Abstract: INTRODUCTION This work shows that 3% (v/v) human urine (HU) in semisolid Liver Infusion Tryptose (SSL) medium favors the growth of Trypanosoma cruzi and T. rangeli. METHODS Parasites were plated as individual or mixed strains on SSL medium and on SSL medium with 3% human urine (SSL-HU). Isolate DNA was analyzed using polymerase chain reaction (PCR) and pulsed-field gel electrophoresis (PFGE). RESULTS SSL-HU medium improved clone isolation. PCR revealed that T. cruzi strains predominate on mixed-strain plates. PFGE confirmed that isolated parasites share the same molecular karyotype as parental cell lines. CONCLUSIONS SSL-HU medium constitutes a novel tool for obtaining T. cruzi and T. rangeli clonal lineages.

IntroductionDetermining the genetic similarities among Trypanosoma cruzi populations isolated from different hosts and vectors is very important to clarify the epidemiology of Chagas disease.MethodsAn epidemiological study was conducted in a Brazilian endemic area for Chagas disease, including 76 chronic chagasic individuals (96.1% with an indeterminate form; 46.1% with positive hemoculture).ResultsT. cruzi I (TcI) was isolated from one child and TcII was found in the remaining (97.1%) subjects. Low-stringency single-specific-primer-polymerase chain reaction (LSSP-PCR) showed high heterogeneity among TcII populations (46% of shared bands); however, high similarities (80-100%) among pairs of mothers/children, siblings, or cousins were detected.ConclusionsLSSP-PCR showed potential for identifying similar parasite populations among individuals with close kinship in epidemiological studies of Chagas disease.

INTRODUÇÃO: Durante muito tempo, foi questionada a importância da doença de Chagas no México onde muitos a consideravam um padecimento exótico. Considerando a grande diversidade genética existente, entre os isolados de Trypanosoma cruzi, a importância da caracterização biológica desses e o escasso número de informações sobre os aspectos clínicos e biológicos da doença de Chagas no México, o objetivo deste trabalho foi realizar a caracterização biológica e molecular de isolados de Trypanosoma cruzi originários de diferentes áreas endêmicas deste país, principalmente do Estado de Jalisco. MÉTODOS: Oito cepas mexicanas de Trypanosoma cruzi foram caracterizadas biologicamente e geneticamente (PCR específica para Trypanosoma cruzi, PCR-multiplex, amplificação do espaço não transcrito dos genes do mini-exon, amplificação das regiões polimórficas do gene do mini-exon, classificação pela amplificação de regiões intergênicas dos genes do spliced leader, RAPD - random amplified polymorphic DNA). RESULTADOS: Foram observados dois tipos de parasitemia: patente com picos máximos de parasitemia entre 4,6x10(6) e 10(7) parasitas/mL e subpatente. Além disso, todos os isolados foram capazes de infectar 100% dos animais. Observou-se tropismo predominante pelo músculo estriado (cardíaco e esquelético). As técnicas de PCR do gene do mini-éxon classificaram as oito cepas como TcI e a técnica de RAPD mostrou variação intra-especifica das mesmas, separando as cepas isoladas de humanos daquelas de triatomíneos e por origem geográfica. CONCLUSÕES: As cepas mexicanas de Trypanosoma cruzi são miotrópicas e correspondem ao TcI.

INTRODUCTION: For a long time, the importance of Chagas disease in Mexico, where many regarded it as an exotic malady, was questioned. Considering the great genetic diversity among isolates of Trypanosoma cruzi, the importance of this biological characterization, and the paucity of information on the clinical and biological aspects of Chagas disease in Mexico, this study aimed to identify the molecular and biological characterization of Trypanosoma cruzi isolates from different endemic areas of this country, especially of the State of Jalisco. METHODS: Eight Mexican Trypanosoma cruzi strains were biologically and genetically characterized (PCR specific for Trypanosoma cruzi, multiplex-PCR, amplification of space no transcript of the genes of the mini-exon, amplification of polymorphic regions of the mini-exon, classification by amplification of intergenic regions of the spliced leader genes, RAPD - (random amplified polymorphic DNA). RESULTS: Two profiles of parasitaemia were observed, patent (peak parasitaemia of 4.6×10(6) to 10(7) parasites/mL) and subpatent. In addition, all isolates were able to infect 100% of the animals. The isolates mainly displayed tropism for striated (cardiac and skeletal) muscle. PCR amplification of the mini-exon gene classified the eight strains as TcI. The RAPD technique revealed intraspecies variation among isolates, distinguishing strains isolated from humans and triatomines and according to geographic origin. CONCLUSIONS: The Mexican T. cruzi strains are myotrophic and belong to group TcI.

