Evaluation of an enzyme immunoassay for hepatitis C virus antibody detection using a recombinant protein derived from the core region of hepatitis C virus genome

This study was undertaken to evaluate an enzyme immunoassay (EIA) for hepatitis C virus antibody detection (anti-HCV), using just one antigen. Anti-HCV EIA was designed to detect anti-HCV IgG using on the solid-phase a recombinant C22 antigen localized at the N-terminal end of the core region of HCV genome, produced by BioMérieux. The serum samples diluted in phosphate buffer saline were added to wells coated with the C22, and incubated. After washings, the wells were loaded with conjugated anti-IgG, and read in a microtiter plate reader (492 nm). Serum samples of 145 patients were divided in two groups: a control group of 39 patients with non-C hepatitis (10 acute hepatitis A, 10 acute hepatitis B, 9 chronic hepatitis B, and 10 autoimmune hepatitis) and a study group consisting of 106 patients with chronic HCV hepatitis. In the study group all patients had anti-HCV detected by a commercially available EIA (Abbott), specific for HCV structural and nonstructural polypeptides, alanine aminotransferase elevation or positive serum HCV-RNA detected by nested-PCR. They also had a liver biopsy compatible with chronic hepatitis. The test was positive in 101 of the 106 (95%) sera from patients in the study group and negative in 38 of the 39 (97%) sera from those in the control group, showing an accuracy of 96%. According to these results, our EIA could be used to detect anti-HCV in the serum of patients infected with hepatitis C virus.     

Immunoenzyme analysis for determining class G and M antibodies to HBsAg

A scheme of the purification of hepatitis B virus surface antigen (HBsAg) as applied to the enzyme immunoassay (EIA) for the detection of antibodies to HBsAg is described. An indirect EIA technique for the detection of IgG and IgM antibodies to HBsAg has been developed and the diagnostic assay system based on the use of immunoreagents and solid-phase carriers produced in the USSR has been obtained. The sensitivity of the indirect EIA technique in the detection of IgG antibodies to HBsAg exceeds that of double immunodiffusion in gel used for this purpose 2,500- to 5,000-fold. The study has shown the possibility of using the indirect EIA technique for the detection of antibodies to HBsAg, both free and bound in immune complexes, of detecting antibodies to HBsAg in patients with acute and chronic viral hepatitis B, as well as of simultaneous detection of IgG and IgM antibodies to HBsAg without pseudonegative results.     

Prevalence of hepatitis B virus in chronic hepatitis B patients

The aim of the current study was to detect HBV by Real time - PCR in chronic hepatitis B patients. Fifty-eight sera of chronic hepatitis B patients were subjected during the period March 2009 to April 2010 in Ilam cities in West of Iran. Sera assayed by real-time PCR and ELISA methods. Twenty serum samples from healthy volunteers and non-hepatitis B patients and negative for hepatitis B seromarkers served as negative controls for the study. Among fifty-eight sera, ELISA showed fifty-five (94.8%) of the samples were positive for HBsAg and three (5.2%) negative results obtained while real-time PCR specified fifty-eight (100%) positive results in chronic hepatitis B patients. HBsAg status did not necessarily reflect HBV DNA level in the serum, as 5.2% of chronic Hepatitis B patients were positive for HBV DNA but negative for HBsAg. HBV DNA was not found to be positive amongst any of the negative controls. Real time - PCR is a sensitive and reproducible assay for HBV DNA quantization.     

Seroepidemiology of hepatitis C virus in Beijing, China

To investigate the seroprevalence of hepatitis C virus (HCV) in China we tested sera from healthy individuals without hepatitis and no history of parenteral blood exposure and from patients admitted to a hepatitis hospital in Beijing. Sera were tested for anti-HCV by first-generation enzyme immunoassay; selected positives were tested with two second-generation EIAs, one utilizing recombinant antigens and the other synthetic peptides. We found anti-HCV with the following frequencies: 10 of 164 (6%) individuals with no disease; 2 of 36 (5.5%) patients with acute non-A non-B hepatitis (NANBH); 26 of 39 (67%) patients with post-transfusion NANBH; 10 of 34 (29%) patients with chronic hepatitis negative for hepatitis B surface antigen (HBsAg); 3 of 30 (10%) patients with chronic HBsAg-positive hepatitis; 0 of 19 patients with acute HBsAg-positive hepatitis. Of 24 repeat-positive sera, 19 were positive by both and 4 by one second-generation tests. We conclude that hepatitis C infection is common in China, that it contributes substantially to the incidence of post-transfusion hepatitis, and that HCV plays a significant role in both acute and chronic hepatitis. Further studies are needed to extend these observations and to define the predominant routes of transmission of HCV in China.     

