In from the cold: M-protein light chain glycosylation is positively associated with cold agglutinin titer levels.

BACKGROUND: Primary cold agglutinin disease (CAD) is a monoclonal antibody (M-protein) and complement-mediated chronic hemolytic disease process. Antibody glycosylation can play a role in both antibody half-life and complement fixation. Recently, M-protein light chain (LC) glycosylation has been shown to be associated with AL amyloidosis. We hypothesized that M-protein LC glycosylation is also associated with cold agglutinin (CA) titers and CA-mediated hemolysis. STUDY DESIGN AND METHODS: A cross-sectional study of patients undergoing CA titer evaluation underwent mass spectrometric analysis for M-proteins and M-protein LC glycosylation. A subset of serum samples also underwent evaluation for the ability to trigger cold hemolysis in vitro. M-protein and M-protein LC glycosylation rates were compared across CA titer groups, clinical diagnosis, direct antiglobulin testing (DAT) results, and cold in vitro hemolysis rates. RESULTS: Both M-protein and M-protein LC glycosylation rates significantly differed across CA titer groups with the highest rates in those with elevated CA titers. M-protein LC glycosylation occurred almost exclusively on IgM kappa M-proteins and was significantly associated with positive DAT results and a clinical diagnosis of CAD. Cold in vitro hemolysis was demonstrated in two patients who both had a CA titer of more than 512 but there was no significant association with CA titer group or M-protein LC glycosylation status. CONCLUSION: M-protein LC glycosylation is significantly associated with higher CA titer levels. Given the role that antibody glycosylation can play in antibody half-life and complement fixation, further studies are needed to clarify the effects of LC glycosylation within the context of CAD.

Comparison of a complement fixation test, a gel diffusion test and two absorbed and unabsorbed ELISAs for the diagnosis of paratuberculosis in sheep.

A complement fixation test for paratuberculosis, a gel diffusion test and two enzyme-linked immunosorbent assays (ELISA) were evaluated using sera from Mycobacterium paratuberculosis infected and non-infected sheep. Gross pathology and histopathology were used as parameters of infection. The two ELISAs, one of which is commercially available for testing cattle, were used before and after sera had been absorbed with a soluble sonicate of Mycobacterium phlei. Differences between the various tests and between ELISAs before and after absorption were non-significant (P > 0.05) in non-infected sheep or in animals with gross or histopathological lesions. The specificity of all the tests was at least 97%. Sensitivity in histopathologically positive sheep was at least 98%. Sheep from infected flocks but without histopathological lesions showed serological results which were poorly correlated between the various tests.

Evaluation of an enzyme-linked immunosorbent assay for the detection of sheep infected and vaccinated with Brucella melitensis.

An indirect enzyme-linked immunosorbent assay (ELISA), using the lipopolysaccharide of the cell wall as an antigen, was used to detect Brucella melitensis antibodies in ovine serum. The test was carried out on 703 samples of field serums, which were also analyzed by the complement fixation (CF) test and the rose Bengal (RB) test. The ELISA results were more similar to those of the CF test (kappa = 0.89) than to the results of the RB test (kappa = 0.73). The ELISA also had high sensitivity (94.7%) and a somewhat lower specificity (90.4%). One group of 139 young brucellosis-free animals 3-6 months of age were vaccinated with B. melitensis rev. 1 at a dose of 1.2 x 10(9) live organisms. The ELISA detected a significantly lower number of reactors than the CF and RB tests (P < 0.001). The ELISA values remained below the cutoff level during the 9 months following vaccination.

[The evaluation of a new complement fixation microtechnic (Garcia & Sotelo, 1991) for the diagnosis of chronic Chagas disease with different antigenic preparations]. / Avaliação de uma nova microtécnica de fixação de complemento (Garcia & Sotelo, 1991) para o diagnóstico da doença de Chagas crônica com diferentes preparações antigênicas.

