Cytokine responses in Trypanosoma cruzi-infected children in Paraguay.

Both parasite and host immune factors may contribute to the development and progression of chronic chagasic cardiomyopathy during Trypanosoma cruzi infections. The present study targeted infected children (5-14 years of age) from an endemic area of Paraguay in an analysis of T. cruzi-specific cytokine profiles. This age group is characteristically the most affected by the early phases of infection. Trypanosoma cruzi-induced cytokine gene expression (interleukin-2 [IL-2], and interferon-gamma [IFN-gamma], IL-4, and IL-10) was studied in 25 seropositive children categorized as being either acute, symptomatic, with Romana's sign (n = 2), or early, indeterminate (postacute, n = 23). Acutely infected children showed a distinct T helper cell-1 (Th1)-type (IFN-gamma) cytokine response to infection. The cytokine pattern that was observed in the seropositive, asymptomatic (early, indeterminate) group was of the Th0 type (expression of both IFN-gamma and IL-4). We hypothesize that selective induction of a Th0-type cytokine pattern is important for development of cell-mediated and humoral immune responses that suppress parasite burden, thereby prolonging the onset or limiting the severity of chronic Chagas' disease later in life.

PIP: Trypanosoma cruzi, the causative agent of Chagas' disease, is an important cause of heart disease in Latin America. Children are the group most likely to be affected by the early phases of infection. A cross-sectional study conducted in elementary schools in two endemic areas of Paraguay (Potrero and Calle-Poi) detected a T. cruzi seroprevalence of 5.3%. 25 seropositive children in the acute (n = 2) or early indeterminate (n = 23) stage of infection participated in a follow-up study aimed at characterizing the T. cruzi-induced cytokine profile. The acute asymptomatic children showed a distinct T-helper cell-1 (Th1)-type cytokine response to infection with minimal interleukin-4 production, while early indeterminate children showed a pattern of the Th0 type regardless of their parasitemic status. These findings suggest that, once acute symptomatic infections with patent parasitemias are cleared by type 1 immune mechanisms, multiple Th0 responses suppress parasite burdens to subpatent levels as parasites sequester in target organs. Infected persons then remain asymptomatic until the cumulative immunopathogenetic effects of persistent, localized inflammatory responses result in chronic disease.

Efficacy of the bisbenzylisoquinoline alkaloids in acute and chronic Trypanosoma cruzi murine model.

We have shown previously that daphnoline and cepharanthine are active against Trypanosoma cruzi and inhibited trypanothione reductase. The effects of oral treatments with daphnoline, cepharanthine and benznidazole were examined in Balb/c mice infected with T. cruzi acutely and chronically. In acute infections, parasitaemia was significantly reduced in the daphnoline-treated mice compared with controls and benznidazole-treated mice. The parasitological cure rate was increased in mice treated with daphnoline. Fifty days after infection, the negative serological response in both models was significantly different for the three tested drugs. Daphnoline showed the highest negative serological rate (48%). In chronically infected mice treated with daphnoline, we were unable to detect parasites in 70% of mice. The results obtained of oral treatment of daphnoline suggest that this bisbenzylisoquinoline may be useful in the treatment of acute and chronic Chagas' disease. This was not seen with cepharanthine, an excellent trypanothione reductase inhibitor.

Differential expression of systemic cytokine profiles in Chagas' disease is associated with endemicity of Trypanosoma cruzi infections.

