Molecular phylogeography of the Chagas' disease vector Triatoma infestans in Argentina.

Triatoma infestans is the main vector of Chagas' disease in South America between latitudes 10°S and 46°S. A multilocus microsatellite data set of 836 individuals from 27 populations of T. infestans, from all its range of distribution in Argentina, was analyzed. Our results favor the hypothesis of two independent migration events of colonization in Argentina and secondary contacts. The majority of the populations of the western provinces of Catamarca, La Rioja, San Juan and the west of Cordoba province, had almost no shared ancestry with the rest of the populations analyzed. Probably those populations, belonging to localities close to the Andean region, could have been established by the dispersal line of T. infestans that would have arrived to Argentina through the Andes, whereas most of the rest of the populations analyzed may have derived from the dispersal line of T. infestans in non-Andean lowlands. Among them, those from the provinces of Formosa, Chaco, Santiago del Estero and Santa Fe shared different percentages of ancestry and presented lower degree of genetic differentiation. The migratory movement linked to regional economies and possibly associated with passive dispersal, would allow a higher genetic exchange among these populations of T. infestans. This study, using microsatellite markers, provides a new approach for evaluating the validity of the different hypotheses concerning the evolutionary history of this species. Two major lineages of T. infestans, an Andean and non-Andean, are suggested.

Inhibitory action of gossypol on enzymes and growth of Trypanosoma cruzi.

Gossypol, a phenolic compound isolated from the cotton plant, is a powerful inhibitor of nicotinamide adenine dinucleotide-linked enzymes (alpha-hydroxyacid dehydrogenase and malate dehydrogenase) of Trypanosoma cruzi, the parasite that causes Chagas' disease. Parasites at the epimastigote stage that were incubated for 5 minutes with 100 micromolar gossypol were completely immobilized. Concentrations of gossypol as low as 0.01 micromolar markedly reduced the growth rate of T. cruzi in culture.

Mitochondrial 16S DNA variation in populations of Triatoma infestans from Argentina.

Variation in the mtDNA 16S ribosomal RNA gene in populations of Triatoma infestans (Klug) was surveyed. DNA sequence comparisons yielded 18 haplotypes among 130 individuals from 16 localities that represent a large proportion of the range of T. infestans in Argentina. The most common genotype in all populations was found in 76.9% of individuals and two other haplotypes were shared among different populations. The remaining 15 haplotypes were present exclusively in one of the populations, suggesting currently low levels of genetic exchange. Analysis of mtDNA 16S sequences uncovered substantial genetic variation among T. infestans populations. Haplotype and nucleotide diversities varied among populations, from 0% to 0.84% and 0% to 0.29%, respectively. It appears that this locus has a low mutation rate. Uncorrected pairwise differences of T. infestans haplotypes ranged from 0% to 1.2%. The molecular phylogeny supported the monophyly of T. infestans haplotypes and clustered two different pairs of haplotypes with a moderate degree of bootstrap support (approximately 60%). Mitochondrial DNA phylogeographic differentiation was not evident, suggesting a recent rapid spread of the species. Analysis of molecular variance showed hierarchical structure in the data. Considerably less variation was found among T. infestans populations from the northwest and northeast regions than among those belonging to the central area. Such a lack of variation may be indicative of one or more past population bottlenecks.

Density gradient purification of human lymphocytes from contaminating trypomastigotes of Trypanosoma cruzi.

Mixtures of normal human lymphocytes and T. cruzi trypomastigotes obtained from infected mice were centrifuged over Ficoll-Hypaque (FH) continuous and discontinuous gradients. Trypomastigotes were confined to the range 1.051-1.057 g/ml while lymphocytes ranged between 1.046 and 1.080 g/ml. Over 80% of the lymphocytes were found at 1.060 g/ml or higher densities. A discontinuous gradient of FH with 2 layers of 1.060 and 1.077 g/ml of density respectively was selected to obtain trypomastigotes-free white blood cells from blood samples. The functional capacity of lymphocytes recovered from the lower interface, where no parasites were found, was assessed. The response to phytohaemagglutinin of these high density lymphocytes was as good as to total lymphocytes, suggesting that low density lymphocytes are not necessary for proliferative responses. It is postulated that high density lymphoid populations, free from T. cruzi forms, may be used to study the presence of T cell-mediated immune response in Chagas' disease patients.