From this present data it has been evaluated a new complement fixation test, comparatively to indirect immunofluorescence to diagnose chronic Chagas' disease, utilizing one watery extract of epimastigotes of Trypanosoma cruzi and three other ethanolic extracts: one from epimastigotes, one from tripomastigotes and a third one of amastigotes obtained from cultures. Utilizing 236 serum samples indirect immunofluorescence test was performed: 109 positives (20 of them with positive parasitologic diagnostic) and 127 negatives (96 of healthy blood donors and 31 with other diseases). The results have showed that is possible a positive reaction in diluted samples up to 1:16. The best limits of reactivity found were the dilutions 1:4 for the ethanolic extract of amastigotes and 1:2 for the others antigens. The correlation index among the new complement fixation test and indirect immunofluorescence test showed that the ethanolic extract from epimastigotes was the best antigen to be utilized to diagnosis purposes. Its co-positivity index with indirect immunofluorescence was 0.92207 and the co-negative index was 0.90000. Concluding, the new complement fixation test showed itself as a fast, sensible, easily applicable semiquantitative microtechnique to the diagnosis of chronic Chagas' disease.

Diagnosis of American trypanosomiasis (Chagas' disease) by the new complement fixation test.

A new immunodiagnostic method of complement fixation was used for serodiagnosis of American trypanosomiasis; 92% sensitivity and 99% specificity were obtained, for an overall accuracy of 97%. This test can be used in field studies, obviating the use of most laboratory equipment and imported reagents; places where economic limitations hinder the use of other immunodiagnostic procedures; and in association with other tests for confirmation of the diagnosis.

Comparison of commercially available enzyme immunoassay with traditional serological tests for detection of antibodies to Coccidioides immitis.

A newly released commercially available enzyme-linked immunosorbent assay (ELISA) was evaluated for its ability to detect immunoglobulin M (IgM) and IgG antibodies against the tube precipitin and complement fixation (CF) antigens of Coccidioides immitis. The ELISA was compared with more traditional diagnostic assays, CF, latex agglutination (LA), and immunodiffusion (ID). When the IgM-specific portion of the ELISA was compared with LA, there was an agreement of 81.8%, a specificity of 75.0%, and a sensitivity of 84.6%. For the determination of the presence of IgG antibodies, the results of the IgG-specific part of the ELISA were compared with the combined results of ID and CF. After resolution of discrepant results, there was an agreement of 95.6%, a specificity of 98.3%, and a sensitivity of 92.6%. When the results of the IgG- and IgM-specific portions of the ELISA combined were compared with the results of the three traditional assays (CF, LA, and ID) there was an agreement of 96.7%, a specificity of 98.5%, and a sensitivity of 94.8%. The ELISA proved to be a reliable assay for the detection of antibodies against the tube precipitin and CF antigens and did not suffer from the objectivity required to interpret the results of the traditional assays and anticomplement interference associated with the traditional assays.

Evaluation of a commercially available complement fixation test for diagnosis of Helicobacter pylori infection and for follow-up after antimicrobial therapy.

Commercially available complement fixation test reagents (Institute Virion Ltd., Rüschlikon, Zurich, Switzerland) available in package format were evaluated for the serodiagnosis of Helicobacter pylori infection. The assay was compared with bacterial culture and histological Giemsa stain of gastric biopsy specimens obtained from 930 patients of different ages and from different ethnic groups, with a variety of upper gastrointestinal tract symptoms. The prevalence, sensitivity, specificity, and positive and negative predictive values, respectively, were 35, 71, 90, 80, and 85% for Belgian patients aged 40 years or younger, 50, 81, 93, 92, and 83% for Belgian patients older than 40 years, and 83, 83, 79, 95, and 48% for Mediterranean patients. Using 645 serum specimens from 226 patients, we also evaluated the complement fixation test for its ability to monitor the eradication of H. pylori following antimicrobial therapy. Overall, H. pylori was eradicated from 122 patients while 104 patients remained infected with the organism. A significant decrease in antibody levels was observed 3 to 6 months after the end of therapy in the group of patients from whom H. pylori was eradicated.

Comparison of two rapid commercial tests with complement fixation for serologic diagnosis of Mycoplasma pneumoniae infections.

