Performance Characteristics of Human Immunodeficiency Virus Type 1 (HIV-1) Genotyping Systems in Sequence-Based Analysis of Subtypes Other than HIV-1 Subtype B

Abstract

Given the diversity of human immunodeficiency virus type 1 (HIV-1) subtypes and the emergence of subtypes other than HIV-1 subtype B in the United States, genotypic assays must be capable of delivering sequence data on diverse HIV-1 subtypes. We evaluated the performance of Visible Genetics TRUGENE HIV-1 genotyping kit and Applied Biosystems ViroSeq HIV-1 genotyping system on a panel of 34 well-characterized HIV-1 viral stocks (subtypes A through H). Both assays perform well on diverse HIV-1 subtypes despite being designed for HIV-1 subtype B. The TRUGENE assay produced sequence data for 31 isolates but not for one C and two G isolates. The TRUGENE assay using prototype 1.5 RT-PCR primers and the ViroSeq assay were both successful for all variants tested, although five isolates lacked double-strand sequence coverage in the ViroSeq assay. The availability of standardized HIV-1 genotyping kits that perform reliably with all HIV subtypes will facilitate broad implementation of HIV-1 resistance testing.

Use of Sequence Data Generated in the Bayer TruGene Genotyping Assay To Recognize and Characterize Non-Subtype-B Human Immunodeficiency Virus Type 1 Strains

“…Our finding that version 1.5 of the TruGene assay enables non-B HIV-1 subtype viruses to be sequenced with a high success rate is consistent with previous studies examining the performance characteristics of this version of the assay (5,15,20). The primary difference between versions 1.0 and 1.5 of the TruGene HIV-1 genotyping assay is a modification to the RT-PCR primers (20).…”

“…Our finding of a significant failure rate of the PR sequencing primer pair of the TruGene assay for most non-B subtypes is also in agreement with previous studies. In our sample cohort, only 34% of protease sequences could be elucidated using the PR primer pair, a result comparable to the 33% reported by Fontaine et al (15) and 22% determined by Jagodzinski and coworkers (20). Interestingly, we obtained a significantly higher rate of success with the PR primers for determining the protease sequence of both subtype D (75%) and CRF02_AG (77%) viruses compared with other non-B viruses.…”

“…The primary difference between versions 1.0 and 1.5 of the TruGene HIV-1 genotyping assay is a modification to the RT-PCR primers (20). This change resulted in a more efficient and robust RT-PCR amplification, which in turn improved the performance of the assay on both low-viral-load samples and samples containing non-B-subtype viruses (20). Our finding of a significant failure rate of the PR sequencing primer pair of the TruGene assay for most non-B subtypes is also in agreement with previous studies.…”

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

“…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.…”

“…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.…”