Análise de técnicas parasitológicas utilizadas no seguimento de pacientes com doença de chagas e tratados por meio do transplante cardíaco / Analysis of parasitological techniques in the monitoring of patients with Chagas disease and treated by heart transplantation

Este estudo teve como objetivo analisar a função da hemocultura, do xenodiagnóstico e do creme leucocitário no acompanhamento e diagnóstico de possível reativação em pacientescrônicos tratados por meio do transplante de coração. Foram examinadas 70 amostras das quais15,7por cento (11/70) se mostraram positivas, sendo 8,5por cento (6/70) na hemocultura e 12,8por cento (9/70) noxenodiagnóstico. Apresentaram concordância nos dois métodos quatro amostras (36,36por cento). Nãohouve positividade no creme leucocitário. Os achados corroboram informações sobre a superiorsensibilidade do xenodiagnóstico em relação à hemocultura. A amostra do paciente 20, positiva noxenodiagnóstico (décimo quinto dia) e que apresentou miocardite com ninhos de amastigotas 15 dias antes dadetecção laboratorial, sinaliza a importância da leitura precoce dos exames parasitológicos comopreditivos de possível reativação da doença.

In vitro action of antiparasitic drugs, especially artesunate, against Toxoplasma gondii.

INTRODUCTION: Toxoplasmosis is usually a benign infection, except in the event of ocular, central nervous system (CNS), or congenital disease and particularly when the patient is immunocompromised. Treatment consists of drugs that frequently cause adverse effects; thus, newer, more effective drugs are needed. In this study, the possible activity of artesunate, a drug successfully being used for the treatment of malaria, on Toxoplasma gondii growth in cell culture is evaluated and compared with the action of drugs that are already being used against this parasite. METHODS: LLC-MK2 cells were cultivated in RPMI medium, kept in disposable plastic bottles, and incubated at 36ºC with 5% CO2. Tachyzoites of the RH strain were used. The following drugs were tested: artesunate, cotrimoxazole, pentamidine, pyrimethamine, quinine, and trimethoprim. The effects of these drugs on tachyzoites and LLC-MK2 cells were analyzed using nonlinear regression analysis with Prism 3.0 software. RESULTS: Artesunate showed a mean tachyzoite inhibitory concentration (IC50) of 0.075µM and an LLC MK2 toxicity of 2.003µM. Pyrimethamine was effective at an IC50 of 0.482µM and a toxicity of 11.178µM. Trimethoprim alone was effective against the in vitro parasite. Cotrimoxazole also was effective against the parasite but at higher concentrations than those observed for artesunate and pyrimethamine. Pentamidine and quinine had no inhibitory effect over tachyzoites. CONCLUSIONS: Artesunate is proven in vitro to be a useful alternative for the treatment of toxoplasmosis, implying a subsequent in vivo effect and suggesting the mechanism of this drug against the parasite.

In vitro action of antiparasitic drugs, especially artesunate, against Toxoplasma gondii / Ação in vitro de drogas antiparasitárias, especialmente artesunato, contra Toxoplasma gondii

INTRODUCTION: Toxoplasmosis is usually a benign infection, except in the event of ocular, central nervous system (CNS), or congenital disease and particularly when the patient is immunocompromised. Treatment consists of drugs that frequently cause adverse effects; thus, newer, more effective drugs are needed. In this study, the possible activity of artesunate, a drug successfully being used for the treatment of malaria, on Toxoplasma gondii growth in cell culture is evaluated and compared with the action of drugs that are already being used against this parasite. METHODS: LLC-MK2 cells were cultivated in RPMI medium, kept in disposable plastic bottles, and incubated at 36ºC with 5% CO2. Tachyzoites of the RH strain were used. The following drugs were tested: artesunate, cotrimoxazole, pentamidine, pyrimethamine, quinine, and trimethoprim. The effects of these drugs on tachyzoites and LLC-MK2 cells were analyzed using nonlinear regression analysis with Prism 3.0 software. RESULTS: Artesunate showed a mean tachyzoite inhibitory concentration (IC50) of 0.075µM and an LLC MK2 toxicity of 2.003µM. Pyrimethamine was effective at an IC50 of 0.482µM and a toxicity of 11.178µM. Trimethoprim alone was effective against the in vitro parasite. Cotrimoxazole also was effective against the parasite but at higher concentrations than those observed for artesunate and pyrimethamine. Pentamidine and quinine had no inhibitory effect over tachyzoites. CONCLUSIONS: Artesunate is proven in vitro to be a useful alternative for the treatment of toxoplasmosis, implying a subsequent in vivo effect and suggesting the mechanism of this drug against the parasite.

