Expertise of laboratories in viral load quantification, genotyping, and precore mutant determination for hepatitis B virus in a multicenter study

A national evaluation study was performed in 14 specialized laboratories with the objective of assessing their capacities to provide (i) hepatitis B virus (HBV) viral loads (VL), (ii) HBV genotypes, and(iii) identification of precore/core mutants. The panel consisted of 12 HBV DNA-positive samples with VLs from 2.8 to 9.1 log(10) copies/ml, different HBV genotypes (A to F), and 3 mutant and 9 wild-type samples at nucleotide 1896. The coefficients of variation of the mean VLs ranged from 2.4% to 10.4% with the Cobas HBV Monitor assay, from 1.8% to 5.5% with the Cobas TaqMan 48, from 1.5 to 26.2% with RealArt HBV PCR, and from 0 to 7% with branched DNA (bDNA). The Cobas Monitor assay underestimated the VLs of genotype F samples, with differences ranging from 1.4 to 2.4 log(10) copies/ml. The accuracies of genotype determinations ranged from 33% to 100%, and those of precore mutant determinations ranged from 25 to 100%. This study showed some drawbacks of two widely used assays: (i) Cobas Monitor has a narrow dynamic range and underestimates genotype F sample VLs and (ii) bDNA shows poor sensitivity and may fail to identify patients with low VLs. With higher performance in terms of analytical sensitivity combined with a larger dynamic range and an ability to quantify the main genotypes equally, real-time PCR methods appear more appropriate for accurate monitoring of HBV DNA quantification. Furthermore, the clinical implications of HBV genotyping and the determination of precore/core mutants need to be clearly stated to justify the standardization of these methods.     

Correlation between detection of antibodies against hepatitis C virus in oral fluid and hepatitis C virus RNA in serum

Reported here are the results of a study designed to determine the correlation between hepatitis C virus (HCV) RNA positivity in serum and the detection of antibodies against HCV in oral fluid by testing paired serum/oral fluid samples. For the 85 serum samples found positive for antibodies against HCV, using a screening assay and a confirmation assay, 70 of the corresponding oral fluid samples tested positive for HCV antibodies using a previously modified screening assay. For 52 of the 59 serum samples found positive for HCV RNA, the corresponding oral fluid samples also tested seropositive for HCV, while 18 of the 26 serum samples that were negative for HCV RNA had corresponding oral fluid samples that tested seropositive for HCV. For the control group of 54 serum samples that were negative for HCV antibodies, all of the corresponding oral fluid samples were also negative for HCV antibodies, while 53 of the serum samples tested negative for HCV RNA. These results suggest that HCV antibody detection in oral fluid has a slightly higher sensitivity when used to test patients whose serum samples are positive for HCV RNA (chi-square test, p=0.035; Mid-P exact, p=0.049).     

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   

Strengths and limitations of commercial tests for hepatitis C virus RNA quantification

The sensitivity of the Roche COBAS Monitor v.2.0 test was slightly better than that of the Bayer bDNA 3.0 test, but the Monitor test underquantified specimens by 2.5- to 10.6-fold even at relatively low hepatitis C virus RNA concentrations. Dilution prior to assay minimally decreased the reproducibility of the Monitor assay, leading to the recommendation for all specimens to be diluted 1:100 prior to testing by this assay.     

Investigation on HPV DNA detection and genotyping practices used in French laboratories in 2009

The French National reference Laboratory for Human papillomavirus (HPV) performed in 2009 a national study in order to review the methods used to detect and identify HR HPV genotypes in microbiology laboratories. Results from this study show a great diversity in volumes of samples treated in laboratories. Among clinical indications, the most frequent is a result of ASC-US at a Pap smear. This indication in the only one covered by the National Public Insurance System and is mostly performed in laboratories from private sector. Other indications mainly correspond to research programs and are performed in public Hospitals. This study allowed also to review the adequacy between the liquid based cytology samples and the assays used for direct detection of HR HPV or identification of the genotypes present in the sample. The right tests were not carried in the right solution storage according to the recommendations from different HPV testing assays. National recommendations should be elaborated in order to improve the performance of the test used.     

