Survey of bovine brucellosis on the island of Fernando de Noronha, Pernambuco, Brazil / Investigação sobre brucelose bovina na Ilha de Fernando de Noronha, Pernambuco, Brasil

Considering the lack of information about livestock diseases on Brazilian oceanic islands, the occurrence of bovine brucellosis was investigated on the island of Fernando de Noronha, state of Pernambuco, Brazil. Serum samples were collected in October 2009, from all the 105 cows raised on the island at that time. These were examined concurrently using the Rose Bengal test and the Complement Fixation Test. All the samples were negative in both tests, indicating that the cows on the island were likely free from infection by smooth forms of Brucella. These results can partly be explained by the prohibition of introduction and importation of both small and large-sized animals that had been implemented through District Decree 19 of February 28, 2004.(AU)

Tendo em vista a inexistência de informações sobre a ocorrência da brucelose bovina em ilhas oceânicas brasileiras, investigou-se a presença da infecção na ilha de Fernando de Noronha, Estado de Pernambuco, Brasil. Soros de todas as 105 fêmeas bovinas existentes, colhidos em outubro de 2009, foram examinados concomitantemente pelo teste do Antígeno Acidificado Tamponado e pela Reação de Fixação de Complemento. Todas as amostras foram negativas em ambos os testes, indicando que provavelmente os animais presentes na ilha encontravam-se livres da infecção por Brucella. Estes resultados podem ser explicados, em partes, pela proibição da introdução e importação de grandes e pequenos animais, implementada pelo Decreto Distrital 19, de 28 de fevereiro de 2004.(AU)

Varicella zoster virus antibody detection: A comparison of four commonly used techniques.

BACKGROUND: Antibody tests for the varicella zoster virus (VZV) include neutralization, fluorescent antibody to membrane antigen (FAMA), immune adherence hemagglutination (IAHA), enzyme immunoassay (EIA), glycoprotein-based enzyme-linked immunosorbent assay (gpELISA), and complement fixation (CF) tests. Of these, FAMA is considered the most sensitive. However, in Japan, the EIA method is most frequently employed. OBJECTIVE: The VZV antibody detection rate of the FAMA, EIA, gpELISA, and IAHA methods was compared. METHODS: Four types of antibody tests were conducted with sera collected from 83 college students. The relationships between two antibody tests were examined using Pearson's correlation coefficients. RESULTS: All 83 subjects were observed to be VZV antibody-positive using the FAMA method. The Pearson correlation coefficients of gpELISA, EIA, and IAHA relative to FAMA were 0.808, 0.782, and 0.356, respectively. The positive agreement rate of IAHA relative to FAMA was 88.0% (73/83), whereas those of gpELISA and EIA were both 97.6% (81/83). Furthermore, EIA showed 100% positive agreement with gpELISA and a high correlation coefficient of 0.911, whereas these values for IAHA compared to gpELISA were much lower (90.1% and 0.530). The calculated Pearson correlation coefficient for comparison of the EIA and IAHA methods was 0.498, with a positive agreement rate of 90.1% (73/81). CONCLUSIONS: The EIA method should be employed in Japan based on the similarity of the positivity between EIA and gpELISA, as it is more available and practical than gpELISA.

Combining complement fixation and luminol chemiluminescence for ultrasensitive detection of avian influenza A rH7N9.

The complement fixation test (CFT) is a serological test that can be used to detect the presence of either a specific antibody or antigen to diagnose infections. In a conventional CFT, the assay result is determined by observing the clarity of the reaction solution or the sediment of red cells by the naked eye. Although the assay conditions are thereafter simplified, the sensitivity of the assay would be sacrificed due to the limitation of bulk observation. Inspired by the forensic scientists to examine blood at the scene of the crime, we rationally argued that the luminol chemiluminescence (CL) reaction could be applied in the CFT to sense physiological complement-mediated haemolytic phenomena for sensitive protein detection. The combination of the CFT and the luminol CL system was demonstrated in detection of rH7N9, a recombinant avian influenza virus protein. The testing can be accomplished within 2.5 h and the linear detection range covers 0.25 fg mL(-1) to 25 ng mL(-1). The feasibility of the CL based CFT in assaying a real biopsy was successfully demonstrated by specifically detecting rH7N9 and the carcinoembryonic antigen (CEA) in human serum. This new type of protein detection approach inherits the beauty of complement-mediated assay, such as being fast, and no protein immobilization, blocking and washing. In addition, the participation of luminol CL enables us to quantitatively analyse the intensity of a haemeolysis process, ameliorating the limitation of bulk observation in traditional CFT. It is anticipated that the luminol CL-CFT assay would be particularly suitable for investigation of small molecules, toxins, and short peptides.

