Comparative study of sensitivity and mortality of Triatoma infestan nymphs III and IV used in the xenodiagnosis of chronic chagasic patients]

Triatoma infestans nymphs have shown a good sensitivity for detecting Trypanosoma cruzi in the blood stream of infected hosts when are used in the xenodiagnosis (XD). This method, with its natural limitations, using seven nymphs III of T. infestans, has been routinely utilized with a satisfactory yield. With the aim of an eventual improving of the yield of XD (with 7 nymphs), two series of 54 XD boxes each, containing a total of 378 nymphs III and 378 nymphs IV respectively, were applied--one of each during three consecutive days--to nine chronic chagasic patients. Each of the nymphs was weighted before and after the application of the boxes, and the intestinal content of them was examined 30, 60 and 90 days later. The main comparative results obtained with nymphs III and IV of T. infestans were: blood ingestion 40 versus 107 mg (2.7 higher), positivity of insects 35.8% versus 50.6% (15.8% higher), positivity of XD boxes (7 nymphs each) 46.3% versus 55.6% (9.3% higher), and mortality rates 28.6% versus 12.2% (16.4% lower). All these results demonstrate that nymphs IV of T. infestans, because their higher capacity of ingesting blood and higher tolerance to examination manipulations, are more suitable for been used in XD.     

Yielding of xenodiagnosis, according to the number of boxes used in 1,181 persons with chronic chagasic infection diagnosed with indirect hemagglutination reaction]

A study for evaluation of the yielding of xenodiagnosis (XD) in 1,181 persons with a previous positive indirect hemagglutination test (IHAT) for Trypanosoma cruzi infection was carried out. The infection of these people was detected during epidemiological surveys performed in rural-periurban and urban sections of the endemo-enzootic area of Chagas' disease in Chile, which involves the first seven, out of the thirteen political-administrative regions of the country. The sex distribution was 75.0% females and 25.0% males, varying the ages between 2 and 80 years. According to individual and geographical possibilities each person was submitted to 1-8 XD, which consisted in cylindrical wooden boxes containing seven third instar nymphs, laboratory reared, of Triatoma infestans. The boxes, covered with a piece of tulle fixed with a rubber band were applied on the skin of the posterior side of the arm of the subject to be examined, held with a linen bracelet during 25-30 min. After the insects were fed the boxes were maintained in the laboratory at 27 degrees C and 85% relative environmental humidity. Posteriorly, all the nymphs of each box were examined at 30, 60 and 90 days after the application. A drop of abdominal content of each of them, homogenized with a drop of saline, was examined at the microscope looking for T. cruzi. XD resulted positive in 503 (42.6%) people. The positiveness of XD showed a trend of increasing according to the number of boxes used, from 11.4% with one to 51.6% with six. Under a practical point of view, the simultaneous application of four XD boxes seems to be advisable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monitoring the efficacy of specific treatment in chronic Chagas disease by polymerase chain reaction and flow cytometry analysis

PCR and FC-ALTA were used to monitor parasite clearance in 54 chronic chagasic patients who had completed therapy with allopurinol (ALLO, n = 31) or itraconazole (ITRA, n = 23) ten years earlier. All patients maintained positive conventional serology. 25 of them showed positive XD (ALLO, n = 11 and ITRA, n = 14) and 29 negative XD (ALLO, n = 20 and ITRA, n = 9). 43 patients were positive by both techniques (ALLO, n = 23 and ITRA, n = 20). Seven of 54 patients were negative by PCR and positive by FC-ALTA and three of 54 were positive by PCR and negative by FC-ALTA. Only one case with both tests negative should be considered cured. Of 29 patients with negative XD, 14 treated ALLO (70 %) and nine with ITRA (77.8 %) showed positive PCR and FC-ALTA. These results do not show differences of efficacy among the drugs, and reinforce the relevance of using sensitive tools such as PCR and FC-ALTA for the follow-up of patients with chronic Chagas disease.     

Evaluation of Chagas' disease transmission through breast-feeding

One hundred milk or colostrum samples from 78 mothers with chronic Chagas' disease were parasitologically studied for Trypanosoma cruzi infection by means of direct examination and inoculation of mice. The mice were submitted to direct blood examination three times a week. At the end of 45 days, xenodiagnosis and indirect immunofluorescent test (IFAT) for T. cruzi antibodies were carried out in the animals. No parasitized sample was observed even though five mothers had parasitemia at milk collection. In addition, 97 breast-fed children of chronic chagasic mothers, born free of infection, were tested for IgG antibodies to T. cruzi using IFAT. No case of T. cruzi infection was detected. The authors conclude that breast-feeding should not be avoided for children of chronic chagasic women. However, as these mothers had intermittent parasitemia, they should avoid nursing when there is nipple bleeding.     

