Evaluation of serological diagnosis of Borna disease virus infection using recombinant proteins in experimentally infected rats

We produced two recombinant Borna disease virus (BDV) proteins, p40 and p24, by using a baculovirus vector as a diagnostic antigen. Antigenicities of these recombinant proteins were evaluated by immune rabbit sera. Recombinant p40 was a more sensitive antigen than p24 for the detection of antibodies in infected rats. Rats inoculated with BDV within 24 hr after birth showed higher detection rates of viral RNA and viral proteins from the brain than rats inoculated at 4 weeks-old. Depending on the age of infection and the time postinfection, the detection of BDV RNA, protein, or anti-BDV antibody did not always correlate in individuals. We suggest both serological and molecular biological methods are needed in the diagnosis of BDV infection.     

Role of magnetic resonance in hemodialysis patients with amyloid shoulder arthropathy]

Monoclonal antibodies (MAbs) were generated by immunizing mice with a truncated recombinant protein corresponding to the immunodominant region (residues 1-120) of hepatitis C virus (HCV) nucleocapsid protein. The specific recognition by either human sera or mouse monoclonal antibodies of overlapping peptides spanning the core region 1-120 as well as the comparison with epitopes described earlier allowed the fine mapping of HCV core. Within the region 1-120, the major antigenic domain could be restricted to the first 45 amino acids. Indeed, the peptide S42G (residues 2-45) allowed the detection of an anti-HCV core response by all anticore-positive human sera examined. According to their epitope localization, three groups of mouse MABs could be evidenced that were directed against different regions of core. Group II MAbs recognized a strictly linear epitope (QDVKF, residues 20-24), whereas group I MABs were directed against a conformational epitope mainly located at the amino acid residues (QIVGG, 29-33). The epitope of group III MABs was also conformational (PRGRRQPI, residues 58-65). These three epitopes appeared close but different from the three major human epitopes RKTKRNTN, VYLLPR, and GRTWAQPGYPWPLY (residues 7-17, 34-39, and 73-86, respectively). Group II MAB 7G12A8 and group I MAB 19D9D6 were used in a sandwich ELISA for the capture and the detection, respectively, of viral core antigen in sera of patients with chronic HCV infection. After treatment of sera with triton x 100 in acidic conditions, amounts of viral antigen as low as 20 pg/ml of sera could be detected.     

Seroprevalence to bovine immunodeficiency virus and lack of association with leukocyte counts in Italian dairy cattle

We report herein on the first serological detection of antibodies to bovine immunodeficiency virus (BIV) in Italy. According to criteria of a stratified-random sampling of dairy cattle reared in the Parma area (a province in the Po Valley, Northern Italy), sera from 3166 cows belonging to 272 herds were collected. In addition, sera of 138 bulls from eight artificial-insemination (AI) centres were sampled. Seventy-eight cows (2.5%) from 16 herds (5.8%) and seven bulls (5.1%) from two AI centres were positive for BIV-R29 antibodies in the IFA-test. IFA-positive sera assayed by Western blot had reaction to different viral proteins: 81 out of 85 sera showed antibody to p26 (considered the BIV major internal core protein); four sera reacted to other viral proteins but not to p26. Peripheral blood leukocytes of 60 seropositive and 60 seronegative animals, belonging to eight BIV-infected herds, were enumerated to assess any effect of BIV infection on white-blood cells. No significant differences were detected between the two groups. These data indicate that BIV infection is present in Italian dairy cattle--but the role of BIV in inducing disease remains unclear.     

