Hepatitis C Virus Antigens Research Articles

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.

Effect of HLA on hepatitis C virus clearance and persistence in anti-HCV-positive end-stage renal disease patients

Background/Aims:The efficacy of immune response against hepatitis C virus (HCV) is determined by human leukocyte antigen (HLA) molecules of the host which present HCV antigens to CD4 + and CD8 + T lymphocytes. In this study, we aimed to investigate the possible relationship between the frequencies of certain HLA class I–II alleles and the natural history of HCV in patients with end-stage renal disease (ESRD).Settings and Design:This is a retrospective cohort study conducted in a university hospital.Patients and Methods:The present study comprised 189 ESRD patients (candidates for renal transplantation) who had positive anti-HCV antibody test. The results concerning HCV and HLA status were gathered from patients’ files. The viral persistence was compared between the groups that were determined by HLA sub-typing.Statistical Analysis:Statistical evaluation was performed using Mann–Whitney U-test, Chi-square test, and Fisher's exact test. Level of error was set at 0.05 for all statistical evaluations, and P values < 0.05 were considered statistically significant.Results:We found possible association between the course of HCV infection and specific HLA alleles. HLA class I Cw*6 and HLA class II DRB*10 alleles were observed more frequently in the viral clearance group (P < 0.05). The HLA class I B*38 allele group was more prone to develop chronic hepatitis C (P < 0.01).Conclusions:These findings suggest that HLA class I Cw*6 and HLA class II DRB*10 alleles may be associated with immunological elimination of HCV in Turkish patients on hemodialysis. HLA sub-typing could help predict the prognosis of HCV infection.

Is universal screening for hepatitis C infection prior to commencing antitumour necrosis factor-α therapy necessary?

Screening for hepatitis C virus (HCV) prior to the commencement of antitumour necrosis factor (anti-TNF)-α therapies for dermatological disease is recommended for all patients. To determine the incidence of HCV infection among dermatology patients who were screened for HCV infection prior to commencing anti-TNF-α therapies. We reviewed the HCV infection status of all patients attending our dermatology department who had been tested for evidence of HCV infection between January 2005 and November 2012. We identified patients who had been tested as part of routine screening prior to commencing anti-TNF-α therapy using dermatology departmental records. In total, 215 patients were screened for HCV infection prior to commencing anti-TNF-α therapies. Among this group, 143 patients (66·5%) were male and 72 (33·5%) were female. None of these patients tested positive for active HCV infection. One patient tested positive for HCV antibody with negative HCV antigen and HCV RNA. This indicated previous HCV infection that had cleared. This patient had abnormal liver function tests and a history of alcohol excess. There were no cases of active HCV infection diagnosed through pretreatment anti-TNF-α screening in our department, which is located in a low-prevalence area for HCV infection. In view of the lack of evidence of harm associated with anti-TNF-α use in HCV-infected patients, we propose that screening for HCV infection in low-prevalence areas should be targeted to those with pre-existing risk factors. This is consistent with current guidelines from the Royal College of General Practitioners. Targeted screening rather than universal screening may be a safe and cost-effective option among patients being evaluated for anti-TNF-α therapies.

Prominent proliferative response of peripheral blood mononuclear cells to a recombinant non-structural (NS3) protein of hepatitis C virus in patients with chronic hepatitis C.

The proliferative response of peripheral blood mononuclear cells (PBMC) to a recombinant non-structural (NS3) protein of hepatitis C virus (HCV) was studied in 41 patients with chronic hepatitis C. Of them, 28 had chronic persistent hepatitis (CPH) and 13 chronic active hepatitis (CAH). The positive proliferation rate of PBMC to the recombinant NS3 protein, T9Ag, was 66% in the 41 patients (77% in CAH versus 61% in CPH; P > 0.05) when stimulation index (SI) = 4 was set as the cut-off value. However, mean SI of CAH patients was significantly higher than that of CPH patients (8.3 +/- 5.2 versus 5.1 +/- 3.6; P < 0.05). Six other chronic hepatitis patients who were repeatedly negative for anti-HCV antibody but positive for serum HCV RNA also had an SI of > or = 4.0. The frequency of cellular immune response to the T9Ag is among the highest results obtained by using HCV antigens tested so far. Our studies thus indicate that NS3 is an immunologically important region of HCV for T cells. Moreover, the proliferative response to T9Ag may help to establish hepatitis C etiology in chronic hepatitis patients who are seronegative with currently available anti-HCV assays.

