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.

Cervical gland area as an ultrasonographic marker for preterm delivery.

OBJECTIVE: To assess the association between spontaneous preterm delivery (SPTD) in the general population and the measurement of the cervix length, cervical funneling, and absence of the cervical gland area (CGA). METHOD: A prospective cohort of 338 women carrying uncomplicated pregnancies was evaluated by transvaginal sonography between 21 and 24 weeks' gestation. RESULTS: Measurement of cervical length with less than 20 mm and the presence of cervical funneling presented a statistically significant association with SPTD before 35 weeks. The non-detection of CGA demonstrated a strong association with SPTD before 37 weeks' (p < 0.001; OR = 194.5) and before 35 weeks' gestation (p < 0.001; OR = 129.6). The multiple logistic regression analysis suggested the non-detection of CGA as the only variable to reveal statistically significance association with SPTD. CONCLUSION: The results seem to indicate that the absence of CGA can be a new and important ultrasound marker for SPTD, to be confirmed by future multicenter investigations.

Clonal structure of Trypanosoma cruzi Colombian strain (biodeme Type III): biological, isoenzymic and histopathological analysis of seven isolated clones.

The clonal structure of the Colombian strain of Trypanosoma cruzi, biodeme Type III and zymodeme 1, was analyzed in order to characterize its populations and to establish its homogeneity or heterogeneity. Seven isolated clones presented the basic characteristics of Biodeme Type III, with the same patterns of parasitemic curves, tissue tropism to skeletal muscle and myocardium, high pathogenicity with extensive necrotic-inflammatory lesions from the 20th to 30th day of infection. The parental strain and its clones C1, C3, C4 and C6, determined the higher levels of parasitemia, 20 to 30 days of infection, with high mortality rate up to 30 days (79 to 100%); clones C2, C5 and C7 presented lower levels of parasitemia, with low mortality rates (7.6 to 23%). Isoenzymic patterns, characteristic of zymodeme 1, (Z1) were similar for the parental strain and its seven clones. Results point to a phenotypic homogeneity of the clones isolated from the Colombian strain and suggest the predominance of a principal clone, responsible for the biological behavior of the parental strain and clones.

Clonal structure of Trypanosoma cruzi Colombian strain (biodeme Type III): biological, isoenzymic and histopathological analysis of seven isolated clones

The clonal structure of the Colombian strain of Trypanosoma cruzi, biodeme Type III and zymodeme 1, was analyzed in order to characterize its populations and to establish its homogeneity or heterogeneity. Seven isolated clones presented the basic characteristics of Biodeme Type III, with the same patterns of parasitemic curves, tissue tropism to skeletal muscle and myocardium, high pathogenicity with extensive necrotic-inflammatory lesions from the 20th to 30th day of infection. The parental strain and its clones C1, C3, C4 and C6, determined the higher levels of parasitemia, 20 to 30 days of infection, with high mortality rate up to 30 days (79 to 100%); clones C2, C5 and C7 presented lower levels of parasitemia, with low mortality rates (7.6 to 23%). Isoenzymic patterns, characteristic of zymodeme 1, (Z1) were similar for the parental strain and its seven clones. Results point to a phenotypic homogeneity of the clones isolated from the Colombian strain and suggest the predominance of a principal clone, responsible for the biological behavior of the parental strain and clones.

A estrutura clonal da cepa Colombiana do Trypanosoma cruzi, biodema Tipo III e zimodema 1, foi analisada com o objetivo de caracterizar as suas populações e estabelecer a homogeneidade ou heterogeneidade das mesmas. Foram isolados sete clones, os quais apresentaram as características básicas do biodema Tipo III, com o mesmo padrão de curvas parasitêmicas, tropismo tecidual para músculo esquelético e miocárdio, alta patogenicidade, com extensas lesões necrótico-inflamatórias, do 20º ao 30º dia de infecção. A cepa parental e os clones C1, C3, C4 e C6 apresentaram os niveis mais elevados de parasitemia entre 20 e 30 dias pós-infecção e alto indice de mortalidade até 30 dias (79 a 100%); os clones C2, C5 e C7 apresentaram niveis mais baixos de parasitemia com baixa mortalidade até 30 dias (7,6 a 23%). Os padrões isoenzimáticos foram característicos do zimodema 1 (Z1) para a cepa parental e os sete clones. Os achados do presente trabalho indicam uma homogeneidade fenotípica entre os clones isolados da cepa Colombiana e sugerem a predominância de um clone principal, responsável pelo padrão de comportamento biológico da cepa parental e dos clones.