Isolation of Trypanosoma cruzi samples by xenodiagnosis and hemoculture from patients with chronic Chagas' disease

Fifty nine chronic chagasic patients were simultaneously submitted to xenodiagnosis and hemoculture for Trypanosoma cruzi samples isolations. The xenodiagnosis was done with 40 Panstrongylus megistus, Triatoma infestans and Dipetalogaster maximus nymphs, performing 120 triatomines. Groups of 10 insects per specie were dissecated and the intestinal content pooled and examined, after previous trituration and homogenization. The microscopically negative material was seed into LIT medium and examined after 20 days. Twenty nine patients were parasitologically proved, being 15 only by xenodiagnosis, 4 only by hemoculture and 10 by both methods. It was discussed the parasitological comprovation difficulties in chronic chagasic patients, the value of the simultaneous utilization of different triatomine species in xenodiagnosis and the hemoculture, in a favorable positive association to the sensitivity increase in the diagnosis' disease. The 49.2% of positivity obtained in this group, visualize approaches like clinic-therapeutic assay and or epidemiological (case-control) with the purpose to investigate a possible association with T. cruzi samples and different clinic forms in Chagas' disease.     

MicroRNA Transcriptome Profiling in Heart of Trypanosoma cruzi-Infected Mice: Parasitological and Cardiological Outcomes

Chagas disease is caused by the parasite Trypanosoma cruzi, and it begins with a short acute phase characterized by high parasitemia followed by a life-long chronic phase with scarce parasitism. Cardiac involvement is the most prominent manifestation, as 30% of infected subjects will develop abnormal ventricular repolarization with myocarditis, fibrosis and cardiomyocyte hypertrophy by undefined mechanisms. Nevertheless, follow-up studies in chagasic patients, as well as studies with murine models, suggest that the intensity of clinical symptoms and pathophysiological events that occur during the acute phase of disease are associated with the severity of cardiac disease observed during the chronic phase. In the present study we investigated the role of microRNAs (miRNAs) in the disease progression in response to T. cruzi infection, as alterations in miRNA levels are known to be associated with many cardiovascular disorders. We screened 641 rodent miRNAs in heart samples of mice during an acute infection with the Colombiana T.cruzi strain and identified multiple miRNAs significantly altered upon infection. Seventeen miRNAs were found significantly deregulated in all three analyzed time points post infection. Among these, six miRNAs had their expression correlated with clinical parameters relevant to the disease, such as parasitemia and maximal heart rate-corrected QT (QTc) interval. Computational analyses identified that the gene targets for these six miRNAs were involved in networks and signaling pathways related to increased ventricular depolarization and repolarization times, important factors for QTc interval prolongation. The data presented here will guide further studies about the contribution of microRNAs to Chagas heart disease pathogenesis.     

Household chagasic endemia detected as a consequence of a case of congenital infection]

During a study on prevalence of parasitic and viral serological markers in pregnant adolescents, a 17-year-old primipara from Polpaico, village near Santiago, gave birth to a normal male newborn in a Santiago hospital. As both of them presented positive an indirect hemagglutination test (IHAT) for Chagas' disease and the corresponding xenodiagnosis (XD), the infection in the infant was considered to be acquired through the placental route. According to recent epidemiological surveys Polpaico is an endemo-enzootic chagasic rural settlement, where 14.7% of dwellings were infested with Triatoma infestans, while triatominae, persons and domestic mammals were found infected with Trypanosoma cruzi. One month later the adolescent mother, her son and other 11 consanguineous members of the family were visited in their homes in order to submit each of them to a physical examination and to IHAT for Chagas' disease, and XD to those whose IHAT resulted positive. Thus, in 7 (53.8%) the IHAT was positive and in 4 (57.1%) out of these 7 presented positive the XD, results that as a whole yielded a household chagasic endemics.     

