Hepatitis C Virus Antibody Assay Research Articles

Management of dialysis patients with hepatitis C virus in the era of direct-acting antiviral therapy.

The clinical use of direct-acting antivirals (DAAs) for hepatitis C virus (HCV) infection has dramatically changed management of patients with HCV liver disease since 2014; this is also true for patients undergoing dialysis. Due to the high tolerability and antiviral efficacy of anti-HCV therapy, most dialysis patients with HCV infection should currently be candidates for this treatment. Many patients with HCV antibodies no longer have HCV infection, and it is difficult to identify patients with actual HCV infection based only on HCV antibody assays. Despite the high rate of successful HCV eradication, the risk of liver-related events such as hepatocellular carcinoma (HCC), the major complication of HCV infection, persists even after HCV cure, and patients at risk of HCC should undergo continuous HCC surveillance. Finally, the rarity of HCV reinfection and the survival benefit of HCV eradication in dialysis patients should be explored in further studies.

Comparison Of Hepatitis C Antibody Assays And Evaluation Of Agreement Between Hepatitis C Antigen And PCR Results.

Hepatitis C is associated with a wide range of health repercussions. Pakistan is one of the highly prevalent countries of Hepatitis C Virus (HCV) infection. The availability of cost-effective, robust, and reliable screening and diagnostic tests for hepatitis C is important to address the disease burden. Standardization of screening and diagnostic assays in clinical laboratories is crucial for achieving big goals. Objectives of this study are to correlate the results of two different HCV antibody (HCV Ab) assays and to examine the correlation of HCV core antigen (HCV c Ag) results with HCV PCR for HCV infection diagnosis. This descriptive cross-sectional study was carried out from November to December 2020 at Dow University of Health Sciences. Total number of 40 HCV Ab samples were analysed by both chemiluminescence (CMIA) and electrochemiluminescence (ECLIA) immunoassays. Tests for HCV RNA PCR and HCV c Ag were performed on all samples. Results of screening and diagnostic assays were correlated and agreements were examined. Statistical analysis for agreement was carried out by using R software version 3.6.3 through AC1 Gwetz Statistic. The study was approved by the institutional ethical review committee. An agreement of 0.73 and 0.95 was found between two different HCV Ab immunoassays and HCV c Ag and HCV PCR, respectively. We found a good correlation between CMIA and ECLIA for HCV Ab. An excellent correlation was found between HCV c Ag and HCV PCR. Based on our study findings, HCV c Ag is a candidate test for the diagnosis of active HCV infection.

Hepatitis C Testing and Linkage to Care Among Adults on Probation in a Large US City.

BackgroundDespite constituting the largest segment of the correctional population, individuals on probation remain largely unstudied with respect to hepatitis C virus (HCV) testing and linkage to care. We implemented an HCV testing and patient navigation program at an adult probation department.MethodsAdults were tested at a local probation department with a rapid point-of-care HCV antibody (Ab) assay followed by a laboratory-based HCV ribonucleic acid (RNA) assay if anti-HCV positive. All individuals received counseling rooted in harm reduction principles. Individuals testing positive for HCV Ab were immediately linked to a patient navigator in person or via telephone. The patient navigator assisted patients through cure unless the patient was lost to follow-up. Study participation involved an optional survey and optional point-of-care human immunodeficiency virus test.ResultsOf 417 individuals tested, 13% were HCV Ab positive and 65% of those tested for HCV RNA (34 of 52) had detectable HCV RNA. Of the 14 individuals who linked to an HCV treatment provider, 4 completed treatment, as measured by pharmacy fill documentation in the electronic medical record, and 1 obtained sustained virologic response. One hundred ninety-three individuals tested for HIV; none tested positive.ConclusionsThe study cohort had a higher HCV seroprevalence than the general population (13% vs 2%), but linkage to care, completion of HCV treatment, and successful test-of-cure rates were all low. This study indicates that HCV disproportionately impacts adults on probation and prioritizing support for testing and linkage to care could improve health in this population. Colocalization of HCV treatment within probation programs would reduce the barrier of attending a new institution and could be highly impactful.

