High similarity of Trypanosoma cruzi kDNA genetic profiles detected by LSSP-PCR within family groups in an endemic area of Chagas disease in Brazil

Introduction Determining the genetic similarities among Trypanosoma cruzi populations isolated from different hosts and vectors is very important to clarify the epidemiology of Chagas disease. Methods An 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). Results T. 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. Conclusions LSSP-PCR showed potential for identifying similar parasite populations among individuals with close kinship in epidemiological studies of Chagas disease. .

Molecular characterization of Trypanosoma cruzi Mexican strains and their behavior in the mouse experimental model / Caracterização molecular de cepas mexicanas de Trypanosoma cruzi e seu comportamento no modelo experimental do camundongo

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 percent 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.

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 por cento 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.

Socioepidemiological screening of serologically ineligible blood donors due to Chagas disease for the definition of inconclusive cases

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 percent, with a predominance of inconclusive reactions (52 percent) 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.

Absence of experimental cross-protection induced by a Trypanosoma cruzi-like strain isolated from bats / Ausência de proteção cruzada experimental induzida por uma cepa Trypanosoma cruzi-like isolada de morcego

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.

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.

Seasonal profile and level of CD4+ lymphocytes in the occurrence of cryptosporidiosis and cystoisosporidiosis in HIV/AIDS patients in the Triângulo Mineiro region, Brazil

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 percent of positivity for coccidian (8.6 percent with Cryptosporidium sp, 10.3 percent with Cystoisospora belli and 0.8 percent 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 percent 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.

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 por cento destas (8,6 por cento de Cryptosporidium sp, 10,3 por cento de Cystoisospora belli e 0,8 por cento 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 por cento 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.

Cytokine serum levels in patients infected by human immunodeficiency virus with and without Trypanosoma cruzi coinfection

Neste estudo foi avaliado o número de linfócitos TCD4, a parasitemia e os níveis séricos de interferon gama (IFN-g), fator de necrose tumoral alfa (TNF-a), interleucina-1 (IL-1), IL-4 e IL-10 em pacientes infectados pelo vírus da imunodeficiência adquirida ou apresentavam co-infeccão pelo HIV/Trypanosoma cruzi. O número de linfócitos T CD4 estava baixo nos dois grupos de pacientes, embora significativamente menor nos pacientes sem a doenca de Chagas. Os níveis séricos de IFN-g, IL-4 e TNF-a foram significativamente maiores nos pacientes com a co-infeccão pelo HIV/Trypanosoma cruzi. A razão IL-4/IFN-g foi maior nos pacientes com a co-infeccão pelo HIV/T. cruzi, que sugere um balanco favorável para perfil Th2 nesse grupo de pacientes. Este balanco 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 infeccão pelo T. cruzi e que o balanco favorável a Th2 estava associado à presenca de parasitemia.

Genitourinary changes in hamsters infected and reinfected with Trypanosoma cruzi

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 2x10 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

Spontaneous B-cell lymphoma in hamster

During anatomopathologic study, including immunohistochemistry, about chagasic pancreatitis experimentally induced in four month aged male non-isogenic hamsters, weighing 107.8 ±: 10.9g, lymphoma infiltration was observed in a 15 month-aged normal control animal. The neoplasia was disclosed on the occasion of necropsy studies, 330 days after the beginning of experiment. Similar lymphoma was not found in the remainder normal controls (n=73), nor in the group of infected hamsters age and weight matched (n=94). The neoplasia histopathologic and immunohistochemical changes were consistent with non-Hodgkin diffuse large B-cell lymphoma; nevertheless, the hypothesis of eventual leukemic origin was not entirely excluded. Experimentally induced lymphomas have been related in laboratory animals; however, cases of spontaneously occurring lymphoma have been infrequently described in hamsters. In the present case, the development of the disease could have some relation with the animal aging process

Hepatocyte metaplasia in experimental chagasic pancreatitis: preliminary report

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.

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.

The hamster (Mesocricetus auratus) as experimental model in chagas'diseases: parasitological and histopathological studies in acute and chronic phases of trypanosoma cruzi infection

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 of Vicentina, 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 of chronic phase. In the acute phase, parasites were easily recovered from 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 lhe 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 zonal fibrosis in the myocardiun. In 50 per cent of infected animals parasites were found in myocardiun and recovered from 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.

Effect of the host specific treatment in the phagocytosis of Trypanosoma cruzi blood forms by mouse peritoneal macrophages

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

Doença de Chagas congênita: avaliaçäo imunocitoquímica do parasitismo em tecidos de feto macerado / Congenital Chagas' disease: immunocytochemistry evaluation the parasitism of fetal of macerated tissues

O método imunocitoquímico de peroxidase anti-peroxídase é aplicado, pela primeira vez, em tecidos de natimorto macerado de II-III graus para coloraçäo 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 avaliaçäo mais precisa da intensidade do parasitismo

Estudo comparativo entre o método convencional e o método da peroxidase anti-peroxidase na pesquisa do parasitismo tissular na cardiopatia chagásica crônica / Comparative study between the conventional method and the peroxidase anti-perioxidase in the search of tissular parasitism in chronic Chagas' cardiopathy

Na maioria dos chagásicos crônicos o Trypanosoma cruzi näo é detectado no tecido ou apresenta-se com extrema raridade, mesmo quando é pesquisado exaustivamente. Sendo os métodos utilizados, até entäo, inespecíficos para a demonstraçäo do T. cruzi, propôs-se no presente traballho proceder ao estudo comparativo entre o método convencional (HE) e o método imunocitoquímico pela peroxidase anti-peroxidase (PAP), na avaliaçäo quantitativa do parasitismo. Selecionaram-se 3 casos de cardiopatia chagásica crônica e, de um mesmo fragmento de cada caso, obtiveram-se cortes que foram corados pelo H.E. (média de 100 cortes por caso) e, consecutivamente, outros que foram corados pelo PAP (média de 70 cortes por caso). O caso n§ 1 foi autopsiado em 1952 e apresentava parasitismo freqüente. Nos demais, o exame rotineiro foi negativo. Obtiveram-se os seguintes resultados expressos em n§ de ninhos/100 cortes, respectivamente, corado pelo H.E. e pelo P.A.P. (HE/PAP). Caso n§ 1 = 80/171; caso n§ 2 = 5/116 e caso n§ 3 = 1/2. Os resultados mostram que o método imunocitoquímico empregado, além de facilitar o diagnóstico do parasitismo, demonstra também pequenos ninhos de amastigotas de dificilmente seriam diagnosticados pelos métodos convencionais; além disso mostrou-se útil mesmo em tecido incluídos em parafina há longo tempo