Automated multiplex assay system for simultaneous detection of hepatitis B virus DNA, hepatitis C virus RNA, and human immunodeficiency virus type 1 RNA

We have developed an automated multiplex system for simultaneously screening hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) in blood donations. The assay, designated AMPLINAT MPX HBV/HCV/HIV-1 Test (AMPLINAT MPX), consists of virus extraction and target sequence-specific probe capture on specimen preparation workstation GT-X (Roche Diagnostics K.K., Tokyo, Japan) and amplification and detection by TaqMan PCR on the ABI PRISM 7700 Analyzer (Perkin-Elmer Applied Biosystems, Foster City, Calif.). An internal control (IC) is incorporated in the assay to monitor the extraction, target amplification, and detection processes. The assay yields qualitative results without discrimination of the three targets. Detection limits (95% confidence interval) are 22 to 60 copies/ml for HBV, 61 to 112 IU/ml for HCV, and 33 to 66 copies/ml for HIV-1, using a specimen input volume of 0.2 ml. The AMPLINAT MPX assay detects a broad range of genotypes or subtypes for all three viruses and has a specificity of 99.6% for all three viruses with seronegative specimens. In an evaluation of seroconversion panels, the AMPLINAT MPX assay detects HBV infection an average of 24 days before the detection of HBsAg by enzyme immunoassay. HCV RNA was detected an average of 31 days before HCV antibody. HIV-1 RNA was detected an average of 14 days before HIV-1 antibody and an average of 9 days before p24 antigen. The Japanese Red Cross has been evaluating the AMPLINAT MPX system since October 1999. The clinical performance indicates that the AMPLINAT MPX system is robust, sensitive, and reproducible, with a high percentage of valid assay runs (96.8%), a low false-positive rate (0.34%), and a low IC failure rate (0.24%).     

Intermittent detection of hepatitis C virus (HCV) in semen from men with human immunodeficiency virus type 1 (HIV-1) and HCV

HCV is usually transmitted via the blood, but HCV RNA has been detected recently in seminal fluid. This study was done to study HCV seminal shedding and factors that could influence the presence of HCV in the seminal fluid of men coinfected with HCV and HIV-1. HCV and HIV-1 genomes were assayed in multiple paired blood and semen samples obtained from 35 men enrolled in an assisted medical procreation protocol. HCV RNA was found intermittently in semen samples from 9 patients (25.7%). Samples from 9 men with HCV RNA in their semen and 26 men without were compared to further analyze these parameters. No correlation was found between HCV RNA in the seminal fluid and age, HCV virus load, the duration of HIV-1 infection, HIV treatment, the CD4(+) cell count, HIV-1 virus load or HIV-1 detection in the semen. The intermittent detection of HCV RNA in semen samples support the systematic search for HCV RNA in semen and the use of processed spermatozoa in assisted medical procreation of infertile HCV serodiscordant couples.     

Evaluation of a new molecular assay for detection of human immunodeficiency virus type 1 RNA, hepatitis C virus RNA, and hepatitis B virus DNA.

BACKGROUND: Rapid, sensitive, specific, and cost-effective screening of donated blood to prevent transmission of infectious agents remains challenging. In recent years, incorporation of nucleic acid testing for HIV-1 and HCV RNA improved blood safety by reducing the window period between infection and serologic detection. For HBV infection, this window period with most serologic assays is 50-60 days. Adding a nucleic acid test (NAT) for HBV DNA with existing NATs for HIV-1 and HCV RNA would further improve blood safety and blood screening efficiency. OBJECTIVE: To evaluate the Procleix Ultrio Assay for simultaneous detection of HIV-1 and HCV RNA and HBV DNA and corresponding discriminatory assays. STUDY DESIGN: The performance of these assays, which utilize the same technology and assay format as the Procleix HIV-1/HCV assay, was determined using relevant clinical specimens and analytical sensitivity and specificity panels. RESULTS: The Procleix Ultrio Assay demonstrated specificity of > or =99.5% in healthy donor blood specimens and in plasma containing potentially interfering substances or other blood-borne pathogens. Assay sensitivity demonstrated >95% detection of 100copies/mL, 30IU/mL, and 15IU/mL for HIV-1 and HCV RNA, and HBV DNA, respectively. The assay detects all known HIV-1 subtypes and HCV and HBV genotypes and is highly reproducible. Statistical analysis using receiver operating characteristic plots demonstrated wide analyte cutoff values for each assay associated with assay specificity and sensitivity of > or =99.5%. CONCLUSIONS: In this investigational study, the Procleix Ultrio Assay sensitivity and specificity were similar to existing NATs used in blood-bank settings to detect HIV-1 and HCV RNA and provided equivalent sensitivity and specificity for detection of HBV DNA. Using this combination assay, blood safety may be improved and the multiplex format enhances blood screening efficiency. The throughput capability of this assay is compatible with large volume processing and the chemistry is adaptable to full automation.     