TNF-alpha is expressed at sites of parasite and tissue destruction in the spleen of mice acutely infected with Trypanosoma cruzi.

Mice infected with a macrophagotropic strain of Trypanosoma cruzi develop progressive splenomegaly due to reactive hyperplasia with increased number of lymphocytes and macrophages, culminating in parasite disintegration and necrosis of parasitized cells. Necrotic changes have been attributed to the liberation of toxic cytokines, including TNF-alpha, from parasitized macrophages. In the present study, the presence of TNF-alpha was investigated in situ. In addition the participation of destroyed parasites in inducing the liberation of TNF-alpha was examined in two highly susceptible mice strains (C3H and Swiss) and a more resistant strain (DBA). Swiss (90) C3H/He (83) and DBA (30) mice were infected with the Peruvian strain of T. cruzi. Nineteen infected Swiss mice, and 22 infected C3H/He were treated with Benznidazole (one or two doses, 100 mg/kg bw/day), on the 8th and 9th days after infection. Necrotic splenic lesions occurred in both susceptible and resistant strains of mice. Although differing in degree, lesions were more intense in C3H and Swiss than in DBA mice. Comparing untreated and treated susceptible mice, necrotic lesions were significantly less intense in the latter. By specific monoclonal antibody immunolabelling, TNF-alpha was demonstrated in the cytoplasm of macrophages and within necrotic areas, from Swiss, C3H/He and DBA mouse spleens. In conclusion, TNF-alpha, probably synthesized by macrophages, was strongly expressed at the sites of parasite and cell destruction, thus appearing to play a pivotal role in splenic necrotic changes associated with severe experimental T. cruzi infection.

Interstitial dendritic cells of the heart harbor Trypanosoma cruzi antigens in experimentally infected dogs: importance for the pathogenesis of chagasic myocarditis.

Heart sections from 16 mongrel dogs, two normal controls and 14 infected with Trypanosoma cruzi, were submitted to immunohistochemical staining with either rabbit anti-cow S100 Protein monoclonal antibody or rabbit anti-T. cruzi purified specific antibody, using the peroxidase technique to investigate the participation of the interstitial dendritic cells of the heart (IDCs) in myocarditis of Chagas disease. Trypanosoma cruzi antigens were revealed as granular and dense deposits in IDC membrane in the heart of infected dogs both during acute and chronic myocarditis, but not in normal controls. Anti-S100 Protein labeled the IDCs, both in normal and infected dogs and a significant increase in the numbers of IDCs occurred in the myocardium, proportionally to the intensity of the inflammatory infiltration. These findings suggest that IDCs, probably by presenting T. cruzi antigens to immune-competent cells, play an important role in the pathogenesis of Chagas disease.

Apoptosis in a canine model of acute Chagasic myocarditis.

