Heart Fail Rev DOI 10.

1007/s10741-010-9211-5

Chronic Chagas heart disease: a disease on its way to becoming a worldwide health problem: epidemiology, etiopathology, treatment, pathogenesis and laboratory medicine
Silvia Gilka Munoz-Saravia Annekathrin Haberland Gerd Wallukat Ingolf Schimke

Springer Science+Business Media, LLC 2010

Abstract Chagas disease, caused by Trypanosoma cruzi infection, is ranked as the most serious parasitic disease in Latin America. Nearly 30% of infected patients develop life-threatening complications, and with a latency of 1030 years, mostly Chagas heart disease which is currently the major cause of morbidity and mortality in Latin America, enormously burdening economic resources and dramatically affecting patients social and labor situations. Because of increasing migration, international tourism and parasite transfer by blood contact, intrauterine transfer and organ transplantation, Chagas heart disease could potentially become a worldwide problem. To raise awareness of this problem, we reect on the epidemiology and etiopathology of Chagas disease, particularly Chagas heart disease. To counteract Chagas heart disease, in addition to the general interruption of the infection cycle and chemotherapeutic elimination of the infection agent, early and effective causal or symptomatic therapies would be indispensable. Prerequisites for this are improved knowledge of the pathogenesis and optimized patient management. From economic and logistics viewpoints, this last prerequisite should be performed using laboratory medicine tools. Consequently, we rst summarize the mechanisms that have been suggested as driving Chagas heart disease, mainly those associated with the presence of autoantibodies against G-protein-coupled receptors; secondly, we indicate new treatment strategies involving autoantibody
S. G. Munoz-Saravia Santa Barbara Hospital Sucre, Sucre, Bolivia S. G. Munoz-Saravia A. Haberland G. Wallukat I. Schimke (&) Charite-Universitatsmedizin Berlin, Chariteplatz 1, 10117 Berlin, Germany e-mail: ingolf.schimke@charite.de

apheresis and in vivo autoantibody neutralization; thirdly, we present laboratory medicine tools such as autoantibody estimation and heart marker measurement, proposed for diagnosis, risk assessment and patient guidance and lastly, we critically reect upon the increase in inammation and oxidative stress markers in Chagas heart disease. Keywords Autoantibodies Chagas heart disease Heart marker Inammation marker Oxidative stress marker Trypanosoma cruzi

Introduction Chagas disease (American: trypanosomiasis) is an endemic parasitic disease that mainly occurs in Latin American countries and is caused by the agellate protozoan Trypanosoma cruzi (T. cruzi).1 Trypanosoma cruzi (Fig. 1) is commonly transmitted to humans and other mammals by the blood-sucking triatomine bug (Reduvidae), also colloquially referred to as the kissing bug (Fig. 2), of the subfamily Triatominae. Among the Triatominae, Triatoma infestans, Rhodnius prolixus, Triatoma brasiliensis and Panstrongylus megistus are of special importance for transmission in the endemic areas of Chagas disease.

The disease was named in honour of the Brazilian physician Carlos Chagas (born in 1879 in Oliveira, died in 1934 in Rio de Janeiro) who, in 1909, discovered a new trypanosome species in the intestine of the triatomine bug, which he named T. cruzi in honour of his mentor Oswaldo Cruz. A biographical sketch of Carlos Chagas was recently published in memory of the discovery of T. cruzi 100 years before (Moncayo A (2010) Carlos Chagas: biographical sketch. Acta Trop 115:14).

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Infection 1 -2 Weeks Acute Stage (asymptomatic/mild) Weeks/Months

life-long

Chronic Stage Latency of 10-20 Years Symptomatic Chagas Disease (25-30 % of infected Subjects)

Asymptomatic Chagas Disease (70-75 % of infected Subjects)

Chagas Heart Disease (85-95 % of symptomatic Subjects)

Chagas Gastrointestinal Disease (5-10 %)

Chagas Heart/Gastrointestinal Disease

Fig. 3 Time course from Trypanosoma cruzi infection to chronic Chagas disease Fig. 1 Electron micrograph image of Trypanosoma cruzi (reproduced with permission of Rubem F. S. Menna-Barreto, Instituto Oswaldo CruzFIOCRUZ, Rio de Janeiro)

Fig. 2 Triatomine bug (Reduvidae)

Both female and male triatomine bugs are able to transmit T. cruzi throughout their lifetimes (up to 2 years). Besides humans, more than 100 mammals, including dogs, cats, rats, sloths, armadillos and bats, are known to be parasite reservoirs. Due to triatomine bug cannibalism, T. cruzi can also be spread throughout triatomine populations. In contrast, birds and reptiles do not carry T. cruzi. Once infected with T. cruzi, subjects become lifelong parasite carriers and pass through several stages of the disease. As illustrated in Fig. 3, which is based on excellent reviews [15], the acute stage of Chagas disease lasts from a few weeks up to a few months and starts nearly 1 week after infection, although symptoms are often not seen or are only mild. Thereafter, different chronic stages follow. Despite lifelong parasite persistence, two-thirds of the patients remain asymptomatic lifelong. Of the remaining third, sometimes only after decades, 90% develop heart disease, and the other 10% are affected by gastrointestinal

diseases. Together, both diseases cause enormous socioeconomic problems in Latin American countries. In 1987 [6], it was reported for Brazil that the treatment of all symptomatic Chagas disease patients (pacemaker implantation and surgical intervention) might require 750 million US dollars per year. For the time interval between 1979 and 1981, it was estimated that death caused by Chagas disease resulted in a loss of working power of about 259,152 years. Consequently, initiatives and control programs were started that include primary initiatives for interrupting the domestic and peridomestic transmission cycles by chemical control of the vectors, animal reservoirs and infected humans, and initiatives for improving housing conditions and health education. In order to prevent parasite transmission via blood bottles prepared from subjects who are infectedwhich is one of the main non-vector routes of transmissionblood donors began being screened for T. cruzi antibodies. Prospectively, in view of the likely number of subjects already infected, it will become increasingly necessary to implement strategies for the effective prevention and treatment of symptomatic Chagas disease patients. In order to follow such strategies, earlier diagnosis, improved monitoring of the progress of chronic Chagas disease and optimal treatment guidance are prerequisite elements. In our view, and considering the economical and logistical options available in endemic areas, laboratory medicine could, in particular, possess the potential to guarantee the implementation of such strategies.

Epidemiology The geographical distribution of the triatomine vector covers an area located between 300 m and 3000 m above sea level and between 42 north and 40 south.