INTRODUÇÃO: Toxoplasmose é geralmente uma infecção benigna, exceto nos eventos de doença ocular, congênito e do sistema nervoso central, e particularmente quando o paciente é imunocomprometido. O tratamento consiste de drogas que frequentemente causam efeitos adversos, então novas drogas, mais efetivas são necessárias. Neste estudo, a possível atividade de artesunato, uma droga usada com sucesso no tratamento da malária, sobre o crescimento de Toxoplasma gondii em cultura celular é avaliado e comparado à ação de drogas que já estão sendo utilizadas contra este parasita. MÉTODOS: Células LLC-MK2 foram cultivadas em meio RPMI, mantidas em garrafas plásticas descartáveis e incubados a 36ºC com 5% CO2. Taquizoítos da cepa RH foram usados. As seguintes drogas foram testadas: artesunato, cotrimoxazol, pentamidina, pirimetamina, quinino e trimetoprima. Os efeitos dessas drogas sobre taquizoítos foram analisados por análise regressiva não linear com o software Prism 3.0. RESULTADOS: Artesunato mostrou uma concentração inibitória media (IC50) de 0,075µM e uma toxicidade sobre células LLC MK2 de 2,003µM. Pirimetamina foi efetiva a uma IC50 de 0,482µM e uma toxicidade de 11,178µM. Trimetoprima sozinha foi efetiva contra o parasita in vitro. Cotrimoxazol também foi efetivo contra o parasita, mas a concentrações mais altas que aquelas observadas para artesunato e pirimetamina. Pentamidina e quinino não tiveram efeitos inibitórios sobre os taquizoítos. CONCLUSÕES: Provou-se que artesunato in vitro pode ser uma alternativa útil para o tratamento da toxoplasmose, implicando um subsequente efeito in vivo e sugerindo o mecanismo desta droga contra o parasita.

Discrepancies and consequences of indirect hemagglutination, indirect immunofluorescence and Elisa tests for the diagnosis of Chagas disease / Discordâncias e consequências de resultados de provas de hemaglutinação indireta, imunofluorescência indireta e Elisa para o diagnóstico da doença de Chagas

Using the indirect hemagglutination (IH), indirect immunofluorescence (IIF) and enzyme linked immunosorbent assay (ELISA) tests for the diagnosis of Chagas disease, 4000 serum samples were examined. This study was conducted with different purposes: clinical interest, research support and parasitological monitoring of those patients with Chagas disease who were treated with heart transplantations. The tests occurred without patient selection and in accordance with the medical requests. The results showed discrepancies and brought about several questions, considering the different results that all three methods showed when considered together. What was found brought about concerns and we suggest the adoption of different measures, aiming to avoid these mismatches in the context of this disease.

Com as provas de hemaglutinação indireta (HI), imunofluorescência indireta (IFI) e Enzyme Linked Immunosorbent Assay (ELISA), para diagnóstico da doença de Chagas, foram examinadas concomitantemente 4000 amostras de soro, com diferentes finalidades, tais como interesse clínico, apoio a pesquisas e acompanhamento parasitológico de pacientes com tal moléstia tratados por meio de transplante de coração. Os testes ocorreram, sem seleção e conforme as solicitações, em Laboratório que essencialmente prestou colaboração. Os resultados mostraram discordâncias, inclusive motivadoras de dúvidas, considerando especificamente o revelado pelos três métodos em conjunto. O que ficou verificado suscita preocupações e sugere a adoção de medidas aptas a evitar essas inadequações no contexto da parasitose.

