Posttherapeutic cure criteria in Chagas' disease: conventional serology followed by supplementary serological, parasitological, and molecular tests.

We performed a critical study of conventional serology, followed by supplementary serological, parasitological, and molecular tests, to assess the response to etiologic treatment of Chagas' disease. A group of 94 Chagas' disease patients treated with benznidazole at least 10 years earlier were evaluated from the laboratory and clinical points of view. When conventional serology (enzyme-linked immunosorbent assay [ELISA], indirect immunofluorescence [IIF], and indirect hemagglutination [IHA]) and classic criteria (consistent results with any two of the three tests) or more rigorous criteria (consistent results from the three tests) were used, 10.6% and 8.5% of patients were considered treated and cured (TC) by classic and rigorous criteria, respectively. Patients were then evaluated using supplementary (recombinant ELISA and Trypanosoma cruzi excreted-secreted antigen blotting [TESA-blot]), parasitological (hemoculture), and molecular (PCR) tests. The results of recombinant ELISA were similar to those with the rigorous criterion (three consistent test results). The TESA-blot group showed a higher percentage (21.3%) of negative results than the groups defined by either cure criterion. Hemoculture and PCR gave negative results for all treated and cured (TC) patients, regardless of the criterion used. Recombinant ELISA and TESA-blot tests showed negative results for 70% and 87.5% of the patients categorized as TC by the classic and three-test criteria, respectively. For patients with discordant conventional serology, the supplementary serological and molecular tests were the decisive factor in determining therapeutic failure. Clinical evaluation showed that 62.5% of TC patients presented with the indeterminate form of the disease. Additionally, treated patients with negative TESA-blot results should be reevaluated later with all methodologies used here to verify whether TESA-blot is a reliable way to determine early parasitological cure of Chagas' disease.

Challenges and opportunities for primary, secondary, and tertiary prevention of Chagas' disease.

A century after its discovery, Chagas' disease still represents a major public health challenge in Latin America. Moreover, because of growing population movements, an increasing number of cases of imported Chagas' disease have now been detected in non-endemic areas, such as North America and some European countries. This parasitic zoonosis, caused by Trypanosoma cruzi, is transmitted to humans by infected Triatominae insects, or occasionally by non-vectorial mechanisms, such as blood transfusion, mother to fetus, or oral ingestion of materials contaminated with parasites. Following the acute phase of the infection, untreated individuals enter a chronic phase that is initially asymptomatic or clinically unapparent. Usually, a few decades later, 40-50% of patients develop progressive cardiomyopathy and/or motility disturbances of the oesophagus and colon. In the last decades several interventions targeting primary, secondary and tertiary prevention of Chagas' disease have been attempted. While control of both vectorial and blood transfusion transmission of T cruzi (primary prevention) has been successful in many regions of Latin America, early detection and aetiological treatment of asymptomatic subjects with Chagas' disease (secondary prevention) have been largely underutilised. At the same time, in patients with established chronic disease, several pharmacological and non-pharmacological interventions are currently available and have been increasingly used with the intention of preventing or delaying complications of the disease (tertiary prevention). In this review we discuss in detail each of these issues.

Are increased frequency of macrophage-like and natural killer (NK) cells, together with high levels of NKT and CD4+CD25high T cells balancing activated CD8+ T cells, the key to control Chagas' disease morbidity?

