Prevalence of specific antibody to hepatitis E virus in the general population of the community of Madrid, Spain.

Hepatitis E virus (HEV) is an infectious agent causing hepatitis among humans. Although hepatitis E has been reported from many European countries, its incidence in Europe is largely unknown, and the prevalence of the HEV infection is also unknown for most countries of the region. Antibody to HEV (anti-HEV) was tested on 2,305 serum samples from the general population of the Community of Madrid (Spain) collected in the year 2008 among people aged 2-60 years. Total anti-HEV was tested by enzyme-immunoassay (EIA), and reactive samples were retested separately for anti-HEV IgG and IgM by recombinant immunoblot test (RIBT). Fifty samples (2.17%) displayed reactivity for total anti-HEV after EIA testing, and anti-HEV IgG was confirmed by RIBT in 25 (1.08%). The frequency of RIBT-confirmed anti-HEV ranged from 0.97% among the youngest to 3.61% among the oldest, and displayed a statistically significant trend to increasing with age. The rate of RIBT confirmation was also significantly higher among the individuals aged above 20 years old than among those younger of 21 years. HEV infection would be less frequent in the Community of Madrid than in Catalonia or the United Kingdom, and contact with HEV would be very uncommon among children and adolescents of the region. Confirmation of EIA-reactive samples by RIBT reduced the final numbers of anti-HEV testing as much as 50%, and some findings of this study suggest that such testing protocol would reflect better the real prevalence of anti-HEV in settings of low endemicity than the single testing by EIA.

Evaluation of an automated complement-fixation test (Seramat) for diagnosis of acute respiratory infections caused by viruses and atypical bacteria.

The complement-fixation test (CFT) permits low-cost screening of serum samples for different agents within a single assay, and is a useful tool for the serological diagnosis of acute respiratory infections. This study evaluated the automated Seramat CFT system with 160 paired serum samples taken from 80 patients with acute respiratory infection in comparison with in-house CFTs against a panel of agents, including influenza A and B, adenovirus, respiratory syncitial virus, cytomegalovirus, Mycoplasma pneumoniae, Coxiella burnetti and Chlamydia spp., and in comparison with indirect immunofluorescence (IIF) against Legionella pneumophila. Overall, the Seramat system identified 75 (88.2%) of the 85 seroconversions recognised by in-house CFTs or IIF. In comparison to the in-house CFTs, the correlation was 89.2% (66/74). For L. pneumophila, the Seramat system detected nine (81.8%) of the 11 cases diagnosed by IIF. The Seramat system also identified eight additional seroconversions that were not detected by the in-house assays; none of these seroconversions was detected by the in-house assay on retesting. The Seramat system represents a significant technical improvement that may enable many clinical laboratories to use the CFT as a routine diagnostic tool.

Multiplex polymerase chain reaction for the simultaneous detection and typing of polyomavirus JC, BK and SV40 DNA in clinical samples.

A novel multiplex nested PCR (nPCR) method was developed for detecting and differentiating simultaneously the DNA of polyomaviruses JC, BK and SV40 in a single tube. In the first amplification step the same set of primers were used to amplify a conserved DNA region of the large T antigen gene of JCV, BKV and SV40. The second round of multiplex nPCR was carried out using a set of primers designed to render products of different size for each related virus. The thermocycling parameters and concentration of each reaction component were optimised systematically to achieve optimal specificity and sensitivity for the nPCR assay. The sensitivity of the method ranged between one and 10 copies of polyomavirus genome. Cerebrospinal fluid (CSF) was examined from AIDS patients with clinical and neuroradiological evidence of progressive multifocal leukoencephalopathy (PML) and CSF from AIDS patients with other neurological alterations. Urine specimens from bone marrow transplant recipients affected by haemorrhagic cystitis were also tested. The results obtained suggest that the assay is a good tool for supporting the diagnosis of polyomavirus infection and could be used for epidemiological purposes and in other studies in order to define better the role of polyomaviruses in human disease.

Use of overlapping synthetic peptides to characterize samples from blood donors with indeterminate results to hepatitis C virus core antigen.

