No abstract

We evaluated the FDA-approved Roche Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) HIV-1 viral load assay for sensitivity, reproducibility, linearity, HIV-1 subtype detection, and correlation to the Roche Amplicor HIV-1 monitor test, version 1.5 (Amplicor). The limit of detection calculated by probit analysis was 23.8 copies/ml using the 2nd International WHO Standard and 30.8 copies/ml using Viral Quality Assurance (VQA) standard material. Serial dilutions of six patient samples were used to determine inter-and intra-assay reproducibility and linearity, which were very good (<8% coefficient of variation [CV]; between ϳ1.7 and 7.0 log 10 copies/ml). Subtype detection was evaluated in the CAP/CTM, Amplicor, and Bayer Versant HIV-1 bDNA 3.0 (Versant) assays using a commercially available panel. Versant averaged 0.829 log 10 copies/ml lower than CAP/CTM and Amplicor averaged 0.427 log 10 copies/ml lower than CAP/CTM for the subtype panel. Correlation with samples previously tested by Amplicor was excellent (R 2 ‫؍‬ 0.884; average difference [Amplicor value minus CAP/CTM value], 0.008 log 10 copies/ml). Of the 305 HIV samples tested, 7 samples generated CAP/CTM titers between 1.0 and 2.75 log 10 copies/ml lower than those for Amplicor. Three of these samples revealed primer and probe mismatches that could account for the discrepancies. Otherwise, the CAP/CTM assay exhibits excellent sensitivity, dynamic range, reproducibility, and correlation with Amplicor in an automated format.Measurement of HIV-1 RNA in the plasma of infected patients is critical for guiding treatment. Over the past decade, several commercial quantitative HIV-1 RNA assays have become available that utilize endpoint PCR, isothermal amplification, or signal amplification techniques. Most recently, Abbott Molecular (Des Plaines, IL) and Roche Molecular Systems (Branchburg, NJ) received FDA approval for their real-time PCR-based systems, the RealTime HIV-1 assay and Cobas AmpliPrep/Cobas TaqMan HIV-1 Test (CAP/CTM), respectively. Each assay has its own advantages and disadvantages in terms of sensitivity, equipment requirements, throughput, dynamic range, subtype detection, and cost (1a, 4, 6, 7, 8, 11, 13, 15, 16).The CAP/CTM test includes a "docked" version that permits automated "sample in-results out" analysis of specimens without user intervention. We evaluated this configuration and compared its performance to those of the Roche Amplicor HIV-1 monitor test, version 1.5 (Amplicor), and the Bayer Versant HIV-1 bDNA 3.0 assay (Versant). Seven samples which gave discrepant Amplicor versus CAP/CTM results were further evaluated by sequencing analysis. MATERIALS AND METHODS Limit of detection.To determine the limit of detection in the CAP/CTM assay, nine dilutions of the WHO reference material (HIV-1 RNA, 2nd International Standard, 97/650; NIBSC, Potters Bar, United Kingdom) or Viral Quality Assurance (VQA) standard (Rush University Medical Center, Chicago, IL) were prepared in Basematrix diluent (SeraCare Life Sciences, Milford, MA). Fourteen replicates a...

Infections with HIV-1 group M subtype B viruses account for the majority of the HIV epidemic in the Western world. Phylogeographic studies have placed the introduction of subtype B in the United States in New York around 1970, where it grew into a major source of spread. Currently, it is estimated that over one million people are living with HIV in the US and that most are infected with subtype B variants. Here, we aim to identify the drivers of HIV-1 subtype B dispersal in the United States by analyzing a collection of 23,588 pol sequences, collected for drug resistance testing from 45 states during 2004–2011. To this end, we introduce a workflow to reduce this large collection of data to more computationally-manageable sample sizes and apply the BEAST framework to test which covariates associate with the spread of HIV-1 across state borders. Our results show that we are able to consistently identify certain predictors of spread under reasonable run times across datasets of up to 10,000 sequences. However, the general lack of phylogenetic structure and the high uncertainty associated with HIV trees make it difficult to interpret the epidemiological relevance of the drivers of spread we are able to identify. While the workflow we present here could be applied to other virus datasets of a similar scale, the characteristic star-like shape of HIV-1 phylogenies poses a serious obstacle to reconstructing a detailed evolutionary and spatial history for HIV-1 subtype B in the US.

