Evaluation of Two Commercial Kits for the Detection of Genotypic Drug Resistance on a Panel of HIV Type 1 Subtypes A Through J

Abstract

We compared the two commercially available sequencing kits for HIV-1 drug resistance testing, the ViroSeq Genotyping System (Applied Biosystems, Foster City, CA, U.S.A.) and the TRUGENE HIV-1 Genotyping Kit (Visible Genetics, Inc., Toronto, Ontario, Canada), with our in-house genotyping system. Fifteen viral isolates from African patients (6 treated and 9 untreated) covering a panel of HIV-1 subtypes A through J and 7 plasma samples from Belgian and African patients (2 treated and 5 untreated) were tested. All the samples could be amplified and sequenced by the three systems; however, for all systems, alternative amplification/sequencing primers had to be used for some samples belonging to subtype B as well as to other subtypes. The consensus sequence was partially derived from only one strand for the in-house system and for the ViroSeq Genotyping System. The TRUGENE HIV-1 Genotyping Kit scored the highest number of ambiguities, followed by the ViroSeq Genotyping System and the in-house system. For 11 samples, these differences in reporting mixtures affected 14 resistance-related positions, which altered the interpretation toward protease inhibitors for 2 samples when using version 1.2 RetroGram software (Virology Networks, Utrecht, The Netherlands). All three systems were able to sequence diluted samples with a viral load down to 10 3 or 10 4 RNA copies/ml. Our data therefore suggest that the performance of amplification and sequencing primers must be improved to allow fast and reliable resistance testing for all HIV-1 subtypes.

Blinded, Multicenter Comparison of Methods To Detect a Drug-Resistant Mutant of Human Immunodeficiency Virus Type 1 at Low Frequency

“…One (Table 1). Other comparative studies of genotypic assays for HIV-1 mutant detection have been performed (4,5,11,15), but none has covered the breadth of technologies evaluated here. Additional panels of virus mixtures and clinical samples need to be tested to assess the potential impacts of different codons, viral heterogeneity, and operator experience on assay performance, as has been noted previously (6,18).…”

Performance of the Celera Diagnostics ViroSeq HIV-1 Genotyping System for Sequence-Based Analysis of Diverse Human Immunodeficiency Virus Type 1 Strains

“…In a previous report, a high level of performance of the ViroSeq system for analysis of 130 plasma samples with HIV-1 non-subtype B strains from Uganda (mostly subtypes A and D) was demonstrated (16). Two recent reports further evaluated the performance of the ViroSeq system for analysis of non-subtype B HIV-1 (10,12). However, those studies used predominantly cultured isolates rather than plasma and included relatively small sample sets.…”

Sensitive Phenotypic Detection of Minor Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase Variants

“…The genotypic assays used to monitor HIV drug resistance include bulk sequencing (6,13,23,29,34), oligonucleotide ligation assay (11,14), line probe assay (37), microarray hybridization (38), heteroduplex formation (1), allele-specific PCR (S. Palmer, 11th Conference on Retroviruses and Opportunistic Infections), and single-genome sequencing (30). Other than allele-specific PCR, these assays are limited in their sensitivities for the detection of drug-resistant variants present at low frequencies.…”