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Heart Fail Rev Table 1 Changes in some epidemiological parameters following the interruption of Chagas disease transmission, 19992006; adapted from [11] and [12] 1990 Annual death (thousand) Cases of infection (million) Annual incidence (thousand) Population at risk (million) Distribution (countries) [45 30 700 100 21 2000 21 18 200 40 19 2006 12.5 15 41.2 28 18

The vector transmission was interrupted in Uruguay (1997) and Chile (1999) and in Brazil in 2005

Fig. 4 Endemic area of Chagas disease

Consequently, the area of infection risk extends from the southern USA down to southern Argentina. However, the main endemic area of Chagas disease covers more than 20 countries in Central and South America, from Mexico down to northern Argentina (Fig. 4). Only a very small number of autochthonous vector-borne cases of infection have been reported [7] in southern USA. Europe is colonized by different subfamilies of the triatomine bug, including species of the Triatominae subfamily. Among these, T. rubrofasciata is known to be a T. cruzi carrier in Latin America. However, there is no indication of T. cruzi transmission by triatomine bugs in Europe. As summarized [8], various species of ea, y, bedbug, mosquito and lice have been suggested as possible candidates for T. cruzi transmission. Ticks (Ixodida) were seen as another potential vector for T. cruzi in Europe. Some tick subspecies can carry T. cruzi; however, there is presently no indication that ticks are able to transfer T. cruzi to humans. In the 1980s [9, 10], about 100 million inhabitants (25% of the total population of Latin America) were living in endemic areas and were under constant threat of infection, while 1618 million people were estimated to be chronically infected. As indicated in Table 1 (adapted from [11, 12]), there were 30 million cases of infection in the 1980s, with an annual rate of 700,000 newly infected subjects and more than 45,000 fatalities. Following the successful multinational initiatives for interrupting Chagas disease transmission, 28 million people at-risk and 15 million infected cases, an annual incidence and mortality of 41,200 and 12,500, respectively, were estimated for 2006. The number of endemic areas decreased from 21 countries in the 1980s to 18 countries at present.

Due to continuous ruralurban migration, Chagas disease, which historically is a disease of poor, rural areas, became widespread throughout urban centers, such as Sao Paulo with about 300,000 infected individuals and Rio de Janeiro and Buenos Aires with more than 200,000 infected individuals [13]. Due to the international migration of Latin Americans, Chagas disease is increasingly becoming a worldwide problem for health systems. Based on the immigrant population from Latin America living in the USA (23 million) and on the prevalence of T. cruzi in the countries of origin, more than 300,000 infected people were estimated to be living in the USA [14]. Figure 5 shows that Europe is not spared from Chagas disease [15]. Of the 500,000 immigrants to Europe (Spain excluded), nearly 3000 were estimated to be infected. In Spain, with nearly 1,700,000 immigrants, 87,000 individuals could be infected. For Australia and Canada with 85,000 and 157,000 immigrants, 3000 and 5000 infected subjects were calculated, respectively [16]. Since unknown T. cruzi carriers may serve as blood donors, about 100 million people are at risk of becoming infected via contaminated blood [17, 18]. Additionally, occupational groups such as social and healthcare employees are at particular risk of becoming infected via contact with the blood of infected persons. Consequently, blood donor screening began in 2007 in the USA [19]. Since 2005 [20], Spanish regulatory law requires that all at-risk donors (persons born in an endemic area, persons born of a mother native to an endemic area and persons who have undergone transfusion in an endemic area) be screened for Chagas disease or otherwise be excluded from donation. Nevertheless, comparable requirements did not yet exist for other European countries at that time, Chagas disease is now increasingly recognized as an emerging public health problem in Europe [21, 22]. International tourism is another route for the worldwide spread of Chagas disease. Other routes of infection by T. cruzi include diaplacental and/or perinatal transfer from the mother to her fetus [23]. Outside Latin America, one

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Epimastigote T. cruzi

Trypomastigote T. cruzi

Amastigote T. cruzi

Fig. 6 Morphological forms of T. cruzi (reproduced with permission of JW Bastien; University of Texas, Arlington)

Fig. 5 Estimated number of Chagas disease (infected) patients in Europe (color code denotes expected frequency) (reproduced from [15] with permission of Oxford University Press)

case of congenital transmission of T. cruzi has been documented in Spain [24].

Etiopathology The vicious cycle of Trypanosoma cruzi infection

From human blood, as well as from any other mammalian blood contaminated with the trypomastigotes, the parasite can be taken up by the triatomine bug when it sucks blood. In the midgut of the bug, the trypomastigote transforms into the epimastigote form, which is better adapted for survival within the insect and is again able to replicate. Epimastigotes retransform in the hindgut of the bug into the infectious trypomastigotes that are excreted with feces and enter humans and other mammals, thus continuing the vicious cycle of T. cruzi infection. The bugs infectious feces pass via the bugs bite or other small wounds into human blood, but the parasite can also pass from the feces through intact mucous membranes, especially those of the mouth and the eyes. The feces remain infectious for a long time, probably also when outside of the bug. Consequently, infection via the ingestion of food contaminated with infected feces has been reported. However, particular genetic variations in the parasite and also in the host are thought of as being responsible for regional differences in the incidence of this disease [25, 26]. Acute Chagas disease

Trypanosoma cruzi exists in three morphological forms that demonstrate the different life cycle phases (Fig. 6). The life cycle of T. cruzi involves stages in the digestive tract of the triatomine bug (secondary host) as the vector and stages in the blood and tissue of mammals (reservoir). The infectious, metacyclic trypomastigote form circulates freely but it is unable to replicate. After cell colonization in phagocytic and non-phagocytic cells of host tissues, the trypomastigotes transform into amastigotes which replicate over many cycles by binary ssion, producing large quantities of amastigotes that now transform again into mobile infectious trypomastigotes. The variety of cells that act as a reservoir for the parasite are large, although a clear tropism exists for muscular and neuronal cells. Of the muscle cells, those of the heart and skeleton, as well as smooth muscle cells are affected to a similar degree. After cell lysis, the trypomastigotes enter the blood circulation.

The acute stage of Chagas disease can be symptomless or it can present with only mild clinical symptoms and therefore remain undiagnosed. A typical sign that is often ignored due to its unspecicity is chagoma, a local infection characterized by swelling around the bugs bite. If the route of parasite entry is through the conjunctiva of the eye, patients present after 412 days with a more typical symptom named Romanas sign [27], which comprises conjunctivitis, unilateral palpebral edema and pre-auricular lymphadenopathy (Fig. 7). However, only 510% of patients present with fever, malaise and lymphadenopathy. In a small number of patients, especially children, hepatosplenomegaly, myocarditis and meningoencephalitis are seen. The mortality rate due to acute Chagas disease is 26%, which is mainly attributed to myocarditis and meningoencephalitis [24, 10, 28]. The low rate of

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Fig. 7 Child with Romanas sign (also named chagoma); unilateral painless periorbital swelling associated with the acute stage of Chagas disease (reproduced from [27] with permission of F. Torrico and M. Castro; Universidad Mayor de San Simon, Cochabamba, Bolivia and E. van der Enden; ITGPRESS, Antwerpen, Belgium)

toward T. cruzi infection than the corresponding wild-type mice [38]. However, no complete parasite elimination has been observed. There is evidence that CD8? T cells only partially control the infection and that they can lose their activity [39]. Immune suppression elements that directly come from T. cruzi could contribute to the ineffectiveness of the immunological system in parasite control. Such incomplete parasite eradication combined with the molecular mimicry by T. cruzi antigens of human antigens of the heart and intestine might be the reason for the induction of an autoimmune response after T. cruzi infection. Autoimmune activities are increasingly thought to be one of the main reasons for the manifestation of lifethreatening complications in a distinct proportion of chronically infected patients. Chronic Chagas disease The result of incomplete parasite control is the lifelong persistence of the parasite and, therefore, patients with chronic infections. This can be demonstrated by antiT. cruzi antibody positivity andby using modern analytical equipment such PCR techniquesby parasite detection in patient tissue samples [40, 41]. Despite this permanent parasitic load, the asymptomatic phase of chronic Chagas disease can be differentiated from the symptomatic stage (Fig. 3). Asymptomatic chronic Chagas disease (latency stage, indeterminate stage) Patients in the asymptomatic stage (indeterminate phase) are symptomless. The heart and gastrointestinal tract have no distinct pathological ndings when ECG, sonography and radiological examinations are used. Asymptomatic patients are only diagnosed by chance or by screening for T. cruzi antibodies, for example in the case of enrollment into the blood donor system or in preparation for surgery. The asymptomatic stage might be interrupted by episodes showing unspecic characteristics of acute infection. In particular, patients with immunosuppressive disorders, such as HIV-positive patients, show such episodes [2, 3, 42]. Small focal inammatory lesions have been detected in tissue samples of the heart, skeletal muscle and gastrointestinal tract from asymptomatic patients [4345]. Symptomatic phase Of the chronically infected patients [15, 10], one-third become symptomatic, sometimes only after decades. Among these subjects, up to 90% develop Chagas heart

