Quantitative detection of serum HBV DNA levels employing a new S gene based cPCR assay

Summary. Hepatitis B virus (HBV) infection is a major public health problem and a leading cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma. Worldwide, there are about 350 million carriers of this pathogen and India bears the second highest carrier pool in the world. Early diagnosis and measurement of viral load in hepatitis B patients is very helpful for the better management of this disease. The existing methods for viral quantification are either cumbersome or expensive. Since viral replication correlate well with HBV DNA levels a new sensitive, reliable and cost effective competitive PCR assay has been developed for quantifying the viral load in the serum of hepatitis B patients. The S gene based cPCR assay was able to detect as low as 100 genome equivalent/ml of HBV DNA from human serum and was applied to determine viral load among inactive and chronic hepatitis B carriers demonstrating the usefulness of the developed test.     

Evaluation of the COBAS amplicor HBV monitor assay and comparison with the ultrasensitive HBV hybrid capture 2 assay for quantification of hepatitis B virus DNA

Performance characteristics of the COBAS Amplicor HBV Monitor test (Roche Diagnostics), which measures hepatitis B virus (HBV) DNA quantitatively, were evaluated and compared with the Ultrasensitive HBV Hybrid Capture 2 (HC2; Digene Corporation) assay. Linearity and within-run precision were assessed for both methods by using eight HBV DNA-positive samples serially diluted to obtain a range of <100 to 500,000 HBV DNA copies/ml and run in triplicate. Agreement between the methods was studied with 100 clinical samples. HC2 assay performance near the limit of detection was investigated through repeat testing of 149 samples with HC2 and testing of 37 samples with HC2 results of <4,700 HBV DNA copies/ml by Amplicor assay and a qualitative PCR assay. The linearity experiment for Amplicor had regression of observed values compared to expected values (y = 1.073x - 0.247; R(2) = 0.993, n = 32; for HC2, y = 0.855x + 0.759, R(2) = 0.729, n = 18). Within-run standard deviation of log HBV DNA copies/ml ranged from 0.003 to 0.348 (Amplicor) and 0.027 to 0.253 (HC2). Agreement assessed by Deming regression was poor [Amplicor = 1.197(HC2) - 0.961; R(2) = 0.799, standard error of the estimate (SEE) = 0.710, n = 94]. Near the lower limit of detection, 32 of 149 repeat HC2 results were <4,700 HBV DNA copies/ml. Of the 37 samples with HC2 results of <4,700 HBV DNA copies/ml, HBV DNA was not detected in 15 samples, while HBV DNA was detected by at least one PCR method in 12 samples. Amplicor is linear from 200 to 200,000 HBV DNA copies/ml with undiluted samples, and this range can be expanded through dilution. Inconsistent HC2 results near the limit of detection justify use of a grey zone.     

Development and assessment of a novel real-time PCR assay for quantitation of HBV DNA

HBV DNA quantitation is used extensively for the monitoring of treatment of hepatitis B virus (HBV) infection. The aim of this study was to develop a highly sensitive and reproducible real-time PCR (RTD-PCR) assay for the quantitation of HBV DNA using the LightCycler system. The performance of this assay was assessed by analyzing serial dilutions of HBV genomic DNA of known concentration and the lower limit of detection was found to be 1 DNA copy/reaction. By using serial dilutions of plasmid standard, RTD-PCR was determined to quantify HBV DNA in a 10-log10 dynamic range. RTD-PCR was found to be more sensitive than the commercially available tests such as the Quantiplex HBV DNA and the AMPLICOR HBV MONITOR assays. The median coefficient of variation of interexperimental variability was 3.2%. The HBV DNA values obtained with RTD-PCR were highly correlated with assays available commercially. These findings suggest that our RTD-PCR assay combines high sensitivity and reproducibility for HBV DNA quantitation in an incomparable high dynamic range of quantitation.     

