The detection of the herpes simplex virus 1 and 2 antigens in clinical specimens by using monoclonal antibodies

The possibility of using monoclonal antibodies (McAb), obtained earlier, for the detection of herpes simplex virus (HSV) in clinical specimens taken from sick and infected persons was studied. The examination of 90 persons revealed that the mixture of McAb 4A and 2C could effectively detect the presence of HSV antigen in the indirect immunofluorescence assay (IFA) directly in cells contained in cytological preparations (smears, scrapes, impressions) obtained from different organs of patients. The search of optimum combinations of McAb for the detection of HSV antigens by the method of the solid-phase enzyme immunoassay (EIA) was carried out. This study, made on purified HSV used as an experimental model, revealed that the maximum sensitivity could be achieved with the use of two McAb (4f6 and 7c4) out of three McAb (4f6, 7c4 and 3d10). The approbation of both variants of EIA on clinical specimens taken from 99 patients (blood clots, seminal fluid, scrapes of cervical canal cells, peripheral blood lymphocytes) showed that the addition of McAb 3d10 made it possible to detect 8 more positive specimens. 754 specimens from 337 patients were studied with the use of McAb-based EIA, and in 204 of these patients (61%) HSV antigen was detected. The results obtained with the use of our McAb were compared with the data obtained with certified commercial test systems. The coincidence of the EIA data with those obtained with the use of the Murex Wellcozyme HSV test system (UK) was registered in 75% of cases (in 15 out of 20 cases). The coincidence of the IFA data with those obtained with the use of the Sanofi test system (France) was observed in all 19 cases (100%).     

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.     

The first large-scale nucleic acid amplification testing (NAT) of donated blood using multiplex reagent for simultaneous detection of HBV, HCV, and HIV-1 and significance of NAT for HBV.

The first nationwide nucleic acid amplification testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) of voluntarily donated blood after serological pre-screening and before release of cellular components and plasma for fractionation was implemented by the Japanese Red Cross Blood Transfusion Services. From February 1, 2000 to April 30, 2001, specimens from 6,805,010 units of serologically negative donation were screened in minipools of 50 samples within 24 hr after blood donation by NAT using multiplex HBV/HCV/HIV-1 reagent for blood transfusion including short shelf-life platelets. Among them, 112 HBV DNA-positives, 25 HCV RNA positives and 4 HIV-1 RNA positives were screened out and we could prevent transfusion of these NAT positive units. Subtypes/genotypes of HBV DNA, adr/C, adw/A, adw/B, adw/C, ayr/C and ayw/D were found and adr/C was predominant. A total of 61.6 % of them (69/112) were negative by overnight EIA. Sixth three of HBV NAT-positive samples carried virus loads less than 10(4) copies/mL and 92.1 % of them (58/63) were negative by overnight EIA. The virus growth curves of HBV in 6 cases obtained by retrospective and prospective follow-up study showed exponential straight lines in the early stage of serological window periods and the log times of HBV growth (10 fold increase) in serological window period were between 4.6 and 7.6 days. NAT screening with highly sensitive reagents in pool of specimens is useful to exclude blood units with low level of HBV and HBV mutants from blood transfusion.     

Hepatitis E virus infection in selected Brazilian populations

A retrospective study on the prevalence of hepatitis E virus (HEV) infection was conducted in selected populations in Rio de Janeiro, Brazil. A total of 1,115 subjects were tested including 146 patients with acute Non-A Non-B Non-C (NANBNC) viral hepatitis, 65 hemodialysis patients, 93 blood donors, 102 intravenous drug users (IVDUs), 304 pregnant women, 145 individuals living in the rural area and 260 individuals living in the urban area. In order to characterize a favorable epidemiological set for enterically transmitted infection in the studied populations we also evaluated the prevalence of anti-HAV IgG (hepatitis A virus) antibodies. Specific antibodies to HEV (anti-HEV IgG) were detected by a commercial EIA and specific antibodies to HAV (anti-HAV IgG) were detected using a competitive "in house" EIA. We found a high prevalence of anti-HAV IgG in these populations, that could indicate some risk for infections transmitted via the fecal-oral route. The anti-HEV IgG prevalence among the different groups were: 2.1% in patients with acute NANBNC viral hepatitis, 6.2% in hemodialysis patients, 4.3% in blood donors, 11.8% in IVDUs, 1% in pregnant women, and 2.1% in individuals form the rural area. Among individuals living in the urban area we did not find a single positive serum sample. Our results demonstrated the presence of anti-HEV IgG in almost all studied populations; however, further studies are necessary to establish the real situation of HEV epidemiology in Rio de Janeiro, Brazil.     

