Evaluation of a Red Cell Agglutination Test for Detection of Australia Antigen (HB Ag)

An investigational red cell agglutination (RCA) test was evaluated for sensitivity in detecting and titering hepatitis B antigen (HB Ag) in comparison with two counterelectrophoresis (CEP) systems and a solid-phase radioimmunoassay (RIA). The RCA procedure was found to be significantly more sensitive than the CEP methods and compares favorably in sensitivity with the solid-phase RIA, detecting even lower concentrations of the HB Ag. Since the RCA test can be completed in 2 to 3 h and requires relatively inexpensive equipment, it offers a highly sensitive and rapid procedure suitable for use in blood banks to screen donors or detect low levels of antigen in serum of patients.     

Production and standardization of Paracoccidioides brasiliensis, Histoplasma capsulatum and Aspergillus fumigatus antigens to be used in immunodiagnosis. Comparison between immunodiffusion and immunoelectrophoresis

Soluble antigens (Ag) from Paracoccidioides brasiliensis, Histoplasma capsulatum and Aspergillus fumigatus were prepared and standardized by double immunodiffusion (DID) and immunoelectroosmophoresis (IEOP). No difference in sensitivity was observed between the two techniques; 100% of standard patient sera were positive with P. brasiliensis and A. fumigatus Ag and 83.3% were positive with H. capsulatum Ag. The specificity of the tests was verified testing 96 sera from patients with paracoccidioidomycosis, histoplasmosis, systemic candidiasis, sporotrichosis, tuberculosis, lung cancer, visceral or cutaneous leishmaniasis and 18 sera from healthy individuals. All the three antigens were 100% specific with the DID (using the identification pattern indicated by the confluence of test serum with standard serum precipitin lines as a positive criterium). However in the IEOP, the specificity varied with each Ag. Positive reactions with P. brasiliensis Ag were observed in 16.7% of histoplasmosis sera and in 10% of cutaneous leishmaniasis sera. On the other hand 31.8% of paracoccidioidomycosis and 10% of cutaneous leishmaniasis sera reacted with H. capsulatum Ag. The high sensitivity and specificity of the DID test, its easy reproducibility and low cost, led us to consider it highly appropriate as a routine procedure for the screening of patients with respiratory infections.     

Relative Sensitivity of Gel Diffusion, Complement Fixation, and Immunoelectroosmophoresis Tests for Detection of Hepatitis-Associated Antigen and Antibody

The immunoelectroosmophoresis (IEOP) test was compared with gel diffusion and complement fixation (CF) tests for sensitivity in detecting hepatitis-associated antigen (HAA) in the sera of hepatitis patients, for titration of HAA, and for detection of antibody to HAA. The IEOP test was found to be slightly more sensitive than either gel diffusion or CF tests for detection of antigen in the patients' sera. Titers of HAA demonstrated by IEOP were higher than those seen in gel diffusion tests but lower than CF titers. The gel diffusion test with an "enhancement" pattern was found to be more reliable than the other two procedures for detection of low levels of anti-HAA, due to the greater inhibitory effect of an antigen excess in the IEOP system and the possible masking of low levels of antibody by anticomplementary activity in the CF test system. Staining of immunoprecipitates in the IEOP test contributed little to the sensitivity of the test for detection of HAA.     

Evaluation of an immunoenzyme test (ELISA) for the determination of an Ag/Ab system correlated to non-A, non-B post-transfusion hepatitis

An ELISA for the detection of a Ag/Ab system related to non-A, non-B post-transfusion hepatitis (NANB-PTH) has been developed. A convalescent serum from a patient suffering, 14 month before, of NANB-PTH was used both as capture and detector antibody in a "sandwich" type immunoassay to detect an Ag (Ag-NANB). A "blocking" immunoassay was used, in the same test, to detect the corresponding antibody (Ab-NANB). All patients with NANB-PTH were positive, alternately, for Ag-NANB or Ab-NANB. The Ag was found in the acute phase sera of 3 out of 22 cases of NANB-PTH (13,6%), but in none of 29 acute hepatitis type B,26 acute hepatitis type A,14 NANB sporadic hepatitis, 5 infectious mononucleosis and 8 healthy subjects. 2/3 Ag-NANB positive subjects became chronic carriers of this Ag, whereas the third seroconverted for Ab-NANB after the first two months of their disease. Furthermore, a significant increase of the Ab-NANB titer was observed in the convalescent phase of the disease in 18/20 NANB-PTH. As expected, a relatively high prevalence for Ab-NANB was found in the other groups of patients, suggesting the high diffusion of the agent, but none of these subjects showed a progressive increase of the antibody titer during the course of their disease.     