The performance of hepatitis C virus (HCV) antibody point-of-care tests on oral fluid or whole blood and dried blood spot testing for HCV serology and viral load among individuals at higher risk for HCV in South Africa.

Background and AimsTo enhance screening and diagnosis in those at‐risk of hepatitis C virus (HCV), efficient and improved sampling and testing is required. We investigated the performance of point‐of‐care (POC) tests and dried blood spots (DBS) for HCV antibody and HCV RNA quantification in individuals at higher risk for HCV (people who use and inject drugs, sex workers and men who have sex with men) in seven South African cities.MethodsSamples were screened on the OraQuick HCV POC test (471 whole blood and 218 oral fluid); 218 whole blood and DBS paired samples were evaluated on the ARCHITECT HCV antibody (Abbott) and HCV viral load (COBAS Ampliprep/COBAS TaqMan version 2) assays. For HCV RNA quantification, 107 dB were analyzed with and without normalization coefficients.ResultsPOC on either whole blood or oral fluid showed an overall sensitivity of 98.5% (95% CI 97.4‐99.5), specificity of 98.2% (95% CI 98.8‐100) and accuracy of 98.4% (95% CI 96.5‐99.3). On the antibody immunoassay, DBS showed a sensitivity of 96.0% (95% CI 93.4‐98.6), specificity of 97% (95% CI 94.8‐99.3) and accuracy of 96.3% (95% CI 93.8‐98.8). A strong correlation (R 2 = 0.90) between viral load measurements for DBS and plasma samples was observed. After normalization, DBS viral load results showed an improved bias from 0.5 to 0.16 log10 IU/mL.ConclusionThe POC test performed sufficiently well to be used for HCV screening in at‐risk populations. DBS for diagnosis and quantification was accurate and should be considered as an alternative sample to test. POC and DBS can help scale up hepatitis services in the country, in light of our elimination goals.

The clinical impact of hepatitis C virus infection in Egyptian multiple myeloma patients

BackgroundMultiple myeloma (MM) is a human B cell neoplasia characterized by the clonal proliferation of malignant plasma cells in the bone marrow. Worldwide, hepatitis C virus (HCV) infection is a public health problem. For MM patients, the clinical impact of preexisting HCV infection is still unclear. We aim to assess the clinical characteristics and the prevalence of the HCV infection in Egyptian MM patients. This observational study included 81 MM patients. HCV antibody assay was performed, and positive cases were confirmed using a reverse transcription-quantitative PCR (RT-PCR) method.ResultsFifteen (18.5%) patients were anti-HCV antibody positive. Only 6/15 (7.4%) patients were HCV RNA positive by RT-PCR. Liver affection in the form of hyperbilirubinemia with grade 4 adverse events was significantly higher in the anti-HCV positive/HCV RNA positive group versus anti HCV negative group (16.7% vs. 1.5%, p value = 0.005). The median HCV-RNA before the initiation of chemotherapy was 2.5 log IU/ml with mean ± SD = 4.25 ± 1.6 with no HCV reactivation. In the univariate and multivariate analysis, HCV infection was not an independent factor related to DFS. Low hemoglobin level < 10 g/dL (HR 0.59, 95% CI, 0.36–0.97, p value = 0.037) and abnormal serum total bilirubin level (HR 1.9, 95% CI 1.03–3.5, p value = 0.039) influenced DFS in the univariate analysis. However, in the multivariate analysis, serum calcium level greater than 12 mg/dL (HR 7.04, 95% CI 1.12–44.45, p value = 0.038) and abnormal serum total bilirubin level (HR 10.9, 95% CI 2.92–41.02, p value = < 0.001) remained statistically significant worse prognostic factors.ConclusionIn conclusion, our study revealed the prevalence of HCV infection in Egyptian MM patients. Serologic tests at diagnosis are necessary to identify these patients, and confirmation of positive cases by molecular techniques should be mandatory, with regular follow-up for liver dysfunction. Finally, further larger studies explaining the molecular mechanisms linking HCV and the MM pathogenesis are warranted.