Interaction between various polymerized human albumins and hepatitis B surface antigen.

A variety of albumin polymers were prepared and tested for binding with hepatitis B surface antigen (HBsAg): synthetic polymers cross-linked by either glutaraldehyde or carbodiimide; heat-aggregated polymers made by heating albumin solutions at 60 degrees C for 10 h with or without albumin stabilizer; and polymers isolated from fresh or long-stored commercial therapeutic albumin solutions. A sensitive solid-phase, competitive-inhibition radioimmunoassay, which can detect as little as 10 ng of glutaraldehyde-cross-linked human albumin polymer (PHALB-G), was developed and used to measure binding. The binding of PHALB-G with HBsAg was 150- to 1,000-fold greater than that of any other albumin polymer. Glutaraldehyde-cross-linked bovine albumin polymer showed no binding. Albumin monomer and dimer fractions produced by glutaraldehyde treatment exhibited some binding, albeit much weaker than PHALB-G. As measured by a direct-binding assay with solid-phase PHALB-G, the attachment of HBsAg particles from sera positive for antibody to the e antigen was less efficient than that from sera positive for e antigen, even when all sera were tested at equal HBsAg concentrations. In protein blot experiments with radiolabeled albumin preparations, PHALB-G bound almost exclusively to HBsAg polypeptide P31 and showed no binding with the major polypeptides P23 and P26. None of the other radiolabeled albumin polymers was reactive. These results indicate that the interaction between PHALB-G and HBsAg is not due to polymerization of albumin per se, but rather is unique and site specific.     

Prognosis for the patients with chronic hepatitis B

The purpose of the research was to determine the influence of the hepatitis B virus on the progression of the chronic liver disease. In the present paper, 127 patients who were followed up for five years and who had histologically verified chronic liver disease, are described. Fifty two of them were carriers of HBsAg, 75 patients were HBsAg negative, but had other markers typical for a previous infection of HBV in the sera. All the patients were nonalcoholics and no drug addicts. In the sera of these 127 patients markers of HBV were prospectively followed up: HBsAg, HBeAg, anti-HBs, anti-HBc, anti-HBe, HBVDNA, antiHCV for C virus and anti-D for D virus. It was proved by these investigations that HBV provokes very severe chronic hepatitis: CAH (chronic active hepatitis) and CH (cirrhosis hepatis). It was also proved that HBV replicated in 44.20% patients, namely, HBVDNA was positive in the sera of those patients. In 26.08% of such patients the mutant form of HBV was present. In spite of progressive liver disease and without any antiviral therapy all the patients with chronic HBV cirrhosis hepatis were, after five year-follow-up, in Child-Pugh A grade. It was found that the patients who were HBsAg negative, but had one or more markers of HBV positive in the sera, had also a severe chronic hepatitis. That group of patients remains our object of further research. The five-years follow-up of all these patients demonstrates that it is necessary to find out an efficient medicament against HBV chronic hepatitis. Obligatory vaccination of the risk population against virus B remains the only prevention against this severe disease.     

乙型肝炎病人重叠感染丙型肝炎的评价

应用ＥＬＩＳＡ和ＰＣＲ法检测５０２例乙肝病人血清,４０１例ＨＢｓＡｇ阳性血清中,有１１４例（２８．４％）抗－ＨＣＶ和ＨＣＶＲＮＡ双项阳性,２５例（６．２％）ＨＣＶＲＮＡ单项阳性;２１例（５．２％）抗－ＨＣＶ单项阳性。将ＨＢｓＡｇ乙肝病人分成ＨＢＶＤＮＡ,ＨＢｅＡｇ阳性组和ＨＢＶＤＮＡ,ＨＢｅＡｇ阴性组。前者抗－ＨＣＶ阳性率为１１．６％～２０．５％,ＨＣＶＲＮＡ阳性率为１６．２％～２０．５％。后者抗－ＨＣＶ阳性率为２０．２％～５５．６％,ＨＣＶＲＮＡ阳性率为２３％～６０．３％。结果说明长期携带ＨＢＶ者和慢性乙肝病人均可重叠ＨＣＶ感染。ＨＢＶＤＮＡ阳性组抗－ＨＣＶ和ＨＣＶＲＮＡ阳性率明显高于ＨＢＶＤＮＡ阳性组     

Hepatitis C antibodies in groups at epidemic risk

Blood sera from patients with acute virus hepatitis, sexually transmitted diseases, psychiatric and oncological diseases and pulmonary tuberculosis were studied for the presence of antibodies to hepatitis C virus (anti-HCV) and HBsAg with the use of solid-phase enzyme immunoassay. Anti-HCV were detected, respectively, in 9.1% and 67.8% of patients with acute virus hepatitis, in 5.7% and 6.1% of patients with sexually transmitted diseases, in 7.5% and 9.2% of psychiatric patients, in 7.5% and 13% with pulmonary tuberculosis and malignant tumors.     