The complement fixation (CF) test is the current reference serologic test for the diagnosis of Mycoplasma pneumoniae infection. However, it is reported to be insensitive and nonspecific, and it is labor intensive. To determine if a faster and more sensitive diagnosis of M. pneumoniae could be obtained, we examined 50 paired serum samples from patients with suspected M. pneumoniae infection by the CF test and two commercial rapid antibody detection kits, the Remel M. pneumoniae immunoglobulin G (IgG)-IgM antibody test system (Remel, Lenexa, Kans.) and the Seradyn Color Vue M. pneumoniae IgG-IgM kit (Seradyn, Indianapolis, Ind.). The Remel test, a 5-min qualitative immunobinding assay, detected antibodies in three patient serum samples with CF titers of 32 and in all but one sample with titers of > or = 64. The Seradyn test, a 40-min qualitative agglutination test, was less sensitive than CF or Remel. The Seradyn test was positive in 68% of cases, compared with 94 and 96% of cases tested by CF or Remel, respectively. Both commercial tests are faster and less technically demanding to perform than is the CF test.

Use of a recombinant Coccidioides immitis complement fixation antigen-chitinase in conventional serological assays.

The coccidioidal complement fixation (CF) antigen has been cloned previously, and the fusion protein has been expressed in Escherichia coli. The recombinant CF (rCF) antigen was affinity purified by adsorption-desorption to chitin, and its reactivity was studied by using sera containing coccidioidal antibodies. The affinity-purified rCF antigen formed a line of identity with an immunodiffusion (ID) CF reference antigen (coccidioidin) derived from mycelial-phase Coccidioides immitis and was reactive with human, canine, and equine sera containing coccidioidal antibody. The affinity-purified rCF antigen yielded no detectable reaction with Blastomyces of Histoplasma antiserum by ID. The affinity-purified rCF antigen fixed complement with positive human sera and, even when used at lower concentrations, yielded titers comparable to those obtained with the coccidioidin. The reactivity of the affinity-purified rCF antigen was further evaluated by enzyme immunoassay, in which it manifested good sensitivity (96.9%) and specificity (100%) when evaluated with 43 human patients' sera. Thus, the affinity-purified rCF antigen has yielded reactions comparable to those of crude coccidioidal antigens in conventional CF, IDCF, and enzyme immunoassay.

Evaluation of a western blot test in an outbreak of acute pulmonary histoplasmosis.

A western blot (WB) test was evaluated for detection of antibodies against native glycosylated and chemically deglycosylated M and H antigens of Histoplasma capsulatum in serum obtained from patients during the acute phase of pulmonary histoplasmosis that occurred during an outbreak. Of 275 serum samples tested by immunodiffusion and complement fixation (CF) samples from 40 patients affected during this outbreak and from 37 negative controls were tested by WB test. A group of patients whose sera were negative for CF antibodies and precipitins early in the acute stage of histoplasmosis but who all seroconverted during convalescence 6 weeks later were tested with the WB test. Antibodies against untreated H and M antigens were detected at a 1:100 dilution by WB test in 45% of the 20 acute-phase serum samples and in all 20 of the convalescent-phase specimens. The WB test's sensitivity for acute-phase specimens increased to 90% (18 of 20 specimens) when H and M antigens were treated by periodate oxidation to inactivate susceptible carbohydrate epitopes. When native glycosylated antigens were used in the WB test, positive reactions were observed in negative control serum specimens (3 of 37 specimens; 8%) and in serum specimens obtained from asymptomatic persons screened as part of the outbreak investigation (13 of 20 specimens; 65%). These positive reactions were also attributed to glycosidic epitopes since the specificity of the WB test increased from 78 to 100% when periodate-treated H and M antigens were used. WB test with deglycosylated H and M antigens of histoplasmin provides a rapid, sensitive, and specific test to diagnose acute pulmonary histoplasmosis before precipitins can be detected.

Serological diagnosis of ovine enzootic abortion by comparative inclusion immunofluorescence assay, recombinant lipopolysaccharide enzyme-linked immunosorbent assay, and complement fixation test.