Evaluation of different enzyme-linked immunosorbent assays for the diagnosis of brucellosis due to Brucella melitensis in sheep.

Six serological assays for the diagnosis of ovine brucellosis, due to Brucella melitensis were evaluated. Reference serum samples from sheep of known B. melitensis infection status (n=118) were assessed using the Rose Bengal test (RBT), complement fixation test (CFT) and four commercial enzyme-linked immunosorbent assays (ELISAs), including two indirect ELISAs (iELISAs), one competitive ELISA (cELISA) and one blocking ELISA (bELISA). The highest differential positive rates (DPR) were obtained with the cELISA and bELISA, while the lowest DPR was estimated using iELISAs. A latent class analysis was performed to estimate the accuracy of the CFT, RBT and bELISA using 1827 sera from sheep undergoing testing as part of a surveillance and control programme. Lower sensitivity and specificity were obtained for the three serological tests when the field samples were used. A higher DPR was achieved by the CFT, compared to bELISA and RBT. The results suggest that ELISAs, and particularly the bELISA, might be suitable for inclusion in the European Union legislation on intra-community trade for diagnosing B. melitensis infection in sheep, as it has a similar test performance compared to the RBT.

[A simple method for quantification of modified low-density lipoproteins].

A simple method for quantification of modified low-density lipoproteins (mLDL) in the blood serum containing 8.9% polyvinylpyrrolidone solution 12600 +/- 2700 has been developed. The results show that 10 min incubation of serum in a buffer containing 8.9% PVP leads to complete aggregation mLDL. Atherogenicity of aggregated mLDLs is experimentally confirmed by two independent tests (mLDLs bind and activate the complement system of a guinea pig (pro-inflammatory effect) and cause platelet hyperaggregation (thrombogenic effect)). The proposed method is simple and involves only two steps: mixing the serum with a solution of PVP and recording the turbidity. The method allows to register the presence of mLDL directly in serum without its prior fractionation.

Clinical usefulness of a novel C1q assay to detect immunoglobulin G antibodies capable of fixing complement in sensitized pediatric heart transplant patients.

BACKGROUND: Donor-specific antibodies (DSA) against human leukocyte antigens complicate transplantation with the potential for acute antibody-mediated rejection (AMR). Complement-fixing antibodies are required to initiate the complement cascade. Not all DSAs, however, can fix complement. METHODS: A novel C1q assay was developed to detect the sub-set of immunoglobulin G (IgG) antibodies capable of fixing complement. Sera from 18 pediatric heart transplant patients were analyzed for DSAs using a Luminex platform (Luminex Inc, Austin, TX) and commercially available single-antigen bead assay kits. Biopsy specimens were assessed for AMR using histopathologic criteria and immunohistochemical staining. RESULTS: During the study period, 5 patients had AMR; of these, 2 were C1q virtual crossmatch positive (VXM+) and had persistent C1q DSAs after transplant, and 3 were C1q VXM- but antibody developed immediately after transplant. A positive C1q assay in the immediate post-transplant period had a positive predictive value (PPV) of 100% and a negative predictive value (NPV) of 100%, with 100% sensitivity and 100% specificity (Fisher exact p = 0.001). Of 11 patients who were IgG VXM+, 5 had AMR; the IgG VXM had a PPV of 45% and NPV of 100%, with 100% sensitivity and 54% specificity (Fisher exact p = 0.101). CONCLUSIONS: The C1q assay can detect a sub-set of antibodies capable of fixing complement and predicts AMR early after transplant. Avoiding only the donor antigens that would be recognized by the C1q assay may accelerate time to transplant by expansion of the donor pool and potentially allows transplantation of previously "incompatible" organs.