Hepatitis B virus surface and core antigens in the liver of primary hepatocellular carcinoma cases in Virginia

Zenker-fixed paraffin-embedded sections of biopsy liver tissue from 64 cases of primary hepatocellular carcinoma (PHC) were stained for hepatitis B surface antigen (HBsAg) and for hepatitis B core antigen (HBcAg) by histochemical and/or immunohistochemical techniques in a retrospective study. PHC arose in livers with postnecrotic cirrhosis in 30 (46.9%) cases. Controls included liver biopsy sections from 123 miscellaneous liver disorders and from 67 randomly selected autopsy specimens, none of which were known to be associated with hepatitis B virus (HBV) infection. HBsAg was detected in tumorous hepatocytes in only one of the 64 cases of PHC. HBsAg was identified in nontumorous hepatocytes of 8 (20%) of 40 specimens that contained adequate nontumorous liver tissue. All of these HBsAg positive cases of PHC were associated with cirrhosis. Thus HBsAg was detected in 8 (33.3%) of 24 cases of PHC with cirrhosis, but in none of the remaining 16 cases without cirrhosis. HBcAg was not detected in the hepatocytes of those HBsAg positive PHC cases tested. Our results suggest that HBV infection may successively lead to chronic hepatitis, cirrhosis and ultimately PHC.     

Seroprevalence of viral hepatitis in riverine communities from the Western Region of the Brazilian Amazon Basin.

The western region of the Brazilian Amazon Basin has long been shown to be a highly endemic area for hepatitis B and hepatitis D viruses. Data concerning the prevalence of hepatitis C and E viruses in this region are still scarce. In this study we investigated the presence of hepatitis A, B, C, D and E viruses infection in communities that live along the Purus and Acre rivers in the states of Acre and Amazonas within the Amazon Basin. A total of 349 blood samples were collected and tested for hepatitis A-E serological markers (antibodies and/or antigens) using commercial enzyme linked immunosorbent assays. Anti-HCV positive sera were further assayed by an immunoblot. HBsAg positive sera were subtyped by immunodifusion. The overall prevalence for hepatitis A, B, C, and E were 93.7%, 66.1%, 1.7%, and 4%, respectively. A very high prevalence of delta hepatitis (66.6%) was found among HBsAg positive subjects. Hepatitis A, B and D viruses were shown to be largely disseminated in this population, while hepatitis C and E viruses infection presented low prevalence rates in this region. The analysis of risk factors for HBV infection demonstrated that transmission was closely associated with sexual activity.     

Detection of hantaviral antibodies among patients with hepatitis of unknown etiology in Japan

Hantaviral antibodies were detected in the sera from patients with hepatic disease of unknown etiology in Japan by several different serological diagnostic methods. A total of 105 sera from diseased patients which were negative to A-G hepatitis virus infections in the Tokyo area were tested. Among them, 3 out of 73 sera from patients with chronic hepatic disease were positive to hantaviral antibody by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent antibody assay (IFA) and Western blot analysis (WB). Neutralizing antibody titers of the 3 sera to Seoul virus (SEO) were 4 to 8 times higher than those to Hantaan virus (HTN). However, all of the 32 sera from patients with acute hepatitis were negative for hantaviral antibody. Among the 60 patients with chronic hepatitis in Hokkaido which were serologically negative to B and C hepatitis virus infection, one was positive for hantaviral antibody by ELISA and WB. In contrast, the sera from healthy adults in Japan, 550 from the Honshu and Kyushu regions, and 1,000 from the Hokkaido region, were negative for hantavirus antibody. These results show that hantaviral antibodies are more frequently detected in patients with hepatic disease than in healthy adults. However, the observation that no positive sera were detected from patients with acute hepatitis implies that hantavirus might not be directly related to hepatitis.     