Anticonvulsant and glutamate release-inhibiting properties of the highly potent metabotropic glutamate receptor agonist (2S,2''R, 3''R)-2-(2'',3''-dicarboxycyclopropyl)glycine (DCG-IV)

IgA has an important function in the gastrointestinal immune system. We investigated IgA anti-ganglioside antibodies in Guillain-Barré syndrome (GBS) and Fisher's syndrome (FS) subsequent to Campylobacter jejuni enteritis. In previous studies, serological diagnosis of C. jejuni infection was based on the detection of IgG, IgA, and IgM anti-C. jejuni antibodies. Our study, however, showed that the detection of IgG anti-C. jejuni antibody alone was sufficient for the serological diagnosis of antecedent C. jejuni enteritis in GBS and FS, when the cut-off level was defined for results of sera from C. jejuni-isolated patients. Serological evidence of C. jejuni infection was found in 62 (31%) of 201 GBS patients and 12 (18%) of 65 FS patients. IgA anti-GMI antibody was detected in sera from 33 (16%) of the GBS patients, 1 (2%) of the FS patients, and none of the 46 normal control subjects. IgA anti-GM1 antibody titers were significantly higher in the GBS patients with positive C. jejuni serology than in those with negative serology (P < 0.0001) or the FS patients with positive C. jejuni serology (P = 0.007). IgA anti-GQ1b antibody was detected in sera from 18 (28%) of the FS patients, 9 (4%) of the GBS patients, and none of the normal control subjects. FS patients with positive C. jejuni serology had significantly higher titers of IgA anti-GQ1b antibody than those with negative serology (P = 0.01) or the GBS patients with positive C. jejuni serology (P < 0.0001). We conclude that anti-GM1 and anti-GQ1b IgA antibodies are closely associated with antecedent C. jejuni enteritis in GBS and FS, respectively.     

Diagnostics of persistent viruses: human cytomegalovirus as an example

Infections with persistent viruses such as herpesviruses have become of significant clinical importance with the increasing number of immunocompromised patients at risk to suffer from severe disease. As antiviral chemotherapy is available for herpesvirus infections, the diagnostic methods for rapid and sensitive detection of symptomatic infection have been developed and recently refined. In human cytomegalovirus (HCMV), the use of recombinant viral antigens provides a rationale to improve serological assays. This may be of use for the discrimination of primary versus secondary infection. Early diagnosis of symptomatic HCMV infection in immunosuppressed patients can be most effectively achieved by the detection of a viral tegument protein, pp65, in peripheral blood leukocytes. This early diagnosis has been shown to be of major importance for the effective treatment of these patients. HCMV infection in solid organs can be demonstrated by immunohistochemistry using monoclonal antibodies against viral proteins. HCMV involvement in diseases of the central nervous system in AIDS patients can be verified by the detection of very small amounts of HCMV DNA in cerebrospinal fluid by polymerase chain reaction. This method may prove useful for monitoring HCMV encephalitis and neuropathy.     

Study of the viral etiology of lower respiratory tract infections in a neonatal unit

OBJECTIVE: To study the possible viral etiology in 139 infants with lower respiratory tract infection who required hospitalization in the Infant Unit of our hospital, from October 1994 to June 1995. PATIENTS AND METHODS: 139 patients were admitted, aged from 13 days to 14 months, during this period. The etiological agent was detected by direct immunofluorescence from nasopharyngeal secretions. Monoclonal antibodies were used against Respiratory Syncitial Virus, Influenza A Virus, Influenza B Virus, Adenovirus and Parainfluenza 3 Virus. Antibody detection against these viruses by Complement Fixation Test was done on 29 of these patients, with paired sera (acute and convalescent phase). RESULTS: In 82 patients (59%) we found at least one viral agents from the nasopharyngeal specimens, but in 64 of these only one was detected, in the remaining 18, there were more than one. Significant levels of antibodies were detected in only six of the 29 patients tested. Serology was negative in the remaining 23 patients. CONCLUSIONS: Syncitial Respiratory Virus is the first virus responsible for the lower respiratory tract infection in this age group (49%). There was no correlation between serological diagnosis and antigen detection.     