COBAS AmpliPrep-COBAS TaqMan hepatitis B virus (HBV) test: a novel automated real-time PCR assay for quantification of HBV DNA in plasma

Success in antiviral therapy for chronic hepatitis B is supported by highly sensitive PCR-based assays for hepatitis B virus (HBV) DNA. Nucleic acid extraction from biologic specimens is technically demanding, and reliable PCR results depend on it. The performances of the fully automatic system COBAS AmpliPrep-COBAS TaqMan 48 (CAP-CTM; Roche, Branchburg, NJ) for HBV DNA extraction and real-time PCR quantification were assessed and compared to the endpoint PCR COBAS AMPLICOR HBV monitor (CAHBM; Roche). Analytical evaluation with a proficiency panel showed that CAP-CTM quantitated HBV DNA levels in one single run over a wide dynamic range (7 logs) with a close correlation between expected and observed values (r = 0.976, interassay variability below 5%). Clinical evaluation, as tested with samples from 92 HBsAg-positive patients, demonstrated excellent correlation with CAHBM (r = 0.966, mean difference in quantitation = 0.36 log(10) IU/ml). CAP-CTM detected 10% more viremic patients and longer periods of residual viremia in those on therapy. In lamivudine (LAM)-resistant patients, the reduction of HBV DNA after 12 months of Adefovir (ADF) was higher in the combination (LAM+ADF) schedule than in ADF monotherapy (5.1 logs versus 3.5 logs), suggesting a benefit in continuing LAM. CAP-CTM detected HBV DNA in liver biopsy samples from 15% of HBsAg-negative, anti-HBcAg-positive graft donors with no HBV DNA in plasma. The amount of intrahepatic HBV DNA was significantly lower in occult HBV infection than in overt disease. CAP-CTM can improve the management of HBV infection and the assessment of antiviral therapy and drug resistance, supporting further insights in the emerging area of occult HBV infection.     

Analytical performance of the VERIS MDx system HCV assay for detecting and quantifying HCV RNA

BackgroundThe diagnosis of HCV relies on the detection of viral RNA.ObjectiveTo evaluate the performance of the VERIS/MDx System HCV Assay, a new automated system for quantifying HCV RNA, and to compare with the COBAS^® Ampliprep/COBAS^® Taqman™ (CAPCTM) HCV Test version 2.0.Study designThe limit of detection was determined by Probit analysis with the 3rd International WHO HCV standard and precision by assaying in duplicate control samples with HCV RNA concentrations of 7.9; 5.0; 3.4; 1.6 and 0 log IU/ml over 20 days. Analytical specificity was assessed by assaying 180 samples from negative anti-HCV and HCV RNA blood donors and linearity with replicates of serial dilutions of a clinical plasma (6.4–0.6 log IU/ml). We compared the VERIS MDx HCV and CAPCTM HCV assays by testing 209 samples.ResultsThe limit of detection was 6.1 IU/ml [CI 95%: 5.0–8.3] and the precision, given by the standard deviation, was ≤0.11 log IU/ml. Specificity was 100%. The linearity ranged from 1.5 to 6.4 log IU/ml. Passing-Bablok regression analysis gave: VERIS log IU/ml = −0.33 + [1.04× CAPCTM] log IU/ml, with biases for the 25th, 50th, 75th percentiles of 0.18, −0.10 and −0.06 log IU/ml. The two assays were well correlated (ρ = 0.92, p < 0.001) and Bland-Altman analysis gave biases of 0.12, log IU/ml for genotype 1, −0.19 for genotype 2, −0.26 for genotype 3, and −0.77 for genotype 4.ConclusionThe VERIS MDx HCV assay performed well. But, we observed an under-quantification of the genotype 4 samples.     