Epidemiological screening combined with serological tests has become an important tool at blood banks for the characterization of donors with or without Trypanosoma cruzi infection. Thus, the objective of the present study was to describe the sociodemographic and epidemiological characteristics of blood donors with non-negative serology for T. cruzito determine possible risk factors associated with serological ineligibility. Sociodemographic and epidemiological data were collected by analysis of patient histories and interviews. The data were analyzed descriptively using absolute and relative frequencies and odds ratio (OR) evaluation. The frequency of serological ineligibility was 0.28%, with a predominance of inconclusive reactions (52%) and seropositivity among first-time donors (OR = 607), donors older than 30 years (OR = 3.7), females (OR = 1.9), donors from risk areas (OR = 4) and subjects living in rural areas (OR = 1.7). The risk of seropositivity was higher among donors who had contact with the triatomine vector (OR = 11.7) and those with a family history of Chagas disease (OR = 4.8). The results demonstrate the value of detailed clinical-epidemiological screening as an auxiliary tool for serological definition that, together with more specific and more sensitive laboratory methods, will guarantee a higher efficacy in the selection of donors at blood centres.

Este trabalho avaliou a possibilidade da inoculação e reinoculação de um tripanossomatídeo isolado de morcego, morfológica, biológica e molecularmente semelhante ao Trypanosoma cruzi, na proteção contra a infecção por cepas virulentas. Camundongos não-isogênicos foram divididos em 24 grupos, que receberam de zero a três inóculos da cepa RM1 de Trypanosoma cruzi-like, na presença ou ausência de adjuvante de Freund e desafiados com as cepas de Trypanosoma cruzi VIC ou JG. Acompanhou-se a parasitemia e a sobrevida e os camundongos foram sacrificados para análise histopatológica. Os animais imunizados com a cepa RM1 de Trypanosoma cruzi-like apresentaram redução da parasitemia, independente do número de inóculos ou presença de adjuvante. Apesar dessa redução, os animais não apresentaram proteção contra lesões histopatológicas e observaram-se intensos infiltrados eosinofílicos que foram correlacionados com o número de inóculos da cepa RM1 de Trypanosoma cruzi-like. Sugere-se que a inoculação prévia dessa cepa, ao invés de proteger contra a infecção, agravou o processo inflamatório tecidual.

This study evaluated the possibility of inoculation and reinoculation with a trypanosomatid isolated from bats that is morphologically, biologically and molecularly similar to Trypanosoma cruzi, to protect against infection by virulent strains. Non-isogenic mice were divided into 24 groups that received from zero to three inoculations of Trypanosoma cruzi-like strain RM1, in the presence or absence of Freund's adjuvant, and were challenged with the VIC or JG strains of Trypanosoma cruzi. Parasitemia and survival were monitored and animals were sacrificed for histopathological analysis. Animals immunized with Trypanosoma cruzi-like strain RM1 presented decreased parasitemia, independently of the number of inoculations or the presence of adjuvant. In spite of this reduction, these animals did not present any protection against histopathological lesions. Severe eosinophilic infiltrate was observed and was correlated with the number of inoculations of Trypanosoma cruzi-like strain RM1. These findings suggest that prior inoculation with this strain did not protect against infection but, rather, aggravated the tissue inflammatory process.

Pacientes com AIDS são particularmente susceptíveis a infecção por coccídios intestinais e nesse estudo foi avaliada a freqüência de Cryptosporidium sp. e Cystoisospora belli entre pacientes HIV/AIDS na região do Triângulo Mineiro, Brasil. No período de julho de 1993 a junho de 2003, amostras de fezes de 359 pacientes foram submetidas à coloração pelo método de Ziehl-Neelsen modificado, sendo detectada a presença de coccídios em 19,7% destas (8,6% de Cryptosporidium sp, 10,3% de Cystoisospora belli e 0,8% de ambos coccídios). Pacientes com diarréia e níveis de linfócitos T CD4+ < 200 células/mm3 apresentaram maior frequência destes protozoários, demonstrando o perfil oportunista destas infecções e a relação com o status imunológico do indivíduo. Não foi possível determinar a influência da HARRT, pois apenas 8,5% dos pacientes positivos para coccídios fazriam uso regular desta terapia. Parasitismo por Cryptosporidium sp foi mais freqüente no período compreendido de dezembro a fevereiro caracterizando padrão sazonal desta infecção, fato não observado com Cystoisospora belli.

