Development of a Novel Multiplex Immunoassay Multi-cruzi for the Serological Confirmation of Chagas Disease

Background

Chagas disease is due to the parasite Trypanosoma cruzi, a protist disseminated by a Triatome vector. This disease is endemic to Latin America and considered by WHO as one of the 17 world’s neglected diseases. In Europe and in North America, imported cases are also detected, due to migration of population outside of the endemic region. Diagnosis of T. cruzi infection is usually made indirectly by the detection of specific antibodies to T. cruzi antigens. Following initial diagnostic evaluation or screening test (qualifying or discarding blood donation), a confirmation test is performed for samples initially reactive. The test presented in this study aims at the confirmation/refutation of the infectious status of human blood samples and will permit taking appropriate clinical measures.

Methodology/Principal Findings

We designed a novel array of twelve antigens and printed these antigens onto 96-well plates. We tested 248 positive samples T. cruzi, 94 unscreened blood donors’ samples from non-endemic area, 49 seronegative blood donors, 7 false-positive and 3 doubtful samples. The observed reactivities were analyzed to propose a decision-tree algorithm that correctly classifies all the samples, with the potential to discriminate false-positive results and sticky samples. We observed that antibodies levels (Sum of all antigens) was significantly higher for PCR positive than for PCR negative samples in all studied groups with Multi-cruzi.

Conclusion/Significance

The results described in this study indicate that the Multi-cruzi improves the serological confirmation of Chagas disease. Moreover the “sum of all antigens” detected by Multi-cruzi could reflect parasitemia level in patients–like PCR signals does—and could serve as an indicator of parasite clearance in longitudinal follow-ups. Validation of this assay is still required on an independent large collection of well characterized samples including typical false-reactive samples such as Leishmaniasis.     

Barriers to Treatment Access for Chagas Disease in Mexico

Background

According to World Health Organization (WHO) prevalence estimates, 1.1 million people in Mexico are infected with Trypanosoma cruzi, the etiologic agent of Chagas disease (CD). However, limited information is available about access to antitrypanosomal treatment. This study assesses the extent of access in Mexico, analyzes the barriers to access, and suggests strategies to overcome them.

Methods and Findings

Semi-structured in-depth interviews were conducted with 18 key informants and policymakers at the national level in Mexico. Data on CD cases, relevant policy documents and interview data were analyzed using the Flagship Framework for Pharmaceutical Policy Reform policy interventions: regulation, financing, payment, organization, and persuasion. Data showed that 3,013 cases were registered nationally from 2007–2011, representing 0.41% of total expected cases based on Mexico''s national prevalence estimate. In four of five years, new registered cases were below national targets by 11–36%. Of 1,329 cases registered nationally in 2010–2011, 834 received treatment, 120 were pending treatment as of January 2012, and the treatment status of 375 was unknown. The analysis revealed that the national program mainly coordinated donation of nifurtimox and that important obstacles to access include the exclusion of antitrypanosomal medicines from the national formulary (regulation), historical exclusion of CD from the social insurance package (organization), absence of national clinical guidelines (organization), and limited provider awareness (persuasion).

Conclusions

Efforts to treat CD in Mexico indicate an increased commitment to addressing this disease. Access to treatment could be advanced by improving the importation process for antitrypanosomal medicines and adding them to the national formulary, increasing education for healthcare providers, and strengthening clinical guidelines. These recommendations have important implications for other countries in the region with similar problems in access to treatment for CD.     

FC-TRIPLEX Chagas/Leish IgG1: A Multiplexed Flow Cytometry Method for Differential Serological Diagnosis of Chagas Disease and Leishmaniasis