Morbidity in Chagas' disease. III. Longitudinal study of 6 years, in Virgem da Lapa, MG, Brazil

In a clinical, radiological and electrocardiographical, follow-up study of the "case control" type performed in Virgem da Lapa, Minas Gerais State, Brazil, 124 chagasic patients were followed during six years. The results of the patients, the majority in the indeterminate form, did not register any change, in 32.2% there was a progress in the disease and in 5.6% the electrocardiogram returned to normal. These results when compared to that achieved by the control group, composed of pairs of non chagasic persons with the same age and sex, was shown to be 27.4% higher than among patients with positive serology. This factor represents the excess risk or exclusively chagasic component in the development of the disease. No differences were observed by sex related to the development of the disease. It was more premature and seven times more frequent however when related to the cardiopathy than to the megaesophagus. Both conditions occurring mainly in slight or moderate degree. In 192 chagasic patients and 188 non chagasic persons observed in that area in the same period, the mortality was 3.6 times higher among the chagasic patients with a letality due to cardiopathy of 8.9% without difference between sexes but more premature among the males. Sudden death was more frequent than that one caused by cardiac insufficiency. The prognostic was good for the patients with indeterminate and digestive forms and reserved for patients with the highest degree of cardiopathy.     

Experimental Chagas' disease in dogs.

This paper describes the development of experimental Chagas' disease in 64 out-bred young dogs. Twenty-nine animals were inoculated with the Be-62 and 35 with Be-78 Trypanosoma cruzi strains. Twenty-six were infected with blood trypomastigotes by different inoculation routes and 38 with metacyclic trypomastigotes from the vector via the conjunctival route. Twenty of the 26 dogs infected with blood trypomastigotes were autopsied during the acute phase. Eleven died spontaneously and nine were sacrificed. Six remained alive until they died suddenly (two) or were autopsied. (four). Twelve of the 38 dogs infected with metacyclic trypomastigotes evolved naturally to the chronic phase and remained alive for 24-48 months. The parasitemia, clinical aspects and serology (IgM and IgG) as well as electrocardiogram, hemogram and heart anatomo-histopathologic patterns of acute and chronic cardiac forms of Chagas' disease as seen in human infections, were reproduced. The most important finding is the reproducibility of diffuse fibrosing chronic chagasic cardiopathy in all dogs infected with Be-78 T. cruzi strain autopsied between the 90th and 864th days of infection. Thus, the dog can be considered as a suitable experimental model to study Chagas' disease according to the requisites of the Word Health Organization (1984). Furthermore the animal is easily obtained and easy to handle and maintain in experimental laboratory conditions.     

Integrate study of a Bolivian population infected by Trypanosoma cruzi,the agent of Chagas disease

A cross section of a human population (501 individuals) selected at random, and living in a Bolivian community, highly endemic for Chagas disease, was investigated combining together clinical, parasitological and molecular approaches. Conventional serology and polymerase chain reaction (PCR) indicated an active transmission of the infection, a high seroprevalence (43.3%) ranging from around 12% in < 5 years to 94.7% in > 45 years, and a high sensitivity (83.8%) and specificity of PCR. Abnormal ECG tracing was predominant in chagasic patients and was already present among individuals younger than 13 years. SAPA (shed acute phase antigen) recombinant protein and the synthetic peptide R-13 were used as antigens in ELISA tests. The reactivity of SAPA was strongly associated to Trypanosoma cruzi infection and independent of the age of the patients but was not suitable neither for universal serodiagnosis nor for discrimination of specific phases of Chagas infection. Anti-R-13 response was observed in 27.5% only in chagasic patients. Moreover, anti-R13 reactivity was associated with early infection and not to cardiac pathology. This result questioned previous studies, which considered the anti-R-13 response as a marker of chronic Chagas heart disease. The major clonets 20 and 39 (belonging to Trypanosoma cruzi I and T. cruzi II respectively) which circulate in equal proportions in vectors of the studied area, were identified in patients' blood by PCR. Clonet 39 was selected over clonet 20 in the circulation whatever the age of the patient. The only factor related to strain detected in patients' blood, was the anti-R-13 reactivity: 37% of the patients infected by clonet 39 (94 cases) had anti-R13 antibodies contrasting with only 6% of the patients without clonet 39 (16 cases).     