The immunological response during early human Trypanosoma cruzi infection is not completely understood, despite its role in driving the development of distinct clinical manifestations of chronic infection. Herein we report the results of a descriptive flow cytometric immunophenotyping investigation of major and minor peripheral blood leucocyte subpopulations in T. cruzi-infected children, characterizing the early stages of the indeterminate clinical form of Chagas' disease. Our results indicated significant alterations by comparison with uninfected children, including increased values of pre-natural killer (NK)-cells (CD3- CD16+ CD56-), and higher values of proinflammatory monocytes (CD14+ CD16+ HLA-DR++). The higher values of activated B lymphocytes (CD19+ CD23+) contrasted with impaired T cell activation, indicated by lower values of CD4+ CD38+ and CD4+ HLA-DR+ lymphocytes, a lower frequency of CD8+ CD38+ and CD8+ HLA-DR+ cells; a decreased frequency of CD4+ CD25HIGH regulatory T cells was also observed. These findings reinforce the hypothesis that simultaneous activation of innate and adaptive immunity mechanisms in addition to suppression of adaptive cellular immune response occur during early events of Chagas' disease. Comparative cross-sectional analysis of these immunophenotypes with those exhibited by patients with late chronic indeterminate and cardiac forms of disease suggested that a shift toward high values of macrophage-like cells extended to basal levels of proinflammatory monocytes as well as high values of mature NK cells, NKT and regulatory T cells, may account for limited tissue damage during chronic infection favouring the establishment/maintenance of a lifelong indeterminate clinical form of the disease. On the other hand, development of an adaptive cell-mediated inflammatory immunoprofile characterized by high levels of activated CD8+ cells and basal levels of mature NK cells, NKT and CD4+ CD25HIGH cells might lead to late chronic pathologies associated with chagasic heart disease.

Benznidazole treatment during early-indeterminate Chagas' disease shifted the cytokine expression by innate and adaptive immunity cells toward a type 1-modulated immune profile.

Trypanosoma cruzi-infected children was treated with benznidazole (Bz) during the early-indeterminate disease (E-IND) and the cytokine pattern of innate and adaptive immune compartments were evaluated prior to the treatment and 1 year after it. At first, we observed that the ex vivo cytokine profile of circulating leukocytes from E-IND (n = 6) resembled the one observed for healthy schoolchildren (n = 7). Additionally, in vitro stimulation with T. cruzi antigens drove the E-IND cytokine pattern toward a mixed immune profile with higher levels of IFN-gamma+, TNF-alpha+ and IL-4+ NK cells, increased numbers of IFN-gamma+, TNF-alpha+ and IL-10+ CD4+ T cells in addition to enhanced frequency of TNF-alpha+/IL-4+ CD19+ lymphocytes. Interestingly, upon T. cruzi antigen in vitro stimulation, E-IND CD8+ lymphocytes displayed a selective enhancement of IFN-gamma expression, accounting for a global type 1-modulated cytokine microenvironment. A shift toward a type 1-modulated profile was also the hallmark of Bz-treated children (E-IND(T)). In this context, despite the mixed overall ex vivo cytokine profile observed for NK and CD8+ T cells, increased ability of these leukocytes to produce IFN-gamma in response to T. cruzi antigens was reported. Most noteworthy was the IL-10 production evidenced at T lymphocytes, mainly CD4+ cells, as well as B lymphocytes, both ex vivo and upon antigen stimulation. Together, these findings gave evidence that NK cells and CD8+ T lymphocytes are the major sources of IFN-gamma, a pivotal cytokine for successful therapeutic response in human Chagas' disease. Moreover, our data have also brought additional information, pointing out IL-10 production by CD4+ cells and B lymphocytes, as the putative key element for parasite clearance in the absence of deleterious tissue damage.

Patterns of antennal sensilla of Panstrongylus megistus from three Brazilian states.

The objective of the present study was to analyze and describe the phenotype of the antennal sensilla of Panstrongylus megistus, one of the epidemiologically most important species of triatomines in Brazil. Specimens from the Brazilian states of Goiás (GO), Minas Gerais (MG), and Rio Grande do Sul (RS) were compared, based on studies of four types of sensilla on three antennal segments: thick-walled trichoid (TK), thin-walled trichoid (TH), bristles (BR), and basiconica (BA). Discriminant analysis allowed the separation of the RS specimens from those of GO and MG. Multivariate discriminant analysis demonstrated that the sensilla of males differed from those of females, the variables with greatest weight being the BA of all three segments and the TK of flagellum 1. The basiconica sensilla were significantly more abundant in females, on all three segments. Antennal sensilla patterns also demonstrated significant differences among P. megistus specimens.