Molecular approaches to diagnosis of Chagas' disease: use of defined antigens and a target ribosomal RNA sequence

We evaluated the performance of two defined antigens in the serological diagnosis of Chagas' disease. One of them is a recombinant protein named B13 isolated from a genomic library of Trypanosoma cruzi in the expression vector lambda gtll. We show that the gene corresponding to B13 is conserved in the evolutive stages of the two "polar" strains of T. cruzi. The protein epitopes cloned in B13 are represented in 140 kDa, 116 kDa and 35 kDa polypeptides of trypomastigotes. The other antigen chosen for serodiagnosis is a lipopeptidophosphoglycan (LPPG). This glycoconjugate is also widely distributed in T. cruzi strains. The use of a rabbit serum to LPPG allowed the demonstration that this molecule bears epitopes in common to LPPG-like components and to 80-90 kDa glycoproteins of trypomastigotes. Both B13 and LPPG were evaluated in serodiagnosis by ELISA and RIA using a panel of normal human, Chagasic and Leishmaniasis sera. It was observed that B13 presents high sensitivity and specificity for Chagasic sera. For LPPG it was also concluded that this reagent discriminates between individuals infected and non-infected with T. cruzi. A heterogeneity in the level of antibodies to LPPG in Chagasic patients was detected. No correlation was found between the clinical form of Chagas' disease and the preferential reactivity to B13 or LPPG. We also report preliminary studies towards the characterization of a 100 bp sequence of the 24S alpha rRNA as a target for DNA-based detection systems for diagnosis. We show that polymerase chain reaction of total DNA of different trypanosomatids lead to the specific amplification of a 100 bp fragment only for T. cruzi. Northern blots confirmed the presence of the target region in the mature 24S alpha rRNA. Titration experiments based on the direct amplification of RNA with Taq DNA polymerase allowed the detection of 50 parasites. Studies are in progress to increase the sensitivity of the proposed system.     

Pneumolysin PCR-based diagnosis of invasive pneumococcal infection in children

Blood-based pneumolysin PCR was compared to blood culture and detection of pneumolysin immune complexes, as well as to detection of antibodies to pneumolysin and to C polysaccharide, in the diagnosis of pneumococcal infection in 75 febrile children. Invasive pneumococcal infection was suspected on clinical grounds in 67 of the febrile children, and viral infection was suspected on clinical grounds in 8 of the febrile children. In addition, 15 healthy persons were examined to test the specificity of the PCR assay. Plasma, serum, and leukocyte fractions were analyzed by PCR. The combination of all test results led to the diagnosis of pneumococcal infection in 25 patients. Pneumolysin PCR was positive in 44% of these children, an increase occurred in the pneumolysin antibodies in 39% and in the C polysaccharide antibodies in 30% of the patients; pneumolysin immune complexes were found in convalescent serum in 30%, pneumolysin immune complexes occurred in acute-phase serum samples in 16%, and a positive blood culture was found in 20% of the patients. None of the healthy controls had positive results by PCR. The results suggest that the diagnosis of Streptococcus pneumoniae infection from blood samples necessitates the use of several different assays. Pneumolysin PCR was the most sensitive assay, but its clinical value is reduced by the fact that three blood fractions are needed.     

Detection of antibodies to bovine viral diarrhoea virus (BVDV) and characterization of genomes of BVDV from Brazil

An ELISA for the detection of antibodies to bovine viral diarrhea virus (BVDV) was developed based on antigens derived from a genotype I BVDV strain isolated in Switzerland. Using monoclonal antibodies we showed that this antigen contained the conserved non-structural protein NS3 whereas it essentially lacked the more strain-specific E2 surface glycoprotein. This ELISA has a sensitivity of 97.5% and a specificity of 99.2% as compared to the serum neutralization test (SNT). Preliminary experiments showed that this ELISA reliably detects antibodies to BVDV strains circulating in Brazil. Serum samples obtained from 430 adult cattle on 19 farms of the State of Rio Grande do Sul (Brazil) and one farm from Corrientes (Argentina) were tested for antibodies by means of this ELISA. We found antibodies in 56% +/- 15.1% of the cattle sera tested, which indicates that, in Brazil, the prevalence of infection with BVDV is similar to that found in Europe and the USA. Our sequence analysis of two BVDV isolates showed that BVDV of both genotypes I and II circulate in Brazil.     

Differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus

The baculovirus expression system was found to be efficient at expressing the 3D, the 3AB and the 3ABC non-structural proteins (NSP) of foot-and-mouth disease virus (FMDV) as antigens recognised by immune sera in ELISA. ELISA's using 3D, 3AB and 3ABC detected antibodies from day 8 and 10 after experimental infection of susceptible cattle and sheep and cattle remained seropositive for more than 395 days. The ELISA's detected antibodies against any of the seven serotypes of FMDV. The 3D ELISA was specific and precise and as sensitive as established ELISA's which measure antibody to structural proteins. The assay may be used as a resource saving alternative to established ELISA's for the detection of antibodies against any of the seven serotypes. The 3AB and the 3ABC ELISA were also specific and precise. FMDV infected cattle could be differentiated from those that had been merely vaccinated as they gave a positive result in both the 3AB and the 3ABC ELISA's. Two cattle that had been both vaccinated and infected also gave positive results in both tests, suggesting that the 3AB and 3ABC ELISA's, but not the 3D ELISA might represent a reliable means of detecting infection in a vaccinated population.     

The use of monoclonal antibody for the immunodiagnosis of Fasciola gigantica infection in cattle

Antigens that were specific to Fasciola gigantica were obtained from the whole worm homogenate of the parasite by immunoaffinity chromatography in cyanogen bromide-activated sepharose 4B columns and used for the production of monoclonal antibodies. The F. gigantica-specific monoclonal antibody was labelled with horseradish peroxidase and used for the detection of circulating antigen by the direct ELISA method in the sera of cattle experimentally infected with the parasite. Circulating antigens were detectable in the sera of the animals as from the third week after infection while negative absorbance values were obtained 2 weeks after the termination of the infection by chemotherapy with oxyclozanide. This immunodiagnostic method offers an attractive alternative as a supplement to the conventional coprological diagnosis of fasciolosis.     

Prevalence of specific anti-Cryptosporidium IgG, IgM and IgA antibodies in cat sera using an indirect immunofluorescence antibody test

Sera from 258 healthy and sick domestic and feral cats were screened for specific anti-Cryptosporidium antibodies using an indirect immunofluorescence antibody test (IFA). Sera were positive for IgG, IgM and IgA antibodies in 192 (74%), 84 (32%) and 67 (26%) samples, respectively. Antibody was not detected at dilutions of 1:10 and 1:20 or greater in any of eight specific pathogen-free kittens. IgM and IgA antibody classes were more prevalent in sick than in healthy domestic cats. The presence of IgM and/or IgA antibodies indicated early infection. However, these antibody classes were present in sera from cats either positive or negative for Cryptosporidium infection by faecal examination. Pronounced polar fluorescence was observed in the sporozoites in positive samples under fluorescence microscopy. The higher prevalence of specific anti-Cryptosporidium antibodies and the absence of Cryptosporidium oocysts in faecal samples from some IFA-positive animals suggests that detection of these antibodies in sera from cats could be helpful for the diagnosis of feline cryptosporidiosis.     

Phospholipase A2 activity in salivary glands and saliva of the lone star tick (Acari: Ixodidae) during tick feeding

The etiology of subacute granulomatous thyroiditis (SAT) is obscure, although it is postulated to be associated with viral infections and genetic factors. In the present study, the possibility of an infectious etiology was prospectively studied in 27 consecutive patients with SAT. Special emphasis was put on the role of enteroviruses. Coupled sera (interval one month) were taken from all patients and single sera from 29 control subjects for virus antibody determinations. Stool samples were collected for virus isolation and fine-needle aspiration samples from thyroid gland for the detection of enterovirus RNA using RT-PCR were taken from SAT patients. Enteroviral antibodies were tested using three different methods: indirect EIA, heavy chain capture RIA, and standard complement fixation (CF) test. Antibodies against other common viral pathogens, including enteroviruses, were screened using the CF test and those against Mycoplasma pneumoniae and Chlamydia pneumoniae using EIA and microimmunofluorescence techniques, respectively. Common respiratory viruses were also screened from nasopharyngeal suction samples by antigen detection EIA. Based on serological findings, one patient had acute Cytomegalovirus infection. All other patients were negative in antibody tests, virus isolation, RT-PCR, and antigen detection. Enterovirus RNA was not detected by PCR in the thyroid tissue in any of the fine-needle aspiration samples. There was no evidence of recent enteroviral infections in SAT patients. The results suggest that SAT is not usually associated with acute infections. No evidence was obtained to support the proposed role of enteroviruses as an important etiologic agent of SAT.     