Morbidity in Chagas' disease. IV. Longitudinal study of 10 years in Pains and Iguatama, Minas Gerais, Brazil

An evolutive study of the "case-control" type was carried out in an endemic area of Chagas' disease in Minas Gerais State, Brazil, using two cross-section evaluations with an interval of ten years between them (1974-1984). Patients were paired for sex and age. In the first cross-section study 264 pairs one with a positive serology and the other with a negative serology for T. cruzi antibodies were included. In the second evaluation, ten years later, 235 patients among those with previous positive serology and 216 with negative serology were located, but only 110 pairs could be recomposed and reexamined (clinical examination, ECG and Rx of the heart and esophagus). The incidence of chagasic cardiopathy in the cases with positive serology but previously assymptomatic was 38.3% during the ten year period. On the other hand there was a deterioration in 24% of the patients with chagasic cardiopathy since the first examination. Considering all clinical forms of the disease in 34.5% of the patients the clinical situation deteriorated, in 57.3% there was no change and in 8.2% the situation improved. The general mortality in the period was 23% in the chagasic group and 10.6% in the control group, but the lethality by cardiopathy was 17% in chagasic group and only 23.3% in the control group. The mortality was twice as high in males than in females, mainly in the age group from 30 to 59 years.     

Absence of interferon-gamma-inducible gene IGTP does not significantly alter the development of chagasic cardiomyopathy in mice infected with Trypanosoma cruzi (Brazil strain)

Interferon-gamma (IFN-gamma) contributes to host resistance during acute infection with Trypanosoma cruzi, the causative agent of Chagas' disease. Inducibly expressed guanosine triphosphatase (IGTP), a 48-kDa guanosine triphosphatase (GTPase), is a member of a family of GTPase proteins inducibly expressed by IFN-gamma. The expression pattern of IGTP suggests that it may mediate IFN-gamma-induced responses in a variety of cell types. IGTP has been demonstrated to be important for control of Toxoplasma gondii infection but not for resistance against Listeria monocytogenes. We evaluated the role of IGTP in development of chronic chagasic cardiomyopathy in IGTP null mice and C57X129sv (wild type [WT]) mice infected with the Brazil strain for 6 mo. There was no significant difference in parasitemia or cardiac histopathology between null and WT mice. Right ventricular remodeling was observed in infected IGTP null mice, suggesting that IGTP does not significantly alter the course of T. cruzi infection.     

Biological characterization of Trypanosoma cruzi strains from different zymodemes and schizodemes.

The development in C3H mice of thirteen strains of Trypanosoma cruzi belonging to different zymodemes and schizodemes was studied. Host mortality, virulence, histiotropism, parasitemia and polymorphism of the parasites were recorded. The strains were grouped into: a) high virulence--causing 100% mortality and characterized by predominance of very broad trypomastigotes in the bloodstream at the end of infection; b) medium virulence--causing no mortality and with a predominance of broad trypomastigotes; c) low virulence--causing no mortality with blood forms not described due to the very low parasitemia. During 18 months maintenance the parasitemia curves were kept constant for all strains except one. A direct correlation between either zymodeme or schizodeme and experimental biological properties of T. cruzi strains was not found. However, the parasitemia was subpatent and patent for strains from zymodeme C and the others respectively. Furthermore the high virulence seems to be related to one of two schizodemes found within zymodeme B strains. All strains presenting patent parasitemia independent of shizodeme and zymodeme showed a myotropism towards heart and skeletal muscle with variable inflammatory intensity. The present study confirmed the heterogeneity found by isoenzyme and k-DNA patterns among the strains of T. cruzi isolated from chagasic patients in Bambuí, Minas Gerais State, Brasil.     

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.     

Rapid determination of Trypanosoma cruzi urinary antigens in human chronic Chagas disease by agglutination test

Detection of Trypanosoma cruzi in man becomes particularly difficult during the chronic stage of Chagas disease because of the low parasitemia. We were able to develop a simple and straightforward method for determining the concentration of T. cruzi antigens in urine using nitrocellulose micellar suspension (Nitrocell-Mr, Polychaco Argentina) and for their subsequent detection through a "latex" type agglutination test. The latex used was an esferocell nitrocellulose suspension (Esferocell-Mr, Polychaco). Specific antigens for T. cruzi were detected in 54 of 58 urine samples from chronic chagasic patients. The antigens characterized by affinity chromatography and SDS-PAGE were glycoproteins with apparent molecular weights (and pIs) of 100 kDa (pI 5 to 5.5), 80 kDa (pI 6.0), and 50 kDa (pI 6.5 to 7.0). This method is practical and fulfills the requirement of large-scale epidemiological studies. It is also helpful in cases of conflictive serology.     