Histologic, ultrastructural and nick end labeling studies were made to evaluate the occurrence of apoptosis in the hearts of dogs with acute myocarditis due to experimental infection with T. cruzi. The best results for the detection of apoptosis by nick end labeling were obtained by a method combining the use of terminal deoxynucleotidyl transferase, CoCl2 and fluorescein-conjugated deoxyuridine triphosphate, followed by counterstaining of DNA with 4'6-diamidino-2-phenylindole (DAPI) and examination by laser scanning confocal fluorescence microscopy. Apoptosis was found in: (1) cardiac myocytes; (2) endothelial cells of capillaries and venules: (3) immune effector cells, including macrophages, interstitial dendritic cells (antigen-presenting cells) and granular and agranular lymphocytes, and (4) intra- and extracellular forms of T. cruzi. The apoptosis in myocytes and endothelial cells affected cells that were not infected by T. cruzi and was probably caused by the release of toxic mediators of inflammation. The apoptosis of immune effector cells could be related either to the subsidence of inflammation or to modulation (and even failure) of the immune response. The finding of apoptosis in T. cruzi confirms the results of other studies showing that this phenomenon occurs during the differentiation of trypomastigotes in vitro. Thus, apoptosis constitutes an important and multifactorial event in the pathogenesis of acute Chagasic myocarditis.

Comparative analysis by polymerase chain reaction amplified minicircles of kinetoplast DNA of a stable strain of Trypanosoma cruzi from São Felipe, Bahia, its clones and subclones: possibility of predominance of a principal clone in this area.

Molecular characterization of one stable strain of Trypanosoma cruzi, the 21 SF, representative of the pattern of strains isolated from the endemic area of São Felipe, State of Bahia, Brazil, maintained for 15 years in laboratory by serial passages in mice and classified as biodeme Type II and zymodeme 2 has been investigated. The kinetoplast DNA (kDNA) of parental strain, 5 clones and 14 subclones were analyzed. Schizodeme was established by comparative study of the fragments obtained from digestion of the 330-bp fragments amplified by polymerase chain reaction (PCR) from the variable regions of the minicircles, and digested by restriction endonucleases Rsa I and Hinf I. Our results show a high percentual of similarity between the restriction fragment length polymorphism (RFLP) for the parental strain and its clones and among these individual clones and their subclones at a level of 80 to 100%. This homology indicates a predominance of the same "principal clone" in the 21SF strain and confirms the homogeneity previously observed at biological and isozymic analysis. These results suggest the possibility that the T. cruzi strains with similar biological and isoenzymic patterns, circulating in this endemic area, are representative of one dominant clone. The presence of "principal clones" could be responsible for a predominant tropism of the parasites for specific organs and tissues and this could contribute to the pattern of clinico-pathological manifestations of Chagas's disease in one geographical area.

The indeterminate phase of Chagas' disease: ultrastructural characterization of cardiac changes in the canine model.

The indeterminate phase of Chagas' disease is defined as the prolonged period of clinically silent infection that follows the phase of acute primary infection with Trypanosoma cruzi. The dog is the only experimental animal model in which the indeterminate phase progresses to the late phase of severe, chronic myocarditis. This report describes the cardiac histologic and ultrastructural findings in dogs that survived the acute phase of infection with T. cruzi, becoming clinically and electrocardiographically normal for up to 3.5 years, while maintaining positive serologic test results during this period of time. Most of the myocardium appeared morphologically normal; however, small foci of mild, chronic myocarditis were present, with interstitial edema, mild fibrosis, and infiltration by lymphocytes, macrophages, and plasma cells. No microvascular lesions and no areas of close contact between immune effector cells and endothelial cells or cardiac myocytes were present. These findings were in sharp contrast to those observed in the canine model during the acute infection with T. cruzi. In this model, acute myocyte damage and lesions in the microcirculation, including fibrin microthrombi, were associated with close contacts between immune effector cells and myocytes or endothelial cells. Focally inflamed interstitial tissue showed increased deposition of amorphous and collagenous extracellular matrix as well as evidence of breakdown of collagen. The features of the inflammatory cells in the indeterminate phase of Chagas' disease were interpreted as indicating a self-limited cycle of focal inflammatory changes, with modulation and suppression of cell-mediated immune responses. Thus, we consider the indeterminate phase of Chagas' disease to be a stage of host-parasite equilibrium rather than a process of progressive damage.

Biodemes and zymodemes of Trypanosoma cruzi strains: correlations with clinical data and experimental pathology.