Discrepancies and consequences of indirect hemagglutination, indirect immunofluorescence and ELISA tests for the diagnosis of Chagas disease.

Using the indirect hemagglutination (IH), indirect immunofluorescence (IIF) and enzyme linked immunosorbent assay (ELISA) tests for the diagnosis of Chagas disease, 4000 serum samples were examined. This study was conducted with different purposes: clinical interest, research support and parasitological monitoring of those patients with Chagas disease who were treated with heart transplantations. The tests occurred without patient selection and in accordance with the medical requests. The results showed discrepancies and brought about several questions, considering the different results that all three methods showed when considered together. What was found brought about concerns and we suggest the adoption of different measures, aiming to avoid these mismatches in the context of this disease.

Evaluation of a recombinant Trypanosoma cruzi mucin-like antigen for serodiagnosis of Chagas' disease.

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi and is one of the most important endemic problems in Latin America. Lately, it has also become a health concern in the United States and Europe. Currently, a diagnosis of Chagas' disease and the screening of blood supplies for antiparasite antibodies are achieved by conventional serological tests that show substantial variation in the reproducibility and reliability of their results. In addition, the specificity of these assays is curtailed by antigenic cross-reactivity with sera from patients affected by other endemic diseases, such as leishmaniasis. Here we used a highly sensitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) to evaluate a recombinant protein core of a mucin-like molecule (termed trypomastigote small surface antigen [TSSA]) for the detection of specific serum antibodies in a broad panel of human sera. The same samples were evaluated by CL-ELISA using as the antigen either a mixture of native T. cruzi trypomastigote mucins or an epimastigote extract and, for further comparison, by conventional serologic tests, such as an indirect hemagglutination assay and indirect immunofluorescence assay. TSSA showed ∼87% sensitivity among the seropositive Chagasic panel, a value which was increased up to >98% when only parasitologically positive samples were considered. More importantly, TSSA showed a significant increase in specificity (97.4%) compared to those of currently used assays, which averaged 80 to 90%. Overall, our data demonstrate that recombinant TSSA may be a useful antigen for the immunodiagnosis of Chagas' disease.

[Alternative transmission mechanisms of Trypanosoma cruzi in Brazil and proposals for their prevention]. / Mecanismos alternativos de transmissão do Trypanosoma cruzi no Brasil e sugestões para sua prevenção.

INTRODUCTION: Following advances in the control of vector and blood transfusion transmission of Chagas disease, alternative mechanisms of transmission have become more relevant. This article discusses the importance of each one of these alternative mechanisms and the measures to prevent them. METHODS: A review was conducted of the scientific literature concerning alternative transmission mechanisms of Trypanosoma cruzi occurring in Brazil and the measures to prevent them. PubMed and BVS databases were consulted. RESULTS: Twenty-five publications describing alternative mechanisms of transmission of Chagas disease were identified. CONCLUSIONS: Oral transmission, through ingestion of contaminated food items has been the most frequent mode of transmission in Brazil in recent years. Other alternative mechanisms of transmission occur less frequently. It is important to understand these occurrences, especially now that vector transmission of the parasite is under control. Preventive measures have been presented, according to each of the situations considered, in line with current knowledge.

