Rita Maria da Silva

A COMPARATIVE EPIDEMIOLOGIC STUDY OF SPECIFIC ANTIBODIES (IgM AND IgA) AND PARASITOLOGICAL FINDINGS IN AN ENDEMIC AREA OF LOW TRANSMISSION OF Schistosoma mansoni

The diagnostic potential of circulating IgM and IgA antibodies against Schistosoma mansoni gut-associated antigens detected by the immunofluorescence test (IFT) on adult worm paraffin sections was evaluated comparatively to the fecal parasitological method, for epidemiological purposes in low endemic areas for schistosomiasis. Blood samples were collected on filter paper from two groups of schoolchildren living in two different localities of the municipality of Itariri (São Paulo, Brazil) with different histories and prevalences of schistosomiasis. The parasitological and serological data were compared to those obtained for another group of schoolchildren from a non-endemic area for schistosomiasis. The results showed poor sensitivity of the parasitological method in detecting individuals with low worm burden and indicate the potential of the serological method as an important tool to be incorporated into schistosomiasis control and vigilance programs for determining the real situation of schistosomiasis in low endemic areas.

Ocorrência de Strongyloides stercoralis em pacientes portadores da síndrome de imunodeficiência adquirida (AIDS)

A comparative study on the occurrence of Strongyloides stercoralis larvae in 554 patients with AIDS and in 142 patients suffering from infectious diseases other than AIDS was conducted during the period from January 1987 to December 1988. The two groups was constituted by male individuals in-patients at Emilio Ribas Hospital - Sao Paulo. Faeces samples from 696 patients were submitted to spontaneous sedimentation method and Rugai method. The data obtained from the present investigation demonstrated a similar prevalence of Strongyloides stercoralis in both groups (p > 0.05) indicating no significant statistical differences.

DETECTION OF IgM ANTIBODIES TO Schistosoma mansoni GUT-ASSOCIATED ANTIGENS FOR THE STUDY OF THE DYNAMICS OF SCHISTOSOMIASIS TRANSMISSION IN AN ENDEMIC AREA WITH LOW WORM BURDEN

For a period of 2 years, five follow-up measures of prevalence and incidence rates were estimated in a prospective study of S. mansoni infection in a group of schoolchildren who were living in a rural area of the Municipality of Itariri (São Paulo, Brazil), where schistosomiasis is transmitted by Biomphalaria tenagophila. Infection was determined by the examination of three Kato-Katz stool slides, and the parasitological findings were analyzed in comparison to serological data. In the five surveys, carried out at 6-month intervals (March-April and September-October), the prevalences were, respectively, 8.6, 6.8, 9.9, 5.8 and 17.2% by the Kato-Katz, and 56.5, 52.6, 60.8, 53.5 and 70.1% by the immunofluorescence test (IFT). Geometric mean egg counts were low: 57.8, 33.0, 35.6, 47.3 and 40.9 eggs per gram of feces, respectively. Of the total of 299 schoolchildren, who submitted five blood samples at 6-month intervals, one for each survey, 40% were IFT-positive throughout the study, and 22% were IFT-negative in all five surveys. Seroconversion from IFT negative to positive, indicating newly acquired S. mansoni infection, was observed more frequently in surveys carried out during March-April (after Summer holidays), than during September-October. Seasonal trends were not statistically significant for detection of S. mansoni eggs in stool. The results indicate that the use of IgM-IFT is superior to parasitological methods for detection of incidence of S. mansoni infection in areas with low worm burden.

A comparative study on IgG-ELISA, IgM-IFT and Kato-Katz methods for epidemiological purposes in a low endemic area for schistosomiasis

The high sensitivity and the possibility of automation of the enzyme-linked-immunosorbent-assay (ELISA) has indicated this technique as one of the most useful serological test for epidemiological studies. In the present study, an ELISA for detection of IgG antibodies against adult worm antigens (IgG-ELISA) was investigated for epidemiological purposes, in a rural area of the municipality of Itariri (São Paulo, Brazil). Blood on filter paper (1,180 samples) from about 650 schoolchildren were submitted to ELISA and the data compared to the results of the parasitological method of Kato-Katz and also to the IgM-IFT (immunofluorescence test for IgM antibodies to gut associated antigens). The prevalence rates respectively of 8.5%, 43.0% and 56.2% by the Kato-Katz, IgG-ELISA, and IgM-IFT methods suggest the poor sensitivity of the parasitological method for detection of Schistosoma mansoni eggs in individuals with low worm burden, situation commonly observed in low endemic areas. These results can partially explain the poor degree of agreement between the IgG-ELISA and the Kato-Katz, as suggested by the Kappa index of 0.170. Otherwise, the Kappa index of 0.675 showed substantial agreement between the two serological tests. Some discrepancy of results between the two serological techniques must be better investigated.