mortality in the acute stage is certainly the reason for the lack of detailed histopathological information about acutely infected patients. The few data available originate from an 18-month-old boy and a 4-month-old girl who both died after the infection [4]. Amastigotes were found in the testis and ovaries. Moreover, T. cruzi was also found in mononuclear phagocytes. Mechanisms of parasite control Both humoral and cellular immune responses participate in parasite control, but the highly complex interactions are far from close to being claried [29]. More severe signs of infection were shown in B-cell-decient mice compared to their wild type, which indicates that the humoral immune response is an essential player in the ght against T. cruzi. On the other hand, a concert of CD8? T cells and macrophages, as well as interferon-c (IFN-c) secretion, also seems to be essential. In this context, the perforin-/granzyme-dependent killing of infected cells and FASmediated apoptosis must be considered [30]. Furthermore, in reaction to the infection, macrophages produce IL-12, which is an inducer of resistance against T. cruzi [31]. Other important players in parasite defense are cytokines, which regulate the immune response as well as T. cruzi replication [32], and NO [33, 34], due to its cytotoxic properties. Among the cytokines, IFN-y and TNF-a are dominant in parasite defense. Consequently, anti-IFN antibodies cause an increase in parasitemia and mortality. Mice that were decient for the IFN-y receptor and inducible NO synthetase (iNOS) showed an increased infection risk [3537]. The protective effect of TNF-a was concluded from experiments that used TNF-R1FcIgG3 transgenic mice, which were found to be more sensitive

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disease. The others present with gastrointestinal disease, mainly megacolon and megaesophagus, and alterations in the peripheral nervous system. A certain proportion of patients manifest both cardiac and gastrointestinal diseases. Genetic variability has been discussed as being responsible [25, 26, 46], but this debate has not yet been concluded [47]. With respect to the relationship between HLA polymorphism and the manifestation of chronic Chagas disease, associations were observed between distinct HLA alleles and an increased risk of developing of chronic Chagas disease in some studies [48, 49], but others denied nding any relationships [50]. Chagas heart disease Chagas heart disease becomes manifested in men and women with a comparable frequency, and it mainly begins between the ages of 30 and 50 years. Abnormalities found in ECG and/or echocardiography are early indications of the development of Chagas heart disease. In line with the diagnostic options available in endemic areas, however, cardiac arrhythmia found by Holter ECG examination is often the rst indication of the development of Chagas cardiomyopathy in chronically infected patients who, from an anamnestic and clinical viewpoint, are clearly thought to be in the asymptomatic stage [51]. Among the potential blood donors who attended the St. Barbara hospital in Sucre, Bolivia, who were symptomless but were diagnosed during blood donor screening as suffering from chronic Chagas disease, as detected by T. cruzi antibody positivity, we found that one-quarter of these patients presented with distinct ECG indications for Chagas heart disease (unpublished data). Of the typical ECG abnormalities in chronic Chagas heart disease, indicating the diagnostic importance of the ECG [52], right bundle branch block, left anterior hemiblock, ventricular extrasystoles, sinus bradycardia, auricular brillation and complete atrioventricular block were found to be the most frequent ones with increasing severity of the disease [2, 4, 27]. According to practical recommendations for the evaluation of newly diagnosed patients with chronic Chagas disease based on T. cruzi antibody positivity [53], patients should undergo a medical history interview, a physical examination and a resting 12-lead ECG with a 30-second lead rhythm strip. In the case of normality, examination should be repeated annually. Where Chagas heart disease is diagnosed, comprehensive cardiac evaluation is recommended, which should include Holter ECG examination, echocardiography [54, 55] and exercise testing. It was previously suggested that cardiac MRI is useful in the diagnosis and management of chronic Chagas disease [56, 57]. From clinical view, myocarditis, thromboembolic events, sudden cardiac death and congestive heart failure are typical of advanced Chagas heart disease. However, about 30% of Chagas

heart patients die from sudden cardiac death without any characteristic signs of advanced Chagas heart disease being been found before. From an anatomical viewpoint, Chagas cardiomyopathy is characterized by progressive heart enlargement due to chamber dilatation. The walls and septum can be thickened with hypokinesia of the septum and the posterior wall. The radiograph illustrated in Fig. 8 shows a typically enlarged chagasic heart. The microscopic detection of parasites in chronic chagasic hearts is successful in only 1020% of tested patients. However, modern molecular diagnostics using amplication tests on DNA show the appearance of parasites in almost all patients with Chagas heart disease [58, 59]. However, the severity of heart disease does not correlate with the occurrence of parasite DNA. This is a clear sign that direct heart damage in relation to parasite load and inammation is not the only mechanism that must be considered. It is assumed that both the parasite and the host exert an inuence on the pathogenesis of Chagas heart disease [60]. Comparable with the ndings in asymptomatic patients but more pronounced in symptomatic patients, the histopathological pattern of the heart shows nests of focal inammation with T cells and varying numbers of B cells and macrophages, diffuse interstitial brosis and a disturbed morphology of the myocytes. The conduction system in the heart also shows alterations [6163]. The often apically aneurysmatic Chagas heart is thought to be the cause of thrombus formation, which may lead to thromboembolic events in the brain and lungs and

Fig. 8 Cardiomegaly of a chronic Chagas patient with implanted pacemaker, demonstrated by thorax radiography (Reproduced with permission of R. Araujo, Santa Barbara Hospital, Sucre, Bolivia)

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which are thought of as being responsible for the high rate of sudden death in Chagas heart disease. However, the main life-threatening complication of chronic Chagas disease is the continuous progress toward severe heart failure. Nearly 60% of patients die due to cardiomyopathy. Chagas gastrointestinal disease (megaesophagus and megacolon) Malnutrition caused by swallowing problems and regurgitation leading to weight loss, as well as obstipation, accompanied by abdominal pain, marks the progress of chronic Chagas disease to megaesophagus and megacolon. Radiological investigation employing barium as the contrast agent can be used for early diagnosis; however, a simple radiological investigation is often sufcient. The nerve system in chronic Chagas disease The damage to the parasympathetic nervous system may be the main driver of alterations in the vegetative nervous system in chronic Chagas disease [64]. It is assumed that the damage to the parasympathetic nervous system starts in the acute phase and proceeds into the chronic phase. The central nervous system, however, is mostly affected during the acute phase of the disease. Rare cases of changes in the psyche of chronically infected Chagas patients are also a sign of nerve damage during the chronic phase of the disease [2].