Optimization of peptide arrays for studying antibodies to hepatitis C virus continuous epitopes

Accurate and in-depth mapping of antibody responses is of great value in vaccine and antibody research. Using hepatitis C virus (HCV) as a model, we developed an affordable and high-throughput microarray-based assay for mapping antibody specificities to continuous antibody epitopes of HCV at high resolution. Important parameters in the chemistry for conjugating peptides/antigens to the array surface, the array layout, fluorophore choice and the methods for data analysis were investigated. Microscopic glass slide pre-coated with N-Hydroxysuccinimide (NHS)-ester (Slide H) was the preferred surface for conjugation of aminooxy-tagged peptides. This combination provides a simple chemical means to orient the peptides to the conjugation surface via an orthogonal covalent linkage at the N- or C-terminus of each peptide. The addition of polyvinyl alcohol to printing buffer gave uniform spot morphology and improved sensitivity and specificity of binding signals. Libraries of overlapping peptides covering the HCV E1 and E2 glycoprotein polypeptides (15-mer, 10 amino acids overlap) of 6 major HCV genotypes and the entire polypeptide sequence of the prototypic strain H77 were synthesized and printed in quadruplets in the assays. The utility of the peptide arrays was confirmed using HCV monoclonal antibodies (mAbs) specific to known continuous epitopes and immune sera of rabbits immunized with HCV antigens. The methods developed here can be easily adapted to studying antibody responses to antigens relevant in vaccine and autoimmune research.

British HIV Association guidelines for the management of hepatitis viruses in adults infected with HIV 2013

British <scp>HIV A</scp>ssociation guidelines for the management of hepatitis viruses in adults infected with <scp>HIV</scp> 2013

Sensitivity of a Ribavirin Resistant Mutant of Hepatitis C Virus to Other Antiviral Drugs

BackgroundWhile ribavirin mono-therapy regimens have minimal effect on patients with chronic hepatitis C virus (HCV) infections, they can be efficacious when combined with interferon. Clinical studies show that interferon-free combination therapies containing ribavirin are also efficacious, suggesting that an interferon-free therapy could be adopted in the near future. However, generation of drug resistant mutants and cross resistance to other drugs could impair the efficacy of the treatment. Therefore, understanding the mechanism of HCV resistance to ribavirin and cross resistance to other antiviral drugs could be of major importance.MethodsWe tested the ability of a J6/JFH1 derived HCV ribavirin resistant mutant to grow in tissue cultured Huh7D cells in the presence of the mutagen 5-Fluorouracil and the nucleoside analog 2′-C-Methylcytidine. Virus replication was assessed by detecting HCV antigens by immunofluorescence and by titrating virus present in the supernatants. Recovered viruses were amplified by RT-PCR and sequenced.ResultsThe sensitivity of HCV-RR relative to parental J6/JFH1 to the tested drugs varied. HCV-RR was more resistant than J6/JFH1 to 5-Fluorouracil but was not more resistant than J6/JFH1 to 2′-C-Methylcytidine. Growth of HCV-RR in 5-Fluorouracil allowed the selection of an HCV-RR derived mutant resistant to 5-Fluorouracil (HCV-5FU). HCV-5FU grows to moderate levels in the presence of high concentrations of 5-Fluorouracil and to parental levels in the absence of the drug. Sequence of its genome shows that HCV-5FU accumulated multiple synonymous and non-synonymous mutations.ConclusionsThese results indicate that determinants of resistance to ribavirin could also confer resistance to other anti-HCV drugs, shedding light toward understanding the mechanism of action of ribavirin and highlighting the importance of combination drug selection for HCV treatment. The results also show that it is possible to select a 5-Fluorouracil HCV resistant mutant that replicates to levels similar to parental virus when grown in the absence of 5-Fluorouracil.