Since the 1950s, serological diagnosis of ovine enzootic abortion (OEA), caused by strains of Chlamydia psittaci, has been based mainly on the complement fixation test (CFT), which is neither particularly sensitive nor specific since antibodies to other chlamydial and enterobacterial pathogens may be detected. In this study. a recombinant enzyme-linked immunosorbent assay (rELISA) (medac, Hamburg, Germany), based on a unique chlamydial genus-specific epitope of Chlamydia trachomatis L2 lipopolysaccharide, was evaluated for sensitivity and specificity as a primary screening assay for OEA by comparison with the CFT. A comparative inclusion immunofluorescence assay (IFA), in which antibody titers to C. psittaci and Chlamydia pecorum were examined, was used as the reference test for 573 serum samples from four flocks. Reactivity to C. pecorum was measured since inapparent intestinal infections by C. pecorum are believed to be common in British flocks. In detecting positive sera from an abortion-affected flock, in which a C. pecorum infection was also suggested by IFA, the rELISA outperformed the CFT with significant evidence for increased sensitivity (P = 0.003). In two flocks in which C. pecorum infections alone were suggested by IFA, the rELISA and CFT were prone to detect low levels of false-positive results, but the values were not significant. The rELISA provided results in one flock in which sera that were anticomplementary could not be resolved by the CFT. In another flock in which abortion had not occurred but infection by both chlamydial species was suspected, no significant difference was found between the sensitivities of the rELISA and CFT. The rELISA could not differentiate ovine C. psittaci and C. pecorum infections but was shown to be a more sensitive primary screening test for OEA than was the CFT, particularly where abortion had occurred and even when antibodies due to additional inapparent infection(s) by C. pecorum were present.

Evaluation of serological tests for diagnosis of Brucella melitensis infection of goats.

Five serological assays were evaluated for the diagnosis of brucellosis in goats: the rose bengal test (RBT), complement fixation test (CFT), radial immunodiffusion (RID) with Brucella and Yersinia enterocolitica O:9 polysaccharides, counterimmunoelectrophoresis (CIEP) with cytosol, and enzyme-linked immunosorbent assay (ELISA) with polyclonal and protein G conjugates and smooth lipopolysaccharide (S-LPS), native hapten polysaccharide (NH), or cytosol antigens. For optimal sensitivity, RBT had to be used with sera-antigen at a 3:1 dilution. In the RID test, Brucella melitensis biotype 1 NH could not be replaced by Brucella abortus biotype 1 or Y. enterocolitica 0:9 polysaccharides. In the ELISA, S-LPS and NH gave similar results and the protein G conjugate increased the specificity. With the sera from 55 B. melitensis culture-positive goats, the sensitivity was 100% for RBT, CFT (titer > or = 4), and ELISA with S-LPS or NH; 94% for RID; and 93% for CIEP. All tests were negative (100% specific) when testing the sera from 127 brucella-free goats. Larger discrepancies among the results of the serological tests were obtained with sera from goats of areas where brucellosis is endemic. When the sera of 20 young goats vaccinated subcutaneously (10(9) CFU of B. melitensis Rev 1) and bled 6 months later were examined, the specificities were as follows: NH ELISA, 60%; CFT and S-LPS ELISA, 75%; RBT, 80%; CIEP, 90%; and RID, 94%. With the sera from 10 young goats vaccinated conjunctivally (10(9) CFU of B. melitensis Rev 1) all tests were 100% specific 4 months after vaccination. The proportion of goats giving a positive reaction after vaccination decreased faster in RID than in other tests.

Monitoring Babesia bovis infections in cattle by using PCR-based tests.