Therapy Therapy in the acute stage concentrates on the elimination of the parasites, preferably by using Nifurtimox and Benznidazol [3, 18, 65]. While Nifurtimox has to be taken for between 50 and 120 days, Benznidazol is administrated for up to 60 days. However, some T. cruzi strains can develop resistance against the drugs. Consequently, only 50% of treated patients are responders to Nifurtimox and Benznidazol. Furthermore, the existing drugs show enormous potential for toxic side effects, which manifest in the liver and as an allergic reaction, mainly after long-term administration. In contrast, a wide range of therapeutic possibilities are available after the manifestation of Chagas heart disease, which are based on common heart failure therapies including drug treatment and pacemaker and cardioverterdebrillator implantation. Heart transplantation is the only therapeutic option in some cases [2, 18]. As is the case for cardiomyopathy in general, cell-based therapy is being increasingly discussed for Chagas cardiomyopathy [57]. Considering the increasing acceptance of an autoimmune background in the pathogenesis of the life-threatening complications of chronic Chagas disease in general, but especially for Chagas heart disease (see the section

Pathogenesis of symptomatic chronic Chagas disease), new treatment regimes similar to those generally used in autoimmune diseases, such as treatment with anti-inammatory drugs and immunosuppressive drugs, could become increasingly important. Another promising option would be the inhibition or elimination of the high percentage of pathogenetic autoantibodies found in chronic Chagas heart disease [66, 67]. It has been shown for patients with dilated cardiomyopathy and also recently suggested for patients with Chagas cardiomyopathy that a signicant benet could be achieved if the autoantibodies against the beta 1-receptor were eliminated by immunoabsorption [6871]. Since the current immunoabsorption techniques for beta 1-receptor autoantibodies in DCM are based on the elimination of the whole IgG fraction, they might be suitable for chronic Chagas disease patients carrying autoantibodies against the beta 1-receptor and also for those carrying autoantibodies against the beta 2- and muscarinergic 2-receptors. However, cost factors and logistical reasons might be the limiting factors preventing the wide use of immunoabsorption techniques for chronic Chagas disease in the near future. The blockade of autoantibodies by specic peptides, homologs to the autoantibody targets of the corresponding receptors, could be an alternative which is presently under investigation [72] with respect to DCM patients, with the focus on inhibiting beta 1-receptor autoantibodies. Recently, we selected chemical antibodies named aptamers for specic neutralization of the autoantibodies found in patients with DCM and Chagas heart disease [73]. This nding could possibly open the door to a totally new treatment strategy. Whatever strategy of autoantibody-directed therapy is administered to patients with Chagas heart disease in the future, asymptomatic but autoantibody-positive patients should be included in the treatment.

Pathogenesis of symptomatic chronic Chagas disease Different hypotheses have been formulated with respect to the pathogenesis of symptomatic chronic Chagas disease. However, these hypotheses are clearly more detailed for Chagas heart disease. This mirrors the greater amount of data available on Chagas heart disease because of its more frequent occurrence. However, the majority of the pathogenetic data found on Chagas heart disease can also be used to explain the main aspects of the gastrointestinal manifestation of chronic Chagas disease. Chagas heart disease As extensively summarized [29, 74], several hypotheses have been formulated based on the direct response of the

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immune system to the parasites in the tissues, as well as on the interactions between inammatory, immunological and autoimmunological mechanisms. A. Primary damage of the neuronal system The key event in this hypothesis is the denervation of the autonomous parasympathetic system in the heart. This neuronal damage could be induced in the acute phase of the disease, and the resulting lesions might accelerate in the chronic stage. B. Cardiomyocyte toxicity due to T. cruzi and/or T. cruziderived products The myocytolysis of host cells following intracellular infection has been documented for the acute stage, and it could also potentially be of some signicance in Chagas heart disease. It was rst assumed that parasites could secrete cytotoxic products in 1955. However, with respect to the generally low parasite titer in the chronic stage, direct parasite cytotoxicity and/or the cytotoxicity of parasite-derived products should be of only limited relevance. This could change for immunosuppressed patients and/or those with a high parasite titer. C. Parasite-induced microvascular alteration This hypothesis is based on the assumption thatwithout producing cell lysisparasites interact with essential metabolic reactions in microvascular cells. Inhibited protein synthesis and changed Ca homeostasis are only some of the interactions documented that can cause hypoperfusion. The subsequent hypoxic/ischemic damage of cardiomyocytes might then produce chronic inammatory conditions in the heart which drive Chagas heart disease. D. Polyclonal B-cell activation Immunosuppression and autoimmune processes following the disruption of normal immune regulation by polyclonal B-cell activation could support pathogenetic events. E. Persistent T. cruzi antigens Antigens might trigger T-cell-mediated responses of the delayed-type hypersensitivity cells or cytotoxic cells, leading to damage in the hosts infected and/or bystander cells. F. Autoimmunity induced by T. cruzi-specic antigens or by host antigens Autoimmunity might result from T. cruzi antigen-associated molecular mimicry, as well as from bystander activation. Presently, none of these listed hypotheses claim exclusivity. Because of the low quantity of parasites in the myocardium of patients with Chagas heart disease, the development of autoimmunity is increasingly accepted as a key event in its pathogenesis. The scheme [29] demonstrated in Fig. 9 shows the potential cooperation between molecular mimicry and bystander activation for T. cruzi induced autoimmune Chagas heart disease.

T. cruzi Immune response Host cell lysis

T. cruzi antigen specific T and B cell stimulation

Inflammation adjuvant effects

Release of host selfantigens and cryptic epitopes

Molecular mimicry

Bystander activation

Anti-parasite response

Pathological autoimmunity

Fig. 9 Suggested mechanisms of T. cruzi pathogenicity by molecular mimicry and bystander activation adapted from [29]

In the bystander activation, parasites produce proinammatory conditions with the release of cytokines, NO/ peroxynitrite and chemokines, which together with intracellular parasite replication result in cardiomyocyte damage and the liberation of autoantigens and cryptic epitopes. Such autoantigens and cryptic epitopes, normally inaccessible to the hosts immune system, are now recognized by autoreactive T cells. The T cells are activated in parallel and proliferate to perpetuate the immune response against the heart structures. In molecular mimicry, the hosts immune response to T. cruzi proteins is directed against cross-reacting proteins of the hosts heart. Molecular mimicry is thought to be more signicant for the autoimmunity of Chagas heart disease than the bystander activation, and it is being increasingly suggested as a key pathogenetic event in Chagas heart disease. This originates from the increasing number of cross-reactivities between T. cruzi and host antigens found in recent years. In Table 2, adapted from [75], cross-reacting T. cruzi and human antigens are listed, which have been suggested as driving Chagas heart disease. In animal experiments, both immunization with T. cruzi antigens and the transfer of T. cruzi-activated T cells resulted in myocardial alterations such as focal myocarditis, demyelination and conduction system defects. This was accompanied by the detection of autoantibodies affecting the essential structures and functions of the heart. Support for the autoimmunity hypothesis of Chagas heart disease has come from the demonstrated long persistence of antigens, T-cell clones and autoantibodies after infection with T. cruzi [76]. Among the human antigens that show molecular mimicry to T. cruzi antigens, G-proteincoupled receptors (GPCRs) are receiving more and more attention. Table 3, adapted from [77], lists a variety of

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Heart Fail Rev Table 2 Cross-reactivity of Trypanosoma cruzi and human antigens favoring molecular mimicry (adapted from [75])

Human antigens Neurons Neuronal 47-kD protein Heart and skeletal muscle Smooth and striated muscle Cardiac myosin heavy chain Sacroplasmatic reticulum antigen (SRA) Glycosphingolipids Microtuboli-associated protein (MAP) (brain) 28-kD lymphocyte membrane protein 23-kD ribosomal protein Beta 1-adrenoreceptor, M2 muscarinic receptor, M2 cholinergic receptor, cardiac Cardiac muscarinic acetylcholine receptor