Synthesis and evaluation of new oxamniquine derivatives

Oxamniquine derivatives were synthesized and evaluated as novel schistosomicide agents. Oxamniquine (1,2,3,4-tetrahydro-2-[[(1-methylethyl)amino]methyl]-7-nitro-6-quinolinemethanol) was submitted to the Mannich reaction, using formaldehyde, paraformaldehyde and acetaldehyde as reagents, and gave three unexpected products: two of them were cyclized on the alkylamine side chain and another etherified on the aminequinolinemethanol group. The three compounds were biologically evaluated using mice infected with Schistosoma mansoni and showed promising activities, but had higher toxicities. For studies on structure-activity relationships, results demonstrate that the side alkylamine group can be modified with preserved activity, but that this modification is associated with increased toxicity.

Utilização de método sorológico como ferramenta diagnóstica para implementação da vigilância e controle da esquistossomose no Município de Holambra, São Paulo, Brasil

The present study aimed to evaluate the incorporation of the immunofluorescence test (IFT) with adult parasite paraffin sections as antigen substrate for the detection of IgM antibodies (IgM-IFT), as a diagnostic method in the schistosomiasis control program in the county (municipality) of Holambra, São Paulo State, Brazil. This city was selected for this study based on its low endemicity for schistosomiasis, the first cases having been reported in 1993, and because of the need to implement a control program with more sensitive diagnostic techniques. 202 individuals underwent IgM-IFT, with 48 serologically positive cases; of these, 28 were tested with the Kato-Katz technique, using three stool samples. Schistosoma mansoni eggs were found in 14 individuals, with egg counts varying from 2.7 to 224 per gram of stool. The results indicate the potential usefulness of IgM-IFT as a screening test, subject to subsequent confirmation using a parasitological method, in low-endemic areas for schistosomiasis.

Dot-ELISA for the detection of IgM and IgG antibodies to Schistosoma mansoni worm and egg antigens, associated with egg excretion by patients

Human schistosomiasis, caused by Schistosoma mansoni, is highly prevalent in Brazil and usually diagnosed by time consuming stool analysis. Serological tests are of limited use in this disease, mainly for epidemiological studies, showing no discrimination between previous contact with the parasite and active infections. In the present study, we standardized and compared a Dot-ELISA for IgM and IgG antibodies against S. mansoni antigens from eggs and worms with a routine IgG and IgM immunofluorescence assay using similar antigens, in the study of sera from 27 patients who had quantified egg stool excretion. The positivity obtained for IgG Dot-ELISA was 96.3% and 88.9% for IgM Dot-ELISA with worm antigen and 92.6% and 90.9% with egg antigen. The IFI presented similar positivities using worm antigen, 92.6% (IgG) and 96.3% (IgM), and lower results with egg antigen, 77.8%(IgG and IgM). The patients studied were divided into two groups according to their egg excretion, with greater positivity of serological tests in higher egg excreters. When comparing the quantitative egg excretion and the serological titers of the patients, we detected a correlation only with IgM Dot-ELISA, with r = 0.552 (p = 0.0127). These data show that Dot-ELISA can be used for the detection of specific antibodies against S. mansoni in sera from suspected patients or in epidemiological studies and, with further purification of egg antigen and larger samples, IgM Dot-ELISA could be a possible tool for rough estimates of parasite burden in epidemiological studies.

