Outbreak of Chagas disease in Brazil: Validation of a molecular diagnostic method.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi (T. cruzi), affects millions of people worldwide. Polymerase Chain Reaction (PCR) and real-time quantitative PCR (qPCR) have been used as tools to monitor parasitic levels in the bloodstream of individuals exposed to infection, thus enabling the monitoring of relapses and the effectiveness of therapy, for example. The aim of this study was to evaluate the TcSAT-IAM system, developed by our research group, on samples from patients with suspected Chagas disease infection. Initially, primer systems were developed for the detection of the nuclear DNA (SAT-DNA) from T. cruzi (TcSAT-IAM). The Cruzi system, predicted in the literature, and TcSAT-IAM were then evaluated in relation to their analytical sensitivity, specificity and efficiency. Afterwards, the applicability of the qPCR technique using both systems (separately) for the diagnosis of acute CD was evaluated in samples from 77 individuals exposed to the outbreak that occurred in Pernambuco-Brazil, relating the results obtained to those of the classical diagnostic methods recommended for this stage of the infection. TcSAT-IAM and Cruzi had a detection limit of 1 fg of target DNA (0,003 parasites). Thirty-eight cases were recorded, 28 by laboratory criteria and 10 by clinical and epidemiological criteria. Blood samples from 77 subjects were submitted to qPCR by both systems, reaching an agreement of 89.61% between them. After analyzes between systems and diagnostic criteria, the TcSAT-IAM showed sensitivity and specificity of 52.36% (CI 37.26-67.52) and 92.31% (CI 79.68-97.35), respectively, accuracy of 72.73% and moderate agreement. The TcSAT-IAM showed an accuracy of 72.58% and 75% in relation to parasitological and serological tests (IgM anti-T. cruzi), respectively. Therefore, quantitative PCR should be incorporated into the diagnosis of suspected acute cases of Chagas disease.

A multicentric evaluation of dipstick test for serodiagnosis of visceral leishmaniasis in India, Nepal, Sri Lanka, Brazil, Ethiopia and Spain.

Visceral leishmaniasis (VL) is one of the leading infectious diseases affecting developing countries. Colloidal gold-based diagnostic tests are rapid tools to detect blood/serum antibodies for VL diagnosis. Lack of uniformity in the performance of these tests in different endemic regions is a hurdle in early disease diagnosis. This study is designed to validate a serum-based dipstick test in eight centres of six countries, India, Nepal, Sri Lanka, Brazil, Ethiopia and Spain with archived and fresh sera from 1003 subjects. The dipstick detects antibodies against Leishmania donovani membrane antigens (LAg). The overall sensitivity and specificity of the test with 95% confidence intervals were found to be 97.10% and 93.44%, respectively. The test showed good sensitivity and specificity in the Indian subcontinent (>95%). In Brazil, Ethiopia, and Spain the sensitivity and specificity of the dipstick test (83.78-100% and 79.06-100%) were better as compared to the earlier reports of the performance of rK39 rapid test in these regions. Interestingly, less cross-reactivity was found with the cutaneous form of the disease in Spain, Brazil, and Sri Lanka demonstrating 91.58% specificity. This dipstick test can therefore be a useful tool for diagnosing VL from other symptomatically similar diseases and against cutaneous form of leishmaniasis.

Chronic Chagas Disease Diagnosis: A Comparative Performance of Commercial Enzyme Immunoassay Tests.

There is a significant heterogeneity in reported performance of serological assays for Chagas disease diagnosis. The conventional serology testing in laboratory diagnosis and in blood banks is unsatisfactory because of a high number of inconclusive and misclassified results. We aimed to assess the quality of four commercially available enzyme-linked immunosorbent assay tests for their ability to detect Trypanosoma cruzi antibodies in 685 sera samples. Cross-reactivity was assessed by using 748 sera from patients with unrelated diseases. Initially, we found that the reactivity index against T. cruzi antigen was statistically higher in sera from Chagas disease patients compared with those from non-chagasic patients, supporting the notion that all evaluated tests have a good discriminatory ability toward the diagnosis of T. cruzi infection in patients in the chronic phase of the disease. Although all tests were similarly sensitive for diagnosing T. cruzi infection, there were significant variations in terms of specificity and cross-reactivity among them. Indeed, we obtained divergent results when testing sera from patient with unrelated diseases, particularly leishmaniasis, with the levels of cross-reactivity being higher in tests using whole T. cruzi extracts compared with those using recombinant proteins. Our data suggest that all four tests may be used for the laboratory diagnosis and routine blood screening diagnose for Chagas disease. We also emphasize that, despite their general good performance, caution is needed when analyzing the results when these tests are performed in areas where other diseases, particularly leishmaniasis, are endemic.