Differential serological diagnosis of Chagas disease and leishmaniasis is difficult owing to cross-reactivity resulting from the fact that the parasites that cause these pathologies share antigenic epitopes. Even with optimized serological assays that use parasite-specific recombinant antigens, inconclusive test results continue to be a problem. Therefore, new serological tests with high sensitivity and specificity are needed. In the present work, we developed and evaluated the performance of a new flow cytometric serological method, referred to as FC-TRIPLEX Chagas/Leish IgG1, for the all-in-one classification of inconclusive tests. The method uses antigens for the detection of visceral leishmaniasis, localized cutaneous leishmaniasis, and Chagas disease and is based on an inverted detuned algorithm for analysis of anti-Trypanosomatidae IgG1 reactivity. First, parasites were label with fluorescein isothiocyanate or Alexa Fluor 647 at various concentrations. Then serum samples were serially diluted, the dilutions were incubated with suspensions of mixed labeled parasites, and flow cytometric measurements were performed to determine percentages of positive fluorescent parasites. Using the new method, we obtained correct results for 76 of 80 analyzed serum samples (95% overall performance), underscoring the outstanding performance of the method. Moreover, we found that the fluorescently labeled parasite suspensions were stable during storage at room temperature, 4°C, and –20°C for 1 year. In addition, two different lots of parasite suspensions showed equivalent antigen recognition; that is, the two lots showed equivalent categorical segregation of anti-Trypanosomatidae IgG1 reactivity at selected serum dilutions. In conclusion, we have developed a sensitive and selective method for differential diagnosis of Chagas disease, visceral leishmaniasis, and localized cutaneous leishmaniasis.     

Opportunity Cost for Early Treatment of Chagas Disease in Mexico

Background

Given current neglect for Chagas disease in public health programs in Mexico, future healthcare and economic development policies will need a more robust model to analyze costs and impacts of timely clinical attention of infected populations.

Methodology/Principal Findings

A Markov decision model was constructed to simulate the natural history of a Chagas disease cohort in Mexico and to project the associated short and long-term clinical outcomes and corresponding costs. The lifetime cost for a timely diagnosed and treated Chagas disease patient is US$ 10,160, while the cost for an undiagnosed individual is US$ 11,877. The cost of a diagnosed and treated case increases 24-fold from early acute to indeterminate stage. The major cost component for lifetime cost was working days lost, between 44% and 75%, depending on the program scenario for timely diagnosis and treatment.

Conclusions/Significance

In the long term, it is cheaper to diagnose and treat chagasic patients early, instead of doing nothing. This finding by itself argues for the need to shift current policy, in order to prioritize and attend this neglected disease for the benefit of social and economic development, which implies including treatment drugs in the national formularies. Present results are even more relevant, if one considers that timely diagnosis and treatment can arrest clinical progression and enhance a chronic patient''s quality of life.     

Historical Perspectives on the Epidemiology of Human Chagas Disease in Texas and Recommendations for Enhanced Understanding of Clinical Chagas Disease in the Southern United States

Chagas disease (Trypanosoma cruzi infection) has recently been identified as an important neglected tropical disease in the United States. Anecdotally referred to as a “silent killer,” it leads to the development of potentially fatal cardiac disease in approximately 30% of those infected. In an attempt to better understand the potential of Chagas disease as a significant underlying cause of morbidity in Texas, we performed a historical literature review to assess disease burden. Human reports of triatomine bites and disease exposure were found to be prevalent in Texas. Despite current beliefs that Chagas disease is a recently emerging disease, we report historical references dating as far back as 1935. Both imported cases and autochthonous transmission contribute to the historical disease burden in Texas. We end by discussing the current knowledge gaps, and recommend priorities for advancing further epidemiologic studies and their policy implications.     

Recent Clinical Trials for the Etiological Treatment of Chronic Chagas Disease: Advances,Challenges and Perspectives

Chagas disease, a chronic systemic parasitosis caused by the Kinetoplastid protozoon Trypanosoma cruzi, is the first cause of cardiac morbidity and mortality in poor rural and suburban areas of Latin America and the largest parasitic disease burden in the continent, now spreading worldwide due to international migrations. A recent change in the scientific paradigm on the pathogenesis of chronic Chagas disease has led to a consensus that all T. cruzi‐seropositive patients should receive etiological treatment. This important scientific advance has spurred the rigorous evaluation of the safety and efficacy of currently available drugs (benznidazole and nifurtimox) as well as novel anti‐T. cruzi drug candidates in chronic patients, who were previously excluded from such treatment. The first results indicate that benznidazole is effective in inducing a marked and sustained reduction in the circulating parasites’ level in the majority of these patients, but adverse effects can lead to treatment discontinuation in 10–20% of cases. Ergosterol biosynthesis inhibitors, such as posaconazole and ravuconazole, are better tolerated but their efficacy at the doses and treatment duration used in the initial studies was significantly lower; such results are probably related to suboptimal exposure and/or treatment duration. Combination therapies are a promising perspective but the lack of validated biomarkers of response to etiological treatment and eventual parasitological cures in chronic patients remains a serious challenge.     

