with internal mismatches near the 3′ ends of the primers or due to a higher total numbers of mismatches (three to five mismatches for primer H).
Overall, the sequencing success rates for the primers were highly concordant between laboratories. However, in five of six cases (primer F, one subtype A specimen; primer A, one subtype A, one subtype D, and two CRF01_AE specimens) in which sequencing failures were observed at Johns Hopkins University (Table ), no mismatches were present in the primer binding sites. In the sixth case, only a single mismatch, near the 5′ end of primer A, was identified. Thus, no obvious molecular basis for failure was evident. For all six specimens, sequences were successfully generated in the second laboratory, suggesting that the failures may have resulted from technical issues.
Primer D is included in the ViroSeq kit as an accessory primer in the event of sequencing failure with primer A. Consistent with the relatively low success rate (30%) for non-subtype B strains, the primer D binding site is the least conserved of the seven ViroSeq sequencing primer binding sites. Nearly all cases of failure with primer D can be attributed to a relatively high number of total mismatches. Our data are consistent with a previous report of a low success rate for primer D with CRF01_AE strains (17
). Nevertheless, primer D was successful in five cases in which primer A failed (one subtype B specimen, one subtype D specimen, two CRF02_AG specimens, and one CRF01_AE specimen) and thus is of value, even with non-subtype B strains.
The performance of the ViroSeq system (version 2.0) with non-subtype B HIV-1 was evaluated in three previous reports. Those reports also examined the performance of another commercial genotyping system, the TRUGENE HIV-1 genotyping kit (Bayer Diagnostics, Tarrytown, N.Y.), which is also cleared by the FDA for clinical use. In the first report (10
), the performance of the ViroSeq system was evaluated with 15 cultured isolates of non-subtype B HIV-1 (3 subtype A, 2 subtype C, 3 subtype D, 2 subtype F, 1 subtype G, 1 subtype J, and 3 recombinant [1 A/D and 2 CRF02_A/G] isolates) and three non-subtype B plasma samples (one each of subtypes C, D, and H). All of the samples were successfully amplified and sequenced with the ViroSeq system (version 2.0). Full bidirectional sequence data were obtained for 11 (73%) of the 18 non-subtype B samples. Primer failures included both the A and D primers for three samples (subtypes F, J, and H), the F and G primers for one subtype A sample, the F primer for one subtype C sample, and the G primer for one subtype D and one CRF02_AG sample. Genotypes were also obtained for all of the non-B samples using the TRUGENE system and supplemental primers provided by the manufacturer. Samples analyzed with the TRUGENE system were noted to have more frequent sequence ambiguities.
In the second report (12
), 34 cultured isolates, 27 of which were non-subtype B isolates (2 subtype A, 2 subtype A/G, 6 subtype C, 2 subtype D, 8 CRF01_AE, 4 subtype F, 2 subtype G, and 1 subtype H), were analyzed. Genotyping was successful for all of the non-subtype B samples with the ViroSeq system. Sequencing with primer A was successful for 24 (89%) of the non-subtype B samples, while sequencing with the alternate primer D was successful for only 8 (30%) of the samples. Both primers A and D failed for three isolates (of subtypes C, E, and H) and one sample for which primer H failed (a subtype C sample). Analysis with the TRUGENE system required the use of the version 1.5 primers for three (11%) of the 27 non-B samples (1 subtype C and 2 subtype G samples). Sequencing primer failures, primarily in the protease region, were described for 22 (81%) of the 27 non-subtype B samples. Samples with subtypes B and D performed better with the PR set of protease sequencing primers, whereas the P2 protease primer set was preferable for the other subtypes. The P2 primer set was successful for all samples tested but provides a shorter sequence in the protease region. Both sets of protease primers are provided with the FDA-cleared version of the TRUGENE system.
In the third report (3
), genotyping was performed by using viral isolates from cell culture. Genotyping with the version 2.0 ViroSeq system was successful for 20 (80%) of 25 non-subtype B isolates. In contrast, genotyping with the TRUGENE system (version 1, without the use of supplemental primers) was successful for only 12 (50%) of 24 non-subtype B isolates. The five non-subtype B isolates that failed with the ViroSeq system included three subtype A isolates, one subtype C isolate, and one subtype J isolate. In all five cases, all seven sequencing primers failed. This type of result was not observed in the present study or in the other two studies cited. It seems unlikely that all five samples diverged genetically at the binding sites of all seven sequencing primers. Instead, these results suggest that a contaminant (e.g., from cell culture) might have interfered with the sequencing reactions. In contrast to the three previous studies described, the present study used only plasma samples for analysis.
The present report confirms the value of the ViroSeq system for the analysis of plasma samples from countries harboring diverse HIV-1 subtypes. In the present study, a high level of interlaboratory assay reproducibility was observed with this system. Further studies are needed to extend this analysis to include non-subtype B samples with low viral loads and non-subtype B samples from patients failing antiretroviral therapy. The FDA-cleared ViroSeq system offers an advantage for drug resistance monitoring in that a single set of PCR and sequencing primers can be used for the analysis of genetically diverse strains of HIV-1. The inclusion of the uracil N-glycosylase contamination control system reduces the likelihood of PCR carryover. The ViroSeq system provides a single, uninterrupted pol region sequence, and sequence data are provided in the standard FASTA format, simplifying phylogenetic analysis for subtype determination and other studies. Moreover, the ViroSeq system has been FDA-cleared for use on multiple platforms, including the high-throughput ABI Prism 3100 and 3700 DNA analyzers, making it an attractive tool for patient monitoring.