T. cruzi antigens Sulphated glycolipid FL-160 Microsomal fraction 150-kDa protein B13 protein SRA Glycosphingolipids MAP 55-kD membrane protein 23-kD ribosomal protein Ribosomal P0, P2 and 150-kD protein Unknown

Table 3 The G-proteincoupled receptors and their functional autoantibodies that were suggested as driving the pathogenesis of heart and circulatory diseases adapted from [77]

Receptor a1-R b1-R b1-R b1-R b2-R b2-R AT1-R AT1-R AT1-R

Disease Hypertension Dilated cardiomyopathy Myocarditis Chagas disease Chagas disease Allergic asthma Preeclamsia Malignant hypertension Vascular renal rejection Chagas disease Dilated cardiomyopathy Systemic lupus erythematodes Myasthenia gravis Rassmussens encephalitis, non-inammatory focal epilepsy, catastrophic epilepsy Graves disease

AAB effect Agonist-like Agonist-like Agonist-like Agonist-like Agonist-like Inhibitory Agonist-like Agonist-like Agonist-like Agonist-like Agonist-like Agonist-like Inhibitory Agonist-like

Corresponding epitope Loop 1, 2 Loop 1, 2 Loop 1, 2 Loop 2 Loop 2 Loop 3 Loop 2 Loop 2 Loop 2 Loop 2 Loop 2 Loop 2 N-terminal extracellular domain aa 372386

a-R a-adrenergic receptor, b-R b-adrenergic receptor, AT1-R angiotensin II-receptor 1, M2-R muscarinic 2-receptor, 5HT4-R serotonin receptor, AcCh-R muscarinic acetylcholine receptor, GluR3 glutamate receptor, TSH-R thyroid-stimulating hormone receptor, aa amino acid, AAB autoantibody

M2-R M2-R 5HT4-R AcCh-R GluR3

TSH-R

Agonist

Extracellular domain, conformational epitope

GPCRs for which autoantibodies have been identied in patients with diseases of the heart and circulatory system. G-protein-coupled receptors belong to the superfamily of heptahelical transmembrane proteins. About 80% of all receptors belong to this receptor family. In animals and humans, GPCRs participate in the perception of senses, in inammatory processes, chemotaxis, endo- and exocytosis, cell growth and differentiation. They are also involved in the regulation of metabolism. The effects of glandular hormones, tissue hormones and neurotransmitters such as catecholamines, glucagon, endothelin, angiotensin, acetylcholine, serotonin and others are mediated via GPCRs. G-protein-coupled receptors sense signal molecules outside the cell. The resulting change in receptor conformation activates the G-protein thatdepending on its type

modulates the cAMP or phosphoinositol pathway to translate the outside signal into an internal cellular response. Because of the central role of GPCRs in the regulation of metabolism, it is not surprising that altering GPCR-dependent signal transduction results in a wide variety of pathogenetic consequences. Agonistically acting autoantibodies are thought to drive GPCR- dependent pathogenetic events. Autoantibodies against G-protein-coupled receptors in Chagas heart disease Among the GPCR autoantibodies with potential relevance for Chagas heart disease, those against the beta1-adrenergic receptor (beta1-AAB), the beta 2-adrenergic receptor

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Heart Fail Rev Table 4 Basic data and percentages of positivity for autoantibodies (AAB) against beta 1-adrenergic, beta 2-adrenergic and muscarinergic 2 receptors (beta1-AAB, beta2-AAB, M2-AAB) of 228 Chagas disease patients and 29 healthy subjects [67] C n (male/female) Age (years), median (min/max) AAB positivity (%) Beta1-AAB Beta2-AAB M2-AAB Beta1-/M2-AAB Beta2-/M2-AAB Beta1-/M2-AAB or beta2-/M2-AAB Categories of cardiomyopathy (%) Mild Moderate Severe 5 44 51 15 56 29 0 3 0 0 0 0 34 33 42 29 24 34 100* 89 98 98 89 98 38 97 100 38 97 99 96 98 100 96 98 96 29 (9/20) 30 (19/61) I 96 (26/70) 30 (18/82) CM 57 (34/23) 47 (18/82) MC 30 (11/19) 46 (19/78) CM ? MC 45 (21/24) 54 (29/81)

C healthy control subjects, CM patients with chronic Chagas disease manifested as cardiomyopathy, CM ? MC patients with chronic Chagas disease manifested as cardiomyopathy combined with megacolon, I patients with Chagas disease in the indeterminate (asymptomatic) state, MC patients with chronic Chagas disease manifested as megacolon only * CM [ MC, P B 0.001

(beta2-AAB) and the muscarinergic-2 receptor (M2-AAB) have received particular attention. This is based on the positivity for beta1-AAB, beta2-AAB and M2-AAB found in a distinct percentage of chronically infected Chagas patients [66, 67, 78]. As published recently [67], we divided chronic Chagas patients by anamnesis, Holter ECG and radiology into groups that were asymptomatic and symptomatic. As indicated in Table 4, we found positivity for beta1-AAB, beta2-AAB and M2-AAB in nearly onethird of all asymptomatic patients. This frequency clearly parallels the epidemiological data on the incidence of Chagas heart disease in chronically infected patients. In symptomatic patients, positivity for beta1-AAB was found in 75% of the cases. Nearly all cases had beta2-AAB and M2-AAB positivity. After subdividing the symptomatic patients, beta1-AAB and M2-AAB were found in nearly all patients with cardiomyopathy. In contrast, only 38% of the cardiomyopathy patients had beta2-AAB. Of the megacolon patients, 90% were positive for all receptors. Patients suffering from cardiomyopathy and megacolon carried all three receptors with a frequency of nearly 100%. The AABs target the negatively charged region of the second extracellular receptor loop, and the binding of AAB enables receptor dimerization, stabilizing active receptor conformation [79, 80]. Figure 10 shows the schematic structure of the beta1-adrenergic receptor in relation to beta1-AAB. Only monovalent Fab fragments did not stabilize the receptors.

There is now growing evidence that AABs are also pathogenetic substrates of Chagas heart disease, particularly beta1-AAB. This is not least because of the similarities between Chagas heart disease and dilated cardiomyopathy (DCM). Both patient groups carry a high percentage of beta1-AAB. Consequently, a cross talk [81] was suggested between the beta1-AAB found in Chagas heart disease and the beta1-AAB found in DCM, which means that the results from animal experiments and human studies that focused on the latter case can consolidate the beta1-AAB-associated history of the former. Most important were the experiments that showed cardiomyopathy-typical heart alterations [82, 83] following the transfer of beta1-AAB to animals. Furthermore, as already indicated before, patients show improvements after the elimination (immunoabsorption) of beta1-AAB [68, 69, 71]. At cellular and subcellular levels, changes in the action potential duration and contractility of cardiomyocytes have been observed following the addition of beta1-AAB and M2-AAB [84, 85]. All three of these antibodies, which are found in Chagas heart disease, are agonistic. However, depending on the different downstream effectsthe activation of adenylate cyclase by beta1-AAB and beta2-AAB and its inhibition by M2-AABpositive or negative chronotropy are evident. In agreement with this, IgG prepared from the blood of Chagas patients, which preferentially contains beta1-AAB and beta2-AAB, activates adenylate cyclase and increases cAMP formation in heart cell membranes and cardiomyocytes. This occurs in parallel with increased contractility [86, 87]. However, the

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esophageal strips and reduces cAMP concentration [93, 94]. It has been assumed that the binding of M2-AAB activates pertussis-sensitive G proteins that inhibit adenylate cyclase. Additionally, it has been suggested that M2-AAB induces receptor desensibilization and sequestering, which would support dysautonomia of the colon and esophagus [95]. However, with respect to the high prevalence of M2AAB in Chagas heart disease, these AAB receptors should not be underestimated as drivers of cardiomyopathy.