Multicenter evaluation of the new Abbott RealTime assays for quantitative detection of human immunodeficiency virus type 1 and hepatitis C virus RNA

The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS TaqMan assays. Two different protocols used during the testing period with and without a pre-m1000 RNA isolation spin were compared. The difference proved to be nonsignificant. A uracil-N-glycosylase (UNG) contamination control option in the HCV test for previous Roche COBAS Amplicor users was evaluated. It proved to decrease amplicon carryover by 100-fold independent of the amplicon input concentration. The protocol including UNG proved to overcome problems with false-positive negative controls. Comparison with other assays revealed only minor differences. The largest difference was observed between the Abbott HCV RealTime assay and the Roche COBAS Amplicor HCV Monitor version 2.0 assay.     

Evaluation of the Abbott RealTime HCV assay for quantitative detection of hepatitis C virus RNA.

BACKGROUND: The Abbott RealTime HCV assay for quantitative detection of HCV RNA has recently been introduced. OBJECTIVES: In this study, the performance of the Abbott RealTime HCV assay was evaluated and compared to the COBAS AmpliPrep/COBAS TaqMan HCV test. STUDY DESIGN: Accuracy, linearity, interassay and intra-assay variations were determined, and a total of 243 routine clinical samples were investigated. RESULTS: When accuracy of the new assay was tested, the majority of results were found to be within +/-0.5 log(10) unit of the results obtained by reference laboratories. Determination of linearity resulted in a quasilinear curve up to 1.0 x 10(6)IU/ml. The interassay variation ranged from 15% to 32%, and the intra-assay variation ranged from 5% to 8%. When clinical samples were tested by the Abbott RealTime HCV assay and the results were compared with those obtained by the COBAS AmpliPrep/COBAS TaqMan HCV test, the results for 93% of all samples with positive results by both tests were found to be within +/-1.0 log(10) unit. The viral loads for all patients measured by the Abbott and Roche assays showed a high correlation (R(2)=0.93); quantitative results obtained by the Abbott assay were found to be lower than those obtained by the Roche assay. CONCLUSIONS: The Abbott RealTime HCV assay proved to be suitable for use in the routine diagnostic laboratory. The time to results was similar for both of the assays.     

Evaluation of the new VERSANT HIV-1 RNA 1.0 Assay (kPCR) for quantitative detection of human immunodeficiency virus type 1 RNA

BackgroundThe VERSANT HIV-1 RNA 1.0 Assay (kPCR) for quantitative detection of HIV-1 RNA has recently been introduced.ObjectivesIn this study, the performance of the VERSANT HIV-1 RNA 1.0 Assay (kPCR) was evaluated and compared to the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, version 2.0.Study designAccuracy, linearity, interassay and intra-assay variations were determined, and a total of 196 routine clinical samples including a high number of HIV-1 subtype non-B samples were investigated.ResultsWhen accuracy of the new kit was tested, all of the quantifiable results were found to be within ?0.5 log₁₀ unit of the expected panel results. Determination of linearity resulted in a quasilinear curve up to the initial concentration of 3.4 × 10⁵ copies/mL. The interassay variation ranged from 12 to 20%, and the intra-assay variation ranged from 8 to 16%. When clinical samples were tested by the VERSANT HIV-1 RNA 1.0 Assay (kPCR) and the results were compared with those obtained by the COBAS AmpliPrep/COBAS TaqMan HIV-1 test, version 2.0, the results for 95% of all samples with positive results by both tests were found to be within ±1.0 log₁₀ unit. The viral loads for all samples measured by the Siemens and Roche assays showed a high correlation (R² = 0.94); quantitative results obtained by the Siemens assay were usually found to be lower than those obtained by the Roche assay.ConclusionsThe new VERSANT HIV-1 RNA 1.0 Assay (kPCR) proved to be suitable for use in the routine diagnostic laboratory. The time to results was similar for both of the assays.     