Isolation and characterization of a gene from Trypanosoma cruzi encoding a 46-kilodalton protein with homology to human and rat tyrosine aminotransferase.

The complete sequence of a gene encoding a 46-kDa protein of Trypanosoma cruzi is presented. The first ATG complies with the consensus sequence for initiation of translation. A single band of 2 kb was highlighted by hybridizing a probe from the 46-kDa protein gene to a Northern filter containing total T. cruzi RNA. The gene is present in 50-80 copies per cell and most of them are contained in 2 tandem arrays on large T. cruzi chromosomes (> 2000 kb). A strong homology with rat and human tyrosine aminotransferase was detected. Homology with a Trypanosoma brucei retrotransposon was found in the nonsense strand of the intergenic region.

Monoclonal antibodies against the flagellar fraction of epimastigotes of Trypanosoma cruzi: immunoprotection against metacyclic trypomastigotes obtained by immunization of mice with an affinity-purified antigen.

Subcellular fractions of Trypanosoma cruzi (epimastigotes) were assayed in their capacity to induce protective or aggressive effects in experimental animals. The flagellar fraction showed the best immunoprotective properties without tissular aggression. Monoclonal antibodies were prepared from mice immunized with this fraction. One of them, FCH-F8-4, was able to neutralize the infectivity of bloodstream trypomastigotes, to produce complement-mediated lysis on cell culture-derived trypomastigotes and to recognize the surface of trypomastigotes and epimastigotes by immunofluorescence. FCH-F8-4 reacted in Western blotting with several epimastigote proteins ranging from 50 to 150 kDa, showing a more intense reactivity with 4 bands while it reacted with two molecules on trypomastigote preparations (15 and 48 kDa). Purified antibody was coupled to CNBr-activated Sepharose and used to purify antigens from epimastigote extracts. These antigens were used to immunize BALB/c mice in the presence of Bordetella pertussis as adjuvant. Animals were protected against a challenge with 10(3) metacyclic forms of T. cruzi (Tulahuén strain). Only 40% of immunized mice presented detectable parasites in blood after challenge. Parasitemia decreased 90% in relation to controls in those animals. Survival of immunized mice was 100% in all immunoprotection experiments. These results suggest that the epitope recognized by FCH-F8-4 present in the purified antigens keeps the protective characteristics of flagellar fraction and could be a candidate for the development of a vaccine against T. cruzi infection.

Cellular immunity in Chagas' disease patients. Lymphoproliferative response to subcellular fractions of Trypanosoma cruzi.

The ability of peripheral mononuclear cells from Chagas' disease patients (CDP) to generate specific proliferative responses in vitro to whole homogenate (WH) and subcellular fractions of T. cruzi was investigated. Flagellar (F), microsomal (Mc) and cell sap (CS) fractions were isolated from a WH obtained by disruption by pressure-depressure. Parasite-free human peripheral mononuclear cells were isolated by centrifugation of leukocyte-rich plasma over discontinuous gradients of Ficoll-Hypaque and cultured for 6 days in the presence of the different fractions of T. cruzi. Eighty-six percent (54/63) of the CDP showed good capacity to respond to at least one of the fractions assayed while only 18% (4/22) of the controls reacted. The Mc fraction showed the best specificity as well as the maximal stimulating effect in CDP (37/55 vs 0/18 in controls) while WH (62% vs 10%), F (54% vs 16%) and CS (52% vs 6%) showed a lesser antigenic capacity. These results suggest that recognition mechanisms and proliferative responses are functional in CDP. No correlation was found between the stimulating capacity of fractions in vitro and their immunogenic capacity previously observed in vivo.

