Challenges to Diagnose Chagas Disease in Endemic Areas

1. Introduction

This chapter addresses Chