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Nuclisens Easy Q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/ml 5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.     

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。     

A rapid RT-PCR assay for the detection of HIV-1 in human plasma specimens

Introduction

The CDC estimates that there are currently over 1 million people living with human immunodeficiency virus (HIV-1) in the United States, with new cases increasing by approximately 50,000 each year. HIV-1 consists of four distinct groups: the major M group, and the rare N, O, and P groups, each comprising of various subtypes. Without proper care, HIV-1 can lead to cardiovascular, kidney, and liver diseases, cancer, and rapid progression into acquired immune deficiency syndrome (AIDS). Here, we describe a novel, rapid, and highly sensitive assay for the detection of HIV-1 using intercalating dye based RT-PCR and melt curve analysis.

Materials and methods

We designed an RT-PCR assay for the detection of the major M subtypes in addition to the rare (O, N, and P) HIV-1 groups, as well as an extraction/RT-PCR control, using melt curve analysis. Viral RNA was extracted using the automated Qiagen EZ1 robotic system (Qiagen, Valencia, CA). To establish the limit of detection (LOD) for this assay, we diluted the AcroMetrix HIV-1 panel (LifeTechnologies, Grand Island, NY) to concentrations ranging from 25 to 500 copies/ml. Armored RNA® BCR/ABL b3/a2 (Asuragen, Austin, Texas) was used as our extraction and RT-PCR control. Specificity and accuracy were assessed by testing plasma specimens from 48 anonymized patients negative for HIV-1.

Results

This assay has a turnaround time of less than 2.5 h and has a limit of detection of 50 copies/ml of plasma. Our assay also demonstrated 100% concordance with 53 previously quantified plasma patient specimens, including 48 negative samples and 5 positive samples. HIV-1 and our extraction/RT-PCR control were consistently identified at 79 °C and 82.5 °C, respectively.

Conclusions

We developed a comprehensive, easy to use assay for the detection of HIV-1 in human plasma. Our assay combines a rapid and cost-effective method for molecular diagnostics with the versatility necessary for widespread laboratory use. These performance characteristics make this HIV-1 detection assay highly suitable for use in a clinical laboratory.     

High failure rate of the ViroSeq HIV-1 genotyping system for drug resistance testing in Cameroon, a country with broad HIV-1 genetic diversity

The ViroSeq HIV-1 genotyping system is used in many African countries for drug resistance testing. In this study, we used a panel of diverse HIV-1 group M isolates circulating in Cameroon to show that the performance of this assay can be altered by the sequence variation of non-B HIV-1 strains that predominate in African settings.     

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.     

Technologies for measuring HIV-1 drug resistance

Abstract

Drug resistance testing significantly improves response to antiretroviral treatment in HIV-1-infected patients, therefore it has recently been implemented into current guidelines for the management of antiretroviral therapy. Knowledge about technologies for measuring drug resistance is important for several reasons: (a) differences exist between different technologies and also between assays based on the same technology; (b) the results of resistance testing are strongly dependent on the reliability and precision of the technology used; and (c) technical aspects have to be considered for a clinically relevant interpretation of drug resistance. The spectrum of genotypic and phenotypic technologies as well as the technical quality is increasing, which shifts the emphasis to the interpretation of resistance profiles. The interpretation is based on the knowledge of drug resistance-associated mutations as well as correlations between genotype and phenotype and clinical response, which are incorporated into rules-based systems. Bioinformatic techiques are used to generate mathematical models for the prediction of drug resistance from genotype. Both approaches are converging toward the prediction of clinical response. Because therapy response is dependent on many additional variables, further efforts are required for the generation of a large clinical database. This will be the basis of a prediction system that will optimize the antiretroviral therapy for each individual patient.     

