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.     

Model for assessment of proficiency of human immunodeficiency virus type 1 sequencing-based genotypic antiretroviral assays

Use of sequencing-based genotyping as a diagnostic assay for human immunodeficiency virus (HIV) antiretroviral resistance is increasing. Periodic evaluation of the proficiency of laboratories performing this assay should be established. It is important to identify components of the assay that influence the generation of reliable sequencing data and that should and can be monitored. A model was developed to determine what parameters were reasonable and feasible for assessing the performance of genotyping assays. Ten laboratories using the genotyping platform, HIV-1 Genotyping System (HGS) v. 1 and software versions 1.1 or 2.0, participated in two rounds of testing. For each round, each group was sent a panel consisting of three clinical samples to sequence in real time. Six months later, seven laboratories using the TRUGENE HIV-1 Genotyping Kit participated in a separate round, working with both panels at the same time. Analysis of the data showed that one main indicator of genotyping proficiency was achievement of > or =98% sequence homology of a sample tested to a group consensus sequence for that sample. A second was concordant identification of codons at sites identified with resistance mutations in the sample, although scoring of these criteria is still undetermined from this study. These criteria are applicable to all sequence-based genotyping platforms and have been used as a baseline for assessing the performance of genotyping for the determination of antiretroviral resistance in our ongoing proficiency program.     

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.     

Human immunodeficiency virus type 1 drug resistance testing: a comparison of three sequence-based methods

The use of genotypic assays for determining drug resistance in human immunodeficiency virus (HIV) type 1 (HIV-1)-infected patients is increasing. These tests lack standardization and validation. The aim of this study was to evaluate several tests used for the determination of HIV-1 drug resistance. Two genotypic tests, the Visible Genetics TruGene HIV-1 Genotyping Kit and the Applied Biosystems HIV Genotyping System, were compared using 22 clinical samples. Genotyping results were also obtained from an independent reference laboratory. The Visible Genetics and Applied Biosystems genotyping tests identified similar mutations when differences in the drug databases and reference strains were taken into account, and 19 of 21 samples were equivalent. The concordance between the two assays was 99% (249 of 252 mutation sites). Mutations identified by the reference laboratory varied the most among those identified by the three genotypic tests, possibly because of differences in the databases. The concordance of the reference laboratory results with the results of the other two assays was 80% (201 of 252). Samples with 500 to 750 HIV RNA copies/ml could be sequenced by the Visible Genetics and Applied Biosystems assays using 1 ml of input. The Visible Genetics and Applied Biosystems assays both generated an accurate sequence. However, the throughput of the Visible Genetics assay is more limited and may require additional instruments. The two assays differ technically but are similar in overall complexity. Data analysis in the two assays is straightforward, but only the reports provided by Visible Genetics contain information relating mutations to drug resistance. HIV drug resistance genotyping by sequencing is a complex technology which presents a challenge for analysis, interpretation, and reporting.     

Performance of human immunodeficiency virus type 1 gp41 assays for detecting enfuvirtide (T-20) resistance mutations

The performance of a gp41 assay, created using reagents designed for use with the OpenGene DNA Sequencing System, was evaluated using a panel of plasma samples obtained from enfuvirtide-naive and -experienced human immunodeficiency virus type 1-infected subjects. Resulting sequence data were highly accurate compared to a "home brew" assay and clonal sequence analysis.     

Efficient extraction of viral DNA and viral RNA by the Chemagic viral DNA/RNA kit allows sensitive detection of cytomegalovirus, hepatitis B virus, and hepatitis G virus by PCR

The Chemagic Viral DNA/RNA kit was evaluated for extraction of cytomegalovirus (CMV), hepatitis B virus (HBV), and hepatitis G virus (HGV) by using the QIAamp DNA Blood Mini kit and the QIAamp Viral RNA Mini kit as reference protocols. The extraction efficiencies of the different kits for CMV DNA and HBV DNA were not distinguishable, but the extraction efficiency for HGV RNA was better with the Chemagen protocol. All clinical specimens tested HBV DNA- or HGV RNA-positive after QIAGEN protocols for extraction were confirmed by using the Chemagen protocol. The Chemagen kit failed to confirm one of 75 CMV DNA-positive specimens. Thus, a new competitive extraction method using a technology with a high potential for automation is available.     