应用血清学方法鉴别急性和慢性病毒性乙型肝炎

本文用ELISA间接法检测急性和慢性乙型肝炎病人血清特异性抗HBcIgG,用ELISA捕捉法检测特异性抗HBcIgM。11例急性乙肝病人急性期抗HBcIgM100％阳性,抗HBcIgG全部阴性;恢复期抗HBcIgM 81.8％阴转,抗HBcIgG则100％阳转。17例慢性乙肝病人抗HBcIgM82.35％阳性,抗HBcIgG 100％阳性。被检血清经密度梯度超速离心,证实抗HBcIgM和抗HBcIgG两类抗体反应在急性和慢性乙肝病人血清中具有不同的动态规律。     

Genotype and subtype analyses of hepatitis B virus (HBV) and possible co-infection of HBV and hepatitis C virus (HCV) or hepatitis D virus (HDV) in blood donors, patients with chronic liver disease and patients on hemodialysis in Surabaya, Indonesia

Four subtypes (adw, adr, ayw, and ayr ) and eight genotypes (A to H) of the hepatitis B virus (HBV) have been identified. They appear to be associated with particular geographic distribution, ethnicity, and possibly clinical outcomes. In this study, hepatitis B surface antigen (HBsAg) subtyping and HBV genotyping were carried out on sera obtained from HBsAg-positive HBV carriers, including healthy blood donors; patients with acute hepatitis, chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma; and patients on hemodialysis all located in Surabaya, Indonesia. We report here that all HBV isolates tested in Surabaya belonged to genotype B, with more than 90% of them being classified into subtype adw. Our results also revealed that prevalence of hepatitis C virus (HCV) co-infection among HBV carriers in Surabaya was approximately 10% for healthy blood donors and patients with chronic liver disease, and approximately 60% for patients on maintenance hemodialysis. Interestingly, HBsAg titers were lower in HBV carriers with HCV co-infection than in those without HCV co-infection. We also found that prevalence of hepatitis D virus (HDV) co-infection was < 0.5% among HBV carriers in Surabaya.     

Dirofilaria immitis: performance and standardization of specific antibody immunoassays for filariasis

The factors associated with the development, optimization, and validation of immunoassays for the detection of parasite-specific antibody in filariasis infections were investigated using the dog heartworm, Dirofilaria immitis as a model. We examined two assays, the Protein A solid-phase radioimmunoassay (SPRIA) and enzyme-linked immunosorbent assay (ELISA), for quantitation of specific antibody to the parasite in canine serum. Precision, reproducibility, and parallelism were examined using response-error relationships and precision profile analyses. A staphylococcal Protein A saturation analysis was applied to the standardization of IgG anti-parasite antibody reference sera in weight per volume units (microgram/ml). Using the mean minimal detectable dose + 3 SD and an intraassay precision profile less than 10% coefficient of variation (CV) as criteria for assay sensitivity, the SPRIA and ELISA displayed comparable positive thresholds of 1 microgram/ml IgG anti-parasite antibody/ml of serum. Both assays demonstrated good reproducibility (less than 15% interassay CV) and parallelism (less than 20% interdilutional CV) over their working ranges (SPRIA: 1-40 micrograms/ml; ELISA: 1-5 micrograms/ml). Specificity of each assay was enhanced by preadsorption of cross-reacting antibodies in canine serum (i.e., specific for Toxocara canis antigens) with solid-phase antigen prior to assay. Methods for comparing different immunoassay designs are considered in relation to the variables that influence the assays' performance characteristics.     

Hepatitis G virus (GBV-C) infection among Brazilian patients with chronic liver disease and blood donors

Background: The recently discovered hepatitis G virus (HGV) belongs, as hepatitis C virus (HCV), to the Flaviviridae family. HGV has been isolated from the serum of patients with non A-E hepatitis. However, the association of HGV with hepatitis is uncertain.Objective: To determine the HGV prevalence in blood donors and in patients with liver disease and to evaluate a possible correlation between HGV infection and liver disease.Study design: Sera from a total of 113 consecutive patients with chronic liver disease were submitted to a series of liver enzymes and function tests and analyzed for the presence of HBsAg, anti-HBs, anti-HBc, anti-HCV, HCV RNA and HGV RNA. Prevalence of HGV RNA was determined in a group of 87 blood donors.Results: Nine (10%) sera from blood donors and 15 (13%) sera from patients with chronic liver disease were HGV RNA positive. Some 28 (25%) patients were HCV RNA positive, with genotypes 1a, 1b and 3 present in 10, 12 and 5 patients, respectively. A total of 20 (18%) patients were HBsAg carriers. Five (4%) patients were double infected (one with HBV+HCV, one with HBV+HGV and three with HCV+HGV).Conclusion: The proportion (10%) of HGV-infected blood donors was very high when compared with other countries. The results did not allow to establish HGV as an etiologic agent for chronic liver disease. The parenteral route was the presumed means of HGV transmission for only one-third of the patients.     