Mecanismos alternativos de transmissão do Trypanosoma cruzi no Brasil e sugestões para sua prevenção / Alternative transmission mechanisms of Trypanosoma cruzi in Brazil and proposals for their prevention

INTRODUÇÃO: Com o avanço no controle da transmissão vetorial e por transfusão sanguínea da doença de Chagas, as formas alternativas de transmissão ganharam relevância. Este artigo de opinião discute a importância de cada uma dessas modalidades e as medidas para sua prevenção. MÉTODOS: Foi realizada uma revisão bibliográfica sobre os mecanismos de transmissão do Trypanosoma cruzi através de modalidades alternativas, vigentes no Brasil, e as possibilidades de sua prevenção. Foram consultadas as bases de dados PubMed e BVS. RESULTADOS: Foram identificadas 25 publicações que discutiam as modalidades alternativas de transmissão da doença de Chagas. CONCLUSÕES: A transmissão oral, pela ingestão de alimentos contaminados, tem sido o modo de transmissão predominante no Brasil nos últimos anos. Os demais modos alternativos de transmissão são de ocorrência menos frequente. É importante conhecer essas ocorrências, sobretudo agora que a veiculação vetorial do parasita está controlada. Conforme os conhecimentos atuais foram apresentadas medidas preventivas, de acordo com cada uma das situações consideradas.

INTRODUCTION: Following advances in the control of vector and blood transfusion transmission of Chagas disease, alternative mechanisms of transmission have become more relevant. This article discusses the importance of each one of these alternative mechanisms and the measures to prevent them. METHODS: A review was conducted of the scientific literature concerning alternative transmission mechanisms of Trypanosoma cruzi occurring in Brazil and the measures to prevent them. PubMed and BVS databases were consulted. RESULTS: Twenty-five publications describing alternative mechanisms of transmission of Chagas disease were identified. CONCLUSIONS: Oral transmission, through ingestion of contaminated food items has been the most frequent mode of transmission in Brazil in recent years. Other alternative mechanisms of transmission occur less frequently. It is important to understand these occurrences, especially now that vector transmission of the parasite is under control. Preventive measures have been presented, according to each of the situations considered, in line with current knowledge.

[Prevention concerning the different alternative routes for transmission of Trypanosoma cruzi in Brazil]. / Prevenção referente às modalidades alternativas de transmissão do trypanosoma cruzi no Brasil.

Vectorial, transfusion and congenital are considered the main transmission mechanisms in human Chagas disease. Alternative mechanisms are accidental, oral and by organ transplantation. Other hypothetic mechanisms could be by other vectors, sexual, criminal and by means of marsupial anal secretions. The present accorded strategies for prevention are: CONGENITAL: early case detection and immediate treatment. If possible, start during the pre natal period, throughout mothers serology, performing parasitological tests in the new born from positive women. For positive cases, immediate treatment; for those negative babies, conventional serology at the 8th month, treating specifically those with positive results. ACCIDENTAL TRANSMISSION: Rigorous training and utilization of protection equipments. IF accident occurs, immediate disinfection, conventional serology and beginning of specific treatment by ten days. Revision of the serology after 30 days: if positive, extend the treatment until the total dose (60 days or more). ORGAN TRANSPLANTATION: previous serology for donor and receptor. When the former is infected and the last negative, cancel the surgery or install the specific treatment by ten days before the surgery for the donor, followed by the receptor during ten days after the transplantation. ORAL TRANSMISSION: Specific measures are not available, food hygiene is recommended, including the cooking of meats delivered from possible reservoirs. Nowadays, the detection and immediate treatment of the case is recommended, followed by active research of new cases around the detected one.

Prevenção referente às modalidades alternativas de transmissão do trypanosoma cruzi no Brasil / Prevention concerning the different alternative routes for transmission of Trypanosoma cruzi in Brazil