Evaluation of HCV RNA by PCR and Signal-to-Cutoff Ratios of HCV Antibody Assays for Diagnosis of HCV Infection

In this study, we assessed whether a hepatitis C virus (HCV) RNA test could replace recombinant immunoblot assay (RIBA) and reduce unnecessary supplemental tests as the signal-to-cutoff (S/Co) ratio from anti-HCV antibody (Ab) tests. Anti-HCV Ab tests were performed to screen for HCV infections, and RIBA and real-time polymerase chain reaction were performed for HCV RNA to confirm HCV infection. Receiver operating characteristic curves were evaluated to determine the optimal S/Co ratios for predicting HCV infection. The cutoff value for the S/Co ratio was 3.63 for predicting RIBA results and 10.6 for predicting HCV RNA results. Our data suggested that an S/Co ratio ≥10.6 indicated a high risk of active HCV infection. An S/Co ratio of 3.63 to 10.6 needed further evaluation and repeat HCV RNA testing. No further testing was required for S/Co ratios <3.63 or ≥10.6. We determined that the S/Co ratio of the anti-HCV Ab test provides useful information to confirm HCV infections, including the need for further laboratory testing or clinical follow-up.

Evaluation of efficacy of serological methods for detection of HCV infection in blood donors: A single centre experience.

Background and Objective:Blood transfusion is an essential and life-saving medical intervention. Despite multiple preventive measures transfusion-transmitted hepatitis C virus (HCV) infection continues to be a major healthcare issue in Pakistan. This study was conducted at National Institute of Blood Diseases & Bone Marrow Transplantation to evaluate the frequency of active HCV infection with or without co-infection in blood donors and also to determine comparative efficacy of Multisure HCV antibody assay (MHAA); a new serological device.Methods:A total of 14652 blood donors visiting National Institute of Blood Diseases & Bone Marrow Transplantation (NIBD) Blood Bank from January 2013 to July 2014 were enrolled and screened for a range of blood borne infections such as HBV, HCV, HIV, malaria and syphilis. The HCV was screened simultaneously by Abbot Architect anti-HCV assay (CLIA) and MHAA. The active HCV infection was confirmed by nucleic acid testing (NAT) in reactive donors. Later; for determination of comparative efficacy of MHAA; all NAT positive samples were further tested using Monolisa™, HCV blot 3.0, Anti-HCV plus V2 and Anti-HCV-MPBIO-EIA.Results:The HCV reactive sera were observed in 1.563% (226) donors. The NAT confirmed active HCV infection in 138 donors. Overall 27.84% of HCV positive donors exhibited co-infection either with HBV (2.57%), syphilis (22.78%). Triple infection was not observed in any donor. The efficacy of MHAA is comparable to all the serological tests with a sensitivity of about 96.89%.Conclusion:Active HCV infection was present in 0.94% donors. With a sensitivity of 96.89% (95% CI: 95.66-98.12) the multi-parametric device MHAA can effectively detect HCV infection in donors. Thus, it can be used in limited health care settings for HCV screening.

The Association Between Viral Infections and Co-stimulatory Gene Polymorphisms in Kidney Transplant Outcomes.