With the objective of establishing biological and biochemical characteristics of a significant number of Trypanosoma cruzi strains from different geographical areas, 138 strains isolated from naturally infected humans, triatomine or vertebrate hosts were studied; 120 were isolated from different areas of Brazil and 18 from other South and Central American countries. Inocula from triatomine or culture forms were injected into suckling Swiss mice, followed by passages into mice 10 to 12 g. Biological characters and histopathological study permitted the inclusion of the strains into three Types or biodemes: I, II, III. Isoenzymic analysis confirmed a correspondence between the biodemes and zymodemes: Type I and Z2b, Type II and Z2, Type III and Z1. Results showed the ubiquitary distribution of the several types of strains. The predominance of the same Type and zymodeme in one geographical area was confirmed: Type II strains among the human cases from eastern Bahia and east of Goiás; Type III strains from humans of north Brazil and Central America and from silvatic vectors or vertebrates from other geographical areas. The biological types of strains correlate with different histopathological lesions considering cardiac involvement and neuronal lesions. These findings suggest that the biological behavior together with isoenzymes patterns and pathological pictures in the vertebrate host can be an important tool for establishing correlations between strains behavior and clinico-pathological manifestations of Chagas' disease in different geographical areas.

Influence of treatment with immunosuppressive drugs in mice chronically infected with Trypanosoma cruzi.

Latent Trypanosoma cruzi infection may be reactivated in immunosuppressed individuals, with unusual clinical patterns, such as meningoencephalitis, pseudo neoplastic lesions in the central nervous system, and myocarditis with numerous parasites in the heart muscle. To investigate this problem 68 Swiss mice chronically infected with different strains of T. cruzi were treated with different combinations of immunosuppressive drugs (azathioprine, cyclosporine and betamethasone), in such a way as to imitate the situation during post transplantation treatment. Mortality varied from 6 to 25% in treated mice. There were no deaths in untreated controls. Normal mice have been submitted to the same schedules of immunosuppression as controls of treatment and no deaths were registered during treatment. Chronically infected mice showed significant elevation of total number of leukocytes and lymphocytes in comparison with intact controls; a significant decrease in blood leukocytes and lymphocytes occurred post-treatment in two of the treated experimental groups. Exacerbation of myocarditis and myositis and a high incidence of brain lesions, with focal necrosis, granulomatous lesions and glial proliferation even in the absence of parasites were present in immunosuppressed mice but not in infected controls. Although differing in some aspects from Chagas' disease in immunosuppressed humans, the murine model did show some features that resembled it, especially the peculiar pattern of central nervous system involvement.

[Clinico-pathological correlation in the indeterminate form of experimental Chagas' disease in dogs]. / Correlação clínico-patológica na forma indeterminada da doença de Chagas experimental do cão.

PURPOSE: To study the functional cardiac component of the indeterminate form of experimental Chagas' disease in dogs. METHODS: Four dogs chronically infected with Trypanosoma cruzi and eight normal controls were used. They were submitted to several invasive procedures, either in the presence of complete autonomic block or not, to test for disturbances in the origin and conduction of electric stimuli and the function of cardiac muscle. Histological examination of the heart and its conduction systems was performed in all animals. RESULTS: Mild to moderate focal myocarditis was found in infected dogs, often involving the conduction system of the heart. Sections of the heart from control dogs were histologically normal. Functional data on excitability, intra and interatrial conduction time and sinus node recovering time were essentially similar for both infected and control animals. CONCLUSION: Focal myocarditis, the hallmark of the indeterminate form of Chagas' disease, did not alter the normal parameters of cardiac function, as seen after investigation with sensitive invasive techniques. It is probable that subjects considered as belonging to the indeterminate form of Chagas' disease, but presenting mild alterations at sensitive exploratory tests, may have more severe lesions than that usually described or may be already in the early progressive cardiac form of the disease.

Characterization of subpopulations (clones and subclones) of the 21 SF strain of Trypanosoma cruzi after long lasting maintenance in the laboratory.