重庆地区乙肝血清特殊模式患者HBV基因分型及临床自然病程分析

目的了解重庆地区乙肝病毒（HBV）血清学标志物为特殊模式的HBV感染患者病毒基因型的分布情况,分析其临床特征及自然病程。方法从1000例HBV感染者中检测到48例乙肝病毒血清学标志物为特殊模式的患者（HBsAg与抗一HBs同时阳性,HBeAg与抗一HBe同时阳性）。采用巢式聚合酶链式反应（nPCR）对特殊模式患者的HBV进行基因分型,同时对两组特殊模式患者的临床资料和HBV感染的自然史进行分析。结果48例乙肝病毒血清学标志物为特殊模式的HBV感染者中,36例患者HBsAg与抗-HBs同时阳性,12例患者HBeAg与抗-HBe同时阳性。HBeAg＋／抗-HBe＋患者组的年龄较HBsAg＋／抗-HBs＋患者组的小（P〈0．05）。HBsAg＋／抗-HBs＋患者中,3例（8．3％）为B2亚型,12例（33．3％）为c2亚型,21例（58．4％）未分型;HBeAg＋／抗-HBe＋患者中,8例（66．7％）为B2亚型,1例（8．3％）为c2亚型,3例（25．0％）未分型,两组在HBV基因型的分布上差异具有统计学意义（Y2=17．44,P〈0．05）。在HBsAg＋／抗-HBs＋患者中,2例（4．2％）处于免疫清除期,14例（29．2％）处于低复制期,7例（14．6％）处于再活动期。HBeAg＋／抗-HBe＋患者中,5例（10．4％）处于免疫清除期。两组在HBV感染的自然病程中的分布差异具有统计学意义（X2=18．26,P〈0．05）。结论重庆地区乙肝病毒血清学标志物为特殊模式的慢性HBV感染者中,HBeAg与抗-HBe同时阳性的HBV感染者中B2亚型为优势基因型;HBsAg与抗-HBs同时阳性的HBV感染者中,HBV基因型以C2亚型为主。     

IgM antibody against hepatitis B core antigen (IgM anti-HBc): diagnostic and prognostic significance in acute HBsAg positive hepatitis.

IgM antibody against hepatitis B core antigen (IgM anti-HBc), a marker of recent hepatitis B virus infection, was sought by radioimmunoassay in sera diluted 1/4000 from 376 patients presenting to four centres in Italy with acute, apparently type B hepatitis (hepatitis B surface antigen (HBsAg) positive). In 320 patients (85%) a positive IgM anti-HBc test result confirmed that hepatitis was due to primary infection with hepatitis B virus. In the remaining 56 patients absence of the IgM marker indicated that they were previously unrecognised long term carriers of HBsAg. Further serum analysis often showed delta infection and occasionally hepatitis A or cytomegalovirus infection as the true cause of their illness. After six to eight months circulating HBsAg persisted in 38 of 45 patients (84%) without IgM anti-HBc but in only six of 150 patients (4%) with the IgM antibody (p less than 0.0001). A negative IgM anti-HBc test result in patients with acute HBsAg positive hepatitis points to a factor other than hepatitis B virus as the cause of the liver damage and predicts the carriage of HBsAg.     

Importance of markers of hepatitis B virus in alcoholic liver disease.

To determine the importance of the presence of serological markers of hepatitis B virus infection in patients with alcohol related liver disease we compared cumulative alcohol intake and clinical and histological features in patients with markers of hepatitis B virus infection and in those without. Hepatitis B surface antigen (HBsAg) was detected in five (2%) out of 285 patients studied and antibody to HBsAg (anti-HBs) in 41 (14%); one patient had antibody to hepatitis B core antigen alone. The combined prevalence of markers of hepatitis B virus infection was similar in patients with alcoholic cirrhosis (18%) and precirrhotic liver disease (13%). Two patients positive for HBsAg had histological features of both alcoholic liver disease and chronic active hepatitis, with stainable HBsAg. Patients with anti-HBs were, however, histologically indistinguishable from patients without markers, and the mean cumulative alcohol intake of patients with anti-HBs was similar to or even higher than that of patients with liver disease of comparable severity who had no evidence of previous infection. The presence of markers of hepatitis B virus infection was related to former residence in countries with a high prevalence of the infection and to previous parenteral treatment and blood transfusions. Infection with hepatitis B virus does not enhance the development of chronic liver disease in heavy drinkers, except in the small number who remain positive for HBsAg.