Modifications and substitutions of the RNA extraction module in the ViroSeq HIV-1 genotyping system version 2: effects on sensitivity and complexity of the assay

Genotypic testing for HIV-1 resistance to anti-retroviral drugs has become accepted widely as a routine method to guide anti-retroviral therapy. However, implementation into routine high-throughput laboratory diagnosis is difficult due to the complexity of the assay. A commercially available assay is the ViroSeq HIV-1 Genotyping System (Applied Biosystems, Weiterstadt, Germany). We modified and substituted the RNA extraction module to optimize the proportion of samples amplified successfully as follows: 1 ml plasma was concentrated by ultracentrifugation and extracted according to the manufacturer's instructions (Kit), by substituting the lysis buffer (Roche, Roche Diagnostics GmbH, Mannheim, Germany), and by using the QIAamp Viral RNA Kit (Qiagen GmbH, Hilden, Germany) with elution volumes of 60 (Q60) or 50 micro l (Q50). Overall Q50 showed a higher success rate (97%) than the other extraction modules used (range 88-91%). In samples with a viral load range of 1,000-4,999 copies/ml, Q50 was superior (95 vs. 65% to 83%), while in samples with a viral load range of 5,000-9,999 copies/ml or those with 10,000 or more copies/ml, the success rate of the extraction procedures showed no significant differences. In 18 samples, which were negative using the Kit or Roche extraction, Q60 resulted in 7/18 positive results; in addition the Q50 was successful in amplifying 7/10 of the Q60 negative samples. When investigating samples with a measurable viral load of less than 1,000 copies/ml or lower, Q50 had the highest success rate with 80% compared to the other procedures (33-63%). A statistically significant new cut-off could be defined for Q50 at a value of 250 copies/ml. The results showed clearly that the ViroSeq System is suitable for analyzing the HIV-1 genotype over a wide range of viral loads but could be improved significantly when substituting the RNA extraction module with Q50 without using a nested PCR protocol. This is of great importance as it avoids further time- and cost-intensive steps.     

Magnetic silica extraction for low-viremia human immunodeficiency virus type 1 genotyping

Nucleic acid extraction and human immunodeficiency virus type 1 (HIV-1) genotyping using the NucliSens miniMAG platform and the TruGene HIV-1 genotyping kit gave HIV-1 sequence data from HIV-1-negative plasma spiked with 100 copies/ml reference HIV-1 RNA and from low-viremia clinical samples (<500 copies/ml) without the need for ultracentrifugation or nested second-round PCR.     

Evaluation of the MagNA Pure LC used with the TRUGENE HBV Genotyping Kit.

BACKGROUND: The current manual sample processing method recommended for use with the TRUGENE HBV Genotyping Kit (TRUGENE HBV; Bayer HealthCare LLC, Tarrytown, NY) is labor-intensive and may be prone to specimen cross-contamination. Recent evaluations of the MagNA Pure LC (MP; Roche Applied Science, Indianapolis, IN) suggest that it is suitable for automated, contamination-free extraction and purification of viral nucleic acids from large-volume (1.0 mL) serum or plasma specimens. OBJECTIVES: We evaluated the MP Total Nucleic Acid Isolation Kit--Large Volume (Roche Applied Science) in conjunction with TRUGENE HBV to establish the analytical sensitivity (threshold titer) of the assay, in HBV DNA International Units (IU)/mL, for obtaining consistent, interpretable sequence data from TRUGENE HBV. STUDY DESIGN: HBV analytical standards, prepared as 10 replicates (1.0 mL each) at each of the following concentrations: 200, 1000, 5000, and 10,000 IU/mL, were processed by MP and analyzed by TRUGENE HBV according to manufacturer's instructions. Performance of TRUGENE HBV used in conjunction with MP sample processing was evaluated further using 22 clinical serum specimens containing low titers of HBV DNA. RESULTS: All replicates of HBV analytical standards at 1000, 5000, and 10,000 IU/mL yielded interpretable TRUGENE HBV sequences, whereas interpretable sequences were obtained in 90% (9 of 10) of the replicates at 200 IU/mL. TRUGENE HBV sequences were interpretable in 86% (19 of 22) of the clinical specimens studied. CONCLUSIONS: MP sample processing is efficient and suitable for use with TRUGENE HBV. When combined with MP sample processing, TRUGENE HBV yielded interpretable sequences from HBV analytical standards and clinical serum specimens with HBV DNA titers of > or =200 IU/mL.     