BackgroundThe surveillance of kidney transplant patients depends on function of different immunologic markers like co-stimulatory molecules. These molecules may also be associated with post kidney transplant viral related outcomes.ObjectivesThe aim of this study was to investigate the possible associations between co-stimulatory molecule gene polymorphisms and viral infections in kidney transplant patients.Patients and MethodsIn total, 172 kidney transplant patients were included in this study. Single nucleotide polymorphisms in loci of co-stimulatory molecules including: PDCD.1, CD28, CTLA4 and ICOS, were analyzed in the studied patients by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. Active Cytomegalovirus (CMV) infection and history of hepatitis C virus (HCV) infection were analyzed in each kidney transplant patient using the CMV antigenemia kit and HCV antibody assay, according to the manufacturer’s instructions.ResultsCMV active infection was found in 31 of 172 (18.02%) kidney transplant patients. HCV infection was only found in two of the 172 (1.16%) studied patients. Significant associations were found between TT and TC genotypes of CTLA4 -1722T/C and T allele with acute rejection in CMV infected kidney transplant patients. A significant association was also found between the T allele of CD28 + 17 C/T genetic polymorphism and acute rejection in CMV infected kidney transplant patients. Significantly higher frequency of AA genotype and A allele of CTLA4 + 49AG polymorphism were found in CMV infected female patients. Also a significantly higher frequency of GG genotype and G allele of PDCD-1.3A/G polymorphisms were found in CMV infected female patients.ConclusionsBased on these results, CTLA4 and CD28 genetic polymorphisms, which regulate T-cell activation, can influence active CMV infection in kidney transplant patients. These results should be confirmed by further investigations.

Birth Cohort Screening Rates for Hepatitis C in an Internal Medicine Clinic: Before and After an Educational Intervention

Introduction: People born during 1945-1965 account for approximately 75% of chronic hepatitis C virus (HCV) infections in the United States. In August 2012, the center for disease control (CDC) advised one-time testing for HCV in adults born from 1945 to 1965 without prior ascertainment of HCV risk. This is a quality improvement project with the aim of raising awareness of birth cohort screening through education of resident and attending physicians. The objective was to increase the screening rate for HCV in an outpatient internal medicine practice. Methods: We determined baseline screening rates by retrospectively measuring the number of HCV antibody assays ordered in a large internal medicine practice for screening purposes only during a 5 month period from January to May 2013. Eligible patients were those new to the practice and born between 1945 and 1965. If they had a prior diagnosis of HCV, they were excluded. We subsequently devoted 1 month to educating the internal medicine housestaff and attendings of the clinic on the updated CDC screening recommendations through lectures and handouts. We then prospectively measured the number of HCV antibody assays ordered during a 5 month period from July to November 2013. The 2 periods were then compared to determine if education had any effect on screening rates. Results: In the pre-intervention period, we examined 139 patient records and 5 were excluded due to a prior history of HCV. In the remaining 134 patients, 3 HCV antibody assays were ordered for a screening rate of 2.2%. In the post-intervention period, 106 patient records were examined and 7 were excluded due to a prior history of HCV. In the remaining 99 patients, 9 HCV antibody assays were ordered for a screening rate of 9.1% which was significantly increased compared to the pre-intervention screening rate (p-value=0.0193). Conclusion: We found a 4-fold increase in screening rates for HCV after our educational intervention. It was notable that the majority of eligible patients were still not screened for HCV. We believe that the education provided to the attending physicians and housestaff contributed to the increase in HCV Ab screening rates.

Current testing strategies for hepatitis C virus infection in blood donors and the way forward