Ocorrência de Taenia sp. na população atendida no laboratório central do Instituto Adolfo Lutz, São Paulo, SP, Brasil (1960/1989)

Foram examinados retrospectivamente os relatorios mensais e anuais da Secao de Enteroparasitoses do Laboratorio Central do Instituto Adolfo Lutz, Sao Paulo, SP, do periodo de 1960 a 1989, perfazendo uma serie historica de 30 anos, com 1.519.730 exames protoparasitologicos e 355 identificacoes de proglotes de Taenia. Pelo metodo da sedimentacao espontânea foram diagnosticados 7.663 (0,5%) casos de presenca de ovos de Taenia sp. nas fezes. Das 355 proglotes enviadas para identificacao, 311 (87,60%) estavam em condicoes de serem especificadas, e dessas, 273 (87,80%) eram proglotes de Taenia saginata e 38 (12,22%) de T. solium.Monthly and yearly reports of the Seção de Enteroparasitoses of the Instituto Adolfo Lutz (São Paulo, SP, Brazil) from 1960 to 1989 with 1,519,730 parasitological stool examinations were studied. There were also 355 identifications of Taenia sp. proglottids. Using Hoffman, Pons & Janers method, 7,663 (0.5%) cases of taeniasis were diagnosed, and 311 (87.60%) of the 355 proglottids were on easy terms to be specified, 273 (87.80%) of them were from Taenia saginata.

TG-ROC analysis of immunofluorescence assays in canine visceral leishmaniasis diagnosis.

OBJECTIVE To analyze the accuracy of the diagnosis of two protocols of indirect immunofluorescence assays for canine visceral leishmaniasis. METHODS Dogs from the seroepidemiological survey conducted in an endemic area of the cities of Araçatuba and Andradina, in Northwestern São Paulo state, in 2003, and in a non-endemic area of the metropolitan region of São Paulo, were used to assess two protocols of indirect immunofluorescence assay (IFA) for leishmaniasis: one using a Leishmania major heterologous antigen (IFA-BM) and another using a Leishmania chagasi homologous antigen (IFA-CH). Two-graph receiver operating characteristic (TG-ROC) analysis was used to estimate accuracy. TG-ROC analysis compared 1:20 dilution readings of the homologous antigen (IFA-CH), considered as reference test, with IFA-BM dilutions (heterologous antigen). RESULTS The 1:20 dilution used in the IFA-CH test showed the best contingency coefficient (0.755) and the highest strength of association between the two variables studied (chi-square=124.3). Thus, it was considered the test reference dilution in comparisons with different IFA-BM test dilutions. The best IFA-BM results were obtained from 1:40 dilutions with the best contingency coefficient (0.680) and highest strength of association (chi-square=80.8). With the change in the cut-off point, recommended for the IFA-BM 1:40 dilution in this analysis, the specificity parameter value rose from 57.5% to 97.7%, even though the 1:80 dilution showed the best sensitivity estimate (80.2%), with the new cut-off point. CONCLUSIONS TG-ROC analysis can provide important information about diagnostic tests, in addition to offering suggestions on cut-off points that can improve test sensitivity and specificity estimates and assessing these tests in terms of the best cost-benefit ratio.OBJETIVO: Analizar la precision de diagnostico de dos protocolos de inmunofluorescencia indirecta para leishmaniasis visceral canina. METODOS: Canes provenientes de pesquisa seroepidemiologico realizado en area endemica en los municipios de Aracatuba y de Andradina, en la region noroeste del estado de Sao Paulo (Brasil), en 2003, y area no endemica de la region metropolitana de Sao Paulo, fueron utilizados para evaluar comparativamente dos protocolos de reaccion de inmunofluorescencia indirecta (RIFI) para leishmaniasis: uno utilizando antigeno heterologo Leishmania major (RIFI-BM) y otro utilizando antigeno homologo Leishmania chagasi (RIFI-CH). Para estimar precision se utilizo el analisis two-graph receiver operating characteristic (TG-ROC). El analisis TG-ROC comparo las lecturas de la dilucion 1:20 del antigeno homologo (RIFI-CH), consideradas como pruebas referencia, con las diluciones de la RIFI-BM (antigeno heterologo). RESULTADOS: La dilucion 1:20 de la prueba RIFI-CH presento mejor coeficiente de contingencia (0,755) y la mayor fuerza de asociacion entre las dos variables estudiadas (Chi-cuadrado=124,3), siendo considerada la dilucion-referencia de la prueba en las comparaciones con las diferentes diluciones de la prueba RIFI-BM. Los mejores resultados del RIFI-BM fueron obtenidos en la dilucion 1:40, con mejor coeficiente de contingencia (0,680) y mayor fuerza de asociacion (Chi-cuadrado= 80,8). Con el cambio del punto de corte sugerido en este analisis para la dilucion 1:40 de la RIFI-BM, el valor del parametro especificidad aumento de 57,5% a 97,7%, a pesar de que la dilucion 1:80 haya presentado la mejor estimativa para sensibilidad (80,2%) con el nuevo punto de corte. CONCLUSIONES: El analisis TG-ROC puede suministrar importantes informaciones sobre las pruebas de diagnostico, asi como presentar sugerencias sobre puntos de cortes que pueden mejorar las estimativas de sensibilidad y especificidad de la prueba, y evaluarlos a la luz del mejor costo-beneficio.