BACKGROUND AND OBJECTIVES: Despite recent improvements in supplemental assays, isolated reactivity to the hepatitis C virus (HCV) core antigen continues as one of the main problems in the confirmation of anti-HCV in blood donors. Reactivity against individual peptides from the c22-3 HCV recombinant antigen has been described as a useful tool for anti-HCV confirmation and donor counseling in such cases. MATERIALS AND METHODS: We used a previously described set of overlapping peptides spanning the entire sequence of the c22-3 antigen to study 87 single serum samples from blood donors with reactivity for c22-3 antigen alone in second generation recombinant immunoblot assay (RIBA-2). All of them had been previously studied by RIBA-3, anti-HCV E2 EIA and HCV PCR. RESULTS: 66 of the 87 samples studied could be classified as positive or negative for anti-HCV core using the multipeptide assay and such classification correlated well with the results obtained with RIBA-3 and the anti-E2 EIA. However, some discrepancies were found. The epitopes located along the N-terminal half of the molecule were mainly responsible for the specific antibody recognition but those enclosed within amino acids 1-15 were frequently involved in nonspecific reactivity. Some 38% of samples were considered to have specific antibody to the c22-3 antigen and a further 9% reacted for both anti-E2 and single core peptides that were often involved in specific antibody recognition. CONCLUSION: Testing of blood donor samples indeterminate to the HCV c22-3 antigen for reactivity against individual core peptides can confirm the presence of specific antibody and recognize nonspecific reactivity with certain cross-reacting epitopes. Third generation supplemental tests have reduced such false reactivity, but confirmation of samples with anticore alone is still necessary. Single reactivity to the HCV core antigen is likely to reflect prior exposure to the virus, but rarely active infection, either acute or chronic.

Detection of antibody to hepatitis C virus E2 recombinant antigen among samples indeterminate for anti-HCV after wide serological testing and correlation with viremia. The Spanish Study Group for Blood Donors at Risk of Transmission of HCV.

The detection of antibody to the second envelope protein (E2) of the hepatitis C virus (HCV) has been hampered by the lack of suitable antigens. A previously described E2 recombinant antigen (CHO-E2) expressed as a non-fused, highly glycosylated protein in mammalian cells was used to detect specific antibody (anti-E2) in samples from blood donors and viraemic patients showing positive or indeterminate results for anti-HCV after a wide serological study. Anti-E2 was detected in 50-75% of the donors positive for anti-HCV, 80% of viraemic immunocompetent patients with anti-NS3 alone and 28% of non-viraemic donors with anticore alone. In donors with anti-NS3 (15 samples) or anti-NS4 (51 samples) alone, anti-E2 was found occasionally (3 cases). Moreover, two anti-E2-positive samples from viraemic patients were misidentified by some commercial assays for screening anti-HCV. These results suggest that testing for anti-E2 may be useful for improving the performance of the current assays for anti-HCV screening and confirmation.

Two different PCR assays to detect enteroviral RNA in CSF samples from patients with acute aseptic meningitis.

Two polymerase chain reaction (RT-PCR) assays were developed to allow rapid detection of enteroviral RNA in cerebrospinal fluid samples (CSF). Primers homologous to the conserved 5' noncoding region of the enterovirus genome were designed. The RT-PCR product size was approximately 500 bp (479 bp for Poliovirus, 500 bp for Coxsackievirus) and was visualized using ethidium bromide-stained gels. Assay 1 utilized Moloney Murine Leukaemia Virus Reverse Transcriptase (MMLV-RTase) for reverse transcription and Taq polymerase for subsequent PCR. Assay 2 utilized a thermoactive DNA polymerase of Thermus thermophilus (rTth enzyme) for both reverse transcription and DNA amplification. In addition, in Assay 2 reverse transcription and PCR were accomplished within the same reaction tube. Both assays detected between 1 and 0.02 TCID50 of prototype strains of Polio and Coxsackie type B viruses propagated in VERO cell and spiked in a pooled preparation of CSF samples from patients with noninfective neurological disorders. However, Assay 1 was 10-fold more sensitive than Assay 2 when applied to the detection of enteroviral RNA in CSF samples from patients with etiologically well characterized acute aseptic meningitis.