Biochemical and genetic characterizations that support the conclusion that the variant BRCA2 IVS7 ϩ 2T AE G represents a deleterious mutation are presented. RNA analysis from a breast cancer patient with BRCA2 IVS7 ϩ 2T AE G showed that the productive message was produced from only one chromosome. A haplotype analysis confirmed that the intronic variant resides on the chromosome that does not produce the normal mRNA. Additionally, an RNA splicing product that deletes exon 7 was produced by the chromosome that carries BRCA2 IVS7 ϩ 2T AE G. The deletion of exon 7 from the RNA alters the open reading frame by removing residues 249-287 and incorporating 18 abnormal amino acids before terminating with an opal stop codon. The experimental approach presented produces strong evidence of the presence of a deleterious mutation, because the contribution by both chromosomes to each RNA species analyzed was tracked using a coding region polymorphism as a marker. Furthermore, a single nucleotide polymorphism (SNP) haplotype analysis that confirms the location of the intronic variant and an associated family history that shows a high incidence of cancer supported these biochemical data.

, were compared to a modified (not FDA-approved) version of the HP assay by automating the DNA extraction using the Total Nucleic Acid Isolation (TNAI) kit on the Cobas AmpliPrep. On average, CAP/CTM measurements were 0.08 log IU/ml higher than HP results (n ‫؍‬ 206), and TNAI results were 0.17 log IU/ml higher than HP results (n ‫؍‬ 166). The limit of detection (LOD), as determined by probit analysis using dilutions of the 2nd HBV international standard, was 10.2 IU/ml for CAP/CTM. The data sets for HP and TNAI were insufficient for probit analysis; however, there was 100% detection at >5 or >10 IU/ml for TNAI and HP, respectively. Linearity was demonstrated between 60 and 2,000,000 IU/ml, with slopes between 0.95 and 0.99 and R 2 values of >0.99 for all assays. Total precision (log percent coefficient of variance [CV]) was between 0.8% and 2.1% at 4.3 log IU/ml and between 1.4% and 4.9% at 2.3 log IU/ml. Correlation of samples, reproducibility, linearity, and LOD were acceptable and similar in all assays. The CAP/CTM assay and, to a lesser extent, the TNAI assay reduced hands-on time due to automation. There were no instances of contamination detected in negative samples during the course of the study, despite testing several samples up to 9.6 log IU/ml. The incidence of false-positive negative controls in HP and CAP/CTM clinical testing was <0.5% over 6 to 7 months of testing. Hepatitis B virus (HBV) is a DNA virus that infects up to 400 million people worldwide and causes up to 5,500 deaths annually in the United States from the resulting liver failure, cirrhosis, and hepatocellular carcinoma (5). In the last decade, significant progress has been made in HBV treatment, with the development of new therapeutics (2,5,8,12) with improved genetic barriers and potency. In addition, the techniques for measuring HBV DNA levels in serum and plasma have improved. HBV DNA levels are used to predict response to therapy, to determine therapy initiation, to monitor resistance to therapy, and to establish treatment success (2,5,8,12). Since HBV DNA levels can vary from very low levels to more than 9 log IU/ml, the most recently approved assays that use real-time PCR to generate results over a large dynamic range are preferred. Other available quantitative methods use signal amplification or conventional PCR. The real-time PCR assays typically incorporate robotic automation, making them attractive for high-throughput laboratories.Roche has released two FDA-approved (in vitro diagnostic [IVD]) HBV real-time PCR viral load assays. The first assay approved was the Cobas TaqMan HBV Test for use with the High Pure System (HP) (11). HP uses a manual 12-well silica-based plate format for DNA extraction, followed by manual inoculation into PCR tubes, which are thermocycled and detected using the TaqMan 48 instrument. Most recently, the Cobas AmpliPrep/Cobas TaqMan HBV Test v2.0 (CAP/CTM) (6) integrates automated sample extraction and PCR assembly using the AmpliPrep instrument with the TaqMan instrument for thermocycling and detect...