High Resolution Esophageal Manometry in Patients with Chagas Disease: A Cross-Sectional Evaluation

Introduction

Gastrointestinal involvement affects 30–40% of the patients with chronic Chagas disease. Esophageal symptoms appear once the structural damage is established. Little is known about the usefulness of high resolution manometry to early identification of esophageal involvement.

Method

We performed a cross-sectional study at the Vall d’Hebron University Hospital (Barcelona, Spain) between May 2011 and April 2012. Consecutive patients diagnosed with Chagas disease in the chronic phase were offered to participate. All patients underwent a structured questionnaire about digestive symptoms, a barium esophagogram (Rezende classification) and an esophageal high resolution manometry (HRM). A control group of patients with heartburn who underwent an esophageal HRM in our hospital was selected.

Results

62 out of 73 patients that were included in the study fulfilled the study protocol. The median age of the Chagas disease group (CG) was 37 (IQR 32–45) years, and 42 (67.7%) patients were female. Twenty-seven (43.5%) patients had esophageal symptoms, heartburn being the most frequent. Esophagogram was abnormal in 5 (8.77%). The esophageal HRM in the CG showed a pathological motility pattern in 14 patients (22.6%). All of them had minor disorders of the peristalsis (13 with ineffective esophageal motility and 1 with fragmented peristalsis). Hypotonic lower esophageal sphincter was found more frequently in the CG than in the control group (21% vs 3.3%; p<0.01). Upper esophageal sphincter was hypertonic in 22 (35.5%) and hypotonic in 1 patient. When comparing specific manometric parameters or patterns in the CG according to the presence of symptoms or esophagogram no statistically significant association were seen, except for distal latency.

Conclusion

The esophageal involvement measured by HRM in patients with chronic Chagas disease in our cohort is 22.6%. All the patients with esophageal alterations had minor disorders of the peristalsis. Symptoms and esophagogram results did not correlate with the HRM results.     

A Scientometric Evaluation of the Chagas Disease Implementation Research Programme of the PAHO and TDR

The Special Programme for Research and Training in Tropical Diseases (TDR) is an independent global programme of scientific collaboration cosponsored by the United Nations Children''s Fund, the United Nations Development Program, the World Bank, and the World Health Organization. TDR''s strategy is based on stewardship for research on infectious diseases of poverty, empowerment of endemic countries, research on neglected priority needs, and the promotion of scientific collaboration influencing global efforts to combat major tropical diseases. In 2001, in view of the achievements obtained in the reduction of transmission of Chagas disease through the Southern Cone Initiative and the improvement in Chagas disease control activities in some countries of the Andean and the Central American Initiatives, TDR transferred the Chagas Disease Implementation Research Programme (CIRP) to the Communicable Diseases Unit of the Pan American Health Organization (CD/PAHO).This paper presents a scientometric evaluation of the 73 projects from 18 Latin American and European countries that were granted by CIRP/PAHO/TDR between 1997 and 2007. We analyzed all final reports of the funded projects and scientific publications, technical reports, and human resource training activities derived from them. Results about the number of projects funded, countries and institutions involved, gender analysis, number of published papers in indexed scientific journals, main topics funded, patents inscribed, and triatomine species studied are presented and discussed.The results indicate that CIRP/PAHO/TDR initiative has contributed significantly, over the 1997–2007 period, to Chagas disease knowledge as well as to the individual and institutional-building capacity.     

Ruthenium Complex with Benznidazole and Nitric Oxide as a New Candidate for the Treatment of Chagas Disease

Background

Chagas disease remains a serious medical and social problem in Latin America and is an emerging concern in nonendemic countries as a result of population movement, transfusion of infected blood or organs and congenital transmission. The current treatment of infected patients is unsatisfactory due to strain-specific drug resistance and the side effects of the current medications. For this reason, the discovery of safer and more effective chemotherapy is mandatory for the successful treatment and future eradication of Chagas disease.

