Evaluation of two commercial kits for the detection of genotypic drug resistance on a panel of HIV type 1 subtypes A through J.

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.     

Performance of two commercially available sequence-based HIV-1 genotyping systems for the detection of drug resistance against HIV type 1 group M subtypes

The use of genotyping assays for the detection and evaluation of drug resistance mutations within the polymerase gene of human immunodeficiency virus type 1 (HIV-1) is becoming increasingly relevant in the clinical management of HIV-1 infection. However, genotypic resistance assays available currently have been optimised for genetic subtype B strains of the virus and many clinical centres are presented with strains from subtypes A, C, and D. In the present report, we compare the performance of two sequence-based commercially available kits, the ViroSeq Genotyping System (Applied Biosystems, Foster City, CA) and the TruGene HIV-1 Genotyping Kit (Visible Genetics, Toronto, Ontario) against a panel of 35 virus isolates from HIV-1 Group M (subtypes A-J). Full-length consensus sequences were generated by the ViroSeq genotyping system for 26 of 31 (83.8%) of the isolates tested, in contrast to the TruGene genotyping system, which generated 16 of 30 (53%) usable sequences overall. Overall, subtype B isolates were sequenced with a greater degree of success than non-subtype B isolates. Discrepancies were found between the consensus sequences reported by each system for each sample (mean difference 1.0%; range 0.0-3.2%), but these appeared to be random and did not affect interpretation of the major resistance codons. In addition, both systems were able to amplify template RNA from low copy viral load plasma samples (10(2)-10(3) RNA copies/ml) taken from a random selection of patient samples encompassing subtypes A-C. While the availability of these genotyping systems should facilitate studies of HIV-1 drug resistance in countries in which these subtypes are prevalent, the performance against subtypes other than B needs to be improved.     

Performance characteristics of human immunodeficiency virus type 1 (HIV-1) genotyping systems in sequence-based analysis of subtypes other than HIV-1 subtype B

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.     

Performance evaluation of an in-house human immunodeficiency virus type-1 protease-reverse transcriptase genotyping assay in Cameroon

Most commercial HIV-1 genotyping assays are hampered by high cost in resource-limited settings. Moreover, their performance might be influenced over time by HIV genetic heterogeneity and evolution. An in-house genotyping protocol was developed, and its sequencing performance and reproducibility were compared to that of ViroSeq?. One hundred ninety plasma samples from HIV-1-infected subjects in Cameroon, a resource-limited setting with a high HIV genetic variability, were processed for pol gene sequencing with an in-house protocol, ViroSeq?, or both. Only non-B subtypes were found. The in-house sequencing performance was 98.7% against 92.1% with ViroSeq?. Among 36 sequence pairs obtained using both assays, the overall rate of discordant amino acid positions was negligible (0.24%). With its high sensitivity and reproducibility, as well as its affordable cost (about half of ViroSeq?: 92 euros vs. 217 euros), this in-house assay is a suitable alternative for HIV-1 genotyping in resource-limited and/or in high-genetic-diversity settings.     

Performance of ViroSeq HIV-1 Genotyping System in routine practice at a Belgian clinical laboratory

Since there are indications of an increasing amount of non-B subtypes in Western Europe it was decided to assess the performance of the ViroSeq HIV-1 Genotyping System on a set of samples from the AIDS Reference Laboratory at the University Hospitals Leuven, a hospital with an increasing number of patients infected with non-B subtypes. The set consisted of 383 samples comprising 12 different subtypes and the genotyping kit was assessed for its amplification capabilities as well as its sequencing capabilities. Amplification failed in 32 samples (8.4%) and there was a tendency of a lower performance of the kit when it concerned the amplification of non-B subtypes. Regarding the sequencing performance of the HIV-1 Genotyping System, three different results could be considered. The performance of the entire set of primers (A, B, C, F, G and H) on the different subtypes showed a significant decrease of positive results for subtypes A, G and the recombinants whereas a tendency to less positive results could be detected for subtypes CRF12_BF, D, H and J. When looking at the performance of the individual primers for the different subtypes, only one result differed significantly: there were less positive results by applying primer F on subtype A. A tendency to less positive results was found for other combinations of primer and subtype, most of which comprised combinations with primers B, C, F and H. A final result was obtained by comparing the overall sequencing results of a certain primer on all the non-B subtypes with the results of the same primer on subtype B. Primer F showed significant less positive results and a tendency to less positive results was found for primer H. The other primers showed comparable results. All of the above results regarding the sequencing primers did not include primer D since this is a back-up primer for primer A. Analysis of the results for primer D showed that less positive results were found for all the non-B subtypes, most of which were significant. The overall performance of primer D on all non-B subtypes was only 15.7%. The use of primer D as a back-up primer was also investigated: it generated a positive result in only 17.3% of the cases where primer A failed. Most of these positive results were subtype B (74%). As a result of sequencing problems 65 out of 351 (18.5%) samples had to be processed with "in-house" procedures.     

河北省HIV-1流行株基因序列测定及亚型分析

目的 了解河北省HIV-1流行株的亚型分布和流行趋势.方法 从感染者的血浆样品中提取病毒RNA,逆转录后采用套式PCR扩增HIV-1 gag和env基因的部分片段,对PCR产物直接进行核苷酸序列测定,所获序列与各亚型国际参考株序列比对,确定基因型并进行系统进化树分析.结果 对154份HIV-1感染者的样品进行扩增,得到了148份样品的HIV-1基因片段.发现6种HIV-1亚型和重组型,以及2例未定型.其中B'亚型61例(41.2%)、CRF01_AE 59例(39.9%)、CRF07_BC 16例(10.8%)、CRF08_BC 6例(4.1%)、C亚和B01亚型各2例(1.4%).在河北省首次发现了B01亚型.结论 2009年河北省存在多种HIV-1亚型和流行重组型,主要是B'亚型和CRF01_AE重组型,应加强对HIV-1毒株亚型变异的监测,及时调整防治策略.     

淮安市经异性性传播的HIV感染者中HIV-1病毒基因亚型特征分析

目的 了解淮安市经异性性传播HIV感染者中HIV-1病毒基因亚型分布及其流行特征.方法 从60名异性性传播HIV-1感染者血液中提取DNA或RNA,用巢式PCR或RT-PCR方法扩增gag、env基因区片段,测定序列并分析.结果 60份标本中,确定了48份标本的基因亚型,发现有4种HIV-1亚型和重组型.其中CRF01_AE亚型占62.5％,CRF07_BC亚型占22.9％,CRF08_BC亚型占6.3％,B亚型占6.3％.结论淮安市异性性传播感染HIV者中,流行着CRF01_AE、CRF07_BC、CRF08_BC、B亚型这4种亚型病毒株,CRF01_AE为主要流行亚型.