Screening tests for blood donations are based upon sensitivity, cost-effectiveness and their suitability for high-throughput testing. Enzyme immunoassay (EIAs) for hepatitis C virus (HCV) antibodies were the initial screening tests introduced. The "first generation" antibody EIAs detected seroconversion after unduly long infectious window period. Improved HCV antibody assays still had an infectious window period around 66 d. HCV core antigen EIAs shortened the window period considerably, but high costs did not lead to widespread acceptance. A fourth-generation HCV antigen and antibody assay (combination EIA) is more convenient as two infectious markers of HCV are detected in the same assay. Molecular testing for HCV-RNA utilizing nucleic acid amplification technology (NAT) is the most sensitive assay and shortens the window period to only 4 d. Implementation of NAT in many developed countries around the world has resulted in dramatic reductions in transfusion transmissible HCV and relative risk is now < 1 per million donations. However, HCV serology still continues to be retained as some donations are serology positive but NAT negative. In resource constrained countries HCV screening is highly variable, depending upon infrastructure, trained manpower and financial resource. Rapid tests which do not require instrumentation and are simple to perform are used in many small and remotely located blood centres. The sensitivity as compared to EIAs is less and wherever feasible HCV antibody EIAs are most frequently used screening assays. Efforts have been made to implement combined antigen-antibody assays and even NAT in some of these countries.

Correlation between serum levels of interleukins 10 and 12 and thrombocytopenia in hepatitis C cirrhotic (class A) patients

Hepatitis C virus (HCV) patients commonly have low platelet counts; however, the exact role of HCV infection in thrombocytopenia is unknown. This work aimed to study the serum levels of interleukins (IL) 10 and 12 in patients with mild and moderate thrombocytopenia associated with chronic hepatitis C infection. Our study included 15 patients with chronic HCV infection and newly diagnosed isolated autoimmune thrombocytopenia (Group I) and 15 patients with chronic HCV infection and normal platelet count as controls (Group II). All patients were examined for personal history and clinical aspects, complete blood count, bone marrow aspiration, liver function tests, HCV antibody assay by ELISA and polymerase chain reaction (PCR), abdominal ultrasound, Helicobacter pylori stool antigen test, evaluation of serum levels of IL-10, IL-12 and platelet specific antibodies. Our results revealed that eight patients from Group l had mild thrombocytopenia and seven patients had moderate thrombocytopenia. Serum IL-10 level was significantly elevated (t = 9.301, p < 0.001) while serum IL-12 showed a significant decrease (t = 6.502, p < 0.001) in Group I compared to the control group. No correlation was detected between platelet counts and the serum levels of either IL-10 [r = 0.454, p = 0.089 (Group I), r = 0.038, p = 0.89 (Group II)] or IL-12 [r = 0.497, p = 0.06 (Group I), r = 0.499, p = 0.058 (Group II)]. However, in Group I, a significant correlation was present only between moderate thrombocytopenia and serum levels of either IL-10 (r = 0.794, p = 0.033) or IL-12 (r = 0.967, p = 0.001), while no correlation was detected between these interleukin parameters and mild thrombocytopenia (r = 0.311 and p = 0.453 for IL-10 and r = -0.08 and p = 0.851 for IL-12). Based on our data, we may conclude that interleukins 10 and 12 are involved in low platelet levels.

Hepatitis C virus window‐phase infections: closing the window on hepatitis C virus

The detection of hepatitis C virus (HCV) infection is of major importance for the prevention of transfusion-transmitted hepatitis. The testing of donations by nucleic acid testing (NAT) techniques may not be feasible or economic. Combined antigen and antibody assays are now available, and the performance of two combined assays on window-phase donations is evaluated. Three panels of antibody-negative plasma samples from HCV NAT-only-positive donors were characterized for HCV status by quantitative reverse transcription-polymerase chain reaction, a commercial third-generation HCV antibody assay (Ortho), and combined antigen and antibody assays (Bio-Rad MONOLISA and Murex). All 142 plasma samples were antibody negative by Ortho third-generation HCV. A total of 112 samples (79%) were found to contain HCV RNA; 32 were detected by the Bio-Rad assay (29%), whereas 56 (50%) were detected by the Murex assay. Of 45 samples with viral loads of greater than 10(6), 32 (71%) were positive in the Bio-Rad combination assay and 44 (98%) were positive in the Murex assay. Interestingly none of the 3a genotypes were detected by the Bio-Rad MONOLISA, including eight donations that were greater than 10(6) IU per mL. Combined antigen and antibody testing provides a useful improvement on the sole reliance on antibody testing for detection of HCV infection; however, it remains less sensitive than NAT for detecting viremic donors and may be genotype susceptible.