Assessment of the target-capture PCR hepatitis B virus (HBV) DNA quantitative assay and comparison with commercial HBV DNA quantitative assays

Recent clinical studies suggest that hepatitis B virus (HBV) load and genotype may be independent predictors of responses to antiviral therapies. However, it is difficult for clinicians to accurately determine viral loads in patient samples because results--both the values and the units of measure--can vary greatly among different tests. Accordingly, the World Health Organization (WHO) has produced the first international standard for HBV DNA for nucleic acid amplification technology (NAT) assays. In the present study, we describe the performance of the target-capture PCR HBV DNA quantitative assay for the quantitation of HBV DNA in clinical samples and reference panels. The range of quantitation was between 50 and 10(10) IU/ml. The sensitivity and accuracy of the target-capture PCR assay were demonstrated by using the HBV panel from Quality Control for Medical Diagnostics (QCMD) and the WHO HBV DNA standard. The target-capture PCR assay quantitated the six genotype A members of the QCMD panel and dilutions of the WHO HBV DNA standard within an accuracy of 74 to 142%. Compared to current serological methods, the assay offers window period reductions of 19 days prior to HBV surface antigen and 26 days prior to HBV e antigen detection. The target-capture PCR assay was also compared with four commercially available NAT assays, and the various units of measure were standardized with respect to the international units of the WHO HBV DNA standard. The target-capture PCR assay is a sensitive, accurate, high-throughput, rapid, and reproducible assay for the determination of HBV loads.     

Evaluation of the COBAS AmpliPrep-total nucleic acid isolation-COBAS TaqMan hepatitis B virus (HBV) quantitative test and comparison to the VERSANT HBV DNA 3.0 assay

Quantitative detection of hepatitis B virus (HBV) in serum or plasma has become the most direct and reliable method for monitoring chronic hepatitis B. Here, we report the performance characteristics of a real-time PCR hepatitis B DNA quantitative assay, the COBAS TaqMan (CTM) HBV test (Roche Diagnostics, Meylan, France), in combination with an automated DNA extraction on the COBAS AmpliPrep (CAP) instrument using the total nucleic acid isolation kit (TNAI kit), a generic reagent for nucleic acid isolation (both from Roche Diagnostics). The linearity, accuracy, and specificity of the CAP-TNAI-CTM HBV test were evaluated using various reference panels and standards (HBV panel 2004 from Quality Control for Molecular Diagnostics, OptiQuant HBV panel from AcroMetrix, WHO International Standard for HBV, and Teragenix hepatitis B genotype panel). Quantitative results show that the CAP-TNAI-CTM HBV test performed well with respect to linearity, accuracy, and reproducibility from at least 100 to 500,000 HBV DNA IU/ml. Based on the log(10) IU of HBV DNA/ml measured, the intra-assay variation ranged from 2.49% to 8.46% and the interassay variation ranged from 1.88% to 7.83%. The test was extremely sensitive and could detect samples containing HBV DNA below the reported quantification threshold (<30 IU/ml). All HBV genotypes were correctly amplified, and no cross-contamination occurred during the automated sample preparation. In addition, 402 human serum samples were tested comparatively to the VERSANT HBV DNA 3.0 assay (bDNA; Bayer Diagnostics, Puteaux, France). The viral load results of the CAP-TNAI-CTM test and bDNA were significantly correlated, but the agreement between the two tests was poor, with large differences between results for individual samples. The hands-on time was estimated to be reduced from 2.30 h with bDNA to 45 min with the CAP-TNAI-CTM test, and up to 84 samples were completely processed within a working day. Overall, the performance characteristics of the CAP-TNAI-CTM test demonstrated that it provides a high-throughput sensitive and reliable method for quantitation of HBV DNA levels in the routine molecular laboratory.     

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.     

Evaluation of the performance of a new automated HIV combination assay

BackgroundMore and more countries test for HIV infection using combination assays that simultaneously detect p24 antigen and HIV antibodies.ObjectiveTo assess the performance of a new HIV combo assay: LIAISON^® XL murex HIV Ab/Ag HT.Study designThe assays were examined with a total of 3090 samples that included 769 selected HIV antibody-negative samples, 1849 unselected HIV samples, 15 HIV-1 p24 Ag reference samples, 90 primary HIV-1 infection (PHI) samples, 167 HIV-1 antibody-positive samples (well characterized of groups M and O), 95 HIV-1 antibody-positive samples and 105 HIV-2 antibody-positive samples.ResultsThe specificity of the LIAISON^® XL murex HIV Ab/Ag HT was 99.7%. The analytical sensitivity of Ag p24 of the LIAISON^® XL murex HIV Ab/Ag HT was 0.58 IU/mL and 9.93 pg/mL when using WHO and French national standards, respectively. All screened HIV subtypes was identified by this assay. Also, 90 PHI specimens were detected by this screening assay.ConclusionThe sensitivity and specificity of the LIAISON^® XL murex HIV Ab/Ag HT assay are high. Hence the assay offers automated high-throughput screening with ability to detect primary infection.     