Several studies have shown a clonal structure of Trypanosoma cruzi and its possible correlation with the behavioral heterogeneity of the parasite strains. In the present study, the 21 SF strain, that have been maintained in laboratory by successive passages in mice, for more than 15 years, showing a stability of biological and isoenzymic characteristics has been cloned, with the objective of establishing the characters of its clones and subclones. With the technique of isolation of a single parasite from the blood of infected mice, 5 clones and 14 subclones have been obtained. After four passages into mice, inoculum of 10(5) was obtained for each clone and subclone and inoculated into mice weighing 10 to 12 g. These were used for the study of the biological behavior of the clones: evolution of parasitemia, morphology of blood forms and host mortality. For isoenzymic characterization, the clones and subclones were analyzed for ALAT, ASAT, GPI and PGM enzymes. Results have shown that the 5 clones and the 14 subclones disclosed a biological behavior similar to the parental strain, with minor variability of the parasitemic profiles and also the same isoenzymic patterns. These results confirm the stability of the 21 SF strain and indicate a clonal homogeneity of its populations. This is compatible with the hypothesis that the T. cruzi strains represent an equilibrium of either homogenous or heterogeneous populations.

Trypanosoma cruzi strains and autonomic nervous system pathology in experimental Chagas disease.

Lesions involving the sympathetic (para-vertebral ganglia) and para-sympathetic ganglia of intestines (Auerbach plexus) and heart (right atrial ganglia) were comparatively analyzed in mice infected with either of three different strain types of Trypanosoma cruzi, during acute and chronic infection, in an attempt to understand the influence of parasite strain in causing autonomic nervous system pathology. Ganglionar involvement with neuronal destruction appeared related to inflammation, which most of the times extended from neighboring adipose and cardiac, smooth and striated muscular tissues. Intraganglionic parasitism was exceptional. Inflammation involving peripheral nervous tissue exhibited a focal character and its variability in the several groups examined appeared unpredictable. Although lesions were generally more severe with the Y strain, comparative qualitative study did not allow the conclusion, under the present experimental conditions, that one strain was more pathogenic to the autonomic nervous system than others. No special tropism of the parasites from any strain toward autonomic ganglia was disclosed.

Trypanosoma cruzi strains: behavior after passage into authoctonous or foreign species of traitomine (biological and biochemical patterns).

The behavior of T. cruzi strains from S. Felipe-BA (19 SF, 21 SF and 22 SF) classified as Type II Zymodeme 2, was investigated after passage through the authoctonous (P. megistus) and foreign vectors (T. infestans and R. prolixus). For each strain Swiss mice were infected: I--with blood forms (control); II--with metacyclic forms (MF) from P. megistus; III--with MF from T. infestans; IV--with MF from R. prolixus. Inocula: MF from the three species of triatomine, 60 to 120 days after feeding in infected mice, adjusted to 10(4). Biological behavior in mice (parasitemia, morphology, mortality, virulence and pathogenicity) after passage through triatomine was compared with data from the same strain in control mice. Isoenzymic electrophoresis (ASAT, ALAT, PGM, GPI) were also performed after culture into Warren medium. The three strains maintained the isoenzyme profiles (zymodeme 2), in the control groups and after passages through different species of triatomine. Biological characterization disclosed Type II strains patterns for all groups. An increased virulence was observed with the 22 SF strain isolated from P. megistus and T. infestans and higher levels of parasitemia and predominance of slender forms in mice inoculated with the 19 SF and 21 SF from these same species. Results indicate that the passage through the two species T. infestans and P. megistus had a positive influence on the virulence of the regional strains of S. Felipe, regardless of being autocthonous (P. megistus) or foreign to the area (T. infestans).

Interferon-gamma levels during the course of Trypanosoma cruzi infection of Calomys callosus (Rodentia-Cricetidae) and Swiss mice.