Chagas' disease is a serious public health problem in Paraguay, however, the immunoepidemiology of the disease has not been well documented. A preliminary cross-sectional survey was carried out in two villages of the Paraguayan Chaco region and in five villages of the Oriental region to assess the endemicity of Trypanosoma cruzi infections. Thereafter, a subset of individuals (ages ranging from 23 to 65 years) participated in a follow-up study to evaluate clinical and parasitological parameters. Physical examinations and electrocardiograms (ECG) were conducted and blood samples collected for parasite detection and serology. The most frequent ECG abnormalities which were observed among chagasic individuals were left anterior hemifascicular block and right bundle branch block. Thirty-two of these subjects, seropositive and non-parasitemic from the high endemic Chaco (n = 16) and low endemic Oriental (n = 16) regions, were randomly selected for an analysis of T. cruzi-induced expression of cytokines IL-2, IFN-gamma, IL-4 and IL-10 by RT-PCR. The individuals were grouped (n = 8) according to the presence or absence of abnormal ECG. In subjects that exhibited abnormal ECG profiles, five of eight (63%) individuals from the high endemic area showed a dominant type 2 (IL-4) response, whereas a comparable number (63%) of subjects from the low endemic area expressed a strong type 1 (IFN-gamma) response; the remainder (37%) presented a Th0-type (IFN-gamma, IL-4) response. Subjects with normal ECG showed a defined cytokine profile according to endemicity. All subjects from the high endemic region showed a Th0 response, whereas 100% of the individuals from the low endemic area demonstrated a type 1 response. In most chagasic patients regardless of ECG profile and endemicity, IL-2 expression was depressed, while IL-10 mRNA transcripts were consistently elevated. Taken together, these data indicate that chronic human chagasic disease is associated with increased systemic production of type 2 cytokines in response to T. cruzi infection and may be involved in the reciprocal down-regulation of IL-2 production.

Genotypic variation among lineages of Trypanosoma cruzi and its geographic aspects.

Isozyme analysis with 18 enzyme loci was conducted on 146 isolates of Trypanosoma cruzi from Mexico, Guatemala, Colombia, Ecuador, Peru, Brazil, Bolivia, Paraguay and Chile. Forty-four different MLGs (groups of isolates with identical multilocus genotypes) were identified and a phylogeny was constructed. The phylogenetic tree consisted of two main groups (T. cruzi I, T. cruzi II), and the latter was further divided into two subgroups (T. cruzi IIa, T. cruzi IIb-e). Evidence of hybridization between different MLGs of T. cruzi II was found, which means that genetic exchanges seem to have occurred in South American T. cruzi. On the other hand, the persistence of characteristic T. cruzi I and T. cruzi II isozyme patterns in single small villages in Bolivia and Guatemala suggested that genetic exchange is very rare between major lineages. A significant difference in genetic diversity was shown between T. cruzi I and T. cruzi II from several indices of population genetics. Two possibilities could explain this genetic variation in the population: differences in evolutionary history and/or different tendencies to exchange genetic material. Broad-scale geographic distributions of T. cruzi I and T. cruzi IIb-e were different; T. cruzi I occurred in Central America and south to Bolivia and Brazil, while T. cruzi IIb-e occurred in the central and southern areas of South America, overlapping with T. cruzi I in Brazil and Bolivia.

Cryptofolione derivatives from Cryptocarya alba fruits.

Cryptofolione (1) and the new cryptofolione derivative 6-(4,6-dimethoxy-8-phenyl-octa-1,7-dienyl)-4-hydroxy-tetrahydro-pyran-2-one (2) were isolated from the fruits of Cryptocarya alba. The structures were elucidated by spectroscopic methods. Cryptofolione showed activity towards Trypanosoma cruzi trypomastigotes, reducing their number by 77% at 250 microg mL(-1). Cryptofolione showed moderate cytotoxicity in both macrophages and T. cruzi amastigotes. It also displayed a mild inhibitory effect on the promastigote form of Leishmania spp. As both cytotoxic and trypanocidal effects are similar, the compound presented little selectivity in our assay models.

Activity of compounds isolated from Chilean lichens against experimental cutaneous leishmaniasis.

Three secondary metabolites isolated from Chilean lichens, (+) usnic acid, pannarine and 1'-chloropannarine, were tested against promastigotes forms of three strains of Leishmania ssp. Pannarine and 1'-chloropannarine exhibited in vitro activity at 50 micrograms/ml and (+) usnic acid at 25 micrograms/ml. BALB/c mice infected with Leishmania amazonensis were treated 4 weeks post-infection with (+) usnic acid by subcutaneous or oral routes for 15 days at 25 mg/kg or by five intralesional injections at interval of 4 days at 25 mg/kg of body weight. The reference drug, N-methylglucamine antimonate (Glucantime), was administered by subcutaneous injections (regimens of 28 mg of pentavalent antimony) for 15 days. The subcutaneous and oral treatments with (+) usnic did not produce any effect, but by intralesional administration we observed a significant effect that reduced by 43.34% the weight lesions and by 72.28% the parasites loads in infected footpads.