Quantitation of hepatitis C virus RNA in saliva and serum of patients coinfected with HCV and human immunodeficiency virus

The presence and the quantity of hepatitis C virus (HCV) RNA were investigated in saliva and serum of patients infected with both HCV and human immunodeficiency virus (HIV). Paired serum and saliva samples were collected from 59 HIV-HCV coinfected patients. HCV RNA was detected by nested-PCR, using primers derived from the 5' non-coding region of HCV, and positive results were quantified using the b-DNA method. HCV RNA was detected in the saliva of 22/59 (37.3%) patients, with a mean level of 1.15 x 10(6) genome equivalents/ml; there was no correlation of salivary positivity with immune status (CD4 cell count), age or HIV risk group, but there was with gender (19/38 [50%] positive results in male, compared to 3/21 [14.3%] in female, P = 0.007). HCV RNA was detected in the serum of 45/59 (76.3%) patients at a higher level (mean of 2.52 x 10(7) genome equivalents/ml) compared to saliva. Positivity was not correlated with age, gender or CD4 + cell count. There was a correlation between qualitative saliva and serum results (P = 0.003), but not between quantifications (P = 0.57). This first study reporting significant amounts of HCV RNA in saliva could have important implications for HCV epidemiology.     

Evaluation of the VERSANT HCV RNA 3.0 assay for quantification of hepatitis C virus RNA in serum

We assessed the performance of a new assay (VERSANT HCV RNA 3.0 [bDNA 3.0] assay [Bayer Diagnostics]) to quantitate HCV RNA levels and compared the results of the bDNA 3.0 assay to results of the Quantiplex HCV RNA 2.0 (bDNA 2.0) assay. Samples used in this study included 211 serum specimens from hepatitis C virus (HCV)-infected persons from two sites (Bordeaux and Marseille, France) with different genotypes; 383 serum specimens from HCV antibody-negative, HCV RNA-negative persons; and serial dilutions of World Health Organization (WHO) HCV RNA standard at a titer of 100,000 IU/ml. The specificity of the bDNA 3.0 assay was 98.2%. A high correlation was observed between expected and observed values in all dilutions of WHO standard (r = 0.9982), in serial dilutions of pooled samples (r = 0.9996), and in diluted sera from different HCV genotypes (r = 0.9930 to 0.9995). The standard deviations (SD) for the within-run and between-run reproducibility of the bDNA 3.0 assay were 相似文献   

Evaluation of the quantiplex human immunodeficiency virus type 1 RNA 3.0 assay in a tertiary-care center

The Quantiplex human immunodeficiency virus type 1 RNA 3.0 Assay (bDNA) (Bayer Diagnostics, Walpole, Mass.) produced linear and reproducible (intra-assay and interassay) results over its quantification range of 50 to 500,000 copies/ml of plasma with 96% specificity. A threefold or 0.5-log(10) change or greater was clinically significant for serial patient samples.     

Multicenter evaluation of the performance characteristics of the NucliSens HIV-1 QT assay used for quantitation of human immunodeficiency virus type 1 RNA

The analytical performance of the NucliSens HIV-1 QT assay, a highly sensitive test based on nucleic acid sequence-based amplification technology, was evaluated in a multicenter trial. Assay specificity was evaluated with 502 plasma (EDTA) specimens from human immunodeficiency virus type 1 (HIV-1)-seronegative volunteer donors. No HIV-1 RNA was reported in any of the donor specimens. Analytical sensitivity and reproducibility were estimated with panels prepared from a high-titer well-characterized HIV-1 RNA stock (5.84 x 10(8) RNA copies/ml). The assay's dynamic range was linear from 10(6) to 10(1) HIV-1 RNA copies, with a lower detectable limit of 25 copies/ml and a 95% detection rate of 176 copies/ml. Sensitivity of the assay to detect HIV-1 RNA in clinical specimens from patients (n = 101) and in commercially available or prepared panels (n = 24) was compared with NASBA HIV-1 RNA QT (an earlier version of NucliSens HIV-1 QT) and with the Food and Drug Administration-approved standard and ultrasensitive AMPLICOR HIV-1 MONITOR, version 1.0, assays. Detection of HIV-1 RNA was reproducible over a 5-log range (mean standard deviation = 0.15 log). The NucliSens and the standard AMPLICOR assays were equivalent in detection of HIV-1 RNA (concentration, 10(3) to 10(5) copies/ml) in 57 clinical specimens. The NucliSens assay was more sensitive in detecting HIV-1 RNA at lower concentrations (相似文献   