Assessment of a DNA vaccine encoding an anchored-glycosylphosphatidylinositol tegumental antigen complexed to protamine sulphate on immunoprotection against murine schistosomiasis.

Protamine sulphate/DNA complexes have been shown to protect DNA from DNase digestion in a lipid system for gene transfer. A DNA-based vaccine complexed to protamine sulphate was used to induce an immune response against Schistosoma mansoni anchored-glycosylphosphatidylinositol tegumental antigen in BALB/c mice. The protection elicited ranged from 33 to 44%. The spectrum of the elicited immune response induced by the vaccine formulation without protamine was characterized by a high level of IgG (IgG1> IgG2a). Protamine sulphate added to the DNA vaccine formulation retained the green fluorescent protein encoding-plasmid longer in muscle and spleen. The experiments in vivo showed that under protamine sulphate effect, the scope of protection remained unchanged, but a modulation in antibody production (IgG1= IgG2a) was observed.

Assessment of a DNA vaccine encoding an anchored-glycosylphosphatidylinositol tegumental antigen complexed to protamine sulphate on immunoprotection against murine schistosomiasis

Protamine sulphate/DNA complexes have been shown to protect DNA from DNase digestion in a lipid system for gene transfer. A DNA-based vaccine complexed to protamine sulphate was used to induce an immune response against Schistosoma mansoni anchored-glycosylphosphatidylinositol tegumental antigen in BALB/c mice. The protection elicited ranged from 33 to 44 percent. The spectrum of the elicited immune response induced by the vaccine formulation without protamine was characterized by a high level of IgG (IgG1> IgG2a). Protamine sulphate added to the DNA vaccine formulation retained the green fluorescent protein encoding-plasmid longer in muscle and spleen. The experiments in vivo showed that under protamine sulphate effect, the scope of protection remained unchanged, but a modulation in antibody production (IgG1= IgG2a) was observed.

Humoral and cellular immune responses in BALB/c and C57BL/6 mice immunized with cytoplasmic (CRA) and flagellar (FRA) recombinant repetitive antigens, in acute experimental Trypanosoma cruzi infection.

In previous studies, cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins induced specific humoral and cellular immune responses in susceptible and resistant mice in the absence of Trypanosoma cruzi infection with a significant induction of the Interferon-gamma (IFN-gamma) production in those animals. In this follow-up paper, the immunostimulatory and protective effects of these proteins were evaluated by immunizing with CRA or FRA antigens, BALB/c and C57BL/6 mice and challenging with a T. cruzi (Y strain). Both proteins induced humoral response with high levels of IgG isotypes as well as cellular immunity with high levels of IFN-gamma when compared to controls. However, the lymphocyte proliferative response was minimal. The survival rate at 30 days post-infection was significant in CRA (60%) or FRA (50%)--immunized BALB/c mice and CRA (83.3%)--immunized C57BL/6 mice. Taken as a whole these findings indicate that CRA and FRA are immunogenic and potentially important for protective immunity.

Mitogenic activity of Cratylia mollis lectin on human lymphocytes.

The mitogenic effect of Cratylia mollis seed lectin preparations containing two (Cramoll 1,4) or one molecular form (Cramoll 1) showed activity similar to the well known T-cell mitogen, concanavalin A (Con A). The effect on human lymphocytes was analyzed through a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Inhibition of lymphocyte proliferation with methyl-alpha-d-mannoside (both preparations) indicated that the mitogenic effect involved carbohydrate lectin binding sites.