Comparison of the radioimmunological and immunoenzymatic methods in detection of HBsAg]

We have compared the solid-phase radioimmunoassay(SPRIA) with a solid-phase enzyme-immunoassay (EIA) in the detection of hepatitis B surface antigen (HBsAg). 708 sera from blood donors and 500 sera from patients with various diseases (acute and chronic hepatitis, chronic renal failure in hemodialytic treatment) were tested for HBsAg with both methods. 208 sera (17,2%) were found to be positive in SPRIA and 209 sera (17,3%) in EIA. Two HBsAg positive sera were tested in dilution series with both methods, too. The results show that the sensitivity and specificity of the EIA compare very favourably with those of the SPRIA.     

Level of Tx1- and Tx2-type cytokines in blood sera of hepatitis C patients

The content of cytokines of type Tx1 (IL-2 and IFN-gamma) and type Tx2 (IL-4) in blood sera of 132 patients with hepatitis C and the combined form of hepatitis B + C was studied. For control, blood sera taken from healthy donors were used. A significant increase, in comparison with the control, in the content of IL-4 in all subgroups of the patients was registered. The content of IFN-gamma reached the maximum level in patients with acute hepatitis C with the positive result of the polymerase chain reaction (PCR) for the virus (216.4 and 46.4 pg/ml respectively) and was somewhat lower in acute hepatitis C with the negative PCR-result (77.7 and 9.6 pg/ml), mean while in the chronic course of hepatitis C these data were within the limits of control values irrespective of the results of PCR. In case of mixed infection in the acute clinical form a significant increase in the concentration of IFN-gamma (34.4 pg/ml) in comparison with the control (25.3 pg/ml) was observed. The content of IFN-gamma in patients with acute hepatitis C and the positive result of the test for NS antibodies also reached the maximum level (207.3 and 42.7 pg/mg respectively). But in contrast to hepatitis C in the acute form with the negative results of PCR in patients with hepatitis C in the acute form and the negative results of the NS test these data were within the limits of control values, as well as in the chronic course of hepatitis C irrespective of the results of the NS antibodies serum test. In case of mixed infection a significant increase in the concentration of IFN-gamma was registered in the subgroup of patients with the acute form of NS+ (39.9 pg/ml). The data obtained in this study were indicative of significant changes in the serum profile of serum cytokines of types Tx1 and Tx2 in different forms and courses of virus hepatitis. This makes it possible to believe that the chronization of the process was associated with the prevalence of the Tx2 function.     

细胞培养、酶联免疫法检测生殖器疱疹病毒的对比性研究

目的分析酶联免疫法（EIA）在检测生殖器疱疹病毒的可行性。方法收集性病门诊患者标本338例进行细胞培养和EIA法检测,另将两法检测结果不相符的标本采用荧光PCR复检,比较分析细胞培养、EIA法与扩大金标准结果的相符性。结果分析表明细胞培养与EIA法检测生殖器疱疹病毒的敏感性均为90．24％,特异性分别为100％和96．29％。结论EIA法检测生殖器疱疹病毒有较高的敏感性而且方法简便、快速,可以用于大批量标本的检测和流行病学的筛查。     

Seroepidemiological characterization of virus hepatitis in the Republic of Armenia