Humoral immune response to cruzipain and cardiac dysfunction in chronic Chagas disease.

The humoral immune response to epitopes expressed on cruzipain was evaluated in 31 Chagas disease patients (CDP) with different degrees of cardiac dysfunction. We took advantage of the availability of anti-Trypanosoma cruzi microsomal fraction monoclonal antibodies (MoAbs) reactive with epitopes that are recognized (5A9B11) or not recognized (1A10C11) by CDP sera. The 5A9B11- and 1A10C11-like epitopes are expressed on cruzipain. The reactivity of 5A9B11 against cruzipain was completely inhibited by sera of severe cardiopathy patients while a partial inhibition was found with sera from chagasic patients with mild disease. CDP sera did not block cruzipain recognition by 1A10C11. The antigenic determinants recognized by CDP sera appeared to be linear and carbohydrate free. When the overall anti-cruzipain immune response was evaluated, 70% of CDP with severe disease showed cruzipain titers higher than 1/800 while none of them displayed titers lower that 1/400. This report shows for the first time that the humoral immune response against epitopes expressed on cruzipain appeared to be related with the severity of chronic Chagas disease.

Immunoprotection of mice against Trypanosoma cruzi with a lyophilized flagellar fraction of the parasite plus adjuvant.

The immunization with the flagellar (F) fraction from epimastigotes of Trypanosoma cruzi has been shown to protect mice against a challenge of bloodstream trypomastigotes of the parasite, both in terms of mortality and decrease in parasitemia. We have compared the immunoprotective properties of the fresh F fraction with those of a lyophilized F (LF) fraction, alone or together with Bordetella pertussis (Bp) as adjuvant. The best results were obtained with LF + Bp: after challenge with 1 X 10(3) metacyclic trypomastigotes, 100% of the mice immunized with LF + Bp survived, and 60% of them showed no signs of parasitemia. Only the animals in which patent parasitemia was demonstrated presented heart and muscle infiltrates.

Purification and properties of anti-Trypanosoma cruzi antibodies isolated from patients with chronic Chagas' disease.

Three purified human IgG antibodies against Trypanosoma cruzi (anti-F, anti-Mc and anti-Cs) showed different reactivities in vitro. While all of them agglutinated specifically sensitized sheep red cells the complement-fixing capacity of anti-Fc and anti-Cs was higher than that of anti-Mc antibody. This one also showed a minimal precipitating activity. When the ability to neutralize mice bloodstream trypomastigotes was analyzed, the results obtained indicated that anti-F antibody was the most effective.

Cell-mediated reactivity against human and Trypanosoma cruzi antigens according to clinical status in Chagas' disease patients.

The presence of cellular reactivity against homologous tissues and subcellular fractions of Trypanosoma cruzi was investigated in Chagas' disease patients (CDP). CDP were grouped in asymptomatic (CDP-1) and with probable (CDP-2) and overt (CDP-3) cardiomyopathy. Healthy and non-Chagasic cardiomyopathic subjects were studied as controls. Lymphoproliferative reactions against heart tissue extracts were detected in 42% of 72 CDP studied, with similar prevalence of positive reactions in all groups, and correlated with reactivity to both liver and kidney homologous tissues (P less than 0.001). These results confirm the existence of cellular immune reactivity against tissues in CDP, and indicate the lack of organ specificity of this reaction as well as the absence of relation with the clinical state of patients. Cellular reactivity to subcellular fractions of T. cruzi showed a definite pattern according to the clinical status of CDP. Although prevalence of T. cruzi stimulation appeared similar in all groups (70% in CDP-1, 82% in CDP-2 and 75% in CDP-3), CDP-3 showed a significantly higher reactivity to flagellar (69%) and cytosol (63%) fractions than CDP-1 (38 and 27%, respectively). These findings suggest a variable modulation of immune response according to the clinical state of T. cruzi infected subjects.