Patients with AIDS are particularly susceptible to infection with intestinal coccidia. In this study the prevalence of infections with Cryptosporidium sp and Cystoisospora belli were evaluated among HIV/AIDS patients in the Triângulo Mineiro region, Brazil. Between July 1993 and June 2003 faecal samples from 359 patients were collected and stained by a modified Ziehl-Neelsen method, resulting in 19.7% of positivity for coccidian (8.6% with Cryptosporidium sp, 10.3% with Cystoisospora belli and 0.8% with both coccidian). Patients with diarrhoea and T CD4+ lymphocyte levels < 200 cells/mm3 presented higher frequency of these protozoans, demonstrating the opportunistic profile of these infections and its relationship with the immunological status of the individual. It was not possible to determine the influence of HAART, since only 8.5% of the patients positive for coccidian received this therapy regularly. Parasitism by Cryptosporidium sp was more frequent between December and February and thus was characterised by a seasonal pattern of infection, which was not observed with Cystoisospora belli.

Neste estudo foi avaliado o número de linfócitos TCD4, a parasitemia e os níveis séricos de interferon gama (IFN-gama), fator de necrose tumoral alfa (TNF-alfa), interleucina-1 (IL-1), IL-4 e IL-10 em pacientes infectados pelo vírus da imunodeficiência adquirida ou apresentavam co-infecção pelo HIV/Trypanosoma cruzi. O número de linfócitos T CD4 estava baixo nos dois grupos de pacientes, embora significativamente menor nos pacientes sem a doença de Chagas. Os níveis séricos de IFN-gama, IL-4 e TNF-alfa foram significativamente maiores nos pacientes com a co-infecção pelo HIV/Trypanosoma cruzi. A razão IL-4/IFN-gama foi maior nos pacientes com a co-infecção pelo HIV/T. cruzi, que sugere um balanço favorável para perfil Th2 nesse grupo de pacientes. Este balanço Th2 foi maior nos pacientes com parasitemia detectável. Conclui-se que, embora tenha sido observado imunossupressão, na maioria com linfócitos T CD4 abaixo de 200/µm³, esses pacientes não reativaram a infecção pelo T. cruzi e que o balanço favorável a Th2 estava associado à presença de parasitemia.

This study assessed the number of CD4 T lymphocytes, the parasitemia and serum levels of interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), IL-4 and IL-10 of patients infected by human immunodeficiency virus (HIV) and human immunodeficiency virus/Chagas' disease coinfection. CD4 T lymphocytes were low in the two groups of patients, although significantly lower in patients without Chagas' disease. Serum levels of IFN-gamma, IL-4 and TNF-alpha were significantly higher in patients with HIV/Chagas' disease. IL-4/IFN-gamma ratios were higher in patients with HIV/Chagas' disease, which showed a clear balance in favor of Th2-like cytokines in this group of patients. This Th2 balance was higher in patients with detectable parasitemia. We conclude that, although immunosuppression was observed, with CD4 T lymphocytes bellow 200/µm³, these patients did not display reactivation of T. cruzi infection and that a balance favorable to Th2 was associated with the presence of parasitemia.

Authors describe genitourinary changes in male hamsters infected and reinfected with Trypanosoma cruzi. Changes in genital organs have been described in human and in experimental chagasic infection. Genital dysfunctions in chronic chagasic patients affect ejaculation, libido and sexual potency, and testis biopsies may show arrested maturation of germ cells, oligozoospermia and azoospermia. Sixty-five male hamsters were inoculated and reinoculated with 2x10³ trypomastigotes of T. cruzi VIC strain, and 22 non-infected animals constituted the control group. Animals were necropsied and fragments from testis, epididymis, seminal vesicle and bladder were collected and stained with hematoxylin-eosin. Peroxidase anti-peroxidase procedure was utilized to detect tissue parasitism. T. cruzi nests were found in testis, epididymis and seminal vesicle of these hamsters. Such parasitism plays a role in the origin of genital lesions observed in humans and laboratory animals during chronic chagasic infection.

Iniciando estudo de alterações patológicas crônicas no pâncreas de hamsters experimentalmente infectados com a cepa Vic de Trypanosoma cruzi, metaplasia oncocítica foi observada em um dos animais infectados. Este é o primeiro relato de oncocitos na doença de Chagas, que poderiam decorrer de resposta regenerativa aberrante ao processo inflamatório pancreático.

Beginning the study of chronic pathologic changes in pancreas of hamsters experimentally infected with Trypanosoma cruzi Vic strain, hepatocyte metaplasia was observed in one animal from infected group. This is the first report of oncocytes in Chagas' disease, which could be due to aberrant regenerative response to pancreas inflammatory process.