Use of Laser Capture Microdissection to Map Hepatitis C Virus–Positive Hepatocytes in Human Liver

Hepatitis C virus (HCV) predominantly infects hepatocytes, but many hepatocytes are not infected; studies have shown that HCV antigens cluster within the liver. We investigated spatial distribution and determinants of HCV replication in human liver samples. We analyzed liver samples from 4 patients with chronic HCV infection (genotype 1, Metavir scores 0-1) to estimate the proportion of infected hepatocytes and the amount of HCV viral RNA (vRNA) per cell. Single-cell laser capture microdissection was used to capture more than 1000 hepatocytes in grids, to preserve geometric relationships. HCV vRNA and interferon-induced transmembrane protein 3 (IFITM3) messenger RNA (the transcript of an interferon-stimulated gene) were measured in the same hepatocytes by quantitative polymerase chain reaction and assembled in maps to identify areas of high and low HCV replication. Patients' serum levels of HCV RNA ranged from 6.87 to 7.40 log10 IU/mL; the proportion of HCV-infected hepatocytes per person ranged from 21% to 45%, and the level of vRNA ranged from 1 to 50 IU/hepatocyte. Infection was not random; we identified clustering of HCV-positive hepatocytes using infected-neighbor analysis (P < .0005) and distance to the kth nearest neighbor compared with random distributions, obtained by bootstrap simulations (P < .02). Hepatocytes that expressed IFITM3 did not appear to cluster and were largely HCV negative. We used single-cell laser capture and high-resolution analysis to show that in human liver HCV infects hepatocytes in nonrandom clusters, whereas expression of antiviral molecules is scattered among hepatocytes. These findings show that quantitative single-cell RNA measurements can be used to estimate the abundance of HCV vRNA per infected human hepatocyte and are consistent with cell-cell propagation of infection in the absence of clustered IFITM3.

Imaging of hepatitis C virus infection in liver grafts after liver transplantation

The detection of native hepatitis C virus (HCV) antigens in liver tissue may be relevant to diagnostic purposes and to better understand the pathogenesis of HCV infection. The aim of our study was to characterize HCV antigens in liver grafts. We selected 32 liver transplant (LT) recipients with recurrent hepatitis C. HCV core and NS5A antigens were detected in formalin-fixed, paraffin-embedded (FFPE) liver biopsies obtained immediately after graft reperfusion (negative controls), during the acute phase of HCV infection (1-6 months) and during follow-up (7-74 months) after LT. Viral antigens were assessed by immunohistochemistry and confocal microscopy. All reperfusion biopsies were negative for both antigens. Core protein was detected in 75% and 33% of acute phase and follow-up biopsies, respectively. HCV antigens were not detected in any of the 10 samples from patients who cleared HCV after antiviral treatment. Immunostaining was hepatocellular, with a granular cytoplasmic pattern and a wide spectrum of intensity. We found a significant association between viral load and the presence of HCV core-positive hepatocytes (p=0.004). NS5A colocalized strongly with core (66%) and adipophilin (36%), supporting the localization of core and NS5A around lipid droplets. A detailed three-dimensional analysis showed that NS5A surrounded the core and adipophilin-positive areas. HCV antigens can be detected in FFPE liver biopsies by immunohistochemistry. The in vivo colocalization of core and NS5A proteins around the lipid droplets supports that the latter may play a role in virus particle production, similar to what reported in vitro.

Detection of viral antigens in renal tissue of glomerulonephritis patients without serological evidence of hepatitis B virus and hepatitis C virus infection

Glomerulonephritis is an important extrahepatic manifestation of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. HBV and HCV infection may be occult, and they are often overlooked by both patients and doctors. The aim of this study was to assess the importance of HBV and HCV infection in glomerulonephritis patients with undetectable HBV surface antigen (HBsAg) and HCV antibody in serum. The HBsAg, the HBV core antigen (HBcAg), and the HCV antigen were detected using immunohistochemistry in frozen renal tissues of 500 glomerulonephritis patients without serological evidence of HBV and HCV infection. Electron microscopy was used to trace the virus particles, and clinicopathological features were also reviewed. HBsAg or HBcAg was positive in nine out of 500 cases (9/500, 1.8%). Three cases were HBsAg-positive and another six cases were HBcAg-positive. The HCV antigen was found in eight cases (8/500, 1.6%). There was one case of HBV and HCV co-infection (1/500, 0.2%). Under electron microscopy, virus particles were found in the base membrane and cytoplasm of endotheliocytes in the glomerulus. The most common clinical manifestation was nephrotic syndrome (9/18), followed by nephritic syndrome (7/18). Membranous nephropathy was the most common pathological diagnosis (5/18), followed by mesangioproliferative glomerulonephritis (4/18) and IgA nephropathy (4/18). Occult HBV and HCV infection might be implicated in HBV- or HCV-associated glomerulonephritis. More attention should be focused on the underlying cause.