Evaluation of a fluorescent DNA hybridization assay for the detection ofNeisseria gonorrhoeae

This study evaluates a four-hour fluorescent DNA hybridization assay using both known bacterial isolates and clinical specimens. A biotinylated oligonucleotide probe from a sequence of the plasmid-encoded genecppB was used. Hybrids were detected by addition of a streptavidin-alkaline phosphatase conjugate, followed by incubation for 30 min in a fluorescent substrate for alkaline phosphatase. The level of detection of the fluorescent assay was 0.1 pg of cryptic plasmid DNA or 200 cfu of the plasmid-containing strain NG 34/85 ofNeisseria gonorrhoeae. A total of 119 reference strains ofNeisseria gonorrhoeae and other related bacteria were tested for reactivity with the probe. AllNeisseria gonorrhoeae strains, including eight plasmid-free strains, hybridized with the probe. Fluorescence ratios were 2.67 for plasmid-free strains and 3.85 for plasmid-containing strains. Of the heterologous microorganisms tested, only one of six strains ofNeisseria cinerea gave a fluorescence ratio above the 2.0 cut-off value for positivity with the probe at a cell density of 1 × 10⁴ cfu. The probe was also evaluated using clinical specimens from 100 patients attending a clinic for sexually transmitted diseases. The sensitivity of the assay was 100 % while the specificity was 97.5 %. Positive and negative predictive values were 91.2 % and 100 %, respectively. The fluorescent DNA hybridization assay for the detection ofNeisseria gonorrhoeae described here thus appears to be a highly specific and sensitive assay.     

Evaluation of a commercial DNA probe assay for the identification ofMycobacterium kansasii

A commercially available DNA probe (the Accu-ProbeMycobacterium kansasii culture identification test, Gen-Probe, USA) for the identification ofMycobacterium kansasii was tested on a panel of 143 fully characterized mycobacterial strains. The isolates included 70Mycobacterium kansasii and 73 mycobacteria other thankansasii. The specificity was 100 % while the sensitivity was 72.8 %. This sensitivity is unusually low in comparison with that of commercial DNA probes for other mycobacteria and confirms a previous study that found genetic heterogeneity within the speciesMycobacterium kansasii. Strains that do not hybridize with the AccuProbe are particularly prevalent in Italy and perhaps elsewhere in Europe.     

Dynamic range and reproducibility of hepatitis B virus (HBV) DNA detection and quantification by Cobas Taqman HBV, a real-time semiautomated assay

The Cobas Taqman assay for hepatitis B virus (HBV) DNA showed linear detection over 7 logs for genotypes A to D. The coefficient of variation was 1.2% at > or =1,000 IU/ml and 22.0% at 10 IU/ml. In 97 clinical samples, the log HBV DNA/ml differed by 0.11 between Cobas Amplicor and Cobas Taqman (r2 = 0.97).     

Rapid quantitation of cytomegalovirus DNA in whole blood by a new molecular assay based on automated sample preparation and real-time PCR

Quantitation of cytomegalovirus (CMV) DNA in whole blood samples has gained significance in the routine diagnostic laboratory. In this study, the analytical performance of the artus™ CMV RG PCR kit in conjunction with automated sample preparation on the new QIAsymphony™ SP instrument was evaluated. Clinically referred samples were tested and results compared to those obtained with the routinely used molecular test system. Accuracy testing showed results to be within ±0.2 log₁₀ unit of the expected panel results. The assay was linear (R = 0.9972) from a lower quantification limit of 148 copies/ml to 1.3 × 10⁷ copies/ml. The between-day imprecision CV was 8–63%, and the within-run imprecision CV was 16–61%. When 100 clinically referred samples were tested, results obtained with the new test system showed an acceptable concordance with those obtained with the routinely used easyMAG™ sample preparation and CMV HHV6,7,8 R-gene™ test system. Discrepant results were found with low-titer samples containing CMV DNA concentrations under the lower limit of quantification or within half a log unit above. The time to results was comparable for both test systems. The QIAsymphony™ sample preparation and artus™ CMV RG PCR test system allows for a rapid, sensitive, precise, and accurate high-throughput quantitation of CMV DNA in whole blood in the routine diagnostic laboratory.     

