ViroSeq HIV genotyping system version 2 is a fully integrated system for analysis of HIV-1 drug resistance mutations. Reagents, protocols, and software are provided for all steps of analysis, and the assay is performed on open-system equipment that can be used for other applications. In addition to providing an interpretive drug resistance report, the software provides an uninterrupted sequence encoding protease and the first 335 amino acids of RT in the universal FASTA format, simplifying phylogenetic analysis for quality control and other studies. ViroSeq system version 2.5 was cleared by the FDA for diagnostic use with the ABI PRISM 377 DNA sequencer in 2002. The data that supported the FDA clearance with the 377 instrument included 100 clinical samples. Fifty samples had viral loads from 1,800 to 10,500 copies/ml. Those 50 low-viral-load samples were characterized by cloning and sequencing individual viral variants prior to analysis with the ViroSeq system. Twenty of the low-viral-load samples were selected for the analysis in this study. Data reported in this study supported the FDA clearance of the ViroSeq system for analysis using the 3100 instrument in 2003. The 3100 instrument is a capillary-based instrument that loads samples automatically from 96-well plates. Lane tracking is not required. The 3100 instrument, which uses a 16-capillary array, can be loaded with two 96-well plates and can process 16 reactions in 2.5 h.
In this study, the all-base accuracy of the 3100 instrument without manual intervention (editing) was 99.9% with the Applied Biosystems BDTSS. For bases 27 to 576, 424 of the 425 sequences met the Applied Biosystems specification of >98.5% accuracy. This high accuracy of base calling, along with the double coverage of ViroSeq sequences and the ViroSeq software's ability to perform manual edits, resulted in highly accurate sample consensus sequences.
In this study, the overall sensitivity and specificity of ViroSeq for detection of drug resistance mutations were 99.65 and 99.95%, respectively, for clinical samples, and were 99.7 and 100%, respectively, for recombinant samples containing mutations as 40% mixtures with viral loads of approximately 5,000 copies/ml. Similar results were obtained at three independent testing sites. These performance characteristics are equivalent to those obtained previously using the 377 instrument (ViroSeq HIV-1 Genotyping System Operator's Manual, 2003, CDx, part number 4341639). The sensitivity for mutation detection was determined for 73 of the 75 mutations included in the ViroSeq resistance interpretation algorithm. The sensitivity was 100% for all but two of the mutations (M41L and A62V in RT), and the specificity was 100% for all but one mutation (L63P in protease). For these mutations, the triplicate sequences were consistent, suggesting that detection of the mutation was dependent on a presequencing event. Since these samples were low-viral-load samples (2,000 to 10,500 copies/ml), it is possible that (i) cDNA from the RT reaction was biased due to a less-than-40% mixture or (ii) the sequence context of the mutation affected dideoxynucleoside triphosphate incorporation, such that some mutations were not proportional to the population mixture. This study suggests that the following criteria are important for the generation of high-quality HIV-1 genotypes: (i) the RT-PCR yield should be at least 20 ng per 5 μl; (ii) total sequencing base signals should be ≥400; (iii) peak spacing should be between 11 and 16; and (iv) the ViroSeq positive control (8E5) should show no mixtures.
When genotyping assays are performed, it is important to minimize contamination of reaction mixtures with previously amplified products. Sample cross-contamination is minimized in the ViroSeq system by using a single nonnested PCR for amplification, and by using a dUTP/UNG contamination control system. There was no evidence of sample cross-contamination in this study or in previous reports (2
This study supports use of the ViroSeq system and ABI PRISM 3100 genetic analyzer for identification of HIV-1 drug resistance mutations. Testing is performed using only 0.5 ml of plasma. Research methods have also been developed to use PCR products generated with ViroSeq for analysis of individual HIV-1 variants and genetic linkage of drug resistance mutations by cloning (5
), for analysis of the HIV-1 gag
), and for detection of minority HIV-1 variants with drug resistance mutations (8
). The system is currently available for diagnostic use in both the United States and Europe. The system is cleared by the FDA for analysis of subtype B HIV-1 only. However, excellent performance of the system has also been documented in research studies for analysis of diverse HIV-1 strains (4
). An updated version of the ViroSeq HIV-1 genotyping system (version 2.6) was recently cleared by the FDA. Version 2.6 includes an updated algorithm for interpretation of drug resistance to all currently licensed protease and RT inhibitors. The results described in this report should be relevant to version 2.6 as well as version 2.5, since these versions are identical in all aspects except for the interpretive report.