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.

Combined treatment with a nitrofuranic and a corticoid in experimental Chagas' disease in the dog.

In order to study the effect of combined treatment of Trypanosoma cruzi infection with a nitrofuranic drug and a corticoid, 27 dogs from three different litters infected with T. cruzi (12 SF strain) were randomly litter-paired in three experimental groups: animals infected and not treated; infected and treated with Bay 2502 (2-methyl-4-[5'nitrofurfurylideneamino]-tetrahydro-4H-1,4-thiazine-1,1-dioxine) (nifurtimox); infected and treated with nifurtimox plus betamethasone. While an enhanced myocarditis appeared in the animals treated with a nitrofuranic drug alone, inflammation was almost abolished when corticoid treatment was added. Both groups showed considerable intracellular parasite destruction. These changes were monitored by serial electrocardiograms and a final histopathologic study which included an investigation of the changes in the conducting tissue by serial sectioning. The survival period was prolonged in animals treated with the combination of the nitrofuranic drug and corticoid, and only in this group did some of the animals reach the chronic phase of the infection. Thus, the association of a nitrofuranic drug with a corticoid in the treatment of acute Chagas' disease produced parasite destruction and inhibited the inflammatory responses that are enhanced by such destruction

Sequential changes of the connective matrix components of the myocardium (fibronectin and laminin) and evolution of cardiac fibrosis in mice infected with Trypanosoma cruzi.

Interstitial matrix alterations due to chronic Trypanosoma cruzi myocarditis were studied in mice by immunofluorescent microscopy with specific purified antibodies against the main different collagen isotypes, laminin and fibronectin. During the early subacute stage (26-30 days postinfection), sarcolemmal and perivascular deposits of laminin and fibronectin were prominent. The presence of fibronectin appeared to correlate with the presence of inflammatory cells. By the late subacute phase and early chronic phase (50-90 and 80-90 days postinfection, respectively), laminin and Type IV collagen were present. These were the principal features, although fibronectin continued to be found among inflammatory cells, and pro-III and III collagens formed irregular bands and periarteriolar deposits. During the late chronic phase (150-200 days postinoculation) the interstitium was enlarged and irregular, with positive staining for laminin, Types III, pro-III, and IV collagens; fibronectin appeared as focal, subendocardial, interstitial, and perivascular deposits. The relative absence of Type I collagen and the apparent positive correlation between interstitial matrix amplification and the presence of mononuclear inflammatory cells suggest that fibrotic changes in chronic T. cruzi myocarditis can be reversed if the inflammatory changes subside.

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.

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.

The influence of the strain of Trypanosoma cruzi in placental infections in mice.

Pregnant female mice were infected with Trypanosoma cruzi strains which differed according to several parameters and were classified as three different types. Mice were killed during either the acute or the chronic phase of infection. Animals' tissues and foetuses together with the placentas, were studied histopathologically. Clear cut differences were noted in the incidence of placental parasitism and in the localization of amastigotes in the vascular sinus of the placenta amongst the animals in the acute phase of the infection with different strains. No parasitism of the foetal tissues was seen. The incidence of placental parasitism reached 98% for the Colombian strain, 18.4% for the Peruvian strain, 17% for the Y strain and 13.2% for the Honorina strain (isolated from a woman that transmitted the infection to twins). The presence of parasites in the vascular part of the placenta was prominent with the Colombian strain and rare with the others. These experimental data seem to show that parasite strain plays a role in congenital T. cruzi infection.

Correlation between isoenzyme patterns and biological behaviour of different strains of Trypanosoma cruzi.

Three strains of Trypanosoma cruzi, used previously as prototypes for a classification based on the host-parasite relationships, as well as several stocks isolated from different geographical areas in Brazil, were submitted to isoenzymic analysis. Their isoenzyme patterns revealed a clear correlation with the biological data. The patterns obtained with the enzymes PGM, GPI, ASAT and ALAT permitted discrimination between each of the described types. Only one type was found in each geographical area studied, indicating a possible relationship between regional patterns and clinical presentation of Chagas' disease.