Chagasic patients with indeterminate clinical form of the disease have high frequencies of circulating CD3+CD16-CD56+ natural killer T cells and CD4+CD25High regulatory T lymphocytes.

Several studies have demonstrated that different clinical manifestations of human Chagas' disease are associated with distinct and complex host-parasite relationships directly involving the immune system. In this context, it has been proposed that tissue damage might be more severe in the absence of regulatory mechanisms that involve both innate and adaptive immune responses. Herein, we describe a descriptive phenotypic profile focusing on the frequency of major regulatory T cells [CD4+CD25high and natural killer T (NKT) lymphocytes] in different clinical forms of Chagas' disease. Ex vivo immunophenotyping of whole blood demonstrated that the indeterminate clinical form displays a higher frequency of both CD4+CD25high and NKT regulatory cells (CD3+CD16-CD56+), associated with increased levels of circulating cytotoxic NK cells (CD3-CD16+CD56+ and CD3-CD16+CD56dim NK cells). By contrast, the increased percentage of activated CD8+HLA-DR+ T-cell subset was exclusively associated with severe clinical forms of Chagas' disease. We hypothesize that regulatory T cells may be able to control the deleterious cytotoxic activity in the indeterminate clinical form by inhibiting the activation of CD8+HLA-DR+ T cells. The lack of regulated populations in cardiac and digestive clinical forms could account for impaired immune response that culminates in strong cytotoxic activity and tissue damage.

The impact of Chagas disease control in Latin America: a review.

Discovered in 1909, Chagas disease was progressively shown to be widespread throughout Latin America, affecting millions of rural people with a high impact on morbidity and mortality. With no vaccine or specific treatment available for large-scale public health interventions, the main control strategy relies on prevention of transmission, principally by eliminating the domestic insect vectors and control of transmission by blood transfusion. Vector control activities began in the 1940s, initially by means of housing improvement and then through insecticide spraying following successful field trials in Brazil (Bambui Research Centre), with similar results soon reproduced in São Paulo, Argentina, Venezuela and Chile. But national control programmes only began to be implemented after the 1970s, when technical questions were overcome and the scientific demonstration of the high social impact of Chagas disease was used to encourage political determination in favour of national campaigns (mainly in Brazil). Similarly, large-scale screening of infected blood donors in Latin America only began in the 1980s following the emergence of AIDS. By the end of the last century it became clear that continuous control in contiguous endemic areas could lead to the elimination of the most highly domestic vector populations - especially Triatoma infestans and Rhodnius prolixus - as well as substantial reductions of other widespread species such as T. brasiliensis, T. sordida, and T. dimidiata, leading in turn to interruption of disease transmission to rural people. The social impact of Chagas disease control can now be readily demonstrated by the disappearance of acute cases and of new infections in younger age groups, as well as progressive reductions of mortality and morbidity rates in controlled areas. In economic terms, the cost-benefit relationship between intervention (insecticide spraying, serology in blood banks) and the reduction of Chagas disease (in terms of medical and social care and improved productivity) is highly positive. Effective control of Chagas disease is now seen as an attainable goal that depends primarily on maintaining political will, so that the major constraints involve problems associated with the decentralisation of public health services and the progressive political disinterest in Chagas disease. Counterbalancing this are the political and technical cooperation strategies such as the "Southern Cone Initiative" launched in 1991. This international approach, coordinated by PAHO, has been highly successful, already reaching elimination of Chagas disease transmission in Uruguay, Chile, and large parts of Brazil and Argentina. The Southern Cone Initiative also helped to stimulate control campaigns in other countries of the region (Paraguay, Bolivia, Peru) which have also reached tangible regional successes. This model of international activity has been shown to be feasible and effective, with similar initiatives developed since 1997 in the Andean Region and in Central America. At present, Mexico and the Amazon Region remain as the next major challenges. With consolidation of operational programmes in all endemic countries, the future focus will be on epidemiological surveillance and care of those people already infected. In political terms, the control of Chagas disease in Latin America can be considered, so far, as a victory for international scientific cooperation, but will require continuing political commitment for sustained success.

