Clinical utility of hepatitis C virus core antigen (HCVcAg) assay to identify active HCV infection in hemodialysis and renal transplant patients.

BACKGROUND: The prevalence of HCV infection is high and it is a major cause of liver-related morbidity and mortality in hemodialysis and renal transplant patients. Diagnosis of hepatitis C virus (HCV) infection requires both HCV antibody screening and confirmatory nucleic acid testing (NAT). Hepatitis C virus core antigen (HCVcAg) is a reliable direct viral marker to identify active HCV infection. AIM: To assess the clinical utility of HCV core antigen to identify active HCV infection in hemodialysis and renal transplant patients. METHODS: A representative total of 231 plasma samples with a predominance of low viral load were included for HCVcAg testing and its performance characteristics were compared with the gold standard HCV RNA. RESULTS: Comparison of HCVcAg with HCV RNA showed an excellent specificity of 99% (95% CI: 94.7 to 100%) and sensitivity of 80.62% (95% CI: 73.59 to 87.7%). Likewise, the PPV and NPV of HCVcAg were 99.1% (95% CI: 93.7% to 99.9%) and 80.2% (95% CI: 74% to 85.2%) respectively. The correlation between HCVcAg and HCV RNA was found to be good (R2 = 0.86, p<0.0001). Among common Indian HCV genotypes (1, 3 & 4), good correlation was observed between HCV RNA and HCVcAg (R2 = 0.81, p <0.0001). CONCLUSIONS: It is the first Indian study to show that HCVcAg is a reliable, cost-effective direct marker to identify active HCV infection in hemodialysis and renal transplant patients. Implementation of HCVcAg testing could improve the accessibility to efficacious and affordable disease management in hemodialysis and renal transplant patients. In HCVcAg negative cases, sequential testing with anti-HCV antibody followed by HCV RNA could be a reliable and cost-effective approach.

Autoantibodies Among HIV-1 Infected Individuals and the Effect of Anti-Retroviral Therapy (ART) on It.

BACKGROUND: Antiretroviral therapy (ART) has led to a decline in autoimmune diseases but lacks studies on its effect on autoantibodies. METHODS: It is a cross-sectional study with archived samples from 100 paired HIV-1 infected ART naïve and experienced individuals and 100 prospectively collected matched blood-donor controls. Antinuclear antibody, IgG anticardiolipin antibody, IgM and IgG ß2 glycoprotein-1 antibodies, and total IgG levels were detected. Results are expressed as mean with standard deviation (SD), median, percentage positivity, and a p<0.05 is considered significant. The study was approved by the Institutional Review Board. RESULTS: The median viral load of the treatment naïve samples was 4.34 Log copies/mL, while all were virally suppressed post ART with a median duration of treatment for 12 months (range: 3-36 months). The percentage of antinuclear antibody positivity was 5% among ART naïve and controls, with a decrease of 2% post ART (p= 0.441). The positivity for anti-cardiolipin antibody was 15% among ART naïve while none of the ART experienced or controls were positive (p<0.05). IgM ß2 glycoprotein-1 were 4%, 1% and 3% among ART naïve, treated and controls, respectively (p<0.05). IgG ß2 glycoprotein-1 was 2% among ART naïve while none of the treated and controls were positive (p<0.05). The mean total IgG level among ART naïve, experienced, and controls were 21.82 (SD 6.67), 16.91 (SD 3.38), 13.70 (SD 2.24) grams/Litre, respectively (p<0.05). CONCLUSION: ART has a significant effect on IgG anti-cardiolipin antibody and total IgG but only a marginal effect on ANA, IgM, and IgG ß2 glycoprotein-1 antibodies.

Evaluation of dried blood spots as a feasible alternative to plasma for the detection and quantification of hepatitis c virus in a tropical setting: A pilot study.

