Diagnosis of Schistosoma mansoni infections: what are the choices in Brazilian low-endemic areas?

The population of Brazil is currently characterised by many individuals harbouring low-intensity Schistosoma mansoni infections. The Kato-Katz technique is the diagnostic method recommended by the World Health Organization (WHO) to assess these infections, but this method is not sensitive enough in the context of low egg excretion. In this regard, potential alternatives are being employed to overcome the limits of the Kato-Katz technique. In the present review, we evaluated the performance of parasitological and immunological approaches adopted in Brazilian areas. Currently, the diagnostic choices involve a combination of strategies, including the utilisation of antibody methods to screen individuals and then subsequent confirmation of positive cases by intensive parasitological investigations.

Serological proteomic screening and evaluation of a recombinant egg antigen for the diagnosis of low-intensity Schistosoma mansoni infections in endemic area in Brazil.

BACKGROUND: Despite decades of use of control programs, schistosomiasis remains a global public health problem. To further reduce prevalence and intensity of infection, or to achieve the goal of elimination in low-endemic areas, there needs to be better diagnostic tools to detect low-intensity infections in low-endemic areas in Brazil. The rationale for development of new diagnostic tools is that the current standard test Kato-Katz (KK) is not sensitive enough to detect low-intensity infections in low-endemic areas. In order to develop new diagnostic tools, we employed a proteomics approach to identify biomarkers associated with schistosome-specific immune responses in hopes of developing sensitive and specific new methods for immunodiagnosis. METHODS AND FINDINGS: Immunoproteomic analyses were performed on egg extracts of Schistosoma mansoni using pooled sera from infected or non-infected individuals from a low-endemic area of Brazil. Cross reactivity with other soil-transmitted helminths (STH) was determined using pooled sera from individuals uniquely infected with different helminths. Using this approach, we identified 23 targets recognized by schistosome acute and chronic sera samples. To identify immunoreactive targets that were likely glycan epitopes, we compared these targets to the immunoreactivity of spots treated with sodium metaperiodate oxidation of egg extract. This treatment yielded 12/23 spots maintaining immunoreactivity, suggesting that they were protein epitopes. From these 12 spots, 11 spots cross-reacted with sera from individuals infected with other STH and 10 spots cross-reacted with the negative control group. Spot number 5 was exclusively immunoreactive with sera from S. mansoni-infected groups in native and deglycosylated conditions and corresponds to Major Egg Antigen (MEA). We expressed MEA as a recombinant protein and showed a similar recognition pattern to that of the native protein via western blot. IgG-ELISA gave a sensitivity of 87.10% and specificity of 89.09% represented by area under the ROC curve of 0.95. IgG-ELISA performed better than the conventional KK (2 slides), identifying 56/64 cases harboring 1-10 eggs per gram of feces that were undiagnosed by KK parasitological technique. CONCLUSIONS: The serological proteome approach was able to identify a new diagnostic candidate. The recombinant egg antigen provided good performance in IgG-ELISA to detect individuals with extreme low-intensity infections (1 egg per gram of feces). Therefore, the IgG-ELISA using this newly identified recombinant MEA can be a useful tool combined with other techniques in low-endemic areas to determine the true prevalence of schistosome infection that is underestimated by the KK method. Further, to overcome the complexity of ELISA in the field, a second generation of antibody-based rapid diagnostic tests (RDT) can be developed.

Diagnosis of Schistosoma mansoni infections: what are the choices in Brazilian low-endemic areas?

The population of Brazil is currently characterised by many individuals harbouring low-intensity Schistosoma mansoni infections. The Kato-Katz technique is the diagnostic method recommended by the World Health Organization (WHO) to assess these infections, but this method is not sensitive enough in the context of low egg excretion. In this regard, potential alternatives are being employed to overcome the limits of the Kato-Katz technique. In the present review, we evaluated the performance of parasitological and immunological approaches adopted in Brazilian areas. Currently, the diagnostic choices involve a combination of strategies, including the utilisation of antibody methods to screen individuals and then subsequent confirmation of positive cases by intensive parasitological investigations.

Leishmanicidal metabolites from Cochliobolus sp., an endophytic fungus isolated from Piptadenia adiantoides (Fabaceae).

