Genetic analyses of Trypanosoma cruzi isolates from naturally infected triatomines and humans in northeastern Brazil.

Trypanosoma cruzi genetic diversity was investigated in 25 isolates (vectors and humans) from the semiarid zone of the State of Rio Grande do Norte, Brazil. Molecular markers (3' region of the 24Salpha rRNA; mitochondrial cytochrome oxidase subunit 2 (COII) gene; spliced leader intergenic region (SL-IR) gene; allelic size microsatellite polymorphism) identified 56% TcIII (100% Panstrongyluslutzi; 50% Triatomabrasiliensis); 40% TcII (91.7% humans; 50% T. brasiliensis) and 4% TcI (human). Microsatellite analysis revealed monoclonal and heterozygous patterns on one or more microsatellite loci in 64% of T. cruzi isolates (92.3% triatomines; 33.3% humans) and 36% putative polyclonal populations (66.7% humans; 7.7% triatomines) by loci SCLE10, SCLE11, TcTAT20, TcAAAT6, all belonging to TcII. Identical T. cruzi polyclonal profiles (88.9%) were detected, mostly from humans. The adaptative natural plasticity of TcII and TcIII and their potential for maintaining human infection in T. brasiliensis were confirmed. Intraspecific and phylogenetic T. cruzi diversity in the sylvatic and domestic transmission cycles in this specific region will provide exclusive control strategies.

A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI.

In an effort to unify the nomenclature of Trypanosoma cruzi, the causative agent of Chagas disease, an updated system was agreed upon at the Second Satellite Meeting. A consensus was reached that T. cruzi strains should be referred to by six discrete typing units (T. cruzi I-VI). The goal of a unified nomenclature is to improve communication within the scientific community involved in T. cruzi research. The justification and implications will be presented in a subsequent detailed report.

A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI

In an effort to unify the nomenclature of Trypanosoma cruzi, the causative agent of Chagas disease, an updated system was agreed upon at the Second Satellite Meeting. A consensus was reached that T. cruzi strains should be referred to by six discrete typing units (T. cruzi I-VI). The goal of a unified nomenclature is to improve communication within the scientific community involved in T. cruzi research. The justification and implications will be presented in a subsequent detailed report.

Trypanosoma rangeli: RAPD-PCR and LSSP-PCR analyses of isolates from southeast Brazil and Colombia and their relation with KPI minicircles.

This study presents the first genetic characterization of five Trypanosoma rangeli isolates from Minas Gerais, in the southeast of Brazil and their comparison with Colombian populations by minicircle classification, RAPD-PCR and LSSP-PCR analyses. Our results demonstrated a homogenous T. rangeli population circulating among Didelphis albiventris as reservoir host in Brazil while heterogeneous populations were found in different regions of Colombia. KP1(+) minicircles were found in 100% isolates from Brazil and in 36.4% of the Colombian samples, whereas the KP2 and KP3 minicircles were detected in both groups. RAPD-PCR and LSSP-PCR profiles revealed a polymorphism within KP1(+) and KP1(-) T. rangeli populations and allowed the division of T. rangeli in two branches. The Brazilian KP1(+) isolates were more homogenous than the KP1(+) isolates from Colombia. The RAPD-PCR were entirely consistent with the distribution of KP1 minicircles while those obtained by LSSP-PCR were associated in 88.9% and 71.4% with KP1(+) and KP1(-) populations, respectively.

Cystoisospora belli: in vitro multiplication in mammalian cells.

Intracellular development of Cystoisospora belli was demonstrated in 4 different mammalian cell lines. Human ileocecal adenocarcinoma (HCT-8), epithelial carcinoma of lung (A549), Madin-Darby bovine kidney (MDBK), and African green monkey kidney (VERO) were exposed in vitro to C. belli sporozoites, which had been isolated from the feces of HIV-AIDS patients. Parasites invaded all the cellular types between 4 and 12h after exposure and multiplication was demonstrated after 24 h. Grater number of merozoites formed in VERO cells, followed by HCT-8. In the MDBK and HCT-8 cells, the parasitophorous vacuole was less evident and immobile merozoites were observed in the cytoplasm. In VERO cells, one or several parasitophorous vacuoles contained up to 16 mobile sporozoites. No oocysts were found in any of the cell types used. VERO cells may be suitable for studies of the interaction between parasite and host cells.

Interstitial cells of Cajal in patients with chagasic megacolon originating from a region of old endemicity.