Laboratory medicine Parasite detection

Fig. 10 The human beta1-adrenergic receptor as a model of G-protein-coupled receptors targeted by the corresponding autoantibody. The N-terminal domain usually contains less than 50 amino acids and is located in the extracellular space, whereas the C-terminal part of the protein varies from 23 (muscarinergic2 receptor) to about 100 amino acids (beta2-adrenergic receptor). The transmembrane regions usually contain between 23 and 24 amino acids, limited by the helical secondary structure and the thickness of the hydrophobic lipid bilayer. The homology varies throughout the whole superfamily. However, the homology is higher (35%; 90%) in the transmembrane helices and functionally important side chains in the transmembrane helices, and the loops are strongly conserved between different vertebrate species. Agonists bind to a hydrophobic cave formed by the transmembrane helices. As investigated so far, the transmembrane regions I, II, VI and VII are particularly important for agonist binding. The extracellular loops as well as outer parts of the transmembrane helices are potential targets of the corresponding autoantibodies (reproduced from [77] with permission of Pabst Science Publishers, Lengerich, Germany)

effects of IgG prepared from animals and humans have not always been consistent [88]. Activation of the L-type calcium canal is a further characteristic reaction in the presence of beta1-AAB. This might result from the protein kinase A-dependent phosphorylation of the canal protein, but it could also result from a direct interaction between the activated G-protein subunits and the canal proteins [89, 90]. It has generally been accepted that changes in the calcium ow caused by autoantibodies are an essential component in the pathogenesis of myocarditis and dilatative cardiomyopathy [91]. As mentioned already before, alterations in the parasympathetic nervous system are a key event in the pathogenesis of both Chagas heart disease and gastrointestinal disease. Agonists of the M2-receptor reduce the contractility of the rat atrium, increasing cGMP and reducing cAMP formation [92]. However, it has been suggested that M2-receptordependent pathogenetic mechanisms could be more profound in gastrointestinal manifestations. The M2-AAB receptor increases the tonus (basal) at the colon and

Trypanosoma cruzi in the circulating blood, especially at a high parasite titer, which is predominantly observed in acute Chagas disease, are detectable by microscopic examinations of thick or thin blood lms stained with Giemsa. In addition, anti-T. cruzi antibodies of the IgM type are detectable during the acute phase of the disease [10, 96]. During the chronic stage, because of the low blood titer, direct parasite detection is more difcult, but PCR-based detection of parasite DNA can also be used in tissue samples [40, 41, 97, 98]. However, chronic Chagas disease is most frequently diagnosed by the detection of anti-T. cruzi antibodies of the IgG type. The World Health Organization [99] recently initiated a multicentric study to compare the sensitivity and specicity of assays used to detect anti-T. cruzi antibodies in chronic Chagas patients. Trypanosoma cruzi antibodies of the IgG type were detectable in hemagglutination assays (sensitivity = 8899%, specicity = 96100%), particle agglutination assays (97100, 9799.5%) and ELISA (94100, 96100%). Indirect immunouorescence (sensitivity 98%, specicity 98%), western blot (100, 100%, respectively), immunoblot (98, 99.5%, respectively) and radioimmunoprecipitation (100, 100%, respectively) served for conrmation. Based on the study data, hemagglutination assays are not recommended for T. cruzi antibody detection in blood donor screening. Risk assessment for Chagas heart disease in asymptomatic patients Up until now, there have been no available laboratory medicine tools for assessing the risk of asymptomatic patients developing Chagas heart disease. However, a patients genetic disposition and genetic diversity of T. cruzi could indicate the risk for the development of Chagas complications [26, 100]. Another hopeful strategy for risk assessment could be based on screening for AABs

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in asymptomatic Chagas patients (see the section Autoantibodies against G-protein coupled receptors in Chagas heart disease; Table 4). We recently presented the rst preliminary data showing that beta1-AAB, beta2-AAB and probably M2-AAB in particular are early indictors of the risk of asymptomatic Chagas patients becoming symptomatic [101]. The use of M2-AAB for risk assessment is also seen as a promising strategy by others [92], as its function in driving alterations in the autonomic nervous system [94, 102] has frequently been discussed as being one of the primary steps in the pathogenesis of symptomatic chronic Chagas disease. However, in order to verify the strategy of AAB measurement for risk assessment in asymptomatic Chagas patients, prospective studies enrolling broad cohorts would be indispensable. Currently, a bioassay is mostly used for AAB measurement [67, 103, 104]. In this assay, cultured neonatal rat cardiomyocytes are the substrate for AABs. The AABs are quantied by monitoring the chronotropic effects induced by the AAB-containing IgG fraction prepared from the patients blood. The addition of specic antagonists enables the differentiation between beta1-, beta2- and M2AABs. The beating frequencyas an integral parameter of cell functionalitymeasured in this bioassay is an advantage as it corresponds to the AAB concentration. Moreover, the bioassay tests beta1-AAB, beta2-AAB and M2-AAB in parallel. However, the limitations of this bioassay include sophisticated assay standardization, a lack of adequate control materials and extensive turnaround times. There are other cell-based AAB quantication methods. In particular, the quantication of beta1-AAB was performed via cAMP formation in cultured cells that were engineered to express the recombinant receptor protein for beta1-AAB. The quantication of cAMP is then possible using RIA or ELISA technology. Quantifying the formation of cAMP using FRET technology might become important in the future [70, 105]. Considering the time, cost and capacity of cell-based assays, the development of less costly but well-standardized and more universally available ELISAs seems to be an essential prerequisite for AAB measurement. Whereas the bioassay quanties AABs by measuring their functionality, ELISAs and RIAs use for the AAB quantication exclusively the AAB binding at small peptides that are homologous to extracellular receptor epitopes recognized by the AABs. ELISAs and RIAs do not indicate the AAB functionality. This might be one of the reasons for previous discrepancies in the results between bioassays and ELISAs [106]. Nevertheless, ELISAs specic for beta1-AAB, beta2-AAB and M2-AAB have been frequently used in different studies [107, 108]. However, to the best of our knowledge, no RIAs or ELISAs for Chagas disease-