Protozoan parasites belonging to genera Leishmania and Trypanosoma are the etiological agents of severe neglected tropical diseases (NTDs) that cause enormous social and economic impact in many countries of tropical and sub-tropical areas of the world. In our screening program for new drug leads from natural sources, we found that the crude extract of the endophytic fungus Cochliobolus sp. (UFMGCB-555) could kill 90% of the amastigote-like forms of Leishmania amazonensis and inhibit by 100% Ellman's reagent reduction in the trypanothione reductase (TryR) assay, when tested at 20 microg mL(-1). UFMGCB-555 was isolated from the plant Piptadenia adiantoides J.F. Macbr (Fabaceae) and identified based on the sequence of the internally transcribed spacer (ITS) regions of its ribosomal DNA. The chromatographic fractionation of the extract was guided by the TryR assay and resulted in the isolation of cochlioquinone A and isocochlioquinone A. Both compounds were active in the assay with L. amazonensis, disclosing EC(50) values (effective concentrations required to kill 50% of the parasite) of 1.7 microM (95% confidence interval = 1.6 to 1.9 microM) and 4.1 microM (95% confidence interval = 3.6 to 4.7 microM), respectively. These compounds were not active against three human cancer cell lines (MCF-7, TK-10, and UACC-62), indicating some degree of selectivity towards the parasites. These results suggest that cochlioquinones are attractive lead compounds that deserve further investigation aiming at developing new drugs to treat leishmaniasis. The findings also reinforce the role of endophytic fungi as an important source of compounds with potential to enter the pipeline for drug development against NTDs.

Altenusin, a biphenyl isolated from the endophytic fungus Alternaria sp., inhibits trypanothione reductase from Trypanosoma cruzi.

Parasitic protozoan species belonging to the genera Trypanosoma and Leishmania are the etiological agents of several diseases in tropical areas of the world, for which there is an urgent need for effective and affordable treatment. In this regard, we are screening the Brazilian biodiversity, especially its flora and mycota, for natural products that could serve as leads for drug development against these diseases. Trypanothione reductase (TR) is an enzyme involved in the protection of Trypanosoma and Leishmania species against oxidative stress, and is considered to be a validated drug target. The endophytic fungus Alternaria sp. (UFMGCB55) was isolated from the plant Trixis vauthieri DC (Asteraceae), known to contain trypanocidal compounds. The organic extract of the culture of Alternaria sp. was able to inhibit TR by 99%, when tested at 20 microg mL(-1). Fractionation of the extract identified altenusin, a biphenyl derivative with an IC50 value of 4.3+/-0.3 microM in the TR assay. This compound is the first in its class to have shown TR inhibitory activity, opening new perspectives for the design of more effective derivatives that could serve as drug leads for new chemotherapeutic agents to treat trypanosomiasis and leishmaniasis.

Serodiagnosis of Trypanosoma cruzi infection using the new particle gel immunoassay - ID-PaGIA Chagas

The ID-Chagas test is a particle gel immunoassay (PaGIA). Red coloured particles are sensitised with three different synthtic peptides representing antigen sequences of Trypanosoma cruzi: Ag2, TcD and TcE. When these particles are mixed with serum containing specific antibodies, they agglutinate. The reaction mixture is centrifuged through a gel filtration matrix allowing free agglutinated particles to remain trapped on the top or distributed within the gel. The result can be read visually. In order to investigate the ability of the ID-PaGIA to discriminate negative and positive sera, 111 negative and 119 positive, collected in four different Brazilian institutions, were tested by each of the participants. All sera were previously classified as positive or negative according to results obtained with three conventional tests (indirect immunofluorescence, indirect hemaglutination, and enzyme linked immunosorbent assay). Sensitivity rates of ID-PaGIA varied from 95.7 per cent to 97.4 per cent with mean sensitivity of 96.8 per cent and specificity rates varied from 93.8 to 98.8 per cent with mean specificity of 94.6 per cent. The overall Kappa test was 0.94. The assay presents as advantages the simplicity of operation and the reaction time of 20 min. In this study, ID-PaGIA showed to be highly sensitive and specific.