UNLABELLED: Megacolon is the second most frequent and most important digestive manifestation of Chagas' disease. It is characterized by motor disorders and dilatation of the distal segments of the colon. Several theories have been presented to explain the physiopathology of chagasic megacolon, e.g. the plexus theory. OBJECTIVE: In the present study the distribution of interstitial cells of Cajal (ICC) was evaluated in colon biopsies from chronic chagasic patients originating from a region of old endemicity for Trypanosoma cruzi and for comparison in subjects with other colon disorders. The chagasic patients had been submitted to colectomy for the investigation of other possible mechanisms underlying the physiopathogenesis of megacolons. DESIGN STUDY: Twenty-two colon biopsies (15 from chagasic patients and 7 from nonchagasic patients) were examined. ICC were identified by immunohistochemistry by using the anti-CD117 antibody. The number of ICC was determined in longitudinal and circular muscle layers and in the myenteric plexus, and the results were analyzed by the Kruskal-Wallis and Student t-tests. RESULTS: A reduced number of ICC was observed in all layers and in the myenteric plexus of patients with chagasic megacolon (P<0.05). CONCLUSIONS: We conclude that the physiopathological manifestations observed in the large bowel of chagasic patients originate from alterations that occur in the ICC, which play an important role in the control of gut motility.

Random amplified polymorphic DNA profiles of Trypanosoma cruzi isolates from chagasic patients with different clinical forms.

The genetic variability of 61 Trypanosoma cruzi isolates from 47 chronic chagasic patients of Minas Gerais state was analyzed by random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) using M13-40, lambdagt11-F, and L15996 primers. Cluster analysis by unweighted pair group method analysis was applied to RAPD profiles, and cluster analysis used to verify a possible correlation among different clinical forms of the disease from these patients. The T. cruzi isolates showed distinct grouping on tree topology, with the isolates not being possible to establish a correlation to the clinical forms of Chagas' disease. These data showed that the T. cruzi isolates from these patients would compose a group of populations well correlated genetically.

Real time PCR strategy for the identification of major lineages of Trypanosoma cruzi directly in chronically infected human tissues.

Two evolutionary lineages, called Trypanosoma cruzi I and II, have been identified in T. cruzi, the etiologic agent of human Chagas disease. Here, we describe a molecular strategy for direct genetic typing of these major groups of T. cruzi directly in human tissues. The protocol is based on heminested PCR amplification of the D7 region of the 24Salpha ribosomal DNA (rDNA), followed by identification of the products using denaturation curves in real time PCR. The repetitive nature of the gene, and the heminested PCR format insured the high sensitivity necessary to detect the presence of the very scarce T. cruzi DNA present in the chronically infected human tissues. There is 80% DNA sequence homology between the two 24Salpha rDNA alleles that define the T. cruzi I and II groups, sufficient to produce different thermal denaturation curves with melting temperature (TM) values of 81.7+/-0.43 and 78.2+/-0.33 degrees C (mean+/-SEM). Using this technical approach, we analysed tissue samples (esophagi, hearts and colon) from 25 different patients with the gastrointestinal or cardiac forms of Chagas disease; in all of them we found only the presence of T cruzi II. Previous epidemiological and immunological findings had already led to the idea that chronic human infections occurring in Brazil and Argentina might be primarily due to T. cruzi II strains, but all the evidence available had been indirect. Our findings provide definitive proof of this hypothesis and will also allow the establishment of which group of T. cruzi is responsible for Chagas disease in other countries.

Relationship between Trypanosoma cruzi and human chagasic megaesophagus: blood and tissue parasitism.

The persistence of Trypanosoma cruzi in tissue and blood of 52 patients in the digestive form of chronic Chagas disease was studied. These patients had chagasic megaesophagus and underwent corrective surgery. Parasitologic (xenodiagnosis, hemoculture, or both), histopathologic (hematoxylin and eosin, and peroxidase-anti-peroxidase staining), and molecular (polymerase chain reaction [PCR] followed by slot-blot hybridization) tests were used in the analysis. The presence of T. cruzi, its genomic fragments, or its antigens could be detected in 98% (51 of 52) of the patients. The parasite was randomly identified in 76.9% of esophageal tissues and in 90.4% by PCR and in 73.1% by parasitologic methods from the blood. Fifty percent (26 of 52) of tissue samples had inflammation, 80.8% of which was associated with the parasite. Trypanosoma cruzi was also identified unassociated with inflammatory alterations. Higher tissue parasitism and intense inflammatory processes were observed in esophageal tissue from patients with Grade IV megaesophagus. These data demonstrate that in the digestive form of Chagas' disease, particularly in cases of megaesophagus, T. cruzi is frequently found, both in blood and tissues and may contribute to the pathogenic mechanisms involved.

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.

Trypanosoma cruzi: characterization of reinfection and search for tissue tropism in hamsters (Mesocricetus auratus).