Virus-like particle-based vaccines against hepatitis C virus infection

HCV infection is a worldwide health problem and much effort is being made to develop novel therapies. New vaccines are designed for preventive and therapeutic use through induction of robust immunity, including neutralizing antibodies and T-cell-mediated immunity. Novel future vaccine approaches include virus-like particle (VLP)-based vaccines that have been successfully employed to prevent infections by hepatitis B virus or human papillomavirus. The HCV-derived VLP approach simplifies the delivery of neutralizing antibody- and core-specific T-cell epitopes in a highly immunogenic single construct resembling mature HCV virions. The size, particulate nature and dense, repetitive structure of VLPs are the basis for their innate immunogenicity. Consequently, VLPs have also been exploited as antigenic platforms. Association of HCV antigens with heterologous structural viral proteins able to form recombinant VLPs (HBsAg, HBcAg, MLV Gag, PapMV coat protein) is also a promising approach for induction of HCV-specific immune responses. This article summarizes the different VLP-based vaccine approaches that are currently under development.

Between Scylla and Charybdis: The role of the human immune system in the pathogenesis of hepatitis C

Hepatitis C virus (HCV) frequently elicits only mild immune responses so that it can often establish chronic infection. In this case HCV antigens persist and continue to stimulate the immune system. Antigen persistence then leads to profound changes in the infected host's immune responsiveness, and eventually contributes to the pathology of chronic hepatitis. This topic highlight summarizes changes associated with chronic hepatitis C concerning innate immunity (interferons, natural killer cells), adaptive immune responses (immunoglobulins, T cells, and mechanisms of immune regulation (regulatory T cells). Our overview clarifies that a strong anti-HCV immune response is frequently associated with acute severe tissue damage. In chronic hepatitis C, however, the effector arms of the immune system either become refractory to activation or take over regulatory functions. Taken together these changes in immunity may lead to persistent liver damage and cirrhosis. Consequently, effector arms of the immune system will not only be considered with respect to antiviral defence but also as pivotal mechanisms of inflammation, necrosis and progression to cirrhosis. Thus, avoiding Scylla - a strong, sustained antiviral immune response with inital tissue damage - takes the infected host to virus-triggered immunopathology, which ultimately leads to cirrhosis and liver cancer - the realm of Charybdis.

Induction of intrahepatic HCV NS4B, NS5A and NS5B-specific cellular immune responses following peripheral immunization.

Numerous studies have suggested that an effective Hepatitis C Virus (HCV) vaccine must induce strong cytotoxic and IFN-γ+ T cell responses targeting the non-structural region of the virus. Most importantly, these responses must be able to migrate into and remain functional within the liver, an organ known to cause T cell tolerance. Using three novel HCV DNA vaccines encoding non-structural proteins NS4B, NS5A and NS5B, we assessed the ability of peripheral immunization to induce functional intrahepatic immunity both in the presence and absence of cognate HCV antigen expression within the liver. We have shown that these constructs induced potent HCV-specific CD4+ and CD8+ T cell responses in the spleen of C57BL/6 mice and that these responses were detected within the liver following peripheral immunization. Additionally, using a transfection method to express HCV antigen within the liver, we showed that intrahepatic HCV-specific T cells remained highly functional within the liver and retained the ability to become highly activated as evidenced by upregulation of IFN-γ and clearance of HCV protein expressing hepatocytes. Taken together, these findings suggest that peripheral immunization can induce potent HCV-specific T cell responses able to traffic to and function within the tolerant environment of the liver.

Suspected and unsuspected factors in the multifaceted immunopathology of viral hepatitis