The sensitivity and specificity of PCR tests based on the small-subunit rRNA gene sequence of Babesia bovis were compared in a blind study of experimentally infected cattle with the corresponding parameters of the complement fixation (CF) test currently used in the United States to screen for bovine babesiosis. Cattle were experimentally infected with a single inoculum of a cloned laboratory strain of B. bovis. Blood samples were collected and tested over a period covering from the day of infection to 10 months postinfection. The level of parasitemia (percent infected erythrocytes) present in each sample was estimated from test results and was plotted as a function of time postinfection. These data are the first describing the course of infection by methods capable of detecting parasitemias in the range of 10(-7)%, which frequently occur in the carrier state. Parasitemias in the samples tested strongly influenced the sensitivity and negative predictive value of the PCR-based tests which varied with time postinfection. The average sensitivities of the three PCR-based tests for B. bovis ranged from 58 to 70% for a single determination, while the sensitivity of the CF test was only 6%. Both PCR-based and CF tests for B. bovis had high specificity values ranging from 96 to 100%.

Avaliação de uma nova microtécnica de fixação de complemento (Garcia & Sotelo, 1991) para o diagnóstico da doença de Chagas crônica com diferentes preparaçoes antigênicas / The evaluation of a new complement fixation microtechnic (Garcia & Sotelo, 1991) for the diagnosis of chronic Chagas disease with different antigenic preparations

No presente trabalho, avalia-se a nova fixação de complemento, comparativamente à imunofluorescência indireta, para o diagnóstico da doença de Chagas crônica, utilizando um extrato aquoso de formas epimastigotas do Trypanosoma cruzi e três extratos etanólicos: um de epimastigotas, um de tripomastigotas e outro de amastigotas obtidas de cultura. Empregaram-se 236 amostras testadas por imunofluorescência indireta: 109 positivas (20 com diagnóstico parasitológico) e 127 amostras negativas (96 de doadores de sangue e 31 de portadores de outras patologias). Os resultados mostraram que é possível obter reação positiva em amostras diluídas até 1:16. Os melhores limiares de reatividade encontrados foram a diluição 1:4 para o extrato etanólico de amastigotas e 1:2 para os demais antígenos. Os índices de correlação entre a nova fixação de complemento e imunofluorescência indireta indicaram o extrato etanólico de epimastigotas como o antígeno mais adequado para fins de diagnóstico entre as preparações testadas, tendo apresentado índice de co-positividade com a imunofluorescência indireta de 0,92207 e índice de co-negatividade de 0,90000. Conclui-se que a nova fixação de complemento mostrou-se ser uma microtécnica semi-quantitativa rápida, sensível, barata e de fácil execução, aplicável ao diagnóstico da doença de Chagas crônica.

From this present data it has been evaluated a new complement fixation test, comparatively to indirect immunofluorescence to diagnose chronic Chagas' disease, utilizing one watery extract of epimastigotes of Trypanosoma cruzi and three other ethanolic extracts: one from epimastigotes, one from tripomastigotes and a third one of amastigotes obtained from cultures. Utilizing 236 serum samples indirect immunofluorescence test was performed: 109 positives (20 of them with positive parasitologic diagnostic) and 127 negatives (96 of healthy blood donors and 31 with other diseases). The results have showed that is possible a positive reaction in diluted samples up to 1:16. The best limits of reactivity found were the dilutions 1:4 for the ethanolic extract of amastigotes and 1:2 for the others antigens. The correlation index among the new complement fixation test and indirect immunofluorescence test showed that the ethanolic extract from epimastigotes was the best antigen to be utilized to diagnosis purposes. Its co-positivity index with indirect immunofluorescence was 0.92207 and the co-negative index was 0.90000. Concluding, the new complement fixation test showed itself as a fast, sensible, easily applicable semiquantitative microtechnique to the diagnosis of chronic Chagas' disease.

Diagnóstico etiológico por Latex en LCR en la meningitis bacteriana / Etiologic diagnosis with latex in CSF on bacterial meningitis

Estudio prospectivo, comparativo y descriptivo donde se evalua la eficacia de la prueba de Latex en L.C.R., como un método diagnóstico etiológico rapido de meningoecefalitis bacteriana comparando los resultados con la tinción de Gram y el cultivo de L.C.R. en pacientes internados en el Centro de Pediatria Albina R.de Patiño.