Consideram-se habituais em doença de Chagas humana os mecanismos vetorial, transfusional e congênito de transmissão. Acidental, oral e por transplantes são ditos alternativos. Possibilidades como por outros vetores, sexual, criminal e por secreção de marsupiais são consideradas excepcionais. A prevenção dos mecanismos alternativos, incluindo o congênito, está hoje consensuada: TRANSMISSÃO CONGÊNITA: detecção precoce do caso e seu tratamento específico. Se possível começar, no pré-natal com sorologia de gestantes. Quando viável, pesquisar parasitologicamente o RN de mães reagentes, tratando-se imediatamente os que resultarem positivos. Sendo negativos, sorologia convencional aos 8 meses de vida, tratando imediatamente os que estiverem reagentes. TRANSMISSÃO ACIDENTAL: Usar treinamento e equipamentos de proteção. Se acidente, desinfecção local, sorologia convencional e inicio de tratamento específico por dez dias. Revisão da sorologia em 30 dias, seguindo-se o tratamento até a dose total, no caso de reação positiva. TRANSPLANTES DE ÓRGÃOS: sorologia prévia no doador e receptor. Sendo o primeiro positivo e o segundo negativo, evitar o transplante ou tratar especificamente o doador por 10 dias antes da cirurgia e o receptor nos dez dias subsequentes à mesma. TRANSMISSÃO ORAL: de modo geral, higiene alimentar e cozimento de carnes de possíveis reservatórios. Hoje se recomenda a detecção precoce e tratamento imediato do caso, com intensa busca ativa entre os circunstantes mais próximos do paciente.

Vectorial, transfusion and congenital are considered the main transmission mechanisms in human Chagas disease. Alternative mechanisms are accidental, oral and by organ transplantation. Other hypothetic mechanisms could be by other vectors, sexual, criminal and by means of marsupial anal secretions. The present accorded strategies for prevention are: CONGENITAL: early case detection and immediate treatment. If possible, start during the pre natal period, throughout mothers serology, performing parasitological tests in the new born from positive women. For positive cases, immediate treatment; for those negative babies, conventional serology at the 8th month, treating specifically those with positive results. ACCIDENTAL TRANSMISSION: Rigorous training and utilization of protection equipments. IF accident occurs, immediate disinfection, conventional serology and beginning of specific treatment by ten days. Revision of the serology after 30 days: if positive, extend the treatment until the total dose (60 days or more). ORGAN TRANSPLANTATION: previous serology for donor and receptor. When the former is infected and the last negative, cancel the surgery or install the specific treatment by ten days before the surgery for the donor, followed by the receptor during ten days after the transplantation. ORAL TRANSMISSION: Specific measures are not available, food hygiene is recommended, including the cooking of meats delivered from possible reservoirs. Nowadays, the detection and immediate treatment of the case is recommended, followed by active research of new cases around the detected one.

Trypanosoma cruzi, hemocultura: uma abordagem prática / Trypanosoma cruzi, hemoculture: a practical approach

JUSTIFICATIVA E OBJETIVOS: A hemocultura permanece útil para diagnosticar a doença de Chagas crônica. O objetivo deste estudo foi comparar a técnica "clássica" e a "modificada" mais usada para essa finalidade no Brasil. MÉTODO: A inoculação de "creme leucocitário" com sedimento de sangue centrifugado caracteriza ambas as técnicas. Amostras provenientes de dois grupos de 137 pacientes chagásicos crônicos foram examinadas. A "clássica" utiliza 10 mL de sangue; a "modificada", 30 mL. RESULTADOS: A técnica "modificada" mostrou-se mais sensível do que a "clássica". CONCLUSÃO: Sugere-se o uso rotineiro da técnica "modificada".

BACKGROUND AND OBJECTIVES: Hemoculture remains a valuable tool for the diagnosis of chronic Chagas disease. We tested the two techniques routinely used in Brazil. METHOD: The "modified" technique includes the inoculation of "buffy coat" and centrifuged blood sediment from a 30 mL sample, the serum being washed out after centrifugation and replaced by equal volume of culture medium; the "classic" one requires the inoculation of centrifuged blood sediment plus "buffy coat" from a 10 mL sample. RESULTS: A higher sensitivity of the "modified" technique was observed when the results of two samples of 137 chronic Chagas disease patients were compared. CONCLUSION: The routine use of the "modified" technique should be preferred.