[Comparative sensitivity of different solid-phase immunoassays for detecting HBsAg and anti-HCV in blood]. / Sensibilidad comparativa de distintos inmunoensayos en fase sólida para detección de HBsAg y anti-VHC en suero.

The sensitivities of seven methods of enzyme-immunoassay for HBsAg detection, as well as of twelve immunoassays for detecting antibody to HCV, were compared, in an attempt to evaluate the need for an official technical validation of such methods prior to its commercialization in Spain. Significant differences were seen for the sensitivities of these methods, which may influence the proficiency of blood bank screening for HBsAg and anti-HCV for preventing cases of post-transfusional viral hepatitis. As a conclusion, it is recommended to establish legal regulations for pre-marketing validation of such assays in Spain, as well as to take the results obtained in these evaluation studies as the basis for selecting those tests which may be more convenient for screening purposes at the blood banks.

Sensitivity of seven commercial assays in the detection of hepatitis B virus type 2-like infection.

Two hundred serum samples taken from patients with hepatitis B virus type 2-like infections were tested with seven commercial enzyme immunoassay methods for detection of HBsAg. Positive results were obtained with all methods in some samples, the rate of detection of HBsAg ranging from 9% to 90% for the different methods. A direct correlation was found between the analytical sensitivity of a method and its ability to yield positive results. It is suggested that these findings should be taken into consideration when selecting HBsAg detection methods in blood banks in order to avoid transmission of the HBV2 agent to recipients of blood or blood products.

Diagnosis of Mycoplasma pneumoniae infection by microparticle agglutination and antibody-capture enzyme-immunoassay.

The performance of two new commercial assays for the serological diagnosis of Mycoplasma pneumoniae infection (microparticle agglutination and antibody-capture enzyme-immunoassay) was studied using a panel of 169 serum samples from patients with Mycoplasma pneumoniae pneumonia and a control group. Both assays were shown to be sensitive and specific for diagnosis. The performance of the capture immunoassay, however, decreased in older patients, probably due to its inability to detect cases of reinfection without IgM antibody response.

[Evaluation of serological criteria for the diagnosis of infectious mononucleosis]. / Valoración de criterios serológicos para el diagnóstico de mononucleosis infecciosa.

We have evaluated the application for the diagnosis of infectious mononucleosis of several serologic criteria, including measurement of concentrations of IgM against viral capsid antigen (VCA) of Epstein-Bar virus (EBV) by ELISA, detection of heterophil antibodies and the presence of anti-VCA IgG in absence of IgG against nuclear antigen of EBV (EBNA). Such criteria were evaluated by using a board of sera obtained from cases who had clinically been diagnosed of infectious mononucleosis and identified in the laboratory as produced by EBV (84 sera) or cytomegalovirus (CMV) (9 sera). The measurement of anti-VCA IgM by any of the two tested methods is the best criteria for the serologic diagnosis of infectious mononucleosis. Positive anti-VCA IgM reactions were detected by IIF and ELISA in sera of patients with infectious mononucleosis-like syndrome due to cytomegalovirus, so an alternative method to the measurement of anti-VCA IgM is needed, the most adequate is the measurement of anti-VCA IgG in the absence of anti-EBNA IgG.

Definition of high-proficiency serological markers for diagnosis of varicella-zoster virus infections by enzyme immunoassay.

The levels of Varicella-zoster virus (VZV)-specific IgG, IgM, and IgA antibodies produced in 22 cases of varicella, 22 cases of cutaneous zoster, and 12 cases of acute aseptic meningitis due to VZV in the absence of cutaneous lesions, were measured by indirect enzyme immunoassay (EIA) and compared with those observed in a group of 34 age-matched controls. The definition of cutoff titres for each serological marker and combinations of them allowed early diagnosis of infection in 82% of varicella patients and 91% of patients with acute aseptic meningitis lacking cutaneous lesions on a single serum sample, the specificity being over 90%. The system was as sensitive as the demonstration of intrathecally produced IgG antibodies for the early diagnosis of the infection in 22 cases of neurological disease due to VZV. A working protocol for the serological diagnosis of VZV infections, using currently available EIA reagents, is described.

Screening of antibodies to varicella-zoster virus in leukemic patients: comparison of commercial methods of enzyme-immunoassay and fluoroimmunoassay.