Opresente trabalho tem como finalidade caracterizar as fases aguda e crônica da doença de Chagas em hamster, através de estudos parasitológicos e histopatológicos. A fase aguda foi induzida em 44 hamsters jovens, inoculados intraperitonealmente, com 100.000 tripomastigotas sanguíneos das cepas Y e Benedito do T. cruzi. Afase crônica foi produzida em 46 hamsters, inoculados intraperitonealmente, com 35.000 tripomastigotas sanguíneos das cepas Y, Vicentina e Benedito. Os animais foram sacrificados a intervalos regulares de 24 horas na fase aguda, durante 15 dias e na fase crônica, a partir do 3° mês até o 10° mês da infecção. Na fase aguda, o parasita foi facilmente recuperado de todos os animais, os quais apresentaram reação inflamatória, de intensidade variável, de células mononucleares e polimorfonucleares, na maioria dos tecidos e órgãos, principalmente nos tecidos conjuntivo frouxo e adiposo, músculo liso, cardíaco e esquelético. Na fase crônica, as lesões ocorreram nos mesmos tecidos e órgãos, mas a resposta inflamatória com infiltrado mononuclear foi mais grave no miocárdio onde adquiriu caráter fibrosante. No miocárdio os parasitas foram encontrados em 50% dos casos. A partir do líquido pericárdico, peritoneal e ascítico desses animais foram recuperados parasitas através do microhematócrito e hemocultura. Regularmente, observaram-se sinais de insuficiência cardíaca, com morte súbita e de dilatação do intestino grosso e atrofia dos genitais internos. Conclui-se que o hamster é um modelo útil para o estudo da doença de Chagas.

This research characterizes the acute and chronic phases of Chagas ' disease in hamster through parasitological and histopathological studies. The acute phase was achieved with 44 young hamsters injected intraperitoneally with 100.000 blood trypomastigotes of Benedito and Y strains of T. cruzi. The chronic phase was induced in 46 hamsters injected intraperitoneally with 35.000 trypomastigotes ofVicentina, Benedito and Y strains. Animals were sacrificed at regular intervals of 24 hours of acute phase and from the 3rd to the 10th month of infection ofchronic phase. In the acute phase, parasites were easily recoveredfrom all animals and there was an inflammatory reaction characterized by mononuclear and polymorphous leukocyte infiltration of variable degree in the majority of tissues and organs, specially in the connective loose and fatty tissues, smooth muscle myocardium and skeletal muscle. In the chronic phase the lesions occurred in the same tissues and organs, but the inflammatory response was less severe and characterized by mononuclear infiltration mainly with focal or zonalfibrosis in the myocardiun. In 50% of infected animals parasites were found inmyocardiun and recoveredfrom pericardic, peritoneal and ascitic fluids in some animals. Signs of heart failure, sudden death and enlargement of bowel were observed regularly. We concluded that the hamster is an useful model for Chagas' disease studies.

Single doses of drugs active aginst Trypanosoma cruzi (megazol, nifurtimox and benznidazole) induce a rapid clearence of the blood parasites in experimentally infected mice. Furthermore, the in vitro phagocytosis and intracellular destruction by mouse peritoneal macrophage of blood forms collected from the treatment animals is strongly enhanced as compared with parasites from untreated controls. The uptake of the blood forms by macrophages is significantly higher with megazol than with benznidazole and nifurtimox, a finding that concurs with data showing that megazol is also the most active compound in the living host. The possibility that macrophages participate in a synergic effect between the host immune response and chemotherapeutic effect is discussed.

O método imunocitoquímico de peroxidase anti-peroxídase é aplicado, pela primeira vez, em tecidos de natimorto macerado de II-III graus para coloração específica de amastigotas do Trypanosoma cruzi, em caso de doença de Chagas congênita. Os resultados mostram que o encontro de ninhos de amastigotas é muito facilitado pelo método, mesmo em tecidos autolisados, onde a morfologia dos parasitas geralmente se mostra muito alterada; o método é recomendável, também nestes casos, para a avaliação mais precisa da intensidade do parasitismo.

In order to specijically stain the Trypanosoma cruzi amastigotes, the immunocytochemical peroxiIn order to specijically stain the Trypanosoma cruzi amastigotes, the immunocytochemical peroxidase anti peroxidase melhod was carried out in advanced macerated premature tissues of u case of human congenital Chagas' disease. The results have shown that the immunocytochemical method markedly improved the identijication o f the tissue parasitism when compared with the hematoxylin and eosin stain, even in advanced autolysed tissues.