Monoclonal antibodies against the flagellar fraction of epimastigotes of Trypanosoma cruzi: complement-mediated lytic activity against trypomastigotes and passive immunoprotection in mice.

Trypanosoma cruzi, the causative agent of Chagas' disease, is widely spread in Central and South America. The present report describes three monoclonal antibodies (mAbs) directed against the flagellar fraction of epimastigotes (Ffe) of the parasite, Tulahuén strain. The three mAbs were of IgG1 isotype. Indirect immunofluorescence assays revealed that the three mAbs bind to epimastigotes, the FCH-F8-1 and -3 bind to blood trypomastigotes (BT) and FCH-F8-1 is the only one that binds to amastigotes. Three different proteins of the parasite were recognized by the mAbs in immunoprecipitation assays: an 85 kDa of BT with the FCH-F8-1 mAb, a 40 kDa of Ffe with the FCH-F8-2 mAb, and a 90 kDa of Ffe and of BT with the FCH-F8-3 mAb. Positive complement mediated lysis (CML) of BT and metacyclic forms, obtained from the insect vector feces, were shown by the FCH-F8-1, while FCH-F8-3 only showed CML against the metacyclic trypomastigotes. FCH-F8-2 did not mediate any CML activity. Passive transference of the mAbs to BALB/c mice conferred protection, in terms of survival, ranging from 50 (FCH-F8-2 and -3) to 80% (FCH-F8-1) against a challenge with 1 X 10(3) BT. These results suggest that FCH-F8-1 may be a useful tool to purify proteins in order to investigate their role in immunoprotection experiments.

Involvement of L3T4+, LYT2.2+ T cell subsets and non-T cells in the resistance of mice against Trypanosoma cruzi infection.

Cellular populations involved in resistance against T. cruzi infection were characterized from mice chronically infected with the parasite. Mice transfused with spleen cells (SC), nylon-wool-non-adherent spleen cells (NWNA) or sera from mice chronically infected with T. cruzi, showed an enhanced resistance against challenge with the parasite. The protective activity of NWNA but not of SC was completely abrogated by treatment with anti-Thy1.2 monoclonal antibodies (mAb) and complement (C). Pretreatment of NWNA cells from chronically infected mice with either anti-L3T4 or anti-Lyt 2.2 mAb partially reduced the transfer of resistance. When both L3T4+ and Lyt2.2+ cells were depleted from NWNA populations, transfer of resistance was abolished. These results appear to indicate that L3T4+, Lyt2.2+ T cell subsets and non-T cells are involved in the immunity to T. cruzi.

Leishmania (Viannia) braziliensis: biological behavior in golden hamsters of isolates from Argentine patients.

This study reports intraspecific variations of native isolates of Leishmania (Viannia) braziliensis from patients with leishmaniasis from Salta, Argentina. These isolates induced skin lesions in golden hamsters, initially showing rapid development, reaching their largest size between 28 and 35 days postinfection (PI). Thereafter, the infections were self-limiting and total regression was observed at 80-150 days PI. The majority of the native isolates were characterized by low infectivity in the experimental animals, and a classic pattern of dissemination to systemic organs was established. However, unusual features for L. braziliensis were displayed by two isolates; one showed evidence of high infectivity in hamsters characterized by a short prepatent period and larger, severe and persistent lesions at the inoculation site. The other isolate, of low infectivity, showed cutaneous metastasis and recurrent systemic dissemination in the same animals, suggesting dissociation between infectivity and pathogenicity. Metastasis has been frequently described in hamsters infected with L. (V) guyanensis and L. (V) panamensis, but not in infections induced by L. (V) braziliensis, as was observed in this study. Active and/or regressive histopathologic lesions were observed, depending on the stage of the infection. An exudative and mixed inflammatory pattern with microabscesses and necrotic areas was observed during early infection, while well-defined granulomas and collagen formation were the predominant features detected at a later time. Amastigotes were easily detected in the tissues, although in low numbers. Schaumann bodies were always detected. The characterization of the unique features of these native isolates, and the verification of their reproducibility in vitro and in vivo will be useful tools in tests related to immunoprophylaxis and chemotherapy.