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.     

Evaluation of the NucliSens EasyQ HIV-1 v1.1 and RealTime HIV-1 kits for quantitation of HIV-1 RNA in plasma

Human immunodeficiency virus type-1 (HIV-1) RNA viral load is an important biomarker to evaluate the therapeutic efficacy of antiretroviral drugs and to monitor disease progression in HIV-infected individuals. We compared HIV-1 RNA quantitation between two different kits, the NucliSens EasyQ^® HIV-1 v1.1 (EasyQ, bioMérieux) and RealTime HIV-1 (RealTime, Abbott), using HIV-1 RNA quality control (QC) materials, cell-cultivated viruses, and the plasma samples of 104 patients with HIV. Correlation between the two kits for HIV RNA-1 quantitation with clinical samples was high (R = 0.91). Based on results obtained with quality control standards, the reproducibility of the RealTime kit was higher than the EasyQ kit: the viral load value and coefficient of variation of each kit was 4.11 ± 0.136 and 3.3% for EasyQ and 3.55 ± 0.042 and 1.2% for RealTime, respectively (P < 0.002).This is the first comparative analysis of the detection limit and reproducibility of two different quantitation kits using clinical plasma samples from Korean HIV-1-infected patients. It will serve a useful reference to determine correction values for each HIV-1 RNA quantitation kits and to choose an appropriate assay kit for each laboratory.     

单核细胞TLR7/8表达与HIV-1感染疾病进程关系的研究

目的 对HIV-1感染者单核细胞TLR7/8表达水平及与疾病进展的相关性进行研究,探讨单核细胞TLR7/8在HIV-1疾病进程中的作用.方法 选取63名HIV-1感染者及18名健康对照,应用MACS磁珠分选法纯化CD14+单核细胞,用2.5μg/ml R848刺激单核细胞TLR7/8,QIAGEN公司的RNA提取试剂盒提取单核细胞总RNA,实时定量RT-PCR测定TLR7/8的mRNA表达.结果 HIV-1感染者单核细胞TLR7和TLR8的表达水平与CIM+T细胞显著正相关(r=0.614,P＜0.01;r=0.419,P＜0.01).TLR7 mRNA表达:缓慢进展组明显高于HIV感染组、AIDS组和健康对照组(P＜0.05),HIV感染组明显高于AIDS组(P＜0.05);TLR8 mRNA表达:缓慢进展组明显高于AIDS组(P＜0.05).体外用R848刺激11LR7后表达显著下调,而TLR8的表达稳定.结论 AIDS疾病进程中HIV-1感染者单核细胞TLR7/8表达水平明显下降,TLR7的表达水平下降更显著.     