Circulating antigens levels in different clinical forms of Schistosoma mansoni infection

With the aim to evaluate the circulating cathodic antigen (CCA) levels in relation to the different clinical phases of Schistosoma sp. infection a sandwich ELISA using monoclonal antibody 5H11 was performed. The sera of three groups of 25 Brazilian patients with acute, instestinal and hepatosplenic forms of S. mansoni infections were tested and compared to a non-infected control group. Patients and control groups were matched for age and sex and the number of eggs per gram of faeces was equally distributed among the three patient groups. Sensitivity of 100 per cent, 72 per cent, 52 per cent of the essays was observed for the intestinal, hepatosplenic and acute toxemic groups respectively. The specificity was 100 per cent. Intestinal and hepatosplenic groups presented CCA levels significantly higher in comparison to those observed for acute patients (F-ratio = 2,524; p = 0.000 and F-ratio = 6.314; p = 0.015 respectively). There was no significant difference of CCA serum levels between hepatosplenic and intestinal groups (F-ratio = 1,026; p = 0.316).

Novas abordagens para o diagnóstico da esquistossomose mansoni humana aguda e crônica / New techniques for the diagnosis of human acute and chronic schistosomiasis mansoni

Säo apresentados quatro artigos publicados e um aceito para publicaçäo em revistas científicas internacionais, que têm como linha de pesquisa comum o diagnóstico da esquistossomose mansoni humana crônica e aguda. Introduz-se o assunto, discutindo-se a importância do diagnóstico da esquistossomose no contexto do controle da endemia e seu estado atual de desenvolvimento e revendo-se a literatura da fase aguda da esquistossomose mansoni. O conteúdo dos trabalhos apresentados inclui a descriçäo de duas novas técnicas eficientes de diagnóstico imunológico, o dot-dye immunoassay e o dye-immunoassay, baseadas na substituiçäo dos sistemas enzima-substrato dos ensaios enzimáticos por um conjugado anti-imunoglobulina humana ligado a corantes têxteis. A técnica dot-dye-immunoassay é aplicada ao diagnóstico da esquistossomose aguda, utilizando o antígeno keyhole limpet haemocyanin (KLH), cuja resposta humoral é específica desta fase, obtendo-se elevadas sensibilidades e especificidade. Estabeleceu-se o valor da ultra-sonografia abdominal no diagnóstico da fase aguda da doença, por comparaçäo com grupos de pacientes crônicos e de pessoas näo infectadas. Por este método, a visualizaçäo de aumento de linfonodos abdominais é característica da fase aguda da doença. Estudando-se um grupo familiar com infecçäo esquistossomótica aguda, determinou-se que os anticorpos séricos IgA contra antígeno solúvel de ovos (SEA) e os anticorpos IgM e IgG contra KLH estäo especificamente elevados na fase aguda da esquistossomose. Estabeleceu-se um critério de morbidade de forma aguda, baseado em intensidade de manifestaçöes clínicas e achados ultra-sonográficos. Através deste critério, os anticorpos IgM e IgA contra SEA e IgM e IgG contra KLH relacionam-se com a morbidade da esquistossomose. A intensidade de manifestaçöes clínicas da forma aguda mostrou-se mais grave em crianças do que em adultos, independentemente da intensidade de contato com água e do número de ovos nas fezes. Os anticorpos IgA contra SEA säo os primeiros a retornarem a valores normais, seguidos pelos anticorpos IgM e IgG contra KLH.

Novas abordagens para o diagnósticos da esquistossomose mansoni humana aguda e crônica / New approaches for the diagnosis of schistosomiasis mansoni human acute and chronic

