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Summary Lack of conservation of the Amazon tropical rainforest has imposed severe threats to its human population living in newly settled villages, resulting in outbreaks of some infectious diseases. We conducted a seroepidemiological survey of 1100 inhabitants of 15 villages of Paco do Lumiar County, Brazil. Thirty-five (3%) individuals had been exposed to Trypanosoma cruzi (Tc), 41 (4%) to Leishmania braziliensis (Lb) and 50 (4.5%) to Leishmania chagasi (Lc) infections. Also, 35 cases had antibodies that were cross-reactive against the heterologous kinetoplastid antigens. Amongst these, the Western blot assays revealed that 11 (1%) had Tc and Lb, that seven (0.6%) had Lc and Tc, and that 17 (1.6%) had Lb and Lc infections. All of these cases of exposures to mixed infections with Leishmania sp, and eight of 11 cases of Tc and Lb were confirmed by specific PCR assays and Southern hybridizations. Two cases had triple infections. We consider these asymptomatic cases showing phenotype and genotype markers consistent with mixed infections by two or more kinetoplastid flagellates a high risk factor for association with Psychodidae and Triatominae vectors blood feeding and transmitting these protozoa infections. This is the first publication showing human exposure to mixed asymptomatic kinetoplastid infections in the Amazon. L'absence de conservation de la foret tropicale de l'Amazonie a impose des menaces graves a sa population humaine vivant dans les villages nouvellement arranges, avec pour resultat des epidemies de certaines maladies infectieuses. Nous avons mene une surveillance seroepidemiologique sur 1100 habitants de 15 villages dans le comte de Paco de Lumiar au Bresil. 35 individus (3%) avaient ete exposes a l'infection aTrypanosoma cruzi (Tc), 41 (4%) aLeishmania braziliensis (Lb) et 50 (4,5%) aLeishmania chagasi (Lc). En outre, 35 cas possedaient des anticorps a reaction croisee avec les antigenes heterologues de kinetoplastides. Parmi ces derniers, les analyses par western blot ont revele 11 (1%) cas de coinfections aTc et Lb, 7 (0,6%) cas aLc et Tc et 17 (1,6%) cas aLb et Lc. Tous ces cas d'expositions a des infections mixtes aLeishmania sp et 8 cas sur 11 de Tc et Lb ont ete confirmes par des analyses de PCR specifiques et des hybridations de d'ADN. Deux cas a infection triple ont ete observes. Nous considerons l'existence ces cas asymptomatiques avec des marqueurs phenotypiques et de genotypiques consistants pour des infections mixtes par deux kinetoplastides flagelles ou plus comme etant un facteur de risque eleve pour l'association avec des Psychodidae et des Triatominae qui sont des vecteurs se nourrissant de sang et transmettant ces infections a protozoaires. Ceci est la premiere publication rapportant l'exposition humaine a des infections asymptomatiques mixtes de kinetoplastides en Amazonie. La falta de conservacion del bosque tropical lluvioso Amazonico ha puesto en grave peligro a las poblaciones humanas que viven en poblados de reciente construccion, resultando en brotes de algunas enfermedades infecciosas. Hemos conducido un estudio seroepidemiologico con 1100 habitantes de 15 poblados del condado Paco do Lumiar, Brasil. 35 (3%) individuos habian estado expuestos a infecciones por Trypanosoma cruzi (Tc), 41 (4%) a Leishmania braziliensis (Lb) y 50 (4.5%) a Leishmania chagasi (Lc). Asi mismo, 35 presentaban anticuerpos con reaccion cruzada frente a antigenos heterologos de kinetoplastidos. Entre estos individuos, los ensayos por ‘‘Western blot’’ revelaron que 11 (1%) tenian infecciones por Tc y Lb, que 7 (0.6%) tenian infecciones por Lc y Tc, y que 17 (1.6%) tenian infecciones por Lb y Lc. Todos los casos de exposicion a infecciones mixtas con Leishmania sp, y 8 de los 11 casos de Tc y Lb fueron confirmados por ensayos especificos de PCR e hibridacion de Southern. Dos casos tenian una infeccion triple. Consideramos estos casos asintomaticos, con marcadores fenotipicos y genotipicos consistentes con infecciones mixtas con dos o mas flagelados kintoplastidos, un factor de alto riesgo en asociacion con vectores Psychodidae y Triatominae que se alimentan de sangre y transmiten estas infecciones protozoarias. Esta es la primera publicacion que muestra exposicion humana a infecciones asintomaticas mixtas de protozoos kinetoplastidos en el Amazonas.