The survey of the population immunological structure with respect to parenteral hepatitis showed awide circulation of hepatitis B (HB) and hepatitis C (HC) viruses among the adult population of Armenia. During the 5 year period of observation the number of persons having antibodies to HC virus increased 2.7-fold. High occurrence of antibodies to HBsAg of HB virus among the healthy population in 2002 (12.0%) in comparison with 1997 (5.4%) reflected a decreased infection rate with HB virus as well. Antibodies to hepatitis A (HA) virus were isolated, on the average, in 64 % of persons. Simultaneously with a decrease in the proportion of HA cases an increased number of HC patients was registered. No circulation of hepatitis E virus was detected. A high percentage of hepatitis cases of mixed etiology was established, as well as an increased number of combined parenteral hepatitis cases was registered (57.1%).     

Diagnostic role of different immunologic methods for laboratory diagnostics of legionellosis

The aim of the study was a comparative analysis of diagnostic value of different laboratoty methods conducted on the basis of results of examination of patients during Legionnaires' disease outbreak in town Verkhnyaya Pyshma. Retrospective analysis of laboratory data from 74 patients with diagnosis of Legionnaires' disease was performed. Complex of laboratory methods was used (polymerase chain reaction (PCR), enzyme immunoassay (EIA), immunochromatography). In group of patients with Legionnaires' disease, the highest proportion of positive results (73%) was obtained by the EIA determining total specific antibodies in urine. Determination of antigen in urine by immunochromatographic express-test yielded 52% of positive results. PCR testing of blood specimens yielded positive results in 65% of samples but was low specific, due to that in 19% of patients from control group false-positive results were obtained. Testing of 3 autopsy samples showed that all specimens contained DNA of the causative agent. Performed analysis allowed to recommend complex use of immunochromatographic express-test of antigen detection and identification of total specific antibodies by EIA during mass people examination.     

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.     

Utility of neutralization test for laboratory diagnosis of suspected mumps

Mumps is an infectious disease caused by mumps virus (MuV), which belongs to the family Paramyxoviridae and genus Rubulavirus. Typical symptoms of mumps include fever and swelling of the parotid glands; however, mumps can be asymptomatic. Mumps is diagnosed by molecular and serological methods (i.e., PCR and Enzyme Immunoassay [EIA]); however, both methods have pros and cons. This study was performed to compare the diagnostic utility of a focus reduction neutralization test (FRNT) to that of MuV‐specific commercial IgM and IgG antibody EIA in patients suspected of having mumps. One hundred‐eighty six samples collected during mumps outbreak in 2012–16 were studied. Samples (n = 80) were tested by all the three serological assays and showed 70.4%, 83% and 92.5% positivity by IgM EIA, IgG and FRNT, respectively. In all, 58.8% samples (n = 47) tested positive in all three assays. Concordance between mumps RT‐PCR and IgM EIA was highest during the first 2–5 days and decreased with increasing time post‐onset. Mumps FRNT results agreed with those of RT‐PCR/IgM EIA from the second week onwards, whereas the results of mumps IgG EIA agreed with those of RT‐PCR/IgM EIA from post‐onset days 3–10. These findings suggest the utility of a FRNT for laboratory diagnosis of mumps in countries whose populations are not immunized against this infection.

     

Development and evaluation of a protein microarray chip for diagnosis of hepatitis C virus

A protein chip diagnostic kit was developed for the diagnosis of hepatitis C virus (HCV) based on the protein chip technique and the immuno-concentration method. This kit was designed for low-density protein chips and also for the availability of multiple sample screening. Applicability of the chip was evaluated using 96 blood specimens and the results were compared to results of an anti-HCV enzyme immunoassay (EIA) test. With further development, the technology associated with the development of this chip could be applied to the simultaneous detection of multiple protein-protein, protein-ligand interactions.     