Patterns of antenal sensilla of Panstrongylus megistus from three Brazilian states

The objective of the present study was to analyze and describe the phenotype of the antennal sensilla of Panstrongylus megistus, one of the epidemiologically most important species of triatomines in Brazil. Specimens from the Brazilian states of Goiás (GO), Minas Gerais (MG), and Rio Grande do Sul (RS) were compared, based on studies of four types of sensilla on three antennal segments: thick-walled trichoid (TK), thin-walled trichoid (TH), bristles (BR), and basiconica (BA). Discriminant analysis allowed the separation of the RS specimens from those of GO and MG. Multivariate discriminant analysis demonstrated that the sensilla of males differed from those of females, the variables with greatest weight being the BA of all three segments and the TK of flagellum 1. The basiconica sensilla were significantly more abundant in females, on all three segments. Antennal sensilla patterns also demonstrated significant differences among P. megistus specimens.

A cost-benefit analisys of chagas disease control

Chagas disease transmission can be effetively interrupted by insecticidal control of its triatomine bug vectors. We present here a simple model comparing the costs and benefits of such a programme, designed to eliminate domestic populations of Triatoma infestans throughout its known area of distribution over the seven southernmost countries of Latin America. The model has been simplified to require only four financial estimates relating to the unit cost of housing spraying and benefits due to avoidance of premature death in the acute phase of the disease, avoidance of supportive treatment and care in the chronic phase of the disease, and avoidance of corrective digestive and cardiac surgery. Exceptfor these direct medical costs, al other potential benefits have been ignored. Nevertheless, the model shows that the direct financial benefits of such a programme would far outweigh the costs, and the project would support a remarkably high internal rate of return under the least optimistic estimates

The impact of Chagas disease control in Latin America: a review

Discovered in 1909, Chagas disease was progressively shown to be widespread throughout Latin America, affecting millions of rural people with a high impact on morbidity and mortality. With no vaccine or specific treatment available for large-scale public health interventions, the main control strategy relies on prevention of transmission, principally by eliminating the domestic insect vectors and control of transmission by blood transfusion. Vector control activities began in the 1940s, initially by means of housing improvement and then through insecticide spraying following successful field trials in Brazil (Bambui Research Centre), with similar results soon reproduced in Säo Paulo, Argentina, Venezuela and Chile. But national control programmes only began to be implemented after the 1970s, when technical questions were overcome and the scientific demonstration of the high social impact of Chagas disease was used to encourage political determination in favour of national campaigns (mainly in Brazil). Similarly, large-scale screening of infected blood donors in Latin America only began in the 1980s following the emergence of AIDS. By the end of the last century it became clear that continuous control in contiguous endemic areas could lead to the elimination of the most highly domestic vector populations - especially Triatoma infestans and Rhodnius prolixus - as well as substantial reductions of other widespread species such as T. brasiliensis, T. sordida, and T. dimidiata, leading in turn to interruption of disease transmission to rural people. The social impact of Chagas disease control can now be readily demonstrated by the disappearance of acute cases and of new infections in younger age groups, as well as progressive reductions of mortality and morbidity rates in controlled areas. In economic terms, the cost-benefit relationship between intervention (insecticide spraying, serology in blood banks) and the reduction of Chagas disease (in terms of medical and social care and improved productivity) is highly positive. Effective control of Chagas disease is now seen as an attainable goal that depends primarily on maintaining political will, so that the major constraints involve problems associated with the decentralisation of public health services and the progressive political disinterest in Chagas disease. Counterbalancing this are the political and technical cooperation strategies such as the "Southern Cone Initiative" launched in 1991...