relevant autoantibodies against GPCRs are presently commercially available. Early diagnosis and monitoring of Chagas heart disease Heart non-specic markers With respect to the impact of inammatory processes in the pathogenesis of the Chagas heart disease, established inammation markers should be appropriate for patient monitoring. With the tight connection between inammation and oxidative stress, the measurement of markers for oxidative stress might be additionally helpful. Inammation markers The typical signs of inammatory processes are obvious, especially during the acute stage after infection with T. cruzi. The infection of animals with T. cruzi and experiments based on demonstrating protection against T. cruzi infection by immunization clearly showed that the measurement of cytokines and acute phase proteins has the potential for monitoring Chagas disease, especially the acute stage of Chagas disease [109, 110]. If compared acute-phase proteins and the serological proles in chagasic children [111], the early acute stage only presented an increase in specic anti-T. cruzi IgM; the intermediate acute stage showed high levels of anti-T. cruzi IgM and/or anti-galactose antibodies (anti-Gal; which has been suggested as being an additional marker of the acute phase), as well as high levels of a2-macroglobulin and specic anti-T. cruzi IgG; and in the late acute stage, the IgM level was already low but a2-macroglobulin, CRP, anti-Gal and specic IgG levels were still high. Additionally, increased a2-macroglobulin and CRP levels were shown when healthy controls and acute T. cruzi-infected children were compared [112]. Furthermore, for cytokines such as TNF-a, IL-2, IL-6, IL-8 and IL-12 and soluble receptors such as sIL-2R, sCD8 and sCD4, Chagas diseaseassociated changes were reported, especially during the acute phase of the infection [113116]. The increased IL-2R concentration agrees with ndings in a mouse model which showed that IL-2R expression was elevated during immune suppression in the acute phase of infection. However, inammation markers such as CRP are also increased in chronic chagasic patients when compared to healthy subjects, especially in those suffering from Chagas heart disease at an advanced stage. However, the values did not often exceed the CRP cutoff used to diagnose acute inammation [117]. In our study [118] analyzing healthy controls and patients suffering asymptomatic chronic Chagas disease or

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heart and gastrointestinal diseases, CRP and IL-6 were signicantly increased, mainly in the patient group suffering heart disease. In this group, CRP and IL-6 were found to be above the inammation-specic cutoffs in 41 and 71% of the patients, respectively. Additionally, CRP and IL-6 concentrations and the percentage of patients with values above the cutoffs increased with the severity of heart disease. However, we also found a distinct number of patients in the asymptomatic and gastrointestinal groups who presented with CRP and IL-6 concentrations above the cutoffs. In contrast, another study failed to demonstrate signicant CRP changes in Chagas heart and gastrointestinal diseases compared to healthy subjects and asymptomatic patients, although there a more pronounced variability in the CRP values in symptomatic patients was found [119]. Recently, it was suggested that patients with Chagas heart disease could be more affected by cytokine release than patients with idiopathic dilated cardiomyopathy. Although this was reected by IL-6 levels, TNF-a levels were not found to be different between the two groups of diseases. However, for both diseases, the markers were higher than in healthy subjects [120], and the IL-6 level correlated with the outcome. For TNF-a, comparable values, which were clearly higher than those for healthy subjects, were found in asymptomatic patients and in those with Chagas heart disease presenting with LVEF [ 50%. Patients with LVEF \ 50% showed even higher values [121]. In addition to this TNF-a relationship with the disease stage, differences between healthy subjects and asymptomatic and symptomatic patients were also seen in nitric oxide levels [122]. In contrast, different TNF-a concentrations between healthy subjects and patients with mild cardiomyopathy were sometimes not evident, whereas differences between mild and severe diseases could be substantiated [123]. Patients suffering from both HIV infection and Chagas disease showed higher TNF-a and IL-6 levels than patients with HIV infection only [124]. In summary, the acute disease stage is detected by profound changes in the pattern of acute-phase proteins and cytokines, which could be of interest as laboratory medicine tools for patient guidance of this stage. Unfortunately, the data for acute-phase protein and cytokine measurements in chronic Chagas disease are inconsistent. Consequently, the impact of these markers as laboratory medicine tools for the guidance of chronic Chagas patients is still restricted, especially for monitoring the progress from the asymptomatic stage to the symptomatic stage. Oxidative stress markers Oxidative stress has been dened as a metabolic condition characterized by imbalances in the equilibrium status of the pro-/antioxidant system in favor of pro-oxidants. Under aerobic conditions,

the formation of pro-oxidants, often named reactive oxygen species (ROS), is strongly associated with physiological processes. Reactive oxygen species are formed as products or by-products of phagocytosis, uric acid formation via xanthine oxidase and oxidative phosphorylation, as well as in the metabolism of eicosanoids, catecholamines and xenobiotics. After the overstimulation of these processes, which have been reported to occur in many pathophysiological situations including, among others, hypoxia, hyperoxia, ischemia, inammation and intoxication, ROS can be produced in excess. In this case, ROS react with molecular structures in all major chemical classes found in living cells to realize their toxic potential, ultimately leading to cell death. In particular, the cell-damaging potency of thiol oxidation, lipid peroxidation and DNA degradation has been extensively documented. Hypochlorite and reactive nitrogen species are generated by the reaction of ROS with chloride or NO, accelerating the toxic potency of ROS. Consequently, oxidative stress has been implicated by a growing body of evidence in human diseases including diseases of all major organs and physiological systems [125, 126]. Therefore, it is not surprising that oxidative stress has also been discussed as a pathogenetic factor in heart failure [127], especially also in Chagas heart disease [128, 129]. With respect to this, treatment with antioxidative agents has been tested to protect patients with Chagas disease [130]. The inammatory processes in Chagas disease seem to be mainly associated with ROS generation. As benecial processes, ROS production of granulocytes and macrophages contributes to parasite phagocytosis. Therefore, neutrophil granulocytes lose their capability of phagocytosis when their ROS production is inhibited [131]. The excessive activation of macrophages (acute inammation) or long-term stimulation during chronic infection, as seen in Chagas disease, has resulted in excessive and uncontrolled ROS production [132, 133]. Mitochondrial damage in Chagas disease was suggested as a further source of excessive ROS generation, especially in the case of heart affection [134, 135]. Treatment with antioxidant compounds prevented mitochondrial alteration and reduced ROS production [136]. In advanced Chagas heart disease, hypoxic and ischemic conditions (e.g., via xanthine oxidaseproduced ROS) could be further sources of ROS. The rst evidence of oxidative stress in Chagas disease came from animal experiments, which indicated an increase in oxidative stress during the acute phase of T. cruzi infection. Increased oxidative stress in the serum became visible via the elevation of lipid peroxidation and protein carboxylation products. In parallel, a compensatory increase in the antioxidant capacity was also observed. The acute infection was followed by a time of reduced

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oxidative stress. However, as with the preceding disease, the chronic stage of Chagas disease was again accompanied by increased oxidative stress alongside a reduction in antioxidant defense [137140]. An increasing number of markers have been suggested as laboratory medicine tools to monitor oxidative stress in humans [125, 126]. Among these markers, myeloperoxidase, which is localized in neutrophil granulocytes, monocytes and tissue macrophages, is one of the molecular producers of the hypochlorite needed for parasite defense, but its excess production can be responsible for tissue damage. In cases of excessive stimulation of inammation cells, myeloperoxidase is released into the serum. In acute coronary syndrome and heart failure, the resulting serum levels of myeloperoxidase are of prognostic value [141]. Elevated myeloperoxidase activity has also been observed in the serum of Chagas patients [142]. Other markers used in human studies were products of lipid peroxidation, protein oxidation and protein nitration. The measurement of antioxidant defense compounds, such as antioxidant enzymes and non-enzymatic antioxidant compounds, has also been used to assess oxidative stress in Chagas disease. It was recently demonstrated that increased oxidative stress, as indicated by malone dialdehyde elevation in the plasma, correlated with mitochondrial dysfunction in Chagas patients [143]. Additionally, in this study, the lower blood activities of antioxidant enzymes (total superoxide dismutase, manganese superoxide dismutase and glutathione peroxidase) and non-enzymatic antioxidant glutathione were found. Decreased antioxidant defense was also demonstrated in other studies [144, 145]. A selenium deciency was found in chronic Chagas patients, especially in those with progressed cardiomyopathy [146]. Although selenium is essential for glutathione peroxidase, which is one of the main constituents of the antioxidant defense system, the activity of this enzyme did not decrease in chagasic patients. Heart-specic markers B-type natriuretic peptides (BNP, NT-proBNP) are now well accepted for diagnosing and monitoring heart failure [147]. Therefore, it is not surprising that these markers have already been tested in patients with Chagas heart disease, where it was found that levels of BNP increase with disease severity [148155]. In patients with chronic Chagas disease, characterized by LVEF \ 40%, levels of BNP were signicantly higher than those in patients with LVEF [ 40%. The last patient group was not signicantly different from healthy controls [148]. The level of BNP was found to be negatively correlated with the LVEF. Based on a specic BNP cutoff point, left ventricular dysfunction was detected with a high sensitivity, specicity