Factors Associated with Spontaneous Clearance of Hepatitis C Virus among Illicit Drug Users

Spontaneous clearance of hepatitis C virus (HCV) occurs in approximately 25% of individuals. To better understand the characteristics associated with clearance, the present study evaluated HCV clearance in a community-based cohort study. The Community Health and Safety Evaluation project recruited 3553 individuals via community organizations and door-to-door canvassing of a random sample of single occupancy hotels in the community to monitor uptake of health services and to estimate the incidence of communicable infections. Cohort data were linked with longitudinal laboratory databases, including HCV antibody and polymerase chain reaction assay results. Overall, 762 individuals had HCV antibody and RNA testing performed between 1999 and 2005. Spontaneous HCV clearance was observed in 179 individuals (23.5%), while HCV persistence was observed in 583 individuals (76.5%). The ability to develop protective immunity against HCV, as demonstrated by viral clearance, occurred more often in individuals of Aboriginal ethnicity (adjusted OR [AOR] 2.9, 95% CI 2.0 to 4.3; P<0.001) and female individuals (AOR 1.6, 95% CI 1.1 to 2.4; P=0.01). The rate of spontaneous HCV clearance was reduced in individuals using any type of illicit drugs (AOR 0.54, 95% CI 0.29 to 1.00; P=0.05) and those with HIV coinfection (AOR 0.58, 95% CI 0.38 to 0.88; P=0.01). Of 218 HIV-infected subjects, 48 of 51 (94%) in whom the order of HCV and HIV infection was established were infected with HCV a median of 2.4 years (range 0.2 to 10 years) before becoming infected with HIV. Aboriginal ethnicity and female sex were associated with increased rates of HCV clearance, while HIV coinfection and illicit drug use were associated with increased HCV persistence.

Design of Novel Conformational and Genotype-Specific Antigens for Improving Sensitivity of Immunoassays for Hepatitis C Virus-Specific Antibodies

The current commercially licensed enzyme-linked immunosorbent assays (ELISAs) for hepatitis C virus (HCV) mainly use recombinant proteins containing linear epitopes. There is evidence, however, that conformational epitopes of HCV are more immunoreactive. Thus, we have designed an HCV antibody assay that employs a conformational protein, NS3NS4a PI (with functional protease and helicase activities), and a linear fusion protein, multiple-epitope fusion antigen 7.1 (MEFA 7.1) or MEFA 7.2. We have shown that NS3NS4a PI detects early-seroconversion conformation-sensitive antibodies better than c33c antigen. The correct conformation of NS3NS4a PI also cross-reacts with different genotype samples better than the c33c antigen. MEFA 7.1 and MEFA 7.2 incorporate all the major immunodominant and genotype-specific epitopes of HCV core, E1, E2 hypervariable region 1 (HVR1), E2 HVR1-plus-HVR2 consensus, NS3, NS4, and NS5. Since MEFA 7.1 is degraded by the active NS3NS4a PI protease, we designed a second MEFA 7.2 construct in which the six protease cleavage sites found in MEFA 7.1 were eliminated by amino acid mutation. We demonstrate here that MEFA 7.2 remains intact in the presence of NS3NS4a PI and preserves the epitopes present in MEFA 7.1. Compared to currently licensed assays, an ELISA incorporating a combination of the two antigens NS3NS4a PI and MEFA 7.1 or 7.2 demonstrates better serotype sensitivity and detects seroconversion earlier in many commercially available panels. We believe that an assay using NS3NS4a PI and MEFA 7.1 or 7.2 may have the potential to replace current HCV immunoassays for better sensitivity.