Herpes simplex meningoencephalitis in an infant observed during an epidemic of enteroviral infections of the nervous system

An infant with Herpes simplex meningoencephalitis which occurred during an epidemic of enteroviral neuroinfections is described. Clinical and laboratory signs of meningitis as well as preceding aphtous oropharyngeal inflammation, initially suggested an enteroviral etiology. The appearance of signs of encephalitis with focal neurologic disturbances and the results of brain imaging by computed tomography, primarily the detection of temporoparietal areas of hypodensity, raised the possibility of HSV infection. Thanks to early specific treatment with acyclovir, the infant recovered from meningoencephalitis and 4 months later presented only minor neurological sequelae (slight left hemiparesis). The diagnosis of Herpes simples meningoencephalitis was confirmed by detecting IgM and IgG anti-HSV antibodies in the serum and cerebrospinal fluid both at the beginning and after 10 days of treatment and also by a eightfold rise of the anti-HSV-1 antibody level in serum. The authors emphasize the role of brain imaging (computed tomography and magnetic resonance) in the differential diagnosis of viral nervous system infections and suggest early treatment with acyclovir in case of suspicion of HSV-encephalitis.     

Antibodies against the measles matrix polypeptide after clinical infection and vaccination

Sera from patients exposed to measles virus were investigated for the presence of antibodies against each of the viral antigens. All sera with measurable neutralizing titers contained antibodies against the two surface proteins (the glycoprotein and fusion protein), the nucleocapsid protein, and one of the internal proteins (P2). However, only sera from individuals with clinical symptoms of measles infection (natural measles and atypical measles) contained antibodies against the measles virus matrix protein. Levels of matrix-specific antibodies were highest in patients with atypical measles infection.     

Toxoplasma gondii oocyst survival under defined temperatures

The benefits shown by the recent introduction of PCR for the in vitro diagnosis of hepatitis C virus (HCV) infection has prompted the development of standardized, ready-to-use assays that can be implemented in routine clinical laboratories. We have evaluated the clinical performance of COBAS AMPLICOR HCV (COBAS), the first instrument system that allows the automation of HCV RNA amplification and detection, to determine its performance in the routine laboratory setting. More than 2,000 specimens collected at five centers were analyzed in parallel by the COBAS and the manual AMPLICOR HCV (AMPLICOR) tests, and the results were compared with the results for biochemical and serological markers of HCV. In this study the two PCR systems showed the same accuracy, with a concordance rate of 99.8%. As expected, the correlation between serology and PCR was not absolute because the presence of anti-HCV antibodies may be associated with a latent or past infection. On the other hand, if the presence of confirmed anti-HCV antibodies and elevated alanine aminotransferase levels are taken as the "gold standard," indicating an active, ongoing infection, the COBAS and AMPLICOR tests show high and comparable sensitivities (100%) and specificities (98%), with positive and negative predictive values of 100 and 97%, respectively. During the study no false-positive reactions were detected. The use of an internal control allowed the identification of inhibitory substances that prevented amplification for 0.3 and 0.4% of samples tested by the COBAS and AMPLICOR tests, respectively. Compared to the manual system, the COBAS system allowed a significant reduction of hands-on time and could improve the overall laboratory work flow. In conclusion, these results support the use of the COBAS and AMPLICOR tests for the molecular diagnosis of active HCV infections.     