In the past decade, our knowledge of the mechanisms producing disease during acute and chronic infections with hepatitis B (HBV) and C (HCV) viruses has solidified the fact that the immune response induced by these viruses plays dominant roles. It is becoming increasingly clear that HBV and HCV can subvert the host immune system to survive, and the viral persistence can also induce autoimmune manifestations that complicate the clinical setting and may contraindicate therapies that indiscriminately augment immune effector mechanisms [1]. The liver is considered as an “immunological organ”, with innate immune functions and cells [2] that aid in the recognition of HBV and HCV antigens, leading to the production of type I interferons (I-IFN) and inflammatory cytokines that inhibit the viral replication [3]. This is potentially beneficial in clearing viral infections, but may also initiate liver fibrosis, increasing the risk of cirrhosis and the subsequent risk of hepatocellular carcinoma (HCC) [4]. In the case of HBV, chronic liver disease afflicts approximately 300– 400 million people worldwide, despite the availability of a safe and effective vaccine [5]. HCV infects up to 170 million persons worldwide and the development of cirrhosis is associated with an annual incidence of HCC of 3–5 % [6, 7]. Currently, HCV infection, resulting in cirrhosis and/or HCC, is the leading indication for orthotopic liver transplantation in developed countries [6, 7]. To provide a comprehensive view of the newest information regarding HBV and HCV infections, the articles presented in this special issue of Seminars in Immunopathology focus on the most controversial immunopathological aspects associated with both infections. These articles provide new evidence of the link between the immune response and virus survival within hepatocytes, elucidate occult infections and the risk of recurrent hepatitis in liver transplanted patients, and describe the autoimmunity associated with chronic HBV and HCV infections. The contributions to this special issue prove a deeper understanding of the mechanisms by which hepatitis and its complications are caused by the viruses themselves as well as by inflammation and immunological mechanisms that culminate in chronic liver damage. For pragmatic purposes, we have arbitrarily subdivided the themes discussed in this issue between suspected and unsuspected factors involved in the pathogenesis (Table 1). One limitation in the study of HBVand HCV infections is the lack of understanding in the replication strategies used by these viruses to persist in infected liver cells. Dandri et al. describe the most recent experimental models developed for the study of HBV, that are surrogates because HBV infection occurs naturally only in human beings and chimpanzee, which limits potential studies due to obvious practical and ethical problems. These numerous in vitro systems and animal models (i.e. murine, duck, woodchuck and tree shrew) have provided a better insight into the progress of HBV infection, and have been helpful in understanding the immunopathogenesis and testing of new therapeutic strategies. It is important to highlight the role of the innate immune system in response to viral hepatitis, and in fact This article is a contribution to the special issue on Immunopathology of Viral Hepatitis _ Guest Editors: C. Selmi and J. M. Vierling C. Selmi :A. Ceribelli Rheumatology and Clinical Immunology, Humanitas Clinical and Research Center, Milan, Italy

Immunization with a Recombinant Vaccinia Virus That Encodes Nonstructural Proteins of the Hepatitis C Virus Suppresses Viral Protein Levels in Mouse Liver

Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid–polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29(+/−)/MxCre(+/−) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.

The use of epitope arrays in immunodiagnosis of infectious disease: Hepatitis C virus, a case study

Serodiagnosis of infectious disease is often based on the detection of pathogen-specific antibodies in a patient’s blood. For this, mixtures of pathogen-related antigens are used as bait to capture corresponding antibodies in solid phase immunoassays such as enzyme immunoassay (EIA). Western blots provide improved diagnostic power as compared with EIA due to the fact that the mixture of markers in the EIA well is resolved and tested as individual antigens on the Western blot. Hence, confirmation of EIA results is accomplished using the antigen arrays of Western blots. Here we took this approach one step further and tested the attributes of using epitope arrays in a diagnostic platform coined “combinatorial diagnostics.” As a case in point, we tested a panel of phage-displayed epitope-based markers in the serodiagnosis of hepatitis C virus (HCV). The repertoire of HCV antigens was deconvoluted into panels of distinct linear and conformational epitopes and tested individually by quantitative EIA. Combinatorial diagnostics proved to be effective for the discrimination between positive and negative sera as well as serotyping of HCV.

Induction of Multi-Functional T Cells in a Phase I Clinical Trial of Dendritic Cell Immunotherapy in Hepatitis C Virus Infected Individuals

We have previously reported a world-first phase I clinical trial to treat HCV patients using monocyte-derived dendritic cells (Mo-DC) loaded with HCV-specific lipopeptides. While the brief treatment proved to be safe, it failed to reduce the viral load and induced only transient cell-mediated immune responses, measured by IFNγ ELIspot. Here we reanalysed the PBMC samples from this trial to further elucidate the immunological events associated with the Mo-DC therapy. We found that HCV-specific single- and multi-cytokine secreting T cells were induced by the Mo-DC immunotherapy in some patients, although at irregular intervals and not consistently directed to the same HCV antigen. Despite the vaccination, the responses were generally poor in quality and comprised of primarily single-cytokine secreting cells. The frequency of FOXP3+ regulatory T cells (Treg) fluctuated following DC infusion and eventually dropped to below baseline by week 12, an interesting trend suggesting that the vaccination may have resulted in a more subtle outcome than was initially apparent. Our data suggested that Mo-DC therapy induced complex immune responses in vivo that may or may not lead to clinical benefit.