Association of sex, hygiene and drug equipment sharing with hepatitis C virus infection among non-injecting drug users in New York City

Background: Hepatitis C virus (HCV) rates are higher in non-injecting drug users (NIDUs) than general population estimates. Whether this elevated HCV rate is due to drug use or other putative risk behaviors remains unclear. Methods: Recent non-injection drug users of heroin, crack and/or cocaine were street-recruited from 2000 to 2003 and underwent an interview and venipuncture for HCV antibody assays. Multiple logistic regression analyses were used to assess correlates for HCV infection. Results: Of 740 enrollees, 3.9% were HCV positive. The median age (intraquartile range) was 30 (35–24) years, 70% were male and 90% were Black or Hispanic. After adjustment, HCV seropositives were significantly more likely than seronegatives to be older than 30 [adjusted odds ratio (AOR) = 5.71], tattooed by a friend/relative/acquaintance [AOR = 3.61] and know someone with HCV [AOR = 4.29], but were less likely to have shared nail or hair clippers, razors or a toothbrush [AOR = 0.32]. Conclusions: Non-commercial tattooing may be a mode of HCV transmission among NIDUs and education on the potential risk in using non-sterile tattooing equipment should be targeted toward this population. While no evidence was found for HCV transmission through NIDU equipment sharing or sexual risk behavior, further research is still warranted.

Performance of three generations of anti-hepatitis C virus enzyme-linked immunosorbent assays in donors and patients.

Prevention of posttransfusion non-A,non-B hepatitis in recipients of blood components improved considerably with the introduction of the second-generation of hepatitis C virus (HCV) antibody tests. In 1993, third-generation HCV antibody assays were introduced in Europe. The performance of three generations of anti-HCV enzyme-linked immunosorbent assay (ELISA) (ELISA-1, -2, -3) was compared in routine blood donor screening (99,394 donations were tested with ELISA-1, 167,999 donations with ELISA-2, and 262,090 donations with ELISA-3) and in serial samples from nine patients with documented acute posttransfusion HCV infection. Eight (0.01%) repeat donors, previously negative in ELISA-1, were found positive in ELISA-2 and were confirmed as positive in second-generation recombinant immunoblot assay and/or cDNA polymerase chain reaction. In the donor population, no difference in the sensitivity of ELISA-2 and -3 was observed. The specificity of the three generations of ELISAs was comparable (99.8, 99.7, and 99.7%). In seroconversion samples, ELISA-2 and -3 detected HCV antibodies at the same time in seven patients, but in two patients, ELISA-3 found HCV antibodies, respectively, 63 and 77 days earlier than ELISA-2 did. In the seroconversion samples, ELISA-2 and -3 were significantly more sensitive than second- and third-generation recombinant immunoblot assays. ELISA-3 did not detect more HCV-infected individuals in a donor population that previously tested negative in ELISA-2, but it did detect HCV antibodies earlier in some patients with acute HCV infection. ELISA-2 and -3 were significantly more sensitive than second- and third-generation recombinant immunoblot assays.

E2 and NS5: new antigens for detection of hepatitis C virus antibodies.

The value of two new hepatitis C virus (HCV) antigens for detection of HCV antibodies was studied. These two recombinant antigens were derived from the nonstructural-5 (NS5) and envelope-2 (E2) region of the HCV genome. In a panel of 33 HCV-RNA positive samples with indeterminate Riba-2 confirmatory test results, 29 samples (88%) showed additional antibody reactivity against E2 and 12 samples (36%) showed additional reactivity against NS5. Among 39 HCV-RNA negative, Riba-2 indeterminate donor samples, no additional E2 or NS5 reactivity was found in 34 samples (87%); while 5 samples (13%) showed additional reactivity against NS5 and/or E2. E2 reactivity thus resolved the majority of hitherto indeterminate samples. In serial samples from nine posttransfusion hepatitis C patients, NS5 and E2 antibodies did not appear earlier than classical HCV antibodies. However, E2 antibodies eventually appeared in all nine patients. The recombinant E2 might be a candidate antigen for future HCV antibody assays.