In this study, we isolated Trypanosoma cruzi from chronic Chagas heart disease and from megaesophagus patients. The parasite stock hSLU239 (heart disease) yielded clones h1 and h2, whereas stock mSLU142 (megaesophagus) yielded clones m1, m2, m3 and m4. The parasite growth kinetics, doubling time and differentiation in axenic liquid medium showed broad behavioral diversity. It was shown that a particular pattern of behavior for a parental stock could not necessarily be assigned for subsequent clones. This study indicates that i) each Chagas disease patient is infected with several T. cruzi populations; ii) clonal lines derived from patient samples may have different biological characteristics from the original isolate; and that iii) additional behavioral and/or molecular markers are required for further characterization of Trypanosoma cruzi stocks and clones derived from Chagas disease patients in order to identify correlations with pathology. Neste estudo, foram obtidos estoques de Trypanosoma cruzi de pacientes chagásicos com a doença cardíaca ou com megaesôfago. O estoque hSLU239 (doença cardíaca) forneceu os clones h1 e h2, enquanto o estoque mSLU142 (megaesôfago) forneceu os clones m1, m2, m3 e m4. A cinética de crescimento do parasito, tempo de duplicação e diferenciação em meio líquido axênico mostraram ampla diversidade comportamental. Observou-se que um padrão particular de comportamento de um estoque parental podia não ser necessariamente encontrado na linhagem subclonal subseqüente. Este estudo indica que i) cada paciente chagásico é infectado com várias subpopulações de T. cruzi; ii) linhagens clonais derivadas de cada estoque do parasito podem ter características biológicas diferentes do isolado original de paciente chagásico; e que iii) marcadores comportamentais e/ou moleculares adicionais são necessários para melhor caracterização de estoques de T. cruzi e seus clones derivados de pacientes com doença de Chagas, a fim de identificar as possíveis correlações com a patologia.

Isoenzyme and RFLP analyses were carried on freshly isolated Trypanosoma cruzi stocks and subsequent clones derived from patients with chronic Chagas disease. The isoenzymes separated the parasite stocks and clones in two groups: The stock hSLU239 (group I), isolated from a heart disease patient, showed the zymodeme 3 (Z3) profile (M. A. Miles et al., 1977, Transactions of the Royal Society of Tropical Medicine and Hygiene 71, 217-225). The stock mSLU142 (group II), isolated from a digestive disease (megaesophagus) patient, showed the Z2 profile. The parasite clones m1, m2, m3, and m4, derived from mSLU142, and clones h1 and h2, derived from hSLU239, showed isoenzyme profiles similar to those of Z2 and ZA (Miles et al. 1977; J. A. Romanha, 1982, Thesis, Universidade Federal de Minas Gerais). Furthermore, the T. cruzi clones derived from the cardiac disease patient differed from those derived from the megacolon patient in 3 of the 13 enzymes analyzed. RFLP analysis showed polymorphism at the EcoRI and PstI restriction fragments of the DNA sequences coding the glycolytic enzymes ALD, GPI, GAPDH, and PYK and separated the T. cruzi stocks and clones in three groups: I, comprising the stock hSLU239 and clone m4, which was classified as homozygous CC, BB, AA, and AA for the ALD, GPI, PYK, and GAPDH genes, respectively; II, formed by the parasite stock mSLU142 and clones h1 and h2 (derived from hSLU239), which was classified as homozygous AA, AA, CC, and BB for ALD, GPI, PYK, and GAPDH genes, respectively . These findings show that the infection of each Chagas disease patient may be produced by genetically diverse mixed parasite populations.

A randomized ten-year follow-up study involving 91 Chagas patients and 41 uninfected controls was undertaken to determine the effectiveness of nitroderivative therapy. Anti-Trypanosoma cruzi antibodies were consistently lower one year after treatment than 10 years thereafter (P0.001). The blood of all treated and 93.7% of untreated Chagas patients yielded polymerase chain reaction (PCR) product from probes annealing to T. cruzi nuclear DNA, indicating active infection. Competitive PCR showed means +/- standard deviations of 20.1+/-22.6 T. cruzi/ml of blood from untreated and 13.8+/-14.9 from treated Chagas patients, but the differences between means were not statistically significant (P0.05). Electrocardiograms recorded a gamut of alterations several-fold more frequent in Chagas patients, regardless of treatment, than in uninfected controls (P0.001). These results show that nitroderivative therapy for T. cruzi infections is unsatisfactory and cannot be recommended since it fails to eradicate the parasite or change the progression of heart disease in chronic Chagas patients.