Technologies for measuring HIV-1 drug resistance

Drug resistance testing significantly improves response to antiretroviral treatment in HIV-1-infected patients, therefore it has recently been implemented into current guidelines for the management of antiretroviral therapy. Knowledge about technologies for measuring drug resistance is important for several reasons: (a) differences exist between different technologies and also between assays based on the same technology; (b) the results of resistance testing are strongly dependent on the reliability and precision of the technology used; and (c) technical aspects have to be considered for a clinically relevant interpretation of drug resistance. The spectrum of genotypic and phenotypic technologies as well as the technical quality is increasing, which shifts the emphasis to the interpretation of resistance profiles. The interpretation is based on the knowledge of drug resistance-associated mutations as well as correlations between genotype and phenotype and clinical response, which are incorporated into rules-based systems. Bioinformatic techniques are used to generate mathematical models for the prediction of drug resistance from genotype. Both approaches are converging toward the prediction of clinical response. Because therapy response is dependent on many additional variables, further efforts are required for the generation of a large clinical database. This will be the basis of a prediction system that will optimize the antiretroviral therapy for each individual patient.     

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.     

Transmission of HIV-1 drug resistance.

BACKGROUND: The use of highly-active anti-retroviral therapy (HAART) for treating HIV infections is increasing. Recent studies have demonstrated that HAART is improving both the length and quality of life in HIV-infected patients. Resistant strains of HIV arise when drug adherence is poor. This can lead to the transmission of drug-resistant strains of HIV to susceptible individuals. This can lead to suboptimal first-line therapy, if the resistance profile of the transmitted virus is unknown. OBJECTIVES: To review the mechanisms of how drug resistance arises; the methods used to characterise drug resistance; the problems arising with compliance leading to the development of drug-resistant HIV strains; the evidence for the incidence, prevalence and trends in the transmission of resistant HIV strains in different risk groups; and the evidence of suboptimal response to first-line therapy where transmission of a resistant HIV strain has occurred. On the basis of this, a case is presented for the routine resistance testing of all newly diagnosed HIV-infected individuals. STUDY DESIGN: Literature review. RESULTS AND CONCLUSIONS: There is evidence, though limited at present, that transmission of drug-resistant HIV strains can lead to suboptimal response to first-line therapy in newly diagnosed HIV-infected individuals. As the use of HAART can only increase in the future, and compliance will always be a problem in such HAART-treated patients, baseline resistance testing should become a routine part of their management.     

A simple competitive RT-PCR assay for quantitation of HIV-1 subtype B and non-B RNA in plasma

An easy, inexpensive competitive RT-PCR assay for HIV-1 RNA quantitation was constructed. A 138-bp sequence in the HIV-1 gag p24 region was selected as the target and co-amplified with competitor RNA containing an internal 44-bp deletion. Quantitation of serial dilutions of control RNA samples prepared from the LAI isolate demonstrated a good linearity (R(2)=0.991) within the range between 10 and 250 copies/sample. The detection limit of the assay was determined to be 3.8 copies/sample by Probit analysis and corresponded to 110 copies/ml in plasma. The intra-assay CV value was 9.1%, and the inter-assay value was 25.9%. Both were comparable to those obtained with commercially available HIV-1 RNA quantitation kits. The correlation efficient for the results obtained in 47 plasma samples from HIV-1-infected individuals (subtype A in 1, subtype B in 25, subtype C in 4, subtype F in 1, and CRF01 AE in 16) with the competitive RT-PCR and Cobas Amplicor HIV-1 Monitor test v1.5 was 0.956 for subtype B and 0.947 for subtype non-B. The assay devised is a good alternative for monitoring antiretroviral therapy in resource-poor countries.     

Development of a real-time quantitative RT-PCR for the detection of HIV-2 RNA in plasma

An assay is described for the quantification of human immunodeficiency virus type 2 (HIV-2) RNA in EDTA plasma based on RT-PCR using the Taqman real-time PCR detection method. As standard, an electron microscopically counted virus stock of HIV-2 strain NIHZ was used. The lower detection limit is 5 # 102 HIV-2 RNA copies per ml of EDTA plasma. The assay is linear within the range required (5 # 102-106 HIV-2 RNA copies/ml of EDTA plasma) with an intra assay variability of 2.5% and an inter-assay variability ranging from 2% at 106 copies to 7.5% at the lower detection limit. Three primer/probe combinations were developed to circumvent false negative samples due to nucleotide variation in the target sequence. Using these primer/probe sets enabled the detection of HIV-2 DNA sequences from all HIV-2 seropositive individuals and two out of five dual human immunodeficiency virus type 1 (HIV-1) and HIV-2 seropositive individuals visiting the University Hospital Rotterdam.