Soluble cell adhesion molecules in human Chagas' disease: association with disease severity and stage of infection.

Formation of inflammatory lesions, one of the pathologic consequences of infection with Trypanosoma cruzi, involves intricate cell-cell interactions in which cell adhesion molecules (CAMs) are involved. Sera from 56 Chagas' disease patients grouped according to disease severity were studied for the presence of soluble intercellular adhesion molecule-1 (s-ICAM-1), soluble endothelial selectin (s-E-selectin), soluble vascular cell adhesion molecule-1 (s-VCAM-1), soluble platelet selectin (s-P-selectin), and s-CD44 were studied to determine if they could be used alone or in different combinations as markers for specific diagnostic procedures. Comparisons were made between congenitally, acutely, and chronically infected patients and aged-matched, noninfected individuals, as well as between patients with chronic Chagas' disease grouped according to the severity of their heart-related pathology. No differences in levels of s-CAMs were detected between sera from children with congenital T. cruzi infection and sera from noninfected infants born from chagasic mothers. In contrast, titers of s-ICAM-1, s-VCAM-1, s-selectin, and s-CD44 but not s-P-selectin were significantly increased in sera from patients during the acute phase of infection with T. cruzi. Titers of s-VCAM-1 and s-P-selectin were increased in chronically infected patients. A positive association with disease severity in sera from patients with chronic disease was observed for the levels of s-P-selectin. In contrast, we found no association between clinical symptoms and levels of s-VCAM-1. Patients with chronic disease with severe cardiopathy also showed diminished levels of s-CD44 in comparison with healthy controls or patients with mild disease. The results are discussed in the context of pathology of Chagas' disease.

Efficacy of chemotherapy with benznidazole in children in the indeterminate phase of Chagas' disease.

A double-blind, randomized, clinical field trial was designed to test the efficacy and tolerance of a specific drug treatment in children in the indeterminate phase of infection by Trypanosoma cruzi. Children were treated with benznidazole at a dose of 5 mg/kg/day for 60 days or placebo and followed-up for 48 months. The treated children showed a significant decrease in geometric mean titers of antibodies against T. cruzi measured by indirect hemagglutination, indirect immunofluorescence, and ELISA. After a four year follow-up, 62% of the benznidazole-treated children and no placebo-treated child were seronegative for T. cruzi when tested by an ELISA using a T. cruzi flagellar calcium-binding protein (F29). Xenodiagnosis carried out after 48 months of follow-up was positive in 4.7% of the benznidazole-treated children and in 51.2% of the placebo-treated children. These results show the tolerance to and efficacy of benznidazole against T. cruzi in seropositive children six to 12 years of age. We used an early serologic marker of cure after treatment, consisting of a recombinant antigen implemented in a rapid, conventional serologic procedure.

Assessment of Leishmaniasis notification system in Santiago del Estero, Argentina, 1990-1993.

Using a capture-recapture method, this study evaluates the completeness of the cutaneous leishmaniasis (CL) surveillance system in four districts of Santiago del Estero province, Argentina, for the period 1990-1993. Four reporting sources were evaluated: medical records kept by health facilities, interviews conducted during a case-control study, and the national and provincial levels of the leishmaniasis surveillance system (LSS). Using the capture-recapture method it was estimated that 210 cases (95% confidence interval [CI]: 202-218) of CL occurred in the four districts during the study period. Completeness of reporting to the leishmaniasis surveillance system at the national level was estimated to be 44.8% (95% CI: 43.2-46.4). The study results indicate that there is substantial underreporting within the LSS, although it did show the appropriate secular trends. The reasons for under-reporting and methods for addressing this problem are discussed.