and negative predictive value in chronic chagasic patients [148, 149]. Recently, it was suggested that the combination of ECG and BNP is the best strategy for detecting left ventricular dysfunction in chronic Chagas patients [156] and that this combination is superior compared to the conventional strategy of ECG and chest X-ray. With respect to the economic and logistics situations in endemic areas, this laboratory medicinesupported strategy was recommended as a rst-line strategy in the recognition of LV dysfunction in Chagas disease. A further nding for BNP in Chagas heart disease was that BNP elevation is related to the 6-min walking distance [157]. However, no relationship with the BNP level was found when the exercise capacity of Chagas heart patients was analyzed [158]. It was recently found in a group of patients with chagasic cardiomyopathy that the BNP level correlated with diastolic function patterns, regardless of the systolic function [159]. Using end points such as death and heart transplantation, BNP predicts the outcome of Chagas patients [154, 155]. Higher values for the marker NTpro-BNP were also demonstrated in chagasic patients with heart disease [160162]. The cardiac troponins I and T (cTnI; cTnT) have become another cornerstone in modern laboratory medicine. Troponins were established as markers for the diagnosis, risk stratication and therapy monitoring of patients with acute coronary syndrome [163]. However, there are numerous other physiological heart stressors, for example exercise [164], general pathologies affecting the heart [165] and cardiac diseases such as stable angina, myocarditis and heart failure [166, 167], that are accompanied by serum cardiac troponin values which are clearly different from those of healthy controls (although they might be below or near the established cutoff values for non-ST elevation infarction). Many of the conditions associated with the mild cardiac troponin release of non-necrotic origin are associated with inammation. This was recently shown for a runners cardiac troponin release [168]. With respect to the inammation background in chronic Chagas disease, especially that derived from focal inammation spots and microlesions found in the chagasic heart, we suggest that chronic Chagas patients with heart disease could present with increased levels of cardiac troponin in their blood. However, there have only been two previous studies and a single patient observation that analyzed cardiac troponin levels in Chagas heart disease [169171]. These studies did not indicate a profound relationship between Chagas heart disease and cardiac troponin release, probably because of the insufcient sensitivity of the assays (below or near the 99th percentile of the reference population). However, the recently available highly sensitive cardiac troponin assays with an almost tenfold increase in

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sensitivity compared to former assays could supply the platform for renewed activities to establish cardiac troponin measurements as a hopeful tool for the diagnosis, risk assessment and monitoring of chronic Chagas patients who develop heart injury. Recently, we presented the rst data that demonstrated increasing cTnT concentrations in the blood of chagasic patients suffering from mild to severe cardiomyopathy [118]. Compared with healthy controls, asymptomatic Chagas patients, patients with chagasic gastrointestinal disease and patients with Chagas heart disease had signicantly higher cTnT values, which were correlated with the severity of the cardiomyopathy. Although cTnT was not measurable in the majority of healthy controls, asymptomatic patients and patients with chagasic gastrointestinal disease, i.e., 37% of the patients with Chagas heart disease, had measurable cTnT concentrations, and 13% showed cTnT values above the cutoff for non-ST elevation myocardial infarction. The lower limit of detection for this highly sensitive assay was the best at distinguishing between patients with and without heart injury. Changes in the levels of CRP and IL-6 were found to be parallel to cTnT changes both in the different Chagas disease groups and in the cardiomyopathy groups separated by disease severity.

Screening or suspicion of chronic Chagas disease Hemagglutination assay/ELISA Anti-T. cruzi IgG measurement

(+)
Chronic Chagas disease Bioassay/ELISA 2 yrs. Measurement of beta1-, beta2- muscarinergic2-receptor autoantibodies (+) (-) Risk of Chronic Chagas heart disease hs Tn assay BNP/NT-proBNp assay

1 yr.

(+)

Measurement of B-type natriuretic peptide, cardiac troponin

(-)

Chronic Chagas heart disease

Fig. 11 Suggested algorithm for combining autoantibody measurements and the measurements of B-type natriuretic peptide and cardiac troponins in chronic Chagas disease patients for risk assessment, diagnostics and monitoring

Summary Chagas disease is the most serious parasitic disease in Latin America. Its manifestation as Chagas heart disease places enormous burdens on the economic resources of these countries and dramatically affects patients social and labor situations. Due to increasing migration of infected patients, international tourism and parasite transfer by blood contact, intrauterine transfer and organ transplantation, Chagas heart disease has the potential to become a worldwide problem. In order to counteract the negative effects of Chagas heart disease, detailed knowledge of its pathogenesis is crucial. In this context, Chagas heart disease is increasingly considered as an autoimmune disease where autoantibodies against GPCRs such as beta1-AAB, beta2-AAB and M2-AAB drive the pathogenesis. Consequently, treatment strategies for the removal or neutralization of these pathogenic autoantibodies could be used to complement or replace the conventional therapy for Chagas heart disease. The detection of beta1-AAB, beta2AAB and M2-AAB in asymptomatic Chagas patients might offer the possibility of identifying patients with a high risk of developing Chagas heart disease. For patients at risk of Chagas heart disease, programs for early detection and the monitoring of progression from the asymptomatic stage to the symptomatic stage must be established. In addition to the modern diagnostic tools of

cardiology, such programs, in our view, could include laboratory medicine tools such as the measurement of brain natriuretic peptides and cardiac troponins, in particular. An algorithm to combine autoantibody measurement, B-type natriuretic peptide and cardiac troponin measurements, and cardiological diagnostics in chronic Chagas patients for risk assessment, diagnostics and monitoring is shown in Fig. 11. Based on sensitivity and specicity, we agree that laboratory medicine guidance of chronic Chagas patients is inferior compared to cardiological diagnostics and monitoring especially using echocardiography and, in particular, MRT. However, cardiological diagnostics is cost and labor intensive, dependents on highly qualied staff, and is only available in special centers that are often far from patients homes; therefore, cardiological diagnostics is attached to important economical and logistics problems that are particularly relevant to its common application in endemic areas of Chagas disease. In contrast, laboratory medicine enables sampling to be performed where patients are situated and subsequent economical investigations of the samples at highly specialized and automated centers, guaranteeing the highest quality service. Therefore, modern tools of laboratory medicine, such as heart markers, should be used to supplement the diagnosis and monitoring of Chagas heart disease, especially for increasing the pretest probability for the cardiological diagnostics.
Acknowledgments We are grateful to Deutsche Gesellschaft fur Klinische Chemie und Laboratoriumsmedizin and Europaischer Fonds fur regionale Entwicklung for supporting S.G. Munoz Saravia.

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