Bioactive arabinogalactans from the leaves of Opilia celtidifolia Endl. ex Walp. (Opiliaceae).

The leaves of the tree Opilia celtidifolia have a long tradition for being used in Mali and other West African countries against various ailments such as for wound healing. Previous studies on polysaccharides from these leaves showed the presence of pectic-like polymers with an effect on the human complement system as well as the ability to activate macrophages. The present study shows that bioactive arabinogalactans isolated by water of 50°C could be separated into two acidic fractions, Oc50A1 and Oc50A2. The former could, by gel filtration on Sephacryl S-400, be separated into two fractions, which were further purified on a Superdex 200 column to give the fractions Oc50A1.I.pur and Oc50A1.II.pur. These fractions were subjected to chemical and biological studies. The polysaccharides consisted mainly of heavily branched type II arabinogalactans and minor amounts of rhamnogalacturonan I regions. The isolated polymers had a high human complement-fixating ability, as well as the ability to stimulate rat macrophages and dendritic cells (DCs) and to induce B cell proliferation. These effects were especially pronounced for the higher molecular weight fraction of Oc50A1.I.pur. The fractions Oc50A1.I.pur and Oc501.II.pur stimulated secretion of pro-inflammatory cytokines from purified B cells or DCs. Collectively, these results indicate that the arabinogalactan type II polymers present in the leaves of O. celtidifolia may be used to develop medical devices for regulating inflammatory processes.

[Modified method for determining circulating immune complexes in the complement fixation test with polyethylene glycol precipitate].

The authors have modified a method for determining circulating immune complexes in the complement fixation test. It is shown that with the 7% concentration of polyethylene glycol (PEG)-6000, there is a more complete precipitation of low- and medium-molecular weight immune complexes. The time and temperature of serum incubation were optimized when PEG-6000 was obtained. The use of the soluble circulating immune complexes (sCIC) prepared from human serum as a standard for the construction of a standard plot could substantially enhance the sensitivity of the method (0.325 microg/ ml). The content of circulating immune complexes was studied in donors and in patients with connective tissue dysplasia (CTD) by the improved procedure. In the group of donors, the mean level of sCIC was 0.62 +/- 0.24 mg/ml. In the CTD group, that was 2.32 +/- 0.93 mg/ml; the serum concentrations of sCIC ranging from 1.1 to 5.0 mg/ml. In the donors and patients, the detection rate of serum sCIC was 100%. The proposed method may be clinically used to measure the human serum levels of sCIC.

Estabilidade do antígeno de célula total de Brucella abortus para uso no diagnóstico sorológico da brucelose bovina pela reação de fixação de complemento / Stability of Brucella abortus whole cell antigen for use in the serological diagnosis of bovine brucellosis by the complement fixation test

A reação de fixação de complemento é um dos testes usados no diagnóstico confirmatório da brucelose bovina, e para sua realização emprega-se o mesmo antígeno usado na prova de soroaglutinação lenta, porém não foi possível encontrar na literatura estudos sobre a estabilidade desse antígeno para uso na prova de fixação de complemento, de modo a estabelecer um prazo de validade para o mesmo. Por isso, esta investigação teve por objetivo avaliar a estabilidade do antígeno de célula total de Brucella conservado sob refrigeração, para uso na reação de fixação de complemento. Analisaram-se 14 partidas de antígeno, preparado com Brucella abortus amostra 1119/3 e padronizado para uso na prova de soroaglutinação lenta, com tempo de fabricação variando de 9 meses a 23 anos e 11 meses. Testaram-se 167 soros bovinos com títulos variáveis de anticorpos contra Brucella, adotando-se a técnica com incubação a 37ºC nas duas fases da reação e 5 unidades hemolíticas 50 por cento de complemento. Considerou-se como positivo o soro com pelo menos 25 por cento de fixação de complemento na diluição 1:4. Compararam-se os resultados obtidos com as 13 partidas de antígeno com aqueles obtidos com a partida com 9 meses de fabricação, usando o teste de chi2 de McNemar e o coeficiente kappa. A grande maioria dos soros apresentou resultados muito próximos quando testados com as diversas partidas de antígeno, e não se observou relação entre tempo de fabricação do antígeno e diferenças nos resultados obtidos.