Serum levels of interferon-gamma (IFN-gamma) were evaluated in Calomys callosus and Swiss mice during the course of infection by four strains of Trypanosoma cruzi. All strains stimulated the production of this interleukine; however, the timing of its onset and permanence varied among strains and between the two animal models. When chronically infected animals with no detectable serum IFN-gamma were challenged with the homologous strain, they produced quantities comparable with those obtained during the acute phase of infection. In C. callosus there was a correlation between H2O2 liberation by peritoneal macrophages and serum IFN-gamma levels, whereas no such correlation was found in mice. C. callosus had a higher capacity to heal histopathological lesions, whereas lesions in mice were progressive. The results obtained suggest that C. callosus develops well-adapted immune mechanisms that may be important for its role as a reservoir of T. cruzi.

Investigation on the possibility of spontaneous cure of mice infected with different strains of Trypanosoma cruzi.

Seventy Swiss mice chronically infected with different strains of Trypanosoma cruzi, with persistently negative parasitemia on routine blood examination were parasitologically investigated to find out whether spontaneous cure occurred. Duration of infection varied from 90 to 250 days in the initial phase of this investigation. Parasitological tests consisted of daily direct blood examination performed during at least 25 days, followed by xenodiagnosis and subinoculation of blood into newborn mice. Mice that persisted negative were treated with Cyclophosphamide with one dose of 250 mg/kg of body weight and then investigated by direct blood examination, xenodiagnosis and subinoculation. A second dose of 250 mg/kg b. w. was given to the persistently negative mice. With one single exception, all mice showed positive parasitological tests in the different stages of the present investigation and we conclude that spontaneous cure did not occur in this group, which is representative of the chronic infection with different strains of T. cruzi.

Biochemical behavior of Trypanosoma cruzi strains isolated from mice submitted to specific chemotherapy.

To investigate the influence of chemotherapy on the biochemical behavior of Trypanosoma cruzi strains, three groups of mice were infected with one of three strains of T. cruzi of different biological and isoenzymic patterns (Peruvian, 21SF and Colombian strains). Each group was subdivided into subgroups: 1-treated with nifurtimox; 2-treated with benznidazole and 3-untreated infected controls. At the end of treatment, that lasted for 90 days, xenodiagnosis, subinocculation of blood into new born mice and hemoculture were performed as tests of cure. From the positive tests, 22 samples of T. cruzi were isolated from all subgroups. Electrophoretic analysis of the isoenzymes PGM, GP1, ALAT and ASAT failed to show any difference between parasite strains isolated from treated and untreated mice, which indicates that no detectable clonal selection or parasite genetic markers alterations concerning the isoenzymes analysed have been determined by treatment with drugs of recognized antiparasitic effect, suggesting stability of the phenotypic characteristics of the three biological types of T. cruzi strains.

Morphological aspects of the myocarditis and myositis in Calomys callosus experimentally infected with Trypanosoma cruzi: fibrogenesis and spontaneous regression of fibrosis.

Calomys callosus a wild rodent, is a natural host of Trypanosoma cruzi. Twelve C. callosus were infected with 10(5) trypomastigotes of the F strain (a myotropic strain) of T. cruzi. Parasitemia decreased on the 21st day becoming negative around the 40th day of infection. All animals survived but had positive parasitological tests, until the end of the experiment. The infected animals developed severe inflammation in the myocardium and skeletal muscle. This process was pronounced from the 26th to the 30th day and gradually subsided from the 50th day becoming absent or residual on the 64th day after infection. Collagen was identified by the picro Sirius red method. Fibrogenesis developed early, but regression of fibrosis occurred between the 50th and 64th day. Ultrastructural study disclosed a predominance of macrophages and fibroblasts in the inflammatory infiltrates, with small numbers of lymphocytes. Macrophages had active phagocytosis and showed points of contact with altered muscle cells. Different degrees of matrix expansion were present, with granular and fibrillar deposits and collagen bundles. These alterations subsided by the 64th days. Macrophages seem to be the main immune effector cell in the C. callosus model of infection with T. cruzi. The mechanisms involved in the rapid fibrogenesis and its regression deserve further investigation.