Avaliação hematológica e histopatológica de camundongos BALB/c e C57BL/6 expostos aos antígenos recombinantes Cytoplasmic Repetitive Antigen e Flagellar Repetitive Antigen de Trypanosoma cruzi / Hematological and histopathological evaluation of BALB/c and C57BL/6 mice exposed to Cytoplasmic Repetitive Antigen and Flagellar Repetitive Antigen recombinant antigens of Trypanosoma cruzi

Os antígenos recombinantes Cytoplasmic Repetitive Antigen e Flagellar Repetitive Antigen de Trypanosoma cruzi foram inoculados em camundongos BALB/c e C57BL/6 e o seu efeito avaliado a nível hematológico e histopatológico. Os resultados mostraram que o padrão histológico normal dos órgãos e o perfil hematológico dos camundongos não foram modificados sugerindo que esses antígenos não parecem causar dano ao animal.

Evaluation of the immune response to CRA and FRA recombinant antigens of Trypanosoma cruzi in C57BL/6 mice.

Humoral and cellular immune responses were evaluated in 44 C57BL/6 mice immunized with the Trypanosoma cruzi recombinant antigens CRA and FRA. Both antigens induced cutaneous immediate-type hypersensitivity response. The levels of IgG1, IgG2a, IgG2b and IgG3 were high in CRA immunized mice. IgG3 was the predominant isotype. Although no difference in antibody levels was observed in FRA-immunized mice when compared to control mice, both antigens were able to induce lymphoproliferation in immunized mice. Significant differences were observed between incorporation of [ H]- thymidine by spleen cell stimulated in vitro with CRA or FRA and the control group. These results suggest that CRA and FRA could be involved in mechanisms of resistance to Trypanosoma cruzi infection.

Evaluation of the immune response to CRA and FRA recombinant antigens of Trypanosoma cruzi in C57BL/6 mice

Humoral and cellular immune responses were evaluated in 44 C57BL/6 mice immunized with the Trypanosoma cruzi recombinant antigens CRA and FRA. Both antigens induced cutaneous immediate-type hypersensitivity response. The levels of IgG1, IgG2a, IgG2b and IgG3 were high in CRA immunized mice. IgG3 was the predominant isotype. Although no difference in antibody levels was observed in FRA-immunized mice when compared to control mice, both antigens were able to induce lymphoproliferation in immunized mice. Significant differences were observed between incorporation of [ H]- thymidine by spleen cell stimulated in vitro with CRA or FRA and the control group. These results suggest that CRA and FRA could be involved in mechanisms of resistance to Trypanosoma cruzi infection

[Hematological and histopathological evaluation of BALB/c and C57BL/6 mice exposed to Cytoplasmic Repetitive Antigen and Flagellar Repetitive Antigen recombinant antigens of Trypanosoma cruzi]. / Avaliação hematológica e histopatológica de camundongos BALB/c e C57BL/6 expostos aos antígenos recombinantes Cytoplasmic Repetitive Antigen e Flagellar Repetitive Antigen de Trypanosoma cruzi.

The Cytoplasmic Repetitive Antigen and Flagellar Repetitive Antigen recombinant antigens of Trypanosoma cruzi were inoculated into BALB/c and C57BL/6 mice and its effects evaluated at hematological and histopathological levels. The results showed that the histological pattern of the organs and the hematological profile of mice were not modified suggesting that these antigens are not harmful for the animal.

Antibody isotype responses to egg antigens in human chronic Schistosomiasis mansoni before and after treatment.

In the present communication we analyzed the levels of IgG1, IgG2, IgG3, IgG4 and IgE isotypes to soluble egg antigen of Schistosoma mansoni by ELISA in individuals from an endemic area for schistosomiasis in Northeast Brazil. The analysis was performed before and after treatment to evaluate the age-dependent pattern, and to identify differences in the reactivities to antigens. Our results suggest that schistosomiasis treatment would not interfere with this sort of immune response.