The complement fixation test is used worldwide in the confirmatory diagnosis of bovine brucellosis. For this technique the antigen is the same as the one used in the tube agglutination test. However, literature is poor in information about the stability of the whole cell Brucella antigen for use in the complement fixation test to establish a time of validity of the antigen. Hence the aim of this investigation was to evaluate the stability of this antigen under refrigeration for use in the complement fixation test. Fourteen batches of antigen prepared with Brucella abortus strain 1119/3, produced from 9 months to 23 years and 11 months before, were analysed. One hundred and sixty-seven cattle sera with varying titres of antibodies to Brucella were tested through the warm complement fixation microtechnique with five 50 percent haemolytic units of complement. Sera with at least 25 percent of complement fixation in dilution 1:4 were considered positive. The results with 13 of the antigen batches were compared with the results obtained with the batch produced 9 months before by the McNemar chi2 test and kappa statistic. The oldest antigen batch gave a higher proportion of sera titres which were exactly the same observed with the 9-month-batch (90.4 percent), and the antigen produced 4 years and 3 months before the test gave de lowest proportion of sera with the same titre of the 9-month-antigen (73.7 percent). The comparison of the results after being classified as positive and negative showed that the highest proportion of agreed results was observed with the antigen produced 21 years and 4 months before (98.8 percent, kappa 0.98). The antigen with the lowest proportion of agreed results was the one produced 3 years and 2 months before (91.6 percent, kappa 0.84). The results of the study show that most sera gave very similar results with all antigen batches evaluated, and that there was no relationship between the period of antigen production...

Prevalence of bovine and human brucellosis in western Algeria: comparison of screening tests.

A serological study was carried out in Tiaret province in western Algeria on 1032 cows distributed in 95 flocks to estimate the prevalence of Brucella infection and to compare the sensitivity and specificity of a range of agglutination tests. Screening tests showed 31.5% of herds positive using the buffered plate antigen test and 26.3% using the rose Bengal test compared with 15.7% with the complement fixation test. Using the complement fixation test as the gold standard for confirmatory tests, the Rivanol test was found to be more sensitive but less specific than tube agglutination in detecting brucellosis infection. Three isolates were identified from 105 blood samples from humans with brucellosis and 50 samples of milk and tissues from infected cows and they were all Brucella melitensis biovar 3.

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.

Diagnostic methods for detection of respiratory RNA viruses.

This article is a comprehensive description of diagnostic methods for detection of RNA respiratory viruses - respiratory syncytial virus RSV, influenza A and B viruses, parainfluenza 1, 2 and 3 viruses, coronaviruses and rhinoviruses--from cell culture to molecular biology methods. Both patients and medical personnel appear to be at risk of viral infection, specially during the winter season. Moreover, many health care units lack viral diagnostic facilities; therefore, it is essential for medical personnel to have an understanding of the etiology, mechanisms of transmission and of all disposable today diagnostic methods of RNA respiratory viruses. Patients at greatest risk of acquiring nosocomial viral respiratory disease are children, patients with immunodeficiency and patients treated in intensive care.

A conglutination test for rapid detection of antibodies against Babesia bigemina

A rapid conglutination test (RCT) with performance comparable to the indirect fluorescent antibody technique (IFAT) was developed to detect antibodies against Babesia bigemina (B. bigemina-RCT). The B. bigemina-RCT is a sensitive, specific, economical, and rapidly performed serological test suitable for field application or minimally equipped laboratories. This test had a sensitivity of 90.9 percent, and specificity of 97.6 percent, compared to IFAT, which showed for the same parameters respectively, 98.3 percent and 99.7 percent. The early detection of anti- B. bigemina immunoglobulins by RCT in experimental infections was nearly parallel to that of IFAT. Cross reactions were observed with sera from calves experimentally infected with Babesia bovis (1.8 percent) and with Anaplasma marginale (1.2 percent). RCT antigen prepared with non parasitized erythrocytes (negative antigen) showed 1.5 percent, 3.5 percent and 2.2 percent of positive reactions with sera from animals experimentally infected with B. bigemina, B. bovis and A. marginale. However, none of the sera from animals of endemic areas for babesia infection resulted in positive reactions with the negative antigen. Considering these results and shelf life over six months, the B. bigemina-RCT could be used for epidemiological surveys and evaluation of control measures against this species of Babesia.