Chronic Chagas' disease in the mouse: contractile response of the isolated myocardium.

The effects of different pharmacological agents on the isometric developed tension (IDT) of right ventricles isolated from chronic chagasic C3HS mice were studied. The IDT in ventricles of chagasic animals was lower than in ventricles of normal mice (P less than 0.01). Chagasic ventricles exhibited hyperreactivity to norepinephrine but not to epinephrine. This hyperreactivity was masked by propranolol and not by dibenamine. Acetylcholine produced a reduction of IDT to similar values in normal and chagasic myocardia. The possibility that the effect of the drugs tested might not result from specially beta adrenoceptor-mediated reactions, but from alterations at other cellular sites is discussed.

Result of a first step toward community-based surveillance of transmission of Chagas' disease with appropriate technology in rural areas.

The objective of this work was to develop an effective methodology for the surveillance of Chagas' disease vectors in rural areas. It was based on the use of sensor boxes and portable mini-pumps to be integrated into the regular health promotion activities of the Primary Health Care (PHC) agents. The proposed methodology involves a continuous passive intradomiciliary detection of triatomines by sensor boxes that are monitored quarterly by PHC agents. Insecticidal treatment of the houses was performed immediately after the detection of triatomines. The more conventional method of vertical surveillance involves a direct entomologic evaluation conducted by trained professionals. The entire house is searched and there is a mandated treatment of the positive houses. The results of the followups obtained in the county of Rio Hondo in Santiago del Estero Province during a 36-month evaluation period immediately following attack phase application of insecticides were analyzed. The initial high domiciliary and peridomiciliary infestations decreased abruptly after the insecticidal treatment in both areas. When the performances of both types of surveillance were compared, the PHC agent method showed a lower percentage of houses reinfested, with fewer triatomines in the former, and a decrease in their rate of Trypanosoma cruzi infection. Evaluations of reinfestations using the man/hour method and the senor box method showed the same sensitivity. A higher sensitivity for detection of low densities of vector populations was achieved using the sensor boxes. The cost of PHC agent/sensor boxes surveillance was five times lower than the classic one. The proposed strategy for the continuous surveillance of Chagas' disease vectors has demonstrated effectiveness, allows community participation, and seems suitable for large scale application.

Household prevalence of seropositivity for Trypanosoma cruzi in three rural villages in northwest Argentina: environmental, demographic, and entomologic associations.

Environmental, demographic, and entomologic variables were analyzed by logistic multiple regression analysis for their association with the likelihood of being seropositive for Trypanosoma cruzi in three highly infested rural villages of northwest Argentina. The prevalence of seropositivity for T. cruzi, as determined by the composite results of three serologic tests, was 34% among 338 persons in 1992. The strongest positive predictors of the adjusted odds of being infected were the household number of dogs, the density of T. cruzi-infected Triatoma infestans in bedroom areas, and each person's age. Dwellers from houses with roofs made completely or partly with a grass called simbol, or which used insecticides rudimentarily and nonsystematically, had a significantly lower odds of being seropositive for T. cruzi than residents from other types of dwellings. The adjusted odds of infection also increased with the number of T. cruzi-infected dogs or cats and the presence of chickens in bedroom areas. No significant effects on the adjusted odds of infection of a community-wide deltamethrin spraying carried out in one of the villages seven years before were detected. Socioeconomic indicators, such as domiciliary area, and numbers of corrals and livestock, were inversely related to being infected. Our study identified several manageable variables suitable for control actions, most of them not examined before in univariate or multivariate analyses. Environmental management based on low-cost housing with appropriate local materials and removal of domestic animals from domiciliary areas have a crucial role to play in the control of Chagas' disease in rural areas.