São apresentados quatro artigos publicados e um aceito para publicação em revistas científicas internacionais, que têm como linha de pesquisa comum o diagnóstico da esquistossomose mansoni humana crônica e aguda. Introduz-se o assunto, discutindo-se a importância do diagnóstico da esquistossomose no contexto do controle da endemia e seu estado atual de desenvolvimento e revendo-se a literatura da fase aguda da esquistossomose mansoni. O conteúdo dos trabalhos apresentados inclui a descrição de duas novas técnicas eficientes de diagnóstico imunológico, o dot-dye immunoassay e o dye-immunoassay, baseadas na substituição dos sistemas enzima-substrato dos ensaios enzimáticos por um conjugado anti-imunoglobulina humana ligado a corantes têxteis. A técnica dot-dye-immunoassay é aplicada ao diagnóstico da esquistossomose aguda, utilizando o antígeno keyhole limpet haemocyanin (KLH), cuja resposta humoral é específica desta fase, obtendo-se elevadas sensibilidade e especificidade. Estabeleceu-se o valor da ultra-sonografia abdominal no diagnóstico da fase aguda da doença, por comparação com grupos de pacientes crônicos e de pessoas não-infectadas. Por este método, a vizibilização de aumento de linfonodos abdominais é característica da fase aguda da doença. Estudando-se um grupo familiar com infecção esquistossomótica aguda, determinou-se que os anticorpos séricos IgA contra antígeno solúvel de ovos (SEA) e os anticorpos IgM e IgG contra KLH estão específicamente elevados na fase aguda da esquistossomose. Estabeleceu-se um critério de morbidade de fase aguda, baseado em intensidade de manifestações clínicas e achados ultra-sonográficos. Através deste critério, os anticorpos IgM e IgA contra SEA e IgM e IgG contra KLH relacionam-se com a morbidade da esquistossomose. A intensidade de manifestações clínicas da forma aguda mostrou-se mais grave em crianças do que em adultos, independentemente da intensidade de contato com água e do número de ovos nas fezes. O~ anticorpos IgA contra SEA são os primeiros a retornarem a valores normais, seguidos pelos anticorpos IgM e IgG contra KLH.

Stool examination and rectal biopsy in the diagnosis and evaluation of therapy of schistosomiasis mansoni

From each of a group of 217 adult males selected through enzyme-immunoassay or skin-test (Group A), six stool samples were examined by both the Lutz/Hoffman, Pons & Janer (Lutz/HPJ) and Kato/Katz methods. In addition, one oogram of the rectal mucosa was performed. By these methods, schistosomiasis was detected in 44.7%, 47.5% and 40.1% of the individuals respectively. To evaluate the methods in the assessment of cure, the last 40 patients from group A, treated with a single oral dose of oxamniquine at 15 mg/kg were followed up for six months (Group B). The criteria for parasitological cure included three stool examinations by Kato/Katz and Lutz/HPJ methods, one, three and six months post-treatment and a rectal biopsy between the fourth and sixth months post-treatment. The examinations were negative in 87.5%, 90% and 95% of the patients, respectively. The efficacy of oxamniquine was 82.5% when the three methods were considered together and there was no statistically significant difference between the sensitivity of the individual methods

Dot-dye-immunoassay for the diagnosis of schistosomiasis mansoni

A new serological assay dot-dye-immunoassay (dot-DIA) was evaluated for the diagnosis of schistosomiasis mansoni. This method consist of four steps: (a) biding of antigens to a nitrocellulose membrane (NC); (b) blocking of free sites of the NC; (c) incubation in specific primary antibody; (d) detection of primary antibody reactivity by color development using second antibody coupled to textile dyes. Sera from 82 individuals, 61 with Schistosoma mansoni eggs in the stool and 21 stool negative were tested by ELISA, dot-ELISA, and dotDIA. A high level of agreement between the methods tested was observed for all sera tested: ELISA x dot-ELISA: 95.1%, ELISA x dot-DIA: 92.7% and dot-ELISA x dot-DIA: 97.6%. In this study, dot-DIA proved to be a feasible, sensitive, rapid and practical test for the diagnosis of shcistosomiasis

Parasitological diagnosis of schistosomiasis mansoni: fecal examination and rectal biopsy

Even with all progress in the search of sensitive and methods for the immunological diagnosis of schistosomiasis, the microscopic detection of eggs of the parasite in the stool still remains the most widely used tool for the actual diagnosis of active infection. Among the coproscopic methods, Kato's technic modified by Katz et al (Kato/Katz) has the advantages of higher sensitivity, the possibility of egg quantification, its low operational cost and its feasibility in areas with minimal infra-structure. The oorgram of the rectal mucosa is valuable in initial clinical trials of schistosomicides, when it is needed to observe egg morphology in tissue. It could be an alternative method for individual diagnosis, being more sensitive than a single stool exam in low intensity infection. However, the increased sensitivity of a higher number of fecal exams makes that invasiveprocedure unnecessary. In the assessment of cure of schistosomiasis, Kato/Katz method (three fecal samples in one, three and six months after treatment) and the rectal biopsy four months after treatment, are equally reliable