Um teste rápido de conglutinação (TCR) com desempenho comparável a imunofluorencência indireta (IFI) foi desenvolvido para detectar anticorpos contra Babesia bigemina. O TCR-B.bigemina é um teste sorológico sensível, econômico e executável rapidamente; apropriado para condições de campo ou laboratórios com estrutura mínima. Este teste tem uma sensibilidade de 90,9% e especificidade de 97,6%, enquanto que a IFI apresentou para os mesmos parâmetros, respectivamente, 98,3% e 99,7%. Nas infecções experimentais a detecção de imunoglobulinas anti-B. bigemina pelo TCR foi aproximadamente a mesma da IFI. As reações cruzadas verificadas nos soros de bezerros experimentalmente infectados com Babesia bovis e Anaplasma marginale foram 1,8% e 1,2%, respectivamente. O antígeno preparado com eritrócitos não parasitados (antígeno negativo) apresentou 1,5%, 3,5% e 2,2% de reações positivas com os soros de animais infectados com B. bigemina, B. bovis e A. marginale. Entretanto, nenhum dos soros dos animais de áreas endêmicas para infecção de babésia resultaram em reações positivas com o antígeno negativo. Considerando estes resultados e o período de viabilidade do antígeno de TCR, acima de seis meses, possibilita o TCR-B. bigemina ser utilizado em levantamentos epidemiológicos e na avaliação das medidas de controle contra esta espécie de Babesia.

Analysis of complement fixation and commercial enzyme immunoassays for detection of antibodies to Mycoplasma pneumoniae in human serum.

The Meridian ImmunoCard (IC), GenBio ImmunoWELL-IgM, and Remel EIA commercial antibody tests are qualitative enzyme immunoassays that detect antibodies to Mycoplasma pneumoniae in serum. These tests were compared to an M. pneumoniae complement fixation (CF) assay, which uses a commercially available antigen component. The Meridian IC and the ImmunoWELL-IgM detect immunoglobulin M (IgM) only; the Remel EIA and the CF test detect both IgM and IgG antibodies. Detection of specific IgM antibody, which appears early in infection, can be, but is not always, indicative of a recent or current infection. Paired serum samples from 64 adult patients with probable M. pneumoniae infection were examined with each of the four tests. Thirty (47%) of the 64 acute-phase sera were IgM positive by Meridian IC, 26 (41%) were positive by Remel EIA, 24 (38%) were positive by CF, and 15 (23%) were positive by ImmunoWELL-IgM. When both the acute- and convalescent-phase serum samples from each patient were examined, 61 (95%) of the 64 patients were positive by CF, 60 patients (94%) were positive by Remel EIA, 52 patients (81%) were IgM positive by the Meridian IC, and 29 patients (45%) were IgM positive by the ImmunoWELL-IgM assay. The Meridian IC was more sensitive than the other tests for early detection of IgM antibodies. However, after examining paired serum samples, we concluded that the detection of IgM alone may not be useful for all cases of mycoplasma infection, especially in an adult population.

[The use of a modified complement fixation reaction for the rapid diagnosis of viral infections in children]. / Ispol'zovanie modifitsrovannoi reaktsii sviazyvaniia komplementa dlia ékspresssnoi diagnostiki virusnykh infektsii u detei.

A system of diagnostic tests (complement enzyme assay) is developed, detecting viral and other antigens, toxins, antibodies, and specific immune complexes in liquid enzyme immunoassay based on the complement fixation test. The system is simple and economic, the results can be transferred into digital data, and the above factors can be detected individually in biological materials from patients. The system is effective, and in many cases (for example, in the diagnosis of enteroviral diseases) is the only method for rapid isolation and typing of the infection agents.