Methods and Findings

We investigated the effect of a ruthenium complex with benznidazole and nitric oxide (RuBzNO₂) against Trypanosoma cruzi both in vitro and in vivo. Our results demonstrated that RuBzNO₂ was more effective than the same concentrations of benznidazole (Bz) in eliminating both the extracellular trypomastigote and the intracellular amastigote forms of the parasite, with no cytotoxic effect in mouse cells. In vivo treatment with the compound improved the survival of infected mice, inhibiting heart damage more efficiently than Bz alone. Accordingly, tissue inflammation and parasitism was significantly diminished after treatment with RuBzNO₂ in a more effective manner than that with the same concentrations of Bz.

Conclusions

The complexation of Bz with ruthenium and nitric oxide (RuBzNO₂) increases its effectiveness against T. cruzi and enables treatment with lower concentrations of the compound, which may reduce the side effects of Bz. Our findings provide a new potential candidate for the treatment of Chagas disease.     

Epidemiological,Clinical and Serological Characteristics of Children with Coronavirus Disease 2019 in Wuhan: A Single-centered,Retrospective Study

In December 2019, SARS-CoV-2 was first detected in the samples obtained from three adult patients who suffered from an unknown viral pneumonia in Wuhan (Li et al. 2020). This unknown viral pneumonia is further named as coronavirus disease 2019 (COVID-19) by the World Health Organization. To date, the number of new COVID-19 cases has continued to skyrocket and the impact of SARS-CoV-2 on humans is far greater than any pathogen of this century in both breadth and depth. Previous studies have shown that adults with COVID-19 have symptoms of fever, dry cough, dyspnea, fatigue and lymphocytopenia. Moreover, COVID-19 is more likely to cause death in the elderly, especially those with chronic comorbidities (Huang et al. 2020). In Wuhan, more than 50, 000 COVID-19 cases have been confirmed, including over 780 pediatric patients, and only one child death case (Lu et al. 2020). Although the number of children cases was far fewer than that of adults, COVID-19 might endanger children's health and the information on children remains limited, especially in serological study. In the retrospective study, the investigators analyzed the epidemiological, clinical and serological characteristics of children with COVID-19 in Wuhan in the early stages of the outbreak, which might provide theoretical and practical help in controlling COVID-19 and similar emerging infectious diseases in the future.     

Chagas Disease,Migration and Community Settlement Patterns in Arequipa,Peru

Background

Chagas disease is one of the most important neglected tropical diseases in the Americas. Vectorborne transmission of Chagas disease has been historically rare in urban settings. However, in marginal communities near the city of Arequipa, Peru, urban transmission cycles have become established. We examined the history of migration and settlement patterns in these communities, and their connections to Chagas disease transmission.

Methodology/Principal Findings

This was a qualitative study that employed focus group discussions and in-depth interviews. Five focus groups and 50 in-depth interviews were carried out with 94 community members from three shantytowns and two traditional towns near Arequipa, Peru. Focus groups utilized participatory methodologies to explore the community''s mobility patterns and the historical and current presence of triatomine vectors. In-depth interviews based on event history calendars explored participants'' migration patterns and experience with Chagas disease and vectors. Focus group data were analyzed using participatory analysis methodologies, and interview data were coded and analyzed using a grounded theory approach. Entomologic data were provided by an ongoing vector control campaign. We found that migrants to shantytowns in Arequipa were unlikely to have brought triatomines to the city upon arrival. Frequent seasonal moves, however, took shantytown residents to valleys surrounding Arequipa where vectors are prevalent. In addition, the pattern of settlement of shantytowns and the practice of raising domestic animals by residents creates a favorable environment for vector proliferation and dispersal. Finally, we uncovered a phenomenon of population loss and replacement by low-income migrants in one traditional town, which created the human settlement pattern of a new shantytown within this traditional community.

Conclusions/Significance

The pattern of human migration is therefore an important underlying determinant of Chagas disease risk in and around Arequipa. Frequent seasonal migration by residents of peri-urban shantytowns provides a path of entry of vectors into these communities. Changing demographic dynamics of traditional towns are also leading to favorable conditions for Chagas disease transmission. Control programs must include surveillance for infestation in communities assumed to be free of vectors.     

Improved Completion Rates and Characterization of Drug Reactions with an Intensive Chagas Disease Treatment Program in Rural Bolivia

Background

Chagas disease treatment is limited by drug availability, adverse side effect profiles of available medications, and poor adherence.