Absence of pericentromeric heterochromatin (9qh-) in a patient with bilateral retinoblastoma

The diagnosis of HIV infection is based on screening of HIV antibodies and confirmed by a more specific supplementary test. The most common confirmation test is Western blot, which is expensive, time consuming and subject to technical skill. The present study was carried out to evaluate whether the anti-HIV-1 antibody titer is valid as a supplementary test for diagnosis of HIV-1 infection. Anti-HIV-1 antibody titers of 2,414 anti-HIV-1 positive sera determined by the particle agglutination (PA) method were analysed in comparison with the Western blot analysis. The Western blot negative result was found in 11 of 2,414 (0.46%) anti-HIV-1 positive sera, these sera also gave negative anti-HIV by ELISA. The PA titers of these sera were found in the range of 16 to 64. Seventeen samples (0.70%) with anti-HIV-1 in the titer range of 16 to 256 showed indeterminate Western blot analysis. The rest, 2,386 of these 2,414 sera (98.84%), were shown to be positive by Western blot. However, all of the 2,356 sera with antibody titers > or = 512 (97.6%) demonstrated positive Western blot results. Five cases among the 17 (29.4%) indeterminate sera were examples of early seroconversion of HIV infection, which were confirmed in follow up specimens. The results suggest that only the samples with antibody titers < 512 are required to be confirmed for HIV infection by Western blot. It is possible that early seroconversion may be inferred from anti-HIV titers. Therefore, in order to reduce time and cost, the PA anti-HIV titer can be used as an alternative supplementary test for diagnosis of HIV-1 infection in most positive screened anti-HIV samples. Western blot is needed for testing in only a few cases.     

Detection of virus infection-associated antigen and 3D antibodies in cattle vaccinated against foot and mouth disease

The analysis of sera obtained from animals vaccinated or revaccinated with inactivated vaccines against foot and mouth disease (FMD) virus showed that these vaccines induced antibodies against the virus infection-associated (VIA) antigen, detectable by agar gel immunodiffusion (AGID). The present study evaluates the antibody response to protein 3D and the VIA antigen (VIAA) of FMD virus induced by different vaccines in a group of 51 calves. This response was detected using AGID and a liquid-phase blocking sandwich enzyme-linked immunosorbent assay (ELISA) for anti-3D antibodies (ELISA-3D). No anti-VIAA or anti-3D antibodies were detected after the initial vaccination. Following revaccination, animals giving positive results were detected by both methods. This immune response disappeared 60-120 days post-revaccination (dprv) according to the AGID method, and 90-180 dprv when ELISA-3D was used. Samples of oesophageal-pharyngeal fluid obtained from animals that remained positive for anti-VIAA antibodies at 90-120 dprv gave negative results for viral isolation, indicating that the transitional antibody response induced by the vaccine was due to the presence of non-structural antigens in the vaccine and not to viral infection. These results indicate that the ELISA-3D method could be used as a complementary method for sero-epidemiological studies as an indirect indicator of viral activity, as long as the age and vaccination status of the animals being sampled are taken into consideration.     

Human cytomegalovirus infection: new methods for virological diagnosis

Recently developed methods have greatly increased the sensitivity and speed of virological diagnosis of cytomegalovirus infection. Virus can be detected in infected cell cultures within 24 or 48 hours of specimen inoculation by using monoclonal antibodies to immediate-early antigens in immunocytochemistry procedures or DNA sequences in hybridisation in situ assays. CMV antigens can also be detected directly in infected cells within clinical specimens. An early antigen can be visualized in nuclei of circulating leukocytes from viremic patients. DNA hybridization is used for CMV analysis in Dot-blot, Southern-blot and in situ hybridization assays. DNA amplification, by polymerase chain reaction (PCR), has proven to be a very sensitive method for diagnosis of CMV infection and should be useful for investigation of CMV pathogenesis and latency. Serologic assays such as ELISA and latex agglutination assays are accurate for screening donors and recipients of blood and organ or marrow graft. Studies of viral protein epitopes recognized by human sera are in progress.