Performance of an antigen–antibody combined assay for hepatitis C virus testing without venipuncture

BackgroundHepatitis C virus (HCV) is underdiagnosed and therefore increasing the opportunities for HCV testing without venipuncture may be useful. ObjectivesWe evaluated the analytical performance of a modified, commercially available, combined HCV antigen–antibody assay (cEIA) (Monolisa® HCV-Ag-Ab-ULTRA) and a commercially available point-of-care (POC) device (OraQuick® HCV) on fingerstick blood (FSB) and oral mucosal transudate (OMT). Study designFSB, OMT and serum samples were collected from 113 cases of HCV-antibody-positive patients and 88 HCV-antibody-negative controls. The HCV-antibody-positive group included 63 patients with quantifiable HCV-RNA (56%) and 17 HIV/HCV co-infected patients (15%). FSB and OMT specimens were collected as dried blood spots (DBSs) or with the OraSure collection system, before testing with cEIA. ResultsWith FSB specimens, the cEIA and the POC device exhibited 100% specificity and 98.2% and 97.4% sensitivity, respectively. The specificity of the cEIA in FSB sharply decreased if stored 3days at room temperature. With OMT specimens, the cEIA sensitivity (71.7%) and specificity (94.3%) were significantly lower than the performance of OraQuick® HCV (sensitivity, 94.6%; specificity, 100%). The optical densities obtained with the cEIA in FSB and OMT were lower in HIV/HCV co-infected patients compared with HCV monoinfected patients. ConclusionThe cEIA using FSB specimens collected on DBSs preserved in appropriate storage conditions was a reliable alternative, equivalent to the POC assay, for HCV testing without venipuncture. The cEIA was not adapted for HCV testing on OMT.

Hepatitis C Virus-Specific Cell-Mediated Immune Responses in Children Born to Mothers Infected with Hepatitis C Virus

ObjectiveTo investigate the association between hepatitis C virus (HCV)-specific cell-mediated immunity (CMI) responses and viral clearance in children born to mothers infected with HCV.Study designA cross-sectional study of children from a mother-infant cohort in Egypt were enrolled to detect CMI responses to recombinant core and nonstructural HCV antigens (nonstructural segments NS3, NS4a/b, and NS5 of the HCV genome) using an interferon-gamma enzyme-linked immunospot assay. Children born to mothers with chronic HCV were enrolled into 3 groups: transiently viremic (n = 5), aviremic (n = 36), and positive control (n = 6), which consisted of 1 child with chronic HCV from this cohort and another 5 children with chronic HCV from a companion study. Children without HCV born to mothers without HCV (n = 27) served as a negative control group. Wilcoxon rank sum test was used to compare the magnitude of CMI responses between groups.ResultsNone of the 6 control children who were positive for HCV responded to any HCV antigen, and 4 (80%) of 5 children with transient viremia responded to at least one HCV antigen, compared with 5 (14%) of 36 and 3 (11%) of 27 children in the aviremic and negative control groups, respectively. Children with transient viremia elicited stronger responses than did negative controls (P = .005), positive controls (P = .011), or children without HCV viremia (P = .012), particularly to nonstructural antigens.ConclusionsHCV-specific CMI responses were significantly higher in magnitude and frequency among transiently infected children compared with those persistently infected. This suggests CMI responses may be associated with past viral clearance and can identify children at high risk of infection, who can be targeted for health education, screening, and follow-up.

Laboratory Diagnostics for Hepatitis C Virus Infection

Identification of prevalent infection by hepatitis C virus (HCV) is based serologically on detecting anti-HCV immunoglobulin G, using immunoassays, immunoblot assays, and, more recently, immunochromatography-based rapid tests. None discriminate between active and resolved HCV infection. Tests for detecting HCV RNA identify active HCV infection but are costly. Serologic assays for HCV antigens have been developed and show potential for diagnosis of active HCV infection, and their performance characteristics are undergoing evaluation. The diagnosis of acute HCV infection without the demonstration of seroconversion remains elusive.