Methods

Adult Chagas disease patients initiating 60-days of benznidazole were randomized to weekly or twice-weekly evaluations of medication adherence and screening for adverse drug events (ADEs). Mid-week evaluations employed phone-based evaluations. Adherence was measured by self-report, pill counts with intentional over-distribution, and Medication Event Monitoring Systems (MEMS). Prospective data were compared to historical controls treated with benznidazole at the same hospital.

Results

162 prospective patients were compared to 172 historical patients. Pill counts correlated well with MEMS data (R = 0.498 for 7-day intervals, R = 0.872 for intervals >7 days). Treatment completion rates were higher among prospective than historical patients (82.1% vs. 65.1%), primarily due to lower abandonment rates. Rates of ADEs were lower among prospective than historical patients (56.8% vs. 66.9%). Twice-weekly evaluations increased identification of mild ADEs, prompting higher suspension rates than weekly evaluations. While twice-weekly evaluations identified ADEs earlier, they did not reduce incidence of moderate or severe ADEs. Many dermatologic ADEs were moderately severe upon presentation (35.6%), were not reduced by use of antihistamines, occurred among adult patients of all ages, and occurred throughout treatment, rather than the first few weeks alone.

Conclusions

Intensive management improved completion and identified more ADEs, but did not reduce moderate or severe ADEs. Risk of dermatologic ADEs cannot be reduced by selecting younger adults or monitoring only during the first few weeks of treatment. Pill counts and phone-based encounters are reliable tools for treatment programming in rural Bolivia.     

Prevalence,Clinical Staging and Risk for Blood-Borne Transmission of Chagas Disease among Latin American Migrants in Geneva,Switzerland

Background

Migration of Latin Americans to the USA, Canada and Europe has modified Chagas disease distribution, but data on imported cases and on risks of local transmission remain scarce. We assessed the prevalence and risk factors for Chagas disease, staged the disease and evaluated attitudes towards blood transfusion and organ transplant among Latin American migrants in Geneva, Switzerland.

Methodology/Principal Findings

This cross-sectional study included all consecutive Latin American migrants seeking medical care at a primary care facility or attending two Latino churches. After completing a questionnaire, they were screened for Chagas disease with two serological tests (Biomérieux ELISA cruzi; Biokit Bioelisa Chagas). Infected subjects underwent a complete medical work-up. Predictive factors for infection were assessed by univariate and multivariate logistic regression analysis.1012 persons (females: 83%; mean age: 37.2 [SD 11.3] years, Bolivians: 48% [n = 485]) were recruited. 96% had no residency permit. Chagas disease was diagnosed with two positive serological tests in 130 patients (12.8%; 95%CI 10.8%–14.9%), including 127 Bolivians (26.2%; 95%CI 22.3%–30.1%). All patients were in the chronic phase, including 11.3% with cardiac and 0.8% with digestive complications. Predictive factors for infection were Bolivian origin (OR 33.2; 95%CI 7.5–147.5), reported maternal infection with T. cruzi (OR 6.9; 95%CI 1.9–24.3), and age older than 35 years (OR 6.7; 95%CI 2.4–18.8). While 22 (16.9%) infected subjects had already donated blood, 24 (18.5%) and 34 (26.2%) considered donating blood and organs outside Latin America, respectively.

Conclusions

Chagas disease is highly prevalent among Bolivian migrants in Switzerland. Chronic cardiac and digestive complications were substantial. Screening of individuals at risk should be implemented in nonendemic countries and must include undocumented migrants.     

Agrochemicals against Malaria,Sleeping Sickness,Leishmaniasis and Chagas Disease

In tropical regions, protozoan parasites can cause severe diseases with malaria, leishmaniasis, sleeping sickness, and Chagas disease standing in the forefront. Many of the drugs currently being used to treat these diseases have been developed more than 50 years ago and can cause severe adverse effects. Above all, resistance to existing drugs is widespread and has become a serious problem threatening the success of control measures. In order to identify new antiprotozoal agents, more than 600 commercial agrochemicals have been tested on the pathogens causing the above mentioned diseases. For all of the pathogens, compounds were identified with similar or even higher activities than the currently used drugs in applied in vitro assays. Furthermore, in vivo activity was observed for the fungicide/oomyceticide azoxystrobin, and the insecticide hydramethylnon in the Plasmodium berghei mouse model, and for the oomyceticide zoxamide in the Trypanosoma brucei rhodesiense STIB900 mouse model, respectively.     