Introduction: Confirmatory diagnosis of hepatitis C virus (HCV) infection (HCV RNA detection) is essential before start of the therapy. HCV RNA detection is not available in many parts of India. Shipment of plasma from distant places to referral laboratories may affect HCV RNA titres. Dried blood spots (DBS) provide an easy alternative for transporting samples to centres where HCV RNA testing is done. Aim: Evaluation of DBS as a feasible alternative to plasma for HCV diagnosis. Methods: In this cross-sectional study, 40 consecutive patients' blood samples were collected from patients referred from the Liver Clinic. Whole blood was spotted onto two Whatman 903TM cards. One card was incubated at ≥37°C and other at 4°C for 15 days, after drying. DBS was eluted and run in Abbott RealTime HCV assay. HCV was also quantified using the Abbott ARCHITECT HCV core antigen assay for 29 of the study patients. Results were compared with normal plasma values. Results: The median log HCV RNA value (in log10IU/mL) of plasma was 5.74, with normalised DBS it was 4.92 (≥37°C) and 4.66 (4°C); difference in plasma and DBS median log values was 0.82 (≥37°C) and 1.08 (4°C) logs, respectively. Interclass correlation values were 0.943, P < 0.0001 (≥37°C) and 0.950, P < 0.0001 (4°C), showing high agreement. The median HCV core antigen value (in fmol/L) for plasma was 325.35, whereas it was 4.77 (≥37°C) and 4.64 (4°C) for DBS samples. Conclusions: DBS can be used for sampling patients from distant resource-limited settings as an alternative to plasma for HCV RNA estimation. Larger studies are required to evaluate the feasibility of DBS in the Indian subcontinent, especially for HCV core antigen estimation.

Evaluating the PGMY-centre hospitalier universitaire vaudois assay as a cost-effective tool for human papillomavirus genotyping in HIV-infected women.

Aims: Cervical cancer is one of the leading causes of cancer among women, worldwide. HIV-positive women tend to have persistent infection and infection with multiple human papillomavirus (HPV) types. There is a need for affordable HPV DNA tests as viable alternatives to the existing costly commercial assays. The aim of the study was to establish PGMY-CHUV reverse hybridization assay as a cost-effective tool for HPV genotyping. Study Design: This was a prospective study conducted in a tertiary care centre from March 2011 to July 2012. Subjects and Methods: Fifty cervical brush samples from HIV-infected women and 43 WHO reference samples were tested by both the CHUV assay and linear array (LA). Results: The CHUV assay in comparison to the LA showed a sensitivity of 91%, specificity of 52% and a moderate agreement for all samples that were compared. However, most high-risk HPV types were identified amongst the clinical samples, and the entire range of genotypes in the WHO reference panel was detected. Statistical Analysis: The accuracy indices such as sensitivity, specificity, positive predictive value and negative predictive value were calculated. The level of agreement (kappa value) between the two assays was also calculated. Conclusion: The CHUV assay had an acceptable sensitivity, but it lacked specificity for HPV detection. Despite the lower rates of detection of multiple infections from clinical samples, better results were obtained with the WHO reference samples and the ability of the assay to identify the entire range of genotypes suggests that it can be an efficient tool for genotyping.

Expanded diagnostic approach to hepatitis E virus detection in patients with acute-on-chronic liver failure: A pilot study.

INTRODUCTION: Acute decompensation of pre-existing chronic liver disease (CLD), known as acute-on-chronic liver failure (ACLF), is associated with high mortality. Hepatitis E virus (HEV) as a potential cause was studied. OBJECTIVES: The objectives of this study are to evaluate the role of HEV in ACLF patients using an IgM anti-HEV antibody enzyme-linked immunosorbent assay (ELISA), HEV antigen ELISA, and a quantitative HEV polymerase chain reaction (PCR). MATERIALS AND METHODS: In this prospective cross-sectional study, blood samples were collected from 50 ACLF (cases) as defined by the standard guidelines (APASL, 2014) and 50 patients with stable CLD (controls) from January 2015 to August 2016, after obtaining informed consent. Two IgM ELISAs (MP Diagnostics HEV IgM ELISA 3.0, Singapore and Wantai HEV IgM ELISA, Beijing, China) were compared using plasma from cases and controls. In addition, an HEV antigen detection by ELISA (Wantai, Beijing, China) and a real-time PCR for quantification of HEV RNA in plasma and stool were employed. RESULTS: Ethanol was the leading cause of acute insult in ACLF (54%) cases. HEV infection accounted for 20% of cases. Ten ACLF patients (20%) had 1-3 markers of HEV versus two (4%) among controls (P = 0.0138). Among ACLF cases, one had HEV viraemia (403 IU/ml), faecal shedding (2790 IU/ml) and detectable HEV antigenaemia. Agreement between the two anti-HEV IgM ELISAs was 0.638 (kappa value). CONCLUSION: This study shows that alcohol is a major contributing factor for both underlying CLD and ACLF while HEV is the most common infectious cause for ACLF, suggesting a need for a vaccination in such patients, whenever made available.