The intraspecific variation that has been described in Trypanosoma cruzi was examined in recent isolates from Chagas' disease patients, using behavioral and molecular markers for characterization of the parasite stocks and derived clones. We used these parasite populations to determine virulence and pathogenicity in vivo. The T. cruzi stocks mSLU142 (megaesophagus) and hSLU239 (heart disease) and the clones h1 and h2 induced very low parasitemias in BALB/c mice, whereas high parasitemias were obtained with clones m1, m2, m3, and m4. Clones m1–m4 also produced heart lesions of higher intensity than those observed in mice infected with the h1 and h2 clones. Furthermore, the heart lesions produced by all of these clones were significantly more intense than those seen in mice infected with either of the T. cruzi parental stocks. In addition, neither the kinetics of growth, doubling time, differentiation in axenic culture, zymodemes, nor DNA restriction length polymorphisms showed correlations with parasitemias and pathogenicity in mice. This study suggests that multiple biochemical and physiological markers are required to enable an association of clinical and pathologic manifestations of the disease with intrinsic characters of the T. cruzi populations.

The ploidy of Trypanosoma cruzi is until now undetermined although analysis of isoenzymes, molecular karyotype and DNA content suggest diploidy in a very plastic genome. Also, there has been no convincing demonstration of genetic exchange and it has been proposed that reproduction is clonal. We have compared 18 T. cruzi stocks and clones from the same area or host by means of isoenzyme analysis (12 loci) and restriction site polymorphisms in and around three glycolytic genes (glyceraldehyde-3-phosphate dehydrogenase, aldolase and glucosephosphate isomerase). The analysis demonstrated the presence of homozygotes and heterozygotes and is compatible with diploidy for these housekeeping genes. This strongly supports the hypothesis of genetic exchange in T. cruzi and further elucidates the genetic diversity within natural T. cruzi populations.

A prevalência da infecção chagásica em 368 garis do Serviço de Limpeza Urbana (SLU) do Distrito Federal foi inicialmente indicada pelos exames de hemaglutinação e imunofluorescência, quando encontrou-se 32,1% e 47,6% de resultados positivos, respectivamente, para cada exame. Em vista das discrepâncias obsevadas. utilizou-se o teste cutâneo com o antígeno T12E para esclarecimento do diagnóstico em 15,5% dos que tinham apenas um teste sorológico positivo. O teste cutâneo foi positivo em 38,6% dos garis. A análise dos resultados de 183 garis mostrou 47% dos casos com três exames concordantes positivos. Mais 19, 7% tiveram dois exames concordantes positivos. Porém, 33,3% dos garis tinham apenas um dos três exames positivo. Empregou-se também o "immunoblot" na tentativa de esclarecer o diagnóstico nos 61 casos com apenas um resultado positivo. Foram encontrados 37,5% (3/8) dos casos positivos pela hemaglutinação, 11,5% (3/26) pelo teste cutâneo, e apenas 3,7% (1/27) pela imunofluorescência. Após a adição desses casos positivos pelo "immunoblot", o total de chagásicos passou a ser de 129, ou seja, 35%/ dos garis da amostra estudada.Seropositivity for Trypanosoma cruzi infection was studied in 308 street-sweepers of the SLU. Federal District. Brazil, with the aid of haemaglutination. immunofluorescence and, also, a delayed-type skin test to the parasite T12E antigen. It showed 32,1%, 42.7% and 38.6% positive results, respectively for cach assay. Among these, however, only 47% were positive with each of three exams performed. In addition, 19.7% were positive with two out of three exams performed. The remaining 33-3% sera yielded one positive result out of three exams employed and were submitted to the immunoblot assay. This analysis confirmed 3 cases (37.5%) positive by hemmaglutination. 3 (11.5%) positive by skin test, and 1 (3-7%) positive by immunofluorescence. At the end of the analysis, it was shown that 129 (35%) individuals yielded at least two positive assays and therefore, they should be cousidered as T. cruzi-infected individuals.

To determine the role of Trypanosoma cruzi superinfections on the outcome of Chagas' disease, groups of BALB/c mice were prime-infected with low virulence clones h1 and h2 and challenged with high virulence clones m3 and m4. All mice injected with the m3 and m4 clones succumbed before or at 16 days postinfection. In contrast, all mice injected with the h1 and h2 clones survived the prime infection and were superinfected with the m3 and m4 clones. Low-level parasitemias were observed in mice after challenge with the virulent clones. The mortality ratios in the superinfected mice were not statistically different from those seen in the mice that received a single T. cruzi injection. The histopathological lesions recorded during the course of the infections showed features in the superinfected mice similar to those seen in the animals receiving a single infection. These data argue that morbidity and mortality in BALB/c mice, infected with T. cruzi clonal lines, are not associated with the frequency of receiving the parasite burden.