Commercial methods of enzyme immunoassay and fluoroimmunoassay for the detection of varicella-zoster antibodies were compared using serum samples from individuals in a program of bone marrow transplantation. The correlation was 93.4%. Enzyme immunoassay showed to be more sensitive. Both methods are applicable to the detection of varicella-zoster antibodies. However, when fluoroimmunoassay is used, we recommend the confirmation of negative results by a sensitive enzyme immunoassay.

Evaluation of a latex agglutination test for screening antibodies to rubella virus.

The performance of a commercial latex agglutination test for screening rubella antibodies was investigated, using two panels of selected serum samples especially designed for a careful evaluation of sensitivity. Three false negative results were obtained on samples with very low levels of antibody. However, the test had higher sensitivity than the hemagglutination inhibition test and fluoroimmunoassay when samples were tested undiluted. False positive results were not obtained, and prozone reactions were not observed. The test is a very practical method for screening rubella antibodies in primary health care units.

Comparison of four methods for screening of cytomegalovirus antibodies in normal donors and immunocompromised patients.

Three commercial methods (enzyme immunoassay, fluoroimmunoassay and latex agglutination) and a complement fixation test were compared for detection of cytomegalovirus antibodies using a panel of 490 serum samples from blood and organ donors, from immunocompromised patients and paired sera from five patients with recent cytomegalovirus infection. An indirect immunofluorescence test for antibodies to cytomegalovirus was used for classifying samples giving discrepant results by any of the four methods. All methods showed high sensitivity and specificity, but the enzyme immunoassay and the latex agglutination tests had the highest sensitivity. Latex agglutination is recommended for large-scale screening of cytomegalovirus antibodies in blood and organ donors. Negative results obtained by latex agglutination should be confirmed by sensitive enzyme immunoassays.

Fluoroimmunoassay for detection of rubella-specific immunoglobulin M: comparison with indirect enzyme immunoassay and mu-chain capture.

The performance of a commercially-available method of fluoroimmunoassay (Rubella M FIAX, International Diagnostic Technology, Santa Clara, Calif.), designed for the detection of rubella-specific immunoglobulin M, was tested with 137 selected sera, including 52 from cases of primary rubella, 29 from healthy pregnant women, 21 containing rheumatoid factor, and 35 from cases of infectious mononucleosis (IM) caused by Epstein-Barr virus. The results were compared with those obtained by commercial indirect enzyme immunoassay (EIA) and EIA anti-mu chain capture tests. The fluoroimmunoassay technique showed a satisfactory level of sensitivity, but low values had to be interpreted with caution as false-positive results were detected with sera with rheumatoid factor and from IM cases, even after preliminary treatment of sera with the anti-human immunoglobulin G antisera provided in the kit. On the other hand, no false-positive results in the analysis of IM sera were seen in the EIA anti-mu chain capture method. Because of its sensitivity and specificity, we recommend the use of the latter technique for the diagnosis of primary rubella.

Evaluation of commercial methods of enzyme immunoassay (EIA) for the measurement of rubella-specific IgM.

Four commercial EIA methods for measuring rubella-specific IgM (three indirect tests and one anti-mu capture test) were evaluated, using sucrose gradient centrifugation and hemagglutination inhibition as the reference method. Evaluation was conducted with the aid of four serum panels, including 53 primary rubella cases, 30 healthy pregnant women, 21 sera positive for rheumatoid factor(s) (RF) and 35 sera from 29 cases of heterophil-positive infectious mononucleosis with EBV-specific IgM detected by immunofluorescence. All EIA methods were more sensitive than the reference method when applied to very early samples (1-5 days post-exanthema) and no differences in sensitivity were found between them. On the other hand, we observed a significant incidence of false-positive results if an indirect EIA method is applied to RF-positive samples. False positivity is significantly reduced, but not totally eliminated, when samples are preabsorbed with anti-human IgG serum and, in all cases, the absorbance values obtained were low. In contrast, there were no false-positive results using an anti-mu capture method, even in sera from cases of infectious mononucleosis. The basis for choosing between an indirect method and an anti-mu capture method for the diagnosis of congenital and post-natal rubella virus infection is discussed.