Chemical constituents of essential oils of muña, Bolivian plants traditionally used as pesticides, and their insecticidal properties against Chagas' disease vectors.

The composition of essential oils from two muña, Bolivian medicinal plants, derived from Minthostachys andina and Hedomea mandonianum, were analyzed by gas chromatography/mass spectrometry. Major differences were observed in their chemical composition. Pulegone was the major component of H. mandonianum oil (44.6%) and M. andina oil (25.5%); menthone and isomenthone were around 33% of these oils. Differences were also observed in their insecticidal activity against the Chagas' disease vector, Rhodnius neglectus or Triatoma infestans bugs exposed on impregnated oil filter papers. While M. andina oil showed 30%-50% of mortality in both triatomine species after a period of 1 week, H. mandonianum oil did not show any insecticidal activity. Nevertheless, both species had insecticidal activity (33.3% and 50%) when oils were topically applied. The significance of these results is discussed in relation to the variability of the chemical composition and their potential use in Chasgas' disease vector control.

Mutagenicity, insecticidal and trypanocidal activity of some Paraguayan Asteraceae.

The insecticidal, moulting inhibition and trypanocidal effects of crude extracts of 7 Paraguayan Asteraceae were evaluated on Triatoma infestans and bloodstream forms of Trypanosoma cruzi, respectively. Both mutagenicity and toxicity were evaluated by sister chromatid exchange (SCE) in human peripheral lymphocyte culture and by the lethality test of Artemia salina. The ethanolic extracts from Chromolaena christieana (stem and bark), Achyrocline satureoides (leaves and flowers) and Mikania cordifolia (root and stem), at a concentration of 250 micrograms/ml, showed the highest percentage of lysis on bloodstream forms of Trypanosoma cruzi. The extracts of Chromolaena christieana and Achyrocline satureoides also presented high mutagenic and toxic capacity when they were evaluated by the SCEs assay and Artemia salina test, respectively. Insecticidal activity was only observed in the hexane extract of flowers of Achyrocline satureoides (45% of mortality), when 0.05 microgram of crude concentration was applied on Triatoma infestans. The ethanolic extracts of stem from Mikania cordifolia and Vernonia brasiliana inhibited the moulting of Triatoma infestans when it was compared with their controls. Since no ethnobotanical information on these plants has been found related to similar use in Paraguay, our findings suggest, for the first time, the potential anti-trypanocidal and moulting inhibition of these Asteraceae.

Leishmanicidal activity of two canthin-6-one alkaloids, two major constituents of Zanthoxylum chiloperone var. angustifolium.

The crude alkaloidal extract of Zanthoxylum chiloperone stem bark exhibited in vitro activity against various strains of Leishmania ssp. at 100 microg/ml. Two active major constituents were isolated and identified as canthin-6-one and 5-methoxycanthin-6-one. The effect of these compounds was also tested in an in vivo assay using BALB/c mice infected with Leishmania amazonensis. The mice were treated for 5 weeks postinfection with these alkaloids by oral (14 days) or intralesional route (4 days) at 10 mg/kg daily. The reference drug, N-methylglucamine antimonate was administered by subcutaneous injections at 100 mg/kg for 10 days. Intralesional administration of canthin-6-one reduced the parasite burden but not significantly when it was compared with the untreated group, while the reference drug reduced by 91% the parasite loads in the lesion.

[Eye involvement in Chagas disease (American trypanosomiasis). 1996/1997 studies in Paraguay]. / Augenbeteiligung bei Chagas-Krankheit (Amerikanische Trypanosomiasis). Untersuchungen in Paraguay 1996/1997.

BACKGROUND: Last year's pilot study, which examined for the first time a representative group of patients with Chagas disease, showed that in this parasitosis changes in the ocular fundus can be expected in the form of defects of the retinal pigment epithelium. Examination of more patients in this study should give further information about the character and extent of the alterations found. MATERIALS AND METHODS: After serological confirmation of the disease, we performed an ophthalmological examination of the anterior and posterior segments. RESULTS: In 21 out of 23 patients the fundus was unobtrusive; 1 patient showed fibrae medullares and 1 patient clear pigment dispersion on the posterior pole. Together with the pilot study, a total of 102 chagasic patients were examined. In 8 patients (7.8%) defects of the retinal pigment epithelium were found, which did not cause any significant loss of vision. CONCLUSIONS: We presume that the described defects are a matter of rare and harmless postinflammatory or immunological changes in the American type of trypanosomiasis.