Specific chemotherapy of Chagas disease: a comparison between the response in patients and experimental animals inoculated with the same strains.

Eleven strains of Trypanosoma cruzi were isolated from patients with Chagas disease in central Brazil by xenodiagnosis and inoculation into newborn mice. Biological characterization and isoenzyme analysis showed that 6 strains were type II (zymodeme 2) and 5 were type III (zymodeme 1). Patients were treated with benznidazole or benznidazole plus nifurtimox. Mice infected with each isolated strain were treated for comparison with the results obtained in the respective patient. Evaluation of cure of the patients was based on the indirect immunofluorescence test, complement fixation reaction and xenodiagnosis. For the mice, haemoculture, indirect immunofluorescence testing, xenodiagnosis and inoculation of blood into newborn mice were used. Tests were performed 3-6 months after the end of treatment. The cure rate was 66-100% in mice infected with type II strains and 0-9% in those infected with type III strains. The correlation between treatment results in patients and mice was 81.8% (9 of 11 cases). Type II strains were more susceptible to treatment, in contrast to type III strains which yielded the majority of therapeutic failures.

Trypanosoma cruzi strain-specific monoclonal antibodies: identification of Colombian strain flagellates in the insect vector.

Spleen cells from mice immunized with insect-derived Trypanosoma cruzi metacyclic trypomastigotes were used to obtain Colombian strain-specific monoclonal antibodies. At least 4 different strain-specific antigens were recognized by the monoclonal antibodies on epimastigotes or metacyclic trypomastigotes. There was no reactivity with other stages of Colombian strain T. cruzi, nor with any stage of 15 other T. cruzi strains or isolates, nor with 22 other Trypanosomatidae. One of the monoclonal antibodies was used to identify, by indirect immunofluorescence, Colombian strain flagellates in cryostat sections or glass-slide smears of the insect vector's intestine.

Experimental Chagas' disease in dogs. A pathologic and ECG study of the chronic indeterminate phase of the infection.

Pathology of the chronic latent phase of Chagas' disease is poorly understood. In our experiment, nine dogs were inoculated with Trypanosoma cruzi and acute disease developed within 15 to 25 days, followed by a chronic asymptomatic period that varied from eight months to three years, at which time the animals were killed. Although ECG changes were minimal, all animals showed a mild focal chronic myocarditis, with a few microscopic foci of fibrosis. Focal or diffuse fibrosis, scleroatrophy, and fatty replacement were present in various parts of the conducting system, especially in the atrioventricular node, the distal portion of His' main bundle, and the initial portion of the right bundle. These lesions were considered to be sequelae from inflammation and necrosis during the acute phase of the infection. In addition to characterizing the lesions of the chronic indeterminate phase of Chagas' disease in the canine model, the present findings suggest that the lesions may be responsible for ECG changes that may appear in subjects with otherwise asymptomatic T cruzi infection.

Electrocardiographic changes in experimental chronic murine Chagas' disease.

An electrocardiographic study was performed on 26 AKR and 32 A/J inbred mice and on 100 Swiss outbred mice, chronically infected with different strains of T. cruzi, characterized as Types I, II and III. The incidence of electrocardiographic alterations in AKR mice was of 87.0%, 80.0% and 83.3% respectively for infection with the Type I, Type II and Type III strains of T. cruzi. In A/J mice the incidence of electrocardiographic alterations was 100% in the infection with the Type I and Type III strains and 26.1% with the Type II strain of T. cruzi. In Swiss mice the electrocardiographic alterations occurred in 53.5% of the mice infected with the Type II strain and in 71.4% of those infected with the Type III strain of T. cruzi. The most frequent electrocardiographic alterations in chronically infected mice, independently of mouse or T. cruzi strain, were first degree AV block, intraventricular conduction abnormalities, sinus tachycardia and bradycardia. The predominant alterations caused by each of the T. cruzi strains varied according to mouse strain. In A/J mice, the electrocardiographic alterations were more frequent in those animals infected with Type I and III strains of T. cruzi and in Swiss mice the alterations were more frequent in those infected with the Type III strain. The results presented in this study demonstrate that the murine model is suitable for electrocardiographic studies related to the heart lesions that occur in Chagas' disease.

