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.     

In vivo emergence of drug-resistant mutations at less than 50 HIV-1 RNA copies/mL that are maintained at viral rebound in longitudinal plasma samples from human immunodeficiency virus type-1-infected patients on highly active antiretroviral therapy

OBJECTIVE: Emergence of human immunodeficiency virus type-1 (HIV-1) genotypic drug resistance is generally attributed to noncompliance, poorly absorbed drugs, or drug-to-drug interaction. Attempts to determine emerging genotypic drug resistance from study subjects on highly active antiretroviral therapy (HAART) relied on insensitive polymerase chain reaction (PCR) techniques, revealing wild type HIV-1 or precursor resistant genotypes from few plasma samples successfully amplified with <50 copies/mL. STUDY DESIGN/METHODS: In this analysis, using Applied Biosystems' ViroSeq HIV-1 Genotyping Systems, Version 2.0 (Applied Biosystems, Foster City, CA, USA) and the supplemental, for research use only, nested PCR primers, genotypic drug resistance was determined in longitudinal plasma samples from 11 study subjects on HAART. RESULTS: In 4 of 11 study subjects, newly emerging genotypic primary resistant mutations were detected in plasma samples with <50 copies/mL. Most of these primary drug-resistant mutations were detected in subsequent longitudinal samples with detectable viral load (viral breakthrough). CONCLUSIONS: This analysis suggests sufficient viral replication <50 copies/mL to generate genotypic drug resistance in study subjects on suppressive HAART.     

Detection of HIV-1 antiretroviral resistance from patients with persistently low but detectable viraemia

We modified the Abbott diagnostics HIV-1 Viroseq version 2 assay trade mark in order to detect the presence of HIV-1 drug resistance mutations in patients with viraemia below 1000 copies/ml of plasma. One hundred and forty-four patients with a detectable HIV-1 plasma viral load below 1000 copies/ml were selected and HIV-1 genetic analysis carried out using a modification of the Abbott Diagnostics Viroseq 2.0 assay trade mark. The procedure differs from the standard protocol in that a nested PCR amplification step was introduced. The oligonucleotide primers for the first round of PCR were those supplied in the RT-PCR module of the kit. The nested PCR primers were primers A and H taken from the sequencing module. One hundred and twenty-eight out of 144 (89%) plasma samples with an HIV-1 viral load of less than 1000 copies/ml (ranging from 54 to 992 copies) were successfully sequenced. HIV-1 genotypes were obtained from 68 out of 81 (84%) samples with a viral load of greater than 50 but less than 300 copies/ml and 60/63 (95%) of samples with a viral load of greater than 300 but less than 1000 copies/ml. Serial dilution of a sample with a high viral load did not affect the detection of resistance mutations. Multiple sequencing of samples with low viral load did not result in detection of additional mutations, although, in one sample the K103N mutation was detected in 3/6 replicates while wild-type was detected in 2/6 and a mixture of wild-type/mutant in 1/6. Samples from patients infected with both clade B and non-B clades of HIV-1 could be genotyped at low copy number. Modification of the Abbott Viroseq assay allows reproducible sequencing of the HIV-1 genome from patients with low, but detectable, plasma virus burden.     

Simple detection of point mutations associated with HIV-1 drug resistance

A novel assay is described for the detection of HIV-1 drug resistance that is simple, cheap and sensitive. HIV-1 drug resistance in B and non-B HIV-1 subtypes was investigated using Mutagenically-Separated PCR (MS--PCR) --- a competitive semi-nested PCR which uses mutagenic primers. The assay was assessed for sensitivity, specificity and its ability to detect mutant virus within a mixed mutant--wild-type population. Gene sequencing was carried out simultaneously for comparison. MS--PCR detected five copies of HIV-1 RNA from laboratory isolates and 50 copies from patient samples. We demonstrate 100% specificity of detection for wild type or mutant virus for clades A, B, C, D and E. For mixed populations of virus, MS--PCR can detect at least a 10% mix of wild type:mutant, or vice-versa. When applied to African patient samples MS--PCR detected 91.6% of the codons tested. Concordance with sequencing data was 88.8% for protease and 97.2% for RT. MS--PCR is sensitive and specific for the detection of mutations in HIV-1, and can be adapted easily to test for resistance at any codon of interest.     

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.     

我国主要HIV-1流行株耐药基因型检测方法的研究

目的研究针对中国主要HIV-1流行株优化的实验室自建耐药基因型实验室自建检测方法（in—house）的扩增效果及检测敏感度。方法选取中国主要流行亚型的B、CRF07-BC、CRF01-AE亚型各10份样本,这些样本为2007—2009年本实验室采集采自河南、新疆、湖南三地已接受抗病毒治疗患者的样本,并已确定亚型。选用生物梅里埃公司的NuclisensEasyQ方法对选取的30份样本的病毒载量平行进行3次病毒载量检测,取平均值作为载量数值。对每份样本用HIV阴性血浆进行5个浓度的梯度稀释,稀释为〉1000、401～1000、101～400、50～100和〈50拷贝／ml5个浓度梯度。提取核酸后,进行RT—PCR和巢式PCR扩增,对扩增结果进行统计扩增结果,确定每种亚型的扩增效果和最低检测限。随机选取12份样本的初浓度和最低浓度进行测序,对测序结果进行耐药结果分析和序列一致性分析。结果所选样本的病毒载量介于2．03×102～5．92×104拷贝／ml之间。自建的In—house法对载量50—1000拷贝／ml范围的样本仍可达到较高的扩增效果（86％）。样本稀释前后耐药位点相似,序列一致性在97％以上,在低病毒载量时优势病毒株的检测则更敏感。结论自建的实验室方法的灵敏度较高,对低病毒载量水平的样本仍有较高的扩增效果。