[Intraocular involvement of Chagas disease (American trypanosomiasis). Studies in Paraguay/South America]. / Beteiligung der Chagas-Krankheit (Amerikanische Trypanosomiasis) am inneren Auge. Untersuchungen in Paraguay/Südamerika.

BACKGROUND: In Central and South America, Chagas' disease is of great epidemiologic importance. The epidemiologic agent is represented by Trypanosoma cruzi, a monocellular parasite, instrumental in human infection is the presence of vectors, which are various species of hematophagous bugs. The eye is one of the most important entrance sites of the parasite, and relatively little information about the relationship between Chagas' disease and eye complications is available. PATIENTS AND METHODS: We examined 79 chagasic patients in order to detect changes in the retina. As a control group, we examined 48 patients with negative serology within the same age range and from the same regions. For every patient we completed a routine ophthalmologic examination, with inspection of the retina using direct and indirect ophthalmoscopy. RESULTS: In most of the chagasic patients, the ocular fundus was unobtrusive; in only 6 out of 79 cases (7.6%) we did observe small parafoveolar retinal pigment epithelium defects and in 1 case (1.3%) distinct pigment epithelium atrophy of the posterior pole. No comparable findings were observed in the control group. CONCLUSION: Compared with the examination results of the control group, in the patients with intermediate and chronic Chagas' disease we observed an accumulation of retinal pigment epithelium defects, which, however, did not cause a significant loss of vision.

Chagas disease prevention through improved housing using an ecosystem approach to health.

This Chagas disease prevention project via housing improvement aims to determine the efficiency of different interventions in vector control. The following study describes the target communities, disease magnitude, and housing improvements. Transmission levels are analysed from an ecological and socioeconomic perspective. Special interest was focused on the peridomicile as the origin of domiciliary reinfestation. In the original project, three intervention programs were proposed, one for each of the three communities: (a) an insecticide spraying program; (b) a housing improvement program; and (c) a combined program of spraying and housing improvement. The three communities currently have different risks of exposure to triatominae reinfestation as a consequence of the type of intervention carried out. A new multidisciplinary approach which integrates participatory, community-based research and socioeconomic dimensions will allow to determine the efficiency of models for territorial ordering, community education, and environmental interventions in Chagas disease control.

Comportamientos que favorecen la dinámica de reinfestación de Triatomainfestans del Chaco paraguayo / Behaviors that favor reinfestation by Triatoma infestans in the Paraguayan Chaco

El Chaco central paraguayo es unaregión de alto nivel de reinfestación porTriatoma infestans. La población indígena que la habita tiene alta vulnerabilidad por factores culturales y medio ambientales que dificultan acceso y trabajo en la zona. Se propusoconocer factores psicosociales asociadosal proceso de reinfestación para desarrollar tareas de vigilanciacomunitaria. Estudio transversal, con enfoques cualitativo y cuantitativo.Treinta y seis punto siete por ciento (96)de la población de estudio realizómejoras en viviendas; 41,6 % (40)mejoró revoque en paredes. Poblaciónubica al vector en el monte, entre leñas,hojas secas, agujeros de árboles, pozosde topos o tatú; alrededor de animales domésticos, techos de viviendas, gallineros, chiquero de cabras y cerdos.Comprometiendo el traslado pasivo de vinchucas se encontró la recolección deleña 98,5 % (266), del monte, 97,7 %(261) el cambio de lugar de ropas, cajas y comida en las viviendas, 54,7 % (146). Se asoció (p< 0,0005) vivienda mejorada con revoque en paredes yno infestación; viviendas con animales(p< 0,03) e infestación; actitud positivapara eliminar el vector (p<0,04) y no infestación. Comportamientos que comprometen traslado y permanencia de vinchucas fueron acarreo de leña, almacenamiento de comidas yacumulación de ropa y cajas. Paredes revocadas y presencia de animales domésticos se correlacionaron a infestación y actitudes positivas paraeliminación de la vinchuca con viviendas sin reinfestación. Todos ellos sonfactores estratégicos para tareas deprevención y vigilancia con participación comunitaria.