Clinical utility of transcription mediated amplification-hybridization protection method(TMA-HPA) for the detection of HBV DNA: a comparison with the branched DNA probe assay

The clinical utility of the TMA-HPA method for the detection of HBV DNA was evaluated by comparing results with the branched DNA probe(b-DNA) assay. Sera from 135 biopsy-confirmed HBV carriers were included in the study. Dilution tests with two representative sera revealed the sensitivity of the TMA-HPA method to be 100-fold higher than that of the b-DNA assay. Among the 135 test sera, HBV DNA measurements obtained by the two assays showed a high degree of correlation(r = 0.861, p < 0.0001). Of the 72 sera which tested positive for HBe antigen, 71 and 67 were positive for HBV DNA when tested by TMA-HPA and b-DNA assay, respectively. In contrast, of the 63 which tested negative for HBe antigen, 42 were positive for HBV DNA when tested by TMA-HPA, and only 19 were positive when tested by b-DNA assay(p < 0.05). Among HBe antigen-positive HBV carriers, HBV DNA levels as determined both TMA-HPA and b-DNA assay showed an inverse relationship with pathological stage, but among HBe antigen-negative HBV carriers, no such tendency was observed. Based on these results, it is felt that the detection of HBV DNA by TMA-HPA is useful for the evaluating anti-viral therapeutic effect in HBV carriers, and in estimating a prognosis.     

Presence of HBV DNA in spermatozoa: a possible vertical transmission of HBV via the germ line

Using molecular hybridization we studied the presence and state of HBV DNA in sperm samples obtained from 17 patients with HBV infection (eight HBsAg chronic carrier, nine acute hepatitis B). Presence of HBV DNA was detected in three samples of seminal fluid from three patients with acute hepatitis. Restriction enzyme patterns of cellular DNA were consistent with presence of integrated HBV DNA sequences in spermatozoa from two of these three patients. The results indicate that HBV DNA may be present in the semen, at least during the acute phase of HBV infection. The presence of HBV DNA in seminal fluid confirms the possibility of venereal transmission. The presence of integrated sequences in spermatozoa suggests the possibility of true vertical transmission of HBV via the germ line.     

Evaluation of the INNO-LiPA HBV genotyping assay for determination of hepatitis B virus genotype

Specific genotypes of hepatitis B virus (HBV) are increasingly recognized for their clinical significance and association with particular viral mutations. Although many HBV genotyping methods exist, there has been no standardized or commercially available method for direct molecular typing of the HBV genome. A newly available line probe assay (INNO-LiPA HBV Genotyping assay; Innogenetics N.V., Ghent, Belgium) that allows the identification of HBV genotypes A to G was assessed by comparison with pre-S1/pre-S2 sequence analysis of the isolates in 188 serum specimens. All seven genotypes were detected by the line probe assay (LiPA), and complete concordance between LiPA and sequence analysis was observed for 152 specimens (81%). LiPA was able to detect 19 mixed genotype infections not detected by amplicon sequencing, which for the most part were confirmed by cloning and sequencing of the pre-S1/pre-S2 amplicon. Four specimens had discrepant results between the two methods, and five specimens had indeterminate results by LiPA. The HBV DNA in four specimens was unable to be amplified by the nested INNO-LiPA HBV DR amplification primers; however, the HBV DNA in six specimens unable to be genotyped by sequencing was clearly genotyped by LiPA. The INNO-LiPA HBV Genotyping assay appears to be useful for the rapid genotyping of HBV, particularly for the sensitive detection of mixed genotype infections.