Study of serological and biological markers in viral hepatitis. 157 hemophiliacs

We observed for a two years period 157 hemophiliacs (138 with hemophilia A whose 13 were severe and 19 with hemophilia B whose 13 were severe) and we studied the incidence of liver dysfunction and the role played by HB and non-A, non-B, viruses. Whereas 32 patients not related had no evidence of serological HB virus markers (by radioimmunoassay), 88 (70,4 %) among the 135 hemophiliacs with large or small exposure to blood products were "HB positive". 90,9 % were positive for anti-HBs and anti-HBc antibodies and only two patients had persistent antigenemia. These results appeared independent of the kind of treatment (factor VIII or factor IX concentrates). Six among 17 children born since 1974, when the antigen was detected by RIA, had the serological HB virus markers, showing that this method is not sufficient to completely eliminate the HB virus. However the amount of viruses injected is too small to induce an acute hepatitis and rather produces specific antibodies which protect hemophiliacs against reinfection. An elevated level of serum transaminases (SGPT) was observed in 9,4 % of non treated hemophiliacs, 15,1 % of treated hemophiliacs with no serological markers of HB virus and 27,7 % of treated hemophiliacs "HB positive". This shows that the use of concentrates and the occurring of HB virus in the patients are not the only factors producing liver dysfunction. The role of non-A, non-B viruses has been recognized in 7 patients out of 9 with transient elevation of serum transaminase levels, by Trepo with an immunodiffusion technique.     

Microtiter Solid-Phase Radioimmunoassay for Hepatitis B Antigen

A micro-solid-phase radioimmunoassay (micro-SPRIA) for hepatitis B antigen (HB Ag) was developed for use with microtiter serological equipment. Radiolabeled immunoglobulin G was prepared from human and animal sera containing hepatitis B antibody (HB Ab); it was not necessary to isolate specific HB Ab by immunochemical means. A micro-SPRIA prepared with guinea pig reagents was approximately as sensitive as the AusRIA radioimmunoassay, but, like the AusRIA test, yielded false positive results. A micro-SPRIA prepared with human reagents was slightly less sensitive but did not yield false positive results. These micro-SPRIA tests offer several advantages, including conservation of reagents, adaptability to other antigen-antibody systems, ease of performance (especially when testing large numbers of specimens), and economy.     

Comparative study on subtypes of hepatitis B surface antigen (HBs Ag) in two regions of southern Japan, Okinawa and Kumamoto. Prevalence and unusual subtypes

A total of 209 hepatitis B surface antigen (HBs Ag)-positive sera from two possibly ethnologically different regions of southern Japan, Okinawa and Kumamoto, were subtyped by counterelectrophoresis using monospecific antisera to a, d, y, w, and r antigenic determinants. There was a marked difference in the prevalence of the w and r determinants in the two regions; the frequency of the HBs Ag/adr subtype was 95% in Kumamoto and 47% in Okinawa, suggesting that HBs Ag subtypes may be one of the markers reflecting an ethnological difference between the peoples of the two regions. Seven and three serum specimens in Okinawa and Kumamoto respectively showed unusual HBs Ag subtypes: four carried adwr (two specimens), aywr, or adyw determinants, five had only a, and the remaining one carried ar.     

Hepatitis B Antigen Inhibitor in Human Faeces and Intestinal Mucosa

Hepatitis B antigen (HB Ag) positive sera became negative after in-vivo incubation with homogenates of human faeces or intestinal mucosa. This was found to occur in all sera tested by various methods. These findings suggest the existence in the human intestine of a substance able to inactivate the HB Ag and that it is not an antibody or of the nature of interferon. The presence of an inhibitor could explain why B-type hepatitis is seldom if ever faecally transmitted and also the low oral infectiousness of the B virus.     