Prevalence of Chagas' disease in Ayoreo communities of the Paraguayan Chaco.

The prevalence of Chagas' disease in Ayoreo amerindians living in their traditional habitat of the Paraguayan Chaco was assessed by ELISA and indirect immunofluorescence techniques. The Ayoreo is the last hunther-gatherer tribe of the Paraguayan Chaco and was "pacified" between 1965 and 1975. They still maintain a seminomadic way of life. Chagas' disease prevalence in the Ayoreo communities probably represents their natural prevalence in Chaco Amerindians before settlement. Triatoma sordida was identified as the insect vector in the settlements of Gesudi and Chovoreca, while the highly domiciliary Triatoma infestans was found in Campo Loro. The prevalence of Chagas' disease in Gesudi and Chovoreca, based on our serological surveys was 12-13%. Other authors reported a 72.9% of Chagas' disease prevalence in amerindians settled in the Chaco since 1930-1940.

Chagas disease vector control through different intervention modalities in endemic localities of Paraguay.

In a field study carried out in three rural communities in Paraguay in a zone endemic for Chagas disease, we implemented three different vector control interventions--spraying, housing improvement, and a combination of spraying plus housing improvement--which effectively reduced the triatomine infestation. The reduction of triatomine infestation was 100% (47/47) in the combined intervention community, whereas in the community where housing improvement was carried out it was 96.4% (53/55). In the community where fumigation alone was used, the impact was 97.6% (40/41) in terms of domiciliary infestation. In all the houses where an intervention was made, an 18-month follow-up showed reinfestation rates of less than 10%. A serological survey of the population in the pre- and post-intervention periods revealed a shift in positive cases towards older age groups, but no significant differences were observed. The rate of seroconversion was 1.3% (three new cases) in the community with housing improvement only, but none of these cases could have resulted from vector transmission. The most cost-effective intervention was insecticide spraying, which during a 21-month follow-up period had a high impact on triatomine infestation and cost US$ 29 per house as opposed to US$ 700 per house for housing improvement.

PIP: Chagas disease is most commonly spread by vectors in areas where inadequate housing, crowding, and poor hygienic conditions facilitate the proliferation of triatomines and help to maintain their domestic cycle. Chagas disease is widely distributed in America, from the southern US to southern Argentina, with a prevalence of 5-60%. In 3 rural communities in Paraguay in a zone endemic for Chagas disease, insecticide spraying, housing improvement, and a combination of spraying plus home improvement were implemented in a field study to control the vectors of Chagas disease. The insecticide applied was lambdacyhalothrin in a wettable powder formulation, while houses were modified to have smooth, flat, crack-free walls and ceiling surfaces, as well as better ventilation and illumination. Each intervention effectively reduced the vector of Chagas disease, with triatomine infestation reduced by 100% in the combined intervention community, 96.4% in the community in which only houses were improved, and 97.6% in terms of domiciliary infestation where only fumigation was used. In all houses in which an intervention was made, an 18-month follow-up showed reinfestation rates of less than 10%. A serological survey of the population during the pre- and post-intervention periods showed a shift in positive cases toward older age groups, but no significant differences were observed. The rate of seroconversion was 1.3% (3 new cases) in the community with housing improvement only, but none of those cases could have resulted from vector transmission. Insecticide spraying was the most cost-effective intervention: US$29 per house compared to US$700 per house for housing improvement.

New prenylated quinones from Peperomia galioides.

Two new prenylated quinones, piperogalone (1) and galopiperone (2), and a new prenylated dihydroquinone, hydropiperone (3), were isolated from Peperomia galioides H.B.K (Piperaceae). Hydropiperone exhibited potent antiparasitic activity against three species of Leishmania.

Local and systemic cytokine expression during experimental chronic Trypanosoma cruzi infection in a Cebus monkey model.