Serologic, Virologic, and Histologic Characteristics of Chronic Phase Hepatitis C Virus Disease in Children Infected by Transfusion

We studied the time course of hepatic dysfunction, seropositivity to hepatitis C virus (HCV) antibodies, viremia, and histologic evidence of hepatic injury to evaluate the course of HCV infection in children infected by blood transfusion. Twenty-nine patients (ages 4 to 18 years) who underwent open-heart surgeries for congenital heart disease were grouped into three categories based on alterations in serum alanine aminotransferase (ALT) levels: Group A, acute infection; Group B, subacute infection; and Group C, chronic infection. In Group C, all 13 patients had detectable HCV RNA in serum. In contrast, all patients in Group A had no detectable HCV RNA: In Group B, one of nine patients had detectable HCV RNA and two of four patients examined had persistent chronic hepatitis by histologic criteria. Antibodies directed against C100-3 antigen or core-antigen were more useful than second-generation HCV antibody assays in determining the relationship between viremia and immunologic response. Infection with HCV genotype II and the presence of higher HCV RNA copy numbers were associated with histologic evidence of hepatic damage. An abnormal ALT value is frequently associated with viremia, and biochemically resolved acute infection reflects clearance of HCV. However, a normal ALT does not always reflect an absence of hepatocyte damage and HCV replication in patients with subacute disease. The measures outlined in this study are useful indicators of disease activity during the chronic

Hepatitis C virus antibodies and virus replication in asymptomatic blood donors.

We assessed hepatitis C virus (HCV) infection in 99 asymptomatic blood donors positive using a first-generation HCV antibody assay. When tested with second-generation assays, 86 (87%) donors were reactive (group 1), 2 (2%) were indeterminate (group 2), and 11 (11%) were non-reactive (group 3). Viraemia was revealed by polymerase chain reaction in all group 1 cases. The 2 group 2 cases and 6 (55%) group 3 cases were also viraemic. Viraemia was confirmed by a branched DNA assay in the 2 group 2 cases and 4 (36%) group 3 cases. Serum HCV RNA levels were further studied using a competitive reverse transcription-polymerase chain reaction assay. All cases in groups 2 and 3 were low viraemic (range 10(4)-10(5.5) copies/ml) compared with the 9 group 1 cases examined (range 10(7)-10(9) copies/ml). No correlation was evident between viraemic levels and antibody cut-off index in the first-generation assay. These findings indicate the possibility that low levels of viraemia can occur in individuals non-reactive in second-generation HCV antibody assays.

The polymerase chain reaction and hepatitis C virus diagnosis

In the absence of tissue culture, electron microscopy or assays for viral antigen, the direct detection of hepatitis C virus (HCV) is by necessity dependent upon nucleic acid hybridisation methods. Of the available methods, amplification of HCV cDNA by polymerase chain reaction (PCR) commends itself by virtue of its extreme sensitivity and its consequent ability to detect the very low levels of HCV-RNA that are present in many clinical samples. In this review the development and evolution of PCR techniques for HCV detection are described and a number of clinical applications are considered in detail. The application include diagnosis of acute infection during the seronegative window period prior to the appearance of HCV antibodies, and diagnosis of HCV infection in the immunosuppressed. PCR also enables identification of chronic viraemic carrier state and it permits accurate monitoring of the antiviral effects of drugs such as interferon. Confirmation of the specificity HCV antibody assays and detection of HCV contamination of blood donations and blood products are other important areas in which PCR techniques have proved invaluable. In addition, PCR-based techniques underlie an increasing number of molecular epidemiological and genotyping studies and they are providing insights into the details of HCV cellular tropism and replication. A number of logistic problems and operational difficulties are also discussed. Despite these limitations it is concluded that PCR will continue to make significant contributions to both clinical practice and to our understanding of the basic biology of HCV infection.