Performance of a quantitative human immunodeficiency virus type 1 p24 antigen assay on various HIV-1 subtypes for the follow-up of human immunodeficiency type 1 seropositive individuals

The heat-denatured signal-amplified p24 antigen assay is a low-cost test allowing the determination of plasma levels of HIV-1 p24 antigen in infected patients. This assay may be appropriate for monitoring disease progression in HIV seropositive patients in developing countries. Only a few data on the clinical validation of the test are available for HIV-1 non-subtypes B viruses that represent the vast majority of virus circulating in Africa. The present study was undertaken to evaluate and compare the performance of a heat-denatured signal-amplified p24 assay for the determination of p24 viral load in the plasma of individuals infected with different subtypes of HIV-1 and using the RT-PCR-based RNA viral load test as the gold standard. A total of 120 plasma samples from individuals infected with HIV-1 strains belonging to group M (subtypes A-->H) and group O, as well as recombinant strains, were tested in parallel with the heat-denatured signal-amplified p24 assay and the RNA viral load. Plasma p24 levels appeared to be correlated significantly with the plasma RNA viral loads (R=0.751, P<0.0001). The heat-denatured p24 antigen assay was capable of measuring the plasma level of p24 derived from all the HIV-1 subtypes and recombinants selected for this study, in contrast to the RNA viral load test which lacked sensitivity towards HIV-1 group O. The heat-denatured signal-amplified p24 assay is a reliable, sensitive and a more affordable tool that can be used for the follow-up of patients infected with B and non-B subtypes as well as recombinant forms of HIV-1 in developing countries.     

Differing patterns of liver disease progression and hepatitis C virus (HCV) quasispecies evolution in children vertically coinfected with HCV and human immunodeficiency virus type 1

Hepatitis C virus (HCV) quasispeciation was studied in two children vertically coinfected with HCV and human immunodeficiency virus type 1 (HIV-1). HCV quasispecies diversification and liver injury were more significant in patient C1, who was immunocompetent with anti-HIV therapy, than in patient C2, who was immunosuppressed, in consistency with modulation of HCV quasispeciation and liver injury by immunocompetence in coinfected children.     

An enhanced-sensitivity branched-DNA assay for quantification of human immunodeficiency virus type 1 RNA in plasma.

The quantification of human immunodeficiency virus type 1 (HIV-1) RNA has facilitated clinical research and expedited the development of antiretroviral drugs. The branched-DNA (bDNA) assay provides a reliable method for the quantification of HIV-1 RNA in human plasma and is considered one of the most reproducible assays ready for use in clinical trials. A series of oligonucleotide probe design and solution changes have been developed to enhance the sensitivity of the bDNA assay while maintaining its performance characteristics. Among the changes incorporated into the enhanced-sensitivity bDNA (ES bDNA) assay to reduce the background level and enhance the signal are the use of shorter overhang sequences of target probes for capture, the cruciform design of target probes for amplification, and the addition of preamplifier molecules. The ES bDNA assay is at least 20-fold more sensitive than the first-generation bDNA assay, yet it maintains a high level of accuracy, linearity, and reproducibility. Further, quantification values obtained with the ES bDNA assay and the first-generation bDNA assay are highly correlated, thus allowing for meaningful comparisons of HIV-1 RNA levels in specimens tested with either assay. The ES bDNA assay may be useful in determining the prognostic value of HIV-1 RNA levels of below 10,000 copies per ml and in assessing the clinical benefit of antiretroviral therapy-induced decreases in plasma HIV-1 RNA sustained at levels of below 10,000 copies per ml.