Hepatitis B Core Antigen: Immunology and Electron Microscopy

TWO DISTINCT VIRAL ANTIGENS ARE ASSOCIATED WITH THE HEPATITIS B VIRUS: the hepatitis B surface antigen (HB(s)Ag, Australia antigen) and the hepatitis B core antigen (HB(c)Ag). HB(s)Ag, purified from the serum of asymptomatic human HB(s)Ag carriers, and HB(c)Ag, purified from the liver of a chimpanzee acutely infected with hepatitis B virus, were examined by serological and immune electron microscopic methods. Antisera raised against HB(s)Ag reacted with the outer, surface component of the Dane particle and with the 20-nm spherical and tubular particles present in HB(s)Ag-positive serum, but not with the internal component of the Dane particle or with purified HB(c)Ag particles. Antisera raised against purified HB(c)Ag particles reacted with the internal component of the Dane particle and with HB(c)Ag, but not with the surface of the Dane particle or with the 20-nm spherical and tubular particles associated with HB(s)Ag. Purified HB(c)Ag particles, 27 nm in diameter, demonstrated distinct subunits. The infectious form of hepatitis B virus appears to be represented by the 42-nm Dane particle composed of a 27-nm nucleocapsid core component (HB(c)Ag) surrounded by an antigenically and morphologically distinct lipoprotein surface component (HB(s)Ag).     

Hepatitis B virus markers, beta 2 microglobulin and anti HTLV in a population of blood donors from a prison environment

212 male prisoners were collected at the prison in March 1983. Anti-HBc and HBs Ag were detected by a combined test (AUSRIA-CORE, Abbott Laboratories). 67 sera (31,5%) were anti-HBc positive, 7 of them HBs Ag positive. Screening for anti-HTLV (anti-p24) was negative for all the donors. Beta 2 microglobulin levels were determined on 115 sera (B2-micro RIA 100, Pharmacia Laboratories). 8 had levels above 2,6 mg/l (greater than 2 SD). These 8 sera were anti-HBc positive and one of them HBs Ag positive 3 HBs Ag positive donors had non elevated Beta 2 microglobulin levels. This survey confirms that prisoners as blood donors should be regarded as carrying a high risk of transmission of HBV and probably other infectious agents with similar epidemiology. The signification of elevated Beta 2 microglobulin levels deserves further investigations since this determination could be of value as an additive test to increase the safety of blood products.     

Prevalence of hepatitis B markers, antibodies to adult T cell leukemia/lymphoma virus and antibodies to human immune deficiency virus in prostitutes in Fukuoka, Japan

Prevalence of hepatitis B (HB) markers, antibodies to human T cell leukemia virus (HTLV-1) and antibodies to human immune deficiency virus 1 (HIV-1) in prostitutes working in Fukuoka city were studied. Sera were collected from 237 prostitutes during January-September, 1986. Among them, 9 (3.8%) were HB virus surface-antigen (HBs Ag) positive, of whom, 3 were HBe antigen positive and the remaining 6 were anti-HBe positive. The positive rate of anti-HBs was 34.2%. The incidence of anti-HTLV-1 in the prostitutes was 5.9%. These incidences are considered to be within the usual range in Kyushu district. No seropositive case for anti-HIV was found.     

乙型肝炎血清标志模式与病毒载量的关系及意义

为探讨乙型肝炎 (以下简称乙肝 )血清标志模式与病毒载量的关系及临床意义 ,作者选择符合 2 0 0 0年《全国病毒性肝炎诊断标准》的慢性肝炎血清 1343份 ,分别用ELISA法、PCR ELISA法检测HB血清标志、HBV -DNA和 1896位点变异株。结果显示 :HBsAg阳性血清 10 97份 (81.6 8%、)HBsAg阴性血清 2 4 6份 (81.31% )。在HBsAg阳性血清中 ,HBsAg、HBeAg、抗 HBc(1 3 5 )阳性组 4 0 4份 (30 .l% ) ,HBV -DNA阳性 347份 (85 .89% ) ,DNA阳性值呈递增趋势 (10 4～ 10 6拷贝 /ml,各占 8.6 5 %、33.71%、5 3.6 1% ) ;而HBsAg、抗 HBe阳性组血清 6 0 6份 (45 .12 % ) ,DNA阳性值 10 5拷贝 /ml,占优势 (6 4 .18% )。在HBsAg阴性血清中 ,抗 HBs、抗 HBe、抗 HBc(2 4 5 )阳性组 2 32份 (17.2 7% ) ,DNA阳性占 7.32 % ,DNA阳性值递减由 10 4～ 10 6拷贝 /ml,各占 5 2 .9%、4 1.l%、5 .9%。结论 ,各血清标志模式中的病毒载量为HBsAg阳性组 >HBsAg阴性组 ,阳性组 1 3 5 >1 4 5 >l 5 >2 4 5。但 1 3 5阳性组中 14 %在界值以下 ,1 4 5阳性组中 1896位点自然变异达 78.6 % ,2 4 5阳性组中仍存在DNA+ 血清 ,以上提示在临床判定和治疗时要慎重对待(注 :l=HBsAg,2 =抗 HBs,3=HBeAg ,4 =抗 HBe ,5 =抗 HBc)     