Evaluation of the xenodiagnosis of chronic Chagas patients infected ten years or over in an area where transmission has been interrupted--Iguatama and Pains, west Minas Gerais State, Brazil.

To evaluate the results of xenodiagnosis in chronic Chagas patients infected for ten years or over in an area where transmission has been stemmed as well as the performance of these tests applied one or more times to determine the presence of the parasite in serum-positive patients for Trypanosoma cruzi infection, 570 xenodiagnosis were performed in 246 patients by exposing each patient to 40 Triatoma infestans nymphs of 3rd/4th stage once, twice or three times, at 30 days intervals. The 570 xenodiagnosis showed overall positive results in 50.7% with a peak 78% in patients under 20 years of age, and 60.5% in those over 60. Of the 158 patients who underwent three xenodiagnosis, 51 (32.3%) had three positive tests, 48 (30.3%) had all negative results, and the remainder had alternating positive and negative findings. There was no difference in number of positive results between the 1st, 2nd and 3rd tests; however, the 1st and 2nd trials added up to 53.2% and the sum total of all three trials was 57.7%.     

Trypanosoma cruzi: host selenium deficiency leads to higher mortality but similar parasitemia in mice

Selenium is an essential trace element and its deficiency was implicated in heart diseases. We recently showed low Se levels in chronic chagasic patients with cardiomyopathy. Herein, mice were depleted in Se by feeding the mothers with chow containing only 0.005 mg Se/kg and maintaining this diet for offspring, that were further infected with Trypanosoma cruzi. Survival rate was significantly lower in Se deficient than in control mice. Parasitemia was similar in all groups. Necrotic heart lesions were found after infection (high CK-MB levels). No outbreaks of parasite growth were detected in chronic survivors submitted or not to a second Se depletion. The present results confirm our hypothesis that a nutritional deficiency in Se is associated to a higher mortality during T. cruzi infection. The potential beneficial effect of Se supplementation is a perspective. Hypothesis to explain the higher susceptibility of Se-depleted mice to T. cruzi infection are discussed.     

Alterations in myocardial gene expression associated with experimental Trypanosoma cruzi infection

Chagas disease, characterized by acute myocarditis and chronic cardiomyopathy, is caused by infection with the protozoan parasite Trypanosoma cruzi. We sought to identify genes altered during the development of parasite-induced cardiomyopathy. Microarrays containing 27,400 sequence-verified mouse cDNAs were used to analyze global gene expression changes in the myocardium of a murine model of chagasic cardiomyopathy. Changes in gene expression were determined as the acute stage of infection developed into the chronic stage. This analysis was performed on the hearts of male CD-1 mice infected with trypomastigotes of T. cruzi (Brazil strain). At each interval we compared infected and uninfected mice and confirmed the microarray data with dye reversal. We identified eight distinct categories of mRNAs that were differentially regulated during infection and identified dysregulation of several key genes. These data may provide insight into the pathogenesis of chagasic cardiomyopathy and provide new targets for intervention.     

Cell mediated immunity in Chagas' disease. Trypanosoma cruzi antigens induce suppression of the in vitro proliferative response of mononuclear cells.

The partial suppression of the cell-mediated immune response by Trypanosoma cruzi antigens in patients with Chagas' disease is demonstrated in a costimulation assay with T. cruzi antigens and Mycobacterium tuberculosis purified protein derivative (PPD) or Tetanus toxoid (TT). Mononuclear cells from 13 patients with chagasic infection without evidence of heart disease, 10 patients with chagasic cardiomyopathy and 7 healthy blood bank donors were stimulated with antigen A (autoclaved epimastigotes), PPD, TT, PPD + A, PPD + TT and TT + A. The average percentage of suppression induced by costimulation of mononuclear cells with PPD and antigen A was 47.1% in patients with chagasic infection without heart disease (INF), 38.8% in patients with chagasic cardiomyopathy (CDM) and 23.3% in healthy controls. Similar values were observed when living trypomastigotes were used. A costimulatory study with PPD and TT, PPD and A and TT and A was carried out in 8 patients with chagasic infection, in order to evaluate the possibility that this difference could be due to a nonspecific inhibitory effect. The mean suppression induced by TT + PPD was -8.9, with TT + A was 52.7 and with PPD + A was 50.1. The data reported show that T. cruzi antigens induce a specific suppression of the proliferative response of mononuclear cells, that might be relevant to the persistence of the parasite in the host.