Detection of Epstein-Barr viral antibodies in patients with acute viral hepatitis

Serum samples obtained from 44 patients with virus A hepatitis, 23 patients with virus B hepatitis, 65 patients with virus non A, non B hepatitis and 100 healthy adults were studied for the presence of Epstein-Barr (EB) virus in the indirect immunofluorescence test. In this work lymphoblastoid cell lines PH3-J-1 and CN37 were used. Among patients with different forms of hepatitis, the statistically significant elevation of the titers of antibodies to EB virus was detected only in the group of patients with virus non A, non B hepatitis, and in 6 cases the etiological role of EB virus was confirmed by serological and hematological methods.     

The results of using the molecular hybridization of nucleic acids in the complex study of nasopharyngeal smears from patients with influenza and other acute respiratory diseases

A complex study of samples obtained from patients with influenza and other acute respiratory diseases has revealed that the laboratory methods used in this study can be rated in the following order according to their sensitivity: isolation of the virus in chick embryos, analysis of seroconversions in the hemagglutination inhibition test, immunofluorescent determination of viral antigens, determination of viral antigens by enzyme immunoassay (EIA), detection of RNA-containing viral structures by means of molecular hybridization. From the point of view of the possibility of documenting influenza A in patients, the best results are achieved by the combination of molecular hybridization and EIA techniques: 90% and more of all cases. A rational scheme for the examination of samples obtained from patients with a view to epidemiological study, including both traditional and new rapid diagnostic methods, is proposed.     

“Silent” Hepatitis B Virus Mutants Are Responsible for Non-A,Non-B,Non-C,Non-D,Non-E Hepatitis

Since the recent introduction of diagnostic kits for hepatitis C and E, some cases of nonA, non-B, non-C, non-D, non-E hepatitis (so-called hepatitis F) have been revealed. We attempted to demonstrate that so-called hepatitis F is caused by hepatitis B virus (HBV) variants. Polymerase chain reaction (PCR) was used to amplify serum HBV DNAs from 20 patients with acute hepatitis and 20 patients with chronic hepatitis who had been diagnosed as having so-called hepatitis F on the basis of conventional serological markers. The PCR technique successfully amplified HBV DNAs in 18 (90%) cases of acute hepatitis and 17 (85%) cases of chronic hepatitis. Sequencing of HBV DNAs of six patients (acute 3, chronic 3) revealed equally a T-to-C mutation of DR2 and an 8-nucleotide deletion of the 3′-terminus of the X gene coding region, giving rise to the generation of a C-terminally truncated X protein and probable damage to the enhancer II/core promoter elements. These mutations of the X gene coding region may lead to suppression of replication and expression of HBV DNAs. Thus virtually all cases of so-called hepatitis F appear to be caused by “silent” HBV mutants, at least in Japan.     

Comparison of a recombinant-antigen enzyme immunoassay with Treponema pallidum hemagglutination test for serological confirmation of syphilis

A recombinant-antigen enzyme immunoassay (EIA), BioSCREEN anti-Treponema pallidum, was compared favorably with the T. pallidum hemagglutination test, in the detection of specific antibodies in different groups of sera from patients with primary (n = 38), secondary (n = 10), early latent (n = 28) and congenital syphilis (n = 2), patients with leptospirosis ( n= 8), infectious mononucleosis (n = 7), hepatitis (n = 9), diabetes mellitus (n = 11), rheumatoid arthritis (n = 13), leprosy (n = 11), tuberculosis (n = 9), HIV/Aids ( n= 12), systemic lupus erythematosus (n = 4), rheumatic fever (n = 3), old-persons (n = 9), pregnant women (n = 29) and blood donors (n = 164). The coincidence between them was 95.1%. The sensitivity and specificity of the EIA were 93.3% and 95.5%, respectively. Fifteen serum specimens belonging to old-persons, pregnant women, blood donors, and patients with human leptospirosis, hepatitis, diabetes mellitus, tuberculosis and rheumatic fever gave false-positive results by Venereal Disease Research Laboratory and/or Rapid Plasma Reagin. The EIA can be used as alternative method for the serological confirmation of syphilis.