Performance of an automated system for quantification of hepatitis C virus RNA

The Amplicor HCV Monitor test for quantitative determination of serum or plasma hepatitis C virus (HCV) RNA was modified recently and introduced onto the Cobas Amplicor instrument to automate fully amplification, detection and calculation of results. This new version (v2.0) was evaluated in a routine diagnostic laboratory. The sensitivity and reproducibility were assessed on well-characterized panels (Eurohep) and clinical samples. HCV RNA levels measured by the v2.0 Monitor test were about 1log(10) higher than those detected by the previous version test, and genotypes 1 and 3 were quantified with equal sensitivity. Within the linear dynamic range of 10(3) to 10(6) copies/ml, the coefficients of variation for both intra- and inter-assay reproducibility ranged from 1.9 to 2.95%. This test system was found to be a reliable and labor saving assay for the quantification of HCV RNA.     

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%).     

Simple and reliable method for detection and genotyping of hepatitis C virus RNA in dried blood spots stored at room temperature

We describe a simple, sensitive, and reproducible method for using whole blood collected onto filter paper (dried blood spots) for detection and genotyping of hepatitis C virus RNA that can be useful in large field studies, particularly in settings where collection, preparation, storage, and shipment of samples at controlled temperature can be difficult.     

Routine detection and quantification of hepatitis B virus DNA in clinical laboratories: performance of three commercial assays

The detection and quantification of hepatitis B virus (HBV) genomes in molecular biology-based assays appear to be the most reliable methods for monitoring HBV infection and assessing responses to antiviral treatment. The aim of this study was to evaluate the performance of three HBV-DNA detection and quantification assays currently used for the management of HBV-infected patients: a solution-hybridization assay based on hybrid-capture (Digene Hybrid-Capture, Murex Diagnostics, Dartford, UK); a signal-amplification assay based on 'branched-DNA' (bDNA) technology (Quantiplex HBV DNA, Bayer Diagnostics, Emeryville, CA); and a target-amplification assay based on competitive polymerase chain reaction (Amplicor HBV Monitor, Roche Molecular Systems, Pleasanton, CA). The Monitor assay was significantly more sensitive than both the hybrid-capture and bDNA methods. This better sensitivity appeared to be clinically relevant. The linear ranges of quantification in the hybrid-capture, bDNA and Monitor methods were 6.5-9 log10 genome copies/ml, 6.5-9.5 log10 genome equivalents/ml, and 3-5.5 log10 genome copies/ml, respectively. However, the HBV-DNA units used in the three assays were not comparable. The specificity of the hybrid-capture, bDNA and Monitor assays was 99.2% (95% confidence interval: 97.7-100.0%), 99.2% (97.7-100.0%), and 97.8% (95.3-100%), respectively. Their within-run coefficients of variation and log10 SDs were 5.5% (+/- 0.025 log10 copies/ml), 6.7% (+/- 0.029 log10 Eq/ml) and 21.0% (+/- 0.093 log10 copies/ml), respectively. Between-run coefficients of variation ranged from 4.4-39.1%, 5-39.5%, and 17.8-96.1%, respectively. The competitive PCR-based Monitor assay appears to be significantly more sensitive but slightly less specific and reproducible than the hybrid-capture and bDNA methods. Given their respective performance, these three assays should be used in complementary fashion in the management of HBV-infected patients.     

In situ detection of hepatitis C virus RNA in salivary glands

Chronic hepatitis C virus (HCV) infection has been associated with several extrahepatic manifestations, among these, to diseases with oral manifestations such as Sj?gren's syndrome or sialadenitis. HCV-RNA has been detected in saliva and in salivary glands from patients with sialadenitis by polymerase chain reaction. However, morphological evidence of HCV replication in salivary gland cells is needed to support a role for HCV in causing sialadenitis or Sj?gren's syndrome. We have used in situ hybridization and immunohistochemistry to analyze the presence of HCV-RNA of sense and antisense polarity and HCV core antigen, respectively, in salivary gland biopsies from 19 patients with chronic sialadenitis or Sj?gren's syndrome (eight anti-HCV-positive; 11 anti-HCV-negative). HCV-RNA of both positive and negative polarity as well as HCV core antigen were detected in the epithelial cells of the salivary gland biopsies from all of the anti-HCV-positive patients but in none of the anti-HCV-negative cases. The percentage of HCV-infected cells ranged from 25 to 48.8% in the patients studied. In conclusion, we have shown that HCV infects and replicates in the epithelial cells from salivary glands of patients with Sj?gren's syndrome or chronic sialadenitis. However, its implication in the pathogenesis of these diseases deserves future research.     