Evaluation of reliability and performance of hepatitis B virus-e-antigen assays in tertiary care setting.

Hepatitis B virus-e-antigen (HBeAg) is a viral marker to assess hepatitis B virus (HBV) replication. We have evaluated the reliability of three commonly available HBeAg immunoassays using World Health Organization-International Standard and clinical samples. In addition the performance of enzyme immunoassays (EIAs) was assessed by kinetic binding and reagent exchange experiments. Analytical and diagnostic sensitivity were significantly different among HBeAg assays (P < 0.01). The affinity of capture/detector antibodies varied significantly between EIAs (P < 0.01). Our findings suggest that significant difference in the affinity of capture/detector antibodies to HBeAg may impact the overall performance and the reliability of currently available HBeAg assays in HBV diagnosis and management.

Cost-Effective In-House Neutralization Assay for the Confirmation of HBeAg.

BACKGROUND & AIM: Hepatitis B virus-e-antigen (HBeAg) is an affordable viral marker to assess viral replication kinetics and response to antiviral therapy. In the absence of confirmatory assays, discrepant or false-positive HBeAg results are resolved by screening for other HBV markers. We standardized an in-house HBeAg neutralization assay (HBeAg-NT) to confirm HBeAg in clinical samples. METHODS: The performance and reliability of this assay were evaluated by first WHO International Standard for HBeAg (first WHO-IS HBeAg) from Paul Ehrlich Institute and clinical samples (n = 150) from chronic HBV carriers. Of these, 71 HBeAg-positive sera were used for HBeAg-NT. RESULTS: Concentrations spanning 0.25-10 U of first WHO-IS HBeAg and clinical samples (S/Co ranges from 1.00 to 10.00) were neutralized completely in the HBeAg-NT. CONCLUSIONS: HBeAg-NT is a simple, cost-effective, and reliable direct approach to confirm HBeAg in clinical samples which precludes the need for screening additional HBV markers in low resource settings.

Performance characteristics and comparison of Abbott and artus real-time systems for hepatitis B virus DNA quantification.

Virological monitoring of hepatitis B virus (HBV) DNA is critical to the management of HBV infection. With several HBV DNA quantification assays available, it is important to use the most efficient testing system for virological monitoring. In this study, we evaluated the performance characteristics and comparability of three HBV DNA quantification systems: Abbott HBV real-time PCR (Abbott PCR), artus HBV real-time PCR with QIAamp DNA blood kit purification (artus-DB), and artus HBV real-time PCR with the QIAamp DSP virus kit purification (artus-DSP). The lower limits of detection of these systems were established against the WHO international standards for HBV DNA and were found to be 1.43, 82, and 9 IU/ml, respectively. The intra-assay and interassay coefficients of variation of plasma samples (1 to 6 log(10) IU/ml) ranged between 0.05 to 8.34% and 0.16 to 3.48% for the Abbott PCR, 1.53 to 26.85% and 0.50 to 12.89% for artus-DB, and 0.29 to 7.42% and 0.94 to 3.01% for artus-DSP, respectively. Ninety HBV clinical samples were used for comparison of assays, and paired quantitative results showed strong correlation by linear regression analysis (artus-DB with Abbott PCR, r = 0.95; Abbott PCR with artus-DSP, r = 0.97; and artus-DSP with artus-DB, r = 0.94). Bland-Altman analysis showed a good level of agreement for Abbott PCR and artus-DSP, with a mean difference of 0.10 log(10) IU/ml and limits of agreement of -0.91 to 1.11 log(10) IU/ml. No genotype-specific bias was seen in all three systems for HBV genotypes A, C, and D, which are predominant in this region. This finding illustrates that the Abbott real-time HBV and artus-DSP systems show more comparable performance than the artus-DB system, meeting the current guidelines for assays to be used in the management of hepatitis B.