Benznidazole/Itraconazole Combination Treatment Enhances Anti-Trypanosoma cruzi Activity in Experimental Chagas Disease

The nitroheterocyclic drugs nifurtimox and benznidazole are first-line drugs available to treat Chagas disease; however, they have limitations, including long treatment courses and toxicity. Strategies to overcome these limitations include the identification of new drugs with specific target profiles, re-dosing regimens for the current drugs, drug repositioning and combination therapy. In this work, we evaluated combination therapy as an approach for optimization of the current therapeutic regimen for Chagas disease. The curative action of benznidazole/itraconazole combinations was explored in an established infection of the mice model with the T. cruzi Y strain. The activities of the benznidazole/itraconazole combinations were compared with the results from those receiving the same dosage of each individual drug. The administration of benznidazole/itraconazole in combination eliminated parasites from the blood more efficiently than each drug alone. Here, there was a significant reduction of the number of treatment days (number of doses) necessary to induce parasitemia suppression with the benznidazole/itraconazole combination, as compared to each compound administered alone. These results clearly indicate the enhanced effects of these drugs in combination, particularly at the dose of 75 mg/kg, as the effects observed with the drug combinations were four times more effective than those of each drug used alone. Moreover, benznidazole/itraconazole treatment was shown to prevent or decrease the typical lesions associated with chronic experimental Chagas disease, as illustrated by similar levels of inflammatory cells and fibrosis in the cardiac muscle tissue of healthy and treated mice. These results emphasize the importance of exploring the potential of combination treatments with currently available compounds to specifically treat Chagas disease.     

阿尔茨海默病的预防与临床治疗及护理

阿尔茨海默病(alzheimer's disease,AD)是一种神经退行性疾病,通常起病缓慢并逐渐恶化,是60％～70％痴呆症的病因.截至2020年,全球约5000万人患有各种形式的痴呆症,最常见的是65岁以上的人,因此,俗称老年痴呆症.迄今为止,对阿尔茨海默病的病因仍知之甚少.一般认为许多环境和遗传风险因素与阿尔茨海默病的发展有关.大量的实验研究和病例证明,没有任何一种治疗可以阻止或逆转阿尔茨海默病的病程,只有少数方法或许可以暂时缓解或改善症状.本文概述了对阿尔茨海默病的基本认识,系统地提出了对阿尔茨海默病的预防、临床治疗及护理对策,期望对医护人员和社会大众提供有益的建议和参考.     

Polymorphic Sites at the Immunoregulatory CTLA-4 Gene Are Associated with Chronic Chagas Disease and Its Clinical Manifestations

Background

Chagas disease affects approximately 10 million people mainly in Latin America. The immune regulation by the host seems to be an essential factor for disease evolution, and immune system inhibitory molecules such as CTLA-4 and PD-1 favor the maintenance of peripheral tolerance. Considering that polymorphisms at the immunoregulatory CTLA-4 and PDCD1 genes may alter their inhibitory function, we investigated the association of alleles, genotypes and haplotypes of polymorphic sites observed at the CTLA-4 and PDCD1 genes with different clinical manifestations of chronic Chagas disease (indeterminate, cardiac, digestive and mixed).

Methods

The polymorphisms at the CTLA-4 (-1722T/C, -318C/T and +49A/G) and PDCD1 (PD-1.3G/A) genes were typed using TaqMan methodology in 277 chronic Chagas disease patients classified into four groups, according to clinical characteristics, and 326 non-infected controls.

Results

Our results showed that CTLA-4 -1722CC genotype (22%), -1722C allele (27%) and CTLA-4 TCG (8.6%), TCA (26%) and CCA (15%) haplotypes were strongly associated with the indeterminate form, while the CTLA-4 -318CT genotype (82%) and CTLA-4 -318T allele (47%) were found mainly in patients with the mixed form of the disease. The CTLA-4 TCG haplotype (10.2%) was associated with the digestive form. On the other hand, the PD-1.3G/A polymorphism was not associated with chronic Chagas disease and its clinical manifestations.

Conclusions

Here, we showed that alleles, genotypes and haplotypes reported to increase the expression of the regulatory molecule CTLA-4 were associated with the indeterminate form of the disease. Taken together, our data support the idea that polymorphic sites at immunoregulatory genes may influence the development of Chagas disease variants.