Análise TG-ROC de testes de imunofluorescência no diagnóstico de leishmaniose visceral canina

OBJECTIVE To analyze the accuracy of the diagnosis of two protocols of indirect immunofluorescence assays for canine visceral leishmaniasis. METHODS Dogs from the seroepidemiological survey conducted in an endemic area of the cities of Araçatuba and Andradina, in Northwestern São Paulo state, in 2003, and in a non-endemic area of the metropolitan region of São Paulo, were used to assess two protocols of indirect immunofluorescence assay (IFA) for leishmaniasis: one using a Leishmania major heterologous antigen (IFA-BM) and another using a Leishmania chagasi homologous antigen (IFA-CH). Two-graph receiver operating characteristic (TG-ROC) analysis was used to estimate accuracy. TG-ROC analysis compared 1:20 dilution readings of the homologous antigen (IFA-CH), considered as reference test, with IFA-BM dilutions (heterologous antigen). RESULTS The 1:20 dilution used in the IFA-CH test showed the best contingency coefficient (0.755) and the highest strength of association between the two variables studied (chi-square=124.3). Thus, it was considered the test reference dilution in comparisons with different IFA-BM test dilutions. The best IFA-BM results were obtained from 1:40 dilutions with the best contingency coefficient (0.680) and highest strength of association (chi-square=80.8). With the change in the cut-off point, recommended for the IFA-BM 1:40 dilution in this analysis, the specificity parameter value rose from 57.5% to 97.7%, even though the 1:80 dilution showed the best sensitivity estimate (80.2%), with the new cut-off point. CONCLUSIONS TG-ROC analysis can provide important information about diagnostic tests, in addition to offering suggestions on cut-off points that can improve test sensitivity and specificity estimates and assessing these tests in terms of the best cost-benefit ratio.OBJETIVO: Analizar la precision de diagnostico de dos protocolos de inmunofluorescencia indirecta para leishmaniasis visceral canina. METODOS: Canes provenientes de pesquisa seroepidemiologico realizado en area endemica en los municipios de Aracatuba y de Andradina, en la region noroeste del estado de Sao Paulo (Brasil), en 2003, y area no endemica de la region metropolitana de Sao Paulo, fueron utilizados para evaluar comparativamente dos protocolos de reaccion de inmunofluorescencia indirecta (RIFI) para leishmaniasis: uno utilizando antigeno heterologo Leishmania major (RIFI-BM) y otro utilizando antigeno homologo Leishmania chagasi (RIFI-CH). Para estimar precision se utilizo el analisis two-graph receiver operating characteristic (TG-ROC). El analisis TG-ROC comparo las lecturas de la dilucion 1:20 del antigeno homologo (RIFI-CH), consideradas como pruebas referencia, con las diluciones de la RIFI-BM (antigeno heterologo). RESULTADOS: La dilucion 1:20 de la prueba RIFI-CH presento mejor coeficiente de contingencia (0,755) y la mayor fuerza de asociacion entre las dos variables estudiadas (Chi-cuadrado=124,3), siendo considerada la dilucion-referencia de la prueba en las comparaciones con las diferentes diluciones de la prueba RIFI-BM. Los mejores resultados del RIFI-BM fueron obtenidos en la dilucion 1:40, con mejor coeficiente de contingencia (0,680) y mayor fuerza de asociacion (Chi-cuadrado= 80,8). Con el cambio del punto de corte sugerido en este analisis para la dilucion 1:40 de la RIFI-BM, el valor del parametro especificidad aumento de 57,5% a 97,7%, a pesar de que la dilucion 1:80 haya presentado la mejor estimativa para sensibilidad (80,2%) con el nuevo punto de corte. CONCLUSIONES: El analisis TG-ROC puede suministrar importantes informaciones sobre las pruebas de diagnostico, asi como presentar sugerencias sobre puntos de cortes que pueden mejorar las estimativas de sensibilidad y especificidad de la prueba, y evaluarlos a la luz del mejor costo-beneficio.