Serodiagnosis of acute hepatitis A

Hepatitis A antibody (anti-HAV) was detected by specific radioimmunoassay (RIA) method in sera from 10 patients with acute icteric hepatitis. Anti-HAV was detectable in many subjects very early before the onset of jaundice, but the diagnosis of type A hepatitis in all patients was confirmed by the demonstration of seroconversion during convalescence. Since the initial antibody detected by RIA is predominantly IgM, while IgG specific anti-HAV appears later reaching peak levels within 1 to 2 months, we treated serum specimens of these patients with 2-mercaptoethanol (2ME) in order to differentiate acute-from convalescent-phase hepatitis A sera. Inactivation of IgM fraction with 2ME produced a significant reduction of anti-HAV titer only in acute-phase sera, so that this procedure may be used for early diagnosis of acute type A hepatitis.     

Experimentation in Lille with the passive hemagglutination test for the detection of Australia antigen. Comparison with other methods]

Among 5347 blood donors sera, the reverse passive hemagglutination test (RPH) enabled to detect 3,55% of HB ag (whereas 2,43% by EID). On the other hand, among 180 patients, the rate of positive reactions was observed to be the same for both techniques, which must be attributed to the presence of higher titer antigens. In the two series all positive reactions found by RPH method were also tested by RIA; this enabled to eliminate a great number (18% and 27%) of false positive reactions, which remain the disadvantage of the method. It must be also noticed that the complement fixation reaction gave the same results as the RIA.     

Comparative study of three commerical preparations for the detection of Australia antigen]

2,944 sera from blood donors (420 of them being new donors) were tested in four techniques: Counterelectrophoresis (CEP) and three commercial tests: Ausria II, Auscell and Hepanosticon. Over 10 HBs Ag detected by RIA, 8 were evidenced by the Auscell test and 6 by the Hepanosticon Test. False positive results (approximately 3%) represent a disadvantage to the reverse hemagglutination method; this rate increased considerably in a group of 386 patients sera also tested. The authors conclude that the results obtained with the reverse hemagglutination technique are nearing those of RIA.     

Phylogeography, risk factors and genetic history of hepatitis C virus in Gabon, central Africa

Background

The epidemiological and molecular characteristics of hepatitis C virus (HCV) infection in the general population have been poorly investigated in Africa. The aim of this study was to determine the prevalence, genotype distribution and epidemic history of HCV in the Gabonese general population.

Methods/Principal Findings

A total of 4042 sera collected from adults in 220 villages in all nine administrative areas of the country were screened for antibodies to HCV. HCV NS5B region sequencing was performed for molecular characterization and population genetic analyses. Of 4042 tested sera, 455 (11.2%) were positive. The seroprevalence of HCV varied significantly by administrative area, with the highest rate in Ogooué-Lolo province (20.4%) and the lowest in Ogooué-Maritine province (3.7%). History of parenteral injections, past hospital admission and age over 55 years were independent risk factors for HCV infection (p<0.0001). Phylogenetic analyses showed that 91.9% of the strains were genotype 4 (HCV-4), 5.7% genotype 1 and 2.2% genotype 2. HCV-4 strains were highly heterogeneous, with more than eight subtypes; subtype 4e predominated (57.3%). Coalescence analyses indicated that subtype 4e was the oldest, with an estimated most recent common ancestor of 1702 [95% CI, 1418–1884]. The epidemic profile indicated that it spread exponentially during the first part of the 20th century, probably by iatrogenic transmission.

Conclusions/Significance

These results confirm the endemicity of HCV subtype 4e in Gabon and show that its spread is due to a cohort effect, with previous, possibly iatrogenic events. More extensive epidemiological studies are needed to better characterize the route of transmission and the dissemination of HCV in Gabon.     