Tissue tropism, the role of reinfection in the development of Chagas' disease, and the selection of subpopulations of Trypanosoma cruzi were evaluated in hamsters inoculated with the VIC strain of T. cruzi. Adult allogeneic male hamsters were inoculated once or reinoculated by the intraperitoneal route up to four times with 2000 blood trypomastigotes. Animals were studied by blood culture, histopathology, immunohistochemistry, and molecular techniques (PCR and low-stringency single specific primer-PCR). Homogeneity of the T. cruzi population observed in different tissues suggests that selective tropism of the VIC strain extends only to various muscle tissues in hamsters and that reinfection is not a factor in the development of the inflammatory processes, although it may aggravate it, possibly due to an increase in tissue parasitism, which might induce autoimmune mechanisms. Reinfection did not induce selection of subpopulations in the tissue or in the blood.

Removal of Trypanosoma cruzi by white cell-reduction filters: an electronmicroscopic study.

White cell (WBC)-reduction filters have been shown to be effective in removing infectious agents from infected blood products. In this study, the mechanisms of Trypanosoma cruzi (T. cruzi) retention by WBC-reduction filters were assessed. Human packed red blood cell (PRBC) and platelet concentrate (PC) samples were contaminated with T. cruzi organisms (Y strain; 3.4 x 10(6)/ml), and then filtered using WBC-reduction experimental filters that provided about 3 log10 WBC removal. Transmission electron microscopy sections showed that T. cruzi parasites were removed from contaminated PRBC and PC samples primarily by mechanical mechanism without interacting with filter fibers or blood cells. In addition, we found that T. cruzi parasites were also removed by a direct fiber adhesion. These data indicate that T. cruzi parasites are removed from infected blood not only by mechanical mechanism but also by biological mechanism probably mediated by parasite surface proteins.

Prevention of transfusion-associated Chagas' disease by sterilization of Trypanosoma cruzi-infected blood with gentian violet, ascorbic acid, and light.

BACKGROUND: Allogeneic blood transfusions are the second most frequent route of Chagas' disease transmission in countries where the disease is endemic. The prevention of transfusion-associated Chagas' disease has been attempted through clinical and serologic screening of blood donors and/or by the addition of trypanomicidal substances such as gentian violet (GV) to stored blood for 24 hours. The present study describes an alternative method of chemoprophylaxis of transfusion-associated Chagas' disease that reduces the sterilization time by using a combination of low-concentration GV, ascorbic acid (AA), and photoradiation with visible light. STUDY DESIGN AND METHODS: To better reproduce the conditions of blood transfusion in developing areas, normal human blood was collected in blood collection bags, infected with different concentrations of Trypanosoma cruzi, and treated with GV, AA, and photoradiation. Mice were then inoculated with the T. cruzi-infected human blood that had been stored at different incubation intervals. Active parasites were sought in mouse blood for parasitologic diagnosis and serologic evaluation (mice inoculation, blood culture, and indirect immunofluorescence). RESULTS: The association of GV (250 micrograms/mL), and photoradiation with visible light (75W) sterilized T. cruzi-infected blood even after treatment for less than 30 minutes and even when chagasic blood was treated with low-concentration GV (62.5 micrograms/mL for 30 min). Moreover, the trypanomicidal activity of GV associated with AA and photoradiation with visible light was found even when blood was infected with a 10-fold parasite concentration. CONCLUSIONS: The proposed alternative prophylactic method is reproducible, easy to perform, and inexpensive, and it may have practical importance in endemic areas where serologic screening of donor blood is not always available. In addition, the reduction of the GV trypanomicidal concentration might further minimize the potential for GV-related side effects.

The hamster (Mesocricetus auratus) as experimental model in Chagas' disease: 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 Vicentia, 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 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 zonal fibrosis in the myocardium. In 50% of infected animals parasites were found in myocardium 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 against Trypanosoma cruzi (megazol, nifurtimox and benznidazole) induce a rapid clearance 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 treated 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.

Effect of protective and non-protective antibodies in the phagocytosis rate of Trypanosoma cruzi blood forms by mouse peritoneal macrophages.

The phagocytosis of Trypanosoma cruzi blood forms by mouse peritoneal macrophages is significantly enhanced by sera from chronic chagasic patients, rabbits and mice presenting 'lytic antibodies' (LA) which are associated with resistance and active infections as well as 'conventional serology antibodies' (CSA) which are immunoglobulins involved in the positivity of serological diagnostic tests. The phagocytosis rate, however, is not influenced by sera from mice immunized with T. cruzi antigen or chagasic patients submitted to specific treatment, both displaying only CSA but not LA. The efficacy of LA in increasing phagocytosis is related to their ability to bind to epitopes of living trypomastigotes, a property lacking in CSA that bind only to fixed parasites. This phenomenon is apparently the reason for the low effectiveness of antigens used for vaccination in Chagas' disease which only induce CSA, immunoglobulins apparently unable to mediate a number of regular effector immune mechanisms such as complement-mediated lysis, antibody-dependent cell cytotoxicity and phagocytosis.