Cebus apella is an acceptable model for chronic chagasic cardiomyopathy (CCC), since it is possible to experimentally induce cardiac lesions after 1 year of Trypanosoma cruzi infection. The T. cruzi Y strain, shown previously to produce CCC in C. apella monkeys, was used to experimentally infect 10 monkeys. Parasitological, serological and clinical parameters were monitored during a 19-month follow-up, and systemic cytokine responses were assessed sequentially in five monkeys selected according to the differential parasitemia pattern exhibited. Ten additional monkeys, infected with the same strain for 5, 10 and 12 years, were analysed cross-sectionally. Three monkeys/time point and one uninfected control animal were sacrificed for gross pathology, histology, presence of parasites, and local cytokine gene expression. Elevated expression of interleukin (IL)-4 was observed throughout the study in monkeys that had persistent, high parasitemias, whereas a high level of interferon (IFN)-gamma was seen in monkeys that controlled parasitemias soon after infection. Chronically infected monkeys expressed a nonpolarized, Th0-type response. Cardiac tissue collected from a monkey that succumbed to acute infection had elevated levels of proinflammatory cytokine [IL-1beta, IL-6, tumour necrosis factor-alpha] and interstitial cell adhesion molecule (ICAM)-1, platelet-derived growth factor (PDGF)-alpha, transforming growth factor (TGF)-beta and IL-10 transcripts. Cytokine production in cardiac tissue of chronically infected monkeys was also characterized by elevated expression of ICAM-1, PDGF-alpha and TGF-beta, which correlated with the detection of T. cruzi DNA by polymerase chain reaction.

Synthesis and in vitro antiprotozoal activity of thiophene ring-containing quinones.

A series of quinones (3a-i, 4-9, 11) and aromatic compounds (2a, 2d, 2g) containing the thiophene ring were tested in vitro against the trypomastigote form of Trypanosoma cruzi and the promastigote forms of Leishmania. The quinones 3a-i, 4, 5a, b, 6 and 9 having the thiophene ring fused to a quinone nucleus were the most active members of the series. The electron affinities of the benzo[b]thiophene-4,7-quinones 3, evaluated by their LUMO energies and halfwave potentials, are reported.

Trypanosoma cruzi: polymerase chain reaction-based detection in dried feces of Triatoma infestans.

PCR was employed to detect Trypanosoma cruzi DNA in Triatoma infestans dry fecal spots collected on filter papers. Both insects fed on experimentally infected monkeys and insects collected in a Paraguayan endemic area for Chagas' disease were examined. When the insects fed on a chronically infected monkey with low parasitemia as revealed by direct microscopic observation (DMO), T. cruzi was detected in the insect feces by PCR as soon as 2 days postfeeding. When the same experiment was performed on monkeys with parasitemia levels below the limit of detection by DMO, the degree of positivity found through PCR-Southern hybridization, applied on Day 8 postfeeding, was superior to that obtained through xenoculture. These results suggest that PCR can be used to speed the xenodiagnosis results with great sensitivity. On the other hand, when applied to the feces of triatomines collected in the field, 84% were positive by PCR-Southern hybridization, whereas only 26% were positive by DMO. Therefore, PCR could also be applied to the monitoring of the infection status of triatomines which infest rural dwellings by examining only the feces left on paper sensors hung on the walls of the houses.

Isoenzyme profiles of Trypanosoma cruzi stocks from different areas of Paraguay.

Twenty one Trypanosoma cruzi stocks from humans, domiciliary triatomines and one sylvatic animal of different areas of Paraguay were subjected to isoenzyme analysis. Thirteen enzyme systems (15 loci in total) were studied. MN cl2 (clonets 39) and SO34 cl4 (clonets 20) were used as references. Relationships between stocks were depicted by an UPGMA dendrogram constructed using the Jaccard's distances matrix. Among the Paraguayan stocks 14 zymodemes were identified (Par1 to Par14), Par 5 being the most frequent. Polymorphism rate and clonal diversity were 0.73 and 0.93, respectively. Average number of alleles per polymorphic locus was 2.5 (range 2-4). These measurements show a high diversity, which is confirmed by the dendrogram topology. All stocks belong to the same lineage, as MN cl2 reference strain (T. cruzi II). Moreover three distinct subgroups were identified and two of them correspond to Brazilian and Bolivian zymodemes, respectively. The third subgroup, the most common in Paraguay, is related to Tulahuen stock. The large geographical distribution of some zymodemes agrees with the hypothesis of clonality for T. cruzi populations. However sample size was not adequate to detect genetic recombination in any single locality.