Development of glutamic acid decarboxylase 65 (GAD65) autoantibody assay using biotin-GAD65 fusion protein

We evaluated a biotin-glutamic acid decarboxylase 65 (GAD65)-based enzyme-linked immunosorbent assay (B-ELISA) to detect GAD65 autoantibodies (GAD65Ab) in 78 sera from individuals with newly diagnosed type 1 diabetes. The GAD65Ab index of patients with type 1 diabetes (mean value of GAD65Ab index of 1.891) was significantly higher than those in 50 sera from healthy control group (mean value of 0.068). The intra- and inter-assay coefficients of variation (CV) were calculated to be 1.042 and 10.703%, respectively. The specificity of the B-GAD65 ELISA was comparable to the standard radioimmunoassay (RIA) which is routinely used in the laboratory. We describe the optimal conditions of the binding kinetics from each assay-step for the detection of GAD65Ab using the WHO standard serum 97/550 as a model autoantibody serum. We concluded that incubation times of 15, 90, and 90 min for step 1 (pre-incubation of Biotin14-GAD65 with serum), step 2 (binding the Ab/Ag complex to NeutrAvidin plate), and step 3 (incubation with HRPO-anti-human IgG), respectively, along with human serum dilutions of 1:50, would provide an optimal assay signal within a relatively short timeframe.     

病毒性肝炎患者抗—HBcIgA 、IgE 、IgG 、IgM 平行检测的临床意义

目的：通过对乙肝病人血清抗-HBcIgA、HBcIgE、HbcIgc、HbcIgM平行检测进一步地探讨其临床应用价值。方法：收集216份乙肝病人血清（均为我院住院病人的血清）,采用ELISA法对病人血清抗-HBcIgA、HBcIgE、HBcIgc、HBcIgM平行检测,并对检测结果作统计学分析。结果：检测结果表明：抗-HBcIgA是HBV感染后肝脏损害的明显标志,抗-HBdgE是HBV感染后乙型肝炎慢性化的标志,抗-HBcIgG持续存在则是以往感染的标志,抗-HBcIgM是HBV感染后病毒持续复制的标志。结论：在急慢性乙型肝炎、慢活肝与慢迁肝鉴别诊断中,平行检测病人血清抗-HBcIgA、HBcIgE、HbcIgG、HbcIgM对乙型肝炎慢性化及其预后估价均有重要临床意义。     

Comparative evaluation of methods for detecting HBsAg in sera

The results of the analysis of 1209 serum samples, made with a view to detecting those containing HBsAg, are presented. This analysis was made by the radioimmunoassay (RIA) on a polyethylene film, by the standard RIA technique with the use of a diagnostic kit obtained from Abbott Laboratories (USA) and by the passive hemagglutination (PHA) test. The RIA film technique was found to have the sensitivity of about 2 ng/ml HBsAg, which is similar to the sensitivity of the kit from Abbott Laboratories and exceeds the sensitivity of the PHA test approximately 50-fold. The percentage of detected HBsAg-positive sera, yielded by analysis with the use of the RIA film technique and the standard RIA technique, is the same. The RIA technique make it possible to detect more positive sera than the PHA test by about 2.5%.     

Association between anti-Sm and anti-ribosomal P protein autoantibodies in human systemic lupus erythematosus and MRL/lpr mice

Anti-Sm and anti-ribosomal P protein antibodies show a high degree of specificity for the disease SLE. To determine whether a relationship between these two autoantibodies existed, the frequency of anti-P was determined in sera with and without anti-Sm activity. Of sera from lupus patients with anti-Sm 18/65 (28%), and 6/55 (11%) of sera without anti-Sm had anti-P as determined by an ELISA using a recombinant P2-beta-galactosidase fusion protein as Ag (p less than 0.05). The levels of anti-P were significantly higher in sera containing anti-Sm (0.37 +/- 0.45) than in sera without anti-Sm antibodies (0.18 +/- 0.20) (p less than 0.01). Similarly, a significantly higher proportion of anti-P positivity was found in autoimmune MRL/Mp-lpr/lpr mice positive for anti-Sm (11/53 = 21%) compared to age- and sex-matched mice without anti-Sm (3/53 = 6%) (p less than 0.05). The IgG subclass distributions for anti-Sm and anti-P antibodies were similar in the MRL mice (IgG2a greater than IgG2b greater than IgG3 greater than IgG1). The association did not reflect polyclonal B cell activation in a proportion of MRL mice because no significant differences were observed in anti-DNA, antichromatin or total serum IgG levels in mice with and without anti-Sm or, in mice positive for both anti-P and anti-Sm compared to mice positive for anti-Sm alone. Cross-inhibition experiments excluded the possibility that the Sm and P protein Ag shared a common epitope. Longitudinal measurement of anti-P and anti-Sm antibody levels by ELISA in three mice indicated that both antibodies first appeared at about 3 to 4 mo of age and fluctuated two- to threefold over 3 to 8 mo with independent peaks of activity. Recent observations regarding a relationship between anti-Sm and autoantibodies to other ribosomal proteins suggest that the association may be explained by an immune response to epitopes coassociated on the ribosome.