Inter-relation of sylvatic and domestic transmission of Trypanosoma cruzi in areas with and without domestic vectorial transmission in Minas Gerais, Brazil

During the period 1980-1986, we captured triatomine bugs and mammalian reservoir hosts from sylvatic and domestic situations in different municipalities of the State of Minas Gerais. Trypanosoma cruzi was isolated from captured bugs, mammals and patients. After cultivation in LIT medium, the electrophoretic enzyme profiles were determined. We obtained atotal of 32 parasite isolates from regions with active domestic transmission, and 24 isolates form areas under control. For the first areas the results suggest introduction of T. cruzi from sylvatic habitats, through incursion of infected opossums and/or sylvatic T. sordida, which appears to have given rise to at least one acute human infection. Of particular interest is the finding of sylvatic opossums and a T. sordida nymph infected with ZB, that could indicate return of parasites from chronic human infections to sylvatic transmission cycles. For the areas under control we also interpret the results as interaction between sylvatic and domestic cycles of transmission, here through the invasion of houses by bugs carrying the Z1 zymodeme from the sylvatic environment. The Multivariate Correspondence Analysis gives a spatial description between the different parasite isolates and confirms the existence of a bridge in the opposite direction in the region with active vectorial transmission including the exporting of Z2 through the peridomestic environment into the sylvatic cycle. For the others areas this bridge corresponds especially to Panstrongylus megistus, importing Z1 into the domestic environment.

IFN-gamma in human Chagas' disease: protection or pathology?

An apparently paradoxical role for IFN-gamma in human Chagas'disease was observed when studying the pattern of cytokine production by peripheral blood mononuclear cells (PBMC) obtained from two groups of chagasic patients after specific stimulation with Trypanosoma cruzi-derived antigens. The groups studied were 1) patients treated with bendnidazole during the acute phase of Trypanosoma cruzi infection and 2) chronically infected untreated patients. In the treated group, higher levels of IFN-gamma were produced by PBMC from individuals cured after treatment when compared to non-cured patients. In contrast, in the chronically infected group (not treated) higher levels of IFN-gamma were produced by PBMC from cardiac patients in comparison with asymptomatic (indeterminate) patients. This apparently paradoxical role for IFN-gamma in human Chagas'disease is discussed in terms of the possibility of a temporal difference in IFN-gamma production during the initial stages of the infection (acute phase) in the presence or absence of chemotherapy. The maintenance of an immune response with high levels of IFN-gamma production during the chronic phase of the infection may favor cure or influence the development of the cardiac form of the disease.

Possible oral transmission of acute Chagas'disease in Brazil

Em outubro de 1986, 7 a 22 dias apos uma reuniao em uma fazenda no estado da Paraiba, 26 pessoas apresentaram doenca febril, associada a edema bipalpebral bilateral, e de membros inferiores, hepatoesplenomegalia leve, linfadenopatia e, mais raramente, a um exantema. Um menino de 11 anos apresentou arritmia atrial ao eletrocardiograma e um paciente de 74 anos desenvolveu insuficiencia cardiaca aguda. Em 2 pacientes hospitalizados em Sao Paulo, foi estabelecido o diagnostico de Doenca de Chagas aguda por observacao de T. cruzi em creme leucocitario. Em autopsia de um caso fatal foi demonstrada cradiomiopatia chagasica aguda. O xenodiagnostico foi positivo em 9 de 14 pacientes testados. Anticorpos especificos de classe IgG foram encontrados em todos os pacientes e da classe IgM em 20 de 22 doentes examinados. Estudo epidemiologico revelou Triatoma brasiliensis nas vizinhacas desta fazenda, porem tal vetor nao foi encontrado na casa onde a maioria dos hospedes pernoitou. Observou-se alta taxa de gambas infectados por Trypanosoma cruzi. Essas observacoes, associadas as informacoes relativas aos alimentos consumidos, sugerem que a contaminacao de alimentos tenha se originado de secrecoes de marsupiais naturalmente infectados ou de triatomineos infectados que poderiam ter sido esmagados durante o preparo do caldo de cana.