不同试剂盒对乙脑病毒RNA提取效能的比较

目的比较常见的3种商售RNA提取试剂对乙脑病毒检测的相对效率、耗时和成本,为实验室应用及检测结果的评价提供依据。方法对乙脑病毒参考株悬液作10^-1～10^-4系列稀释,选用QIAGEN公司的QIAamp Viral RNA Mini Kit、Sangon生物公司的RNA提取试剂盒和Invitrogen公司的Trizol试剂3种核酸提取方法提取上述样本RNA．用TaqMan实时荧光定量反转录聚合酶链反应对其提取效率进行评价,并比较3种试剂（盒）的耗费时间和价格。结果对不同稀释度乙脑病毒样本的检测,均以QIAGEN公司QIAamp Viral RNA Mini Kit检测的敏感度最高。以该试剂盒的提取效率为100％,则Invitrogen和Sangon生物公司RNA提取试剂（盒）的提取效率分别为77．4％～86．8％和64．2％～74．1％。3种试剂（盒）对cDNA起始模板量的估计也有明显的影响,差别达到1～3个数量级,以QIAGEN公司的试剂盒最敏感。3种试剂（盒）提取RNA耗时分别为60min、100min和70min,价格效率比分别为46元、23元和20元。结论RNA提取试剂可影响JEV荧光定量PCR检测的结果,各实验室应根据实验目的和实验室条件．合理选择病毒RNA提取试剂盒。     

Distribution of human immunodeficiency virus type 1 subtypes in the State of Amazonas,Brazil, and subtype C identification

Few studies have reported the molecular epidemiological characterization of HIV-1 in the Northern region of Brazil. The present study reports the molecular and epidemiological characterization of 31 HIV-1 isolates from blood donors from the State of Amazonas who donated blood between April 2006 and March 2007. Serum/plasma samples from all donors were screened for HIV antibodies by ELISA and the results confirmed by Western blot analysis. Genomic DNA was extracted from the buffy coat using the Super Quik-Gene-DNA Isolation kit. Nested PCR was performed on the env, gag, and pol regions of HIV-1 using the Gene Amp PCR System 9700. Sequencing reactions were performed using the inner PCR primers and the DYEnamic™ ET Dye Terminator Kit, and phylogenetic analysis was performed using the gag, pol, and env gene sequences. We collected samples from 31 blood donors who tested positive for HIV-1 in confirmatory experiments. The male:female ratio of blood donors was 3.4:1, and the mean age was 32.4 years (range: 19 to 61 years). Phylogenetic analysis showed that subtype B is the most prevalent among Northern Brazilian HIV-1-seropositive blood donors. One HIV-1 subtype C and one circulating recombinant form (CRF_BF) of HIV-1 were identified in the State of Amazonas. This is the first study showing the occurrence of a possible “homogenous” subtype C in this region of Brazil. This finding could contribute to a better characterization of the HIV-1 strains that circulate in the country. Key words: HIV-1; Subtypes; Phylogenetic analysis; Blood donors; Molecular and epidemiological characterization     

Human T-cell lymphotropic virus antibody prevalence in HIV-1-infected individuals attending a sexual health clinic in South-East London

Human immunodeficiency virus (HIV) and human T-cell lymphotropic virus (HTLV) are both retroviruses with similar routes of transmission. A number of reports suggest variable clinical outcomes in HIV and HTLV co-infected individuals. There is no published information regarding the prevalence of HIV and HTLV co-infection in the UK. We therefore carried out an unlinked anonymised retrospective study to investigate the prevalence of HTLV co-infection in HIV infected patients attending a sexual health clinic in South-East London. We identified sera from 777 HIV-1 positive adults (504 male, 273 female) who had attended our sexual health clinic between January 2000 and March 2001. Serum samples stored at -20 degrees C were initially tested by HTLV-1 and HTLV-2 antibody enzyme-linked immunoassay (EIA). An immunoblot assay was carried out on reactive samples to discriminate between viral subtypes. Samples with indeterminate results were also analyzed by Western blot. The prevalence of HTLV antibody was 0.8% (five patients with HTLV-1 and one with HTLV-2). Four of the HTLV-1 co-infected patients were females born abroad, of Black African or Caribbean origin. The other HTLV-1-infected patient was a male in the Black Other ethnic group born in the UK, demonstrating that transmission may occur outside recognized areas of high endemicity. The HTLV-2 co-infected individual was a White male born in Italy, and was likely to have been infected through intravenous drug use. The results suggest HTLV antibody screening should be considered in the local HIV infected population of south London.