Antibody isotype responses to egg antigens in human chronic Schistosomiasis mansoni before and after treatment

In the present communication we analyzed the levels of IgG1, IgG2, IgG3, IgG4 and IgE isotypes to soluble egg antigen of Schistosoma mansoni by ELISA in individuals from an endemic area for schistosomiasis in Northeast Brazil. The analysis was performed before and after treatment to evaluate the age-dependent pattern, and to identify differences in the reactivities to antigens. Our results suggest that schistosomiasis treatment would not interfere with this sort of immune response

Serodiagnosis of chronic Chagas infection by using EIE-Recombinant-Chagas-Biomanguinhos kit

A kit based on an enzyme immunoassay, EIE-Recombinant-Chagas-Biomanguinhos, developed by the Oswaldo Cruz Foundation, was evaluated for the serodiagnosis of chronic Chagas disease. Evaluation was performed with 368 serum samples collected from individuals living in an endemic area for Chagas disease: 131 patients in the chronic phase with confirmed clinical, epidemiological, and serological diagnosis (indirect immunofluorescence, indirect hemagglutination or enzyme-linked immunosorbent assay) and 237 nonchagasic seronegative individuals were considered negative control. The EIE-Recombinant-Chagas-Biomanguinhos kit showed high sensitivity, 100 percent (CI 95 percent: 96.4-100 percent) and high specificity, 100 percent (CI 95 percent: 98-100 percent). The data obtained were in full agreement with clinical and conventional serology data. In addition, no cross-reaction was observed with sera from patients with cutaneous (n=14) and visceral (n=3) leishmaniasis. However, when these sera were tested by conventional serological assays for Chagas disease, cross-reactions were detected in 14.3 percent and 33.3 percent of the patients with cutaneous and visceral leishmaniasis, respectively. No cross-reactions were observed when sera from nonchagasic seronegative patients bearing other infectious disease (syphilis, n=8; HTLV, n=8; HCV, n=7 and HBV, n=12) were tested. In addition, sera of patients with inconclusive results for Chagas disease by conventional serology showed results in agreement with clinical evaluation, when tested by the kit. These results are relevant and indicate that the refered kit provides a safe immunodiagnosis of Chagas disease and could be used in blood bank screening

Immunodiagnosis of chronic Chagas'disease using the Tc 46 and Tc 58 antigens

Os polipeptídeos de 46 e 58kDa foram reconhecidos em diferentes cepas de T. cruzi (Y, WSL e Colombiana) pelo soro de todos os pacientes chagásicos estudados. Estes polipeptídeos foram isolados da cepa Y e usados em ELISA. A sensibilidade e especificidade foram 97,6 por cento [CI 95 por cento: 86-100 por cento] e 100 por cento [CI 95 por cento: 89,3-100 por cento], respectivamente quando Tc 46 foi usada. Quando Tc 58 foi utilizada a sensibilidade e especificidade foram de 100 por cento [CI 95 por cento: 89,6-100 por cento] e 90,2 por cento [CI 95 por cento: 75,9-96,8 por cento], respectivamente

Partial protection of mice against Trypanosoma cruzi after immunizing with the TcY 72 antigenic preparation

A 72 kDa Trypanosoma cruzi glycoprotein recognized by the 164C11 monoclonal antibody (IgM isotype) was purified by preparative electrophoresis. The antigenic preparation obtained, named TcY 72, was used to immunize C57Bl/10 mice. The following results were observed after immunization: (1) induction of higher titres of IgG than IgM antibodies, as evaluated by indirect immunofluorescence; (2) significant DTH after infection of epimastigotes in mice footpads: (3) peak parasitemia in immunized mice was significantly reduced and animals were negative by 13 days post-infection, although the mice still succumb to infection: (4) the phenotypic analysis of spleen cell populations showed a decrease in the CD4/CD8 ratio in immunized mice. Taken as a whole, these findings indicate that TcY 72 is immunogenic and potentially important for protective immunity.

Demonstraçäo de atividade pestosa no foco da Serra dos Orgäos (Rio de Janeiro, Brasil) no período de 1983 a 1984, através de exames sorológicos em roedores / Detection of plague activity in the focus of the Serra dos Orgäos (Rio de Janeiro, Brazil) by serological examination of rodents during the period from 1983 to 1984

No período de 1983 a 1984 foram encontrados no foco pestoso da Serra dos Orgäos (Rio de Janeiro, Brasil) 6 roedores com títulos de anticorpos hemaglutinantes contra a fraçäo antigênica 1A da Yersinia pestis, >=1/16. A pesquisa do bacilo nos roedores e pulicídeos desse foco é recomendável