GEMHEP multicenter quality control study of PCR detection of GB virus C/hepatitis G virus RNA in serum.

PCR is, to date, the only available tool for the detection of GB virus C (GBV-C) and hepatitis G virus (HGV) RNAs. Twenty-two French laboratories participated in a quality control study to assess the sensitivity and specificity of their procedures. The panel included 13 positive controls and 7 negative controls. The laboratories used either in-house PCR techniques adapted from the literature or partly standardized commercial tests. Three laboratories performed faultlessly with the entire panel. Most laboratories had excellent specificity (100% in 20 of 22 laboratories). Sensitivity was acceptable (85 to 100%) in 15 centers and insufficient (38 to 77%) in 7. As with nonstandardized in-house PCR, the commercial assays gave discrepant performances in different laboratories. These results suggest that laboratories willing to use PCR for detection of GBV-C/HGV RNA for research or diagnostic purposes should participate in multicenter quality control trials.     

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.     

Comparative evaluation of the VERSANT HCV RNA 3.0, QUANTIPLEX HCV RNA 2.0, and COBAS AMPLICOR HCV MONITOR version 2.0 Assays for quantification of hepatitis C virus RNA in serum

A comparison of quantitative results expressed in hepatitis C virus (HCV) international units per milliliter, obtained from the VERSANT HCV RNA 3.0 (bDNA-3.0) assay, the QUANTIPLEX HCV RNA 2.0 (bDNA-2.0) assay, and the COBAS AMPLICOR HCV MONITOR version 2.0 (HCM-2.0) test was performed. A total of 168 patient specimens submitted to the Mayo Clinic Molecular Microbiology Laboratory for HCV quantification or HCV genotyping were studied. Of the specimens tested, 97, 88, and 79% yielded quantitative results within the dynamic range of the bDNA-3.0, bDNA-2.0, and HCM-2.0 assays, respectively. Overall, there was substantial agreement between the results generated by all three assays. A total of 15 out of 29 (52%) of the specimens determined to contain viral loads of <31,746 IU/ml by the bDNA-3.0 assay were categorized as containing viral loads within the range of 31,746 to 500,000 IU/ml by the bDNA-2.0 assay. Although substantial agreement was noted between the results generated by the bDNA-2.0 and bDNA-3.0 assays, a bias toward higher viral titer by the bDNA-2.0 assay was noted (P = 0.001). Likewise, although substantial agreement was noted between the results generated by the HCM-2.0 and bDNA-3.0 assays, a bias toward higher viral titer by the bDNA-3.0 assay was noted (P < or = 0.001). The discrepancy between the HCM-2.0 and bDNA-3.0 results was more pronounced when viral loads were >500,000 IU/ml and resulted in statistically significant differences (P < or = 0.001) in determining whether viral loads were above or below 800,000 IU/ml of HCV RNA, the proposed threshold value for tailoring the duration of combination therapy. The expression of quantitative values in HCV international units per milliliter was a strength of both the bDNA-3.0 and HCM-2.0 assays.     

Simultaneous amplification and detection of specific hepatitis B virus and hepatitis C virus genomic sequences in serum samples

A fluorescent antibody (FA) assay for hepatitis E virus antigen (HEVAg) in infected liver tissue was used to confirm the presence of virus-specific antigens in hepatocytes during the course of infection. With the cloning of the HEV genome it is now possible to determine which viral antigens are recognized by this FA assay. Recombinant HEV proteins covering the carboxyl half of HEV open reading frame 2 (ORF2) were used in this study to demonstrate that some of the most immunoreactive virus-specific antigens detected by FA are contained within this region of ORF2 (nucleotides 6169–7126).