Patterns of resistance of inbred mice to Trypanosoma cruzi are determined by parasite strain.

Host response and parasite behavior of three different T. cruzi strains (I-Peruvian, II-21SF, III-Colombian) were investigated by evaluating the course of infection in six inbred strains of mice (A/J, AKR, C3H/He, BALB/c, C57BL/10, DBA/1). Resistance was evaluated in terms of the harmonic mean survival time and the infection was monitored by parasitemia, histopathology and immunological parameters (immunoglobulin subclass levels and antibody titers). All six mouse strains showed high susceptibility to the Peruvian strain (Type I). However, they displayed a different spectrum of susceptibility to Types II and III. Each T. cruzi strain maintained its basic features in the different mouse strains. Despite different maximum levels, the parasitemic curves were characteristic for each type of T. cruzi strain. There was a correlation between the degree of resistance of strains DBA and B10 and their high levels of IgG2a and IgG2b, as well as the presence of the H-2d haplotype, indicating that the genetic background of the mice is also important. The inflammatory process varied with each mouse strain and was correlated with the levels of IgG2a, with resistant mice showing predominance of neutrophilic infiltration with a rise in IgG2a. The susceptible strains responded with a mild inflammatory process with predominance of mononuclear cells. These data suggest that the parasite strain is the most important factor determining the resistance of the different mouse strains to infection with 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.

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).

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.

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.

[Behavior of Trypanosoma cruzi strains after passage in different triatominae species]. / Estudo do comportamento de cepas de Trypanosoma cruzi após passagem em diferentes espécies de triatomíneos.

To study the influence of the intermediate host stage on the course of mouse infection, Trypanosoma cruzi belonging to the Peruvian (Type I), 12 SF (Type II) and Colombian (Type III) strains were passaged through either Rhodnius prolixus, Panstrongylus megistus or Triatoma infestans. T. cruzi metacyclic forms (dose 10(4)) from the different strains were obtained from each bug and inoculated into 8-10 gm mice. Comparison was made in mice inoculated with blood forms. Parasitaemia curves were plotted in the peripheral blood for each strain, reaching more elevated peaks with Peruvian strain parasites from P. megistus and R. prolixus, 12 SF strain from P. megistus and Colombian strain from R. prolixus. Tissue tropism and histopathological patterns were those usually seen in mice infected with each respective strain type. Peruvian virulence was the same for all groups. Slender forms predominate among mice inoculated with metacyclic forms of Colombian and 12 SF strains, probably an adaptative parasite change related to the intermediate host.

[Therapy of the chronic phase of the experimental infection by Trypanosoma cruzi with benzonidazole and nifurtimox]. / Terapêutica da fase crônica da infeccao experimental pelo Trypanosoma cruzi com o benzonidazol e o nifurtimox.

Fifty-eight mice, chronically infected with different T. cruzi strains (Types II and III) were submitted to chemotherapy either with Nifurtimox (Bay 2502) or Benznidazole (Ro 7-1051). Twenty one mice were not treated and were used as infected controls. The duration of infection was from 90 to 400 days. Inocula varied from 1 x 10(4) to 5 x 10(4) blood forms. Treatment lasted for 90 days, doses being 200mg/kg/day during 4 days, followed by 50mg/kg/day for Nifurtimox and 100mg/kg/day for Benznidazole. Parasitological tests (xenodiagnosis, inoculations into baby mice and hemoculture) showed 85.3% negativation for Type II strains and 43% for Type III in animals treated with Benznidazole. As for Nifurtimox, there were 71.4% of parasitological negativation for the animals infected with Type II strains and 66% for those infected with Type III. IFA tests remained positive in 90% of treated and cured animals. Disappearance or marked regression of myocardial and skeletal muscle lesions was seen in the treated and parasitologically negative animals. The conclusion is that the treatment in the chronic phase of T. cruzi infection can result in parasitological cure in a high percentage of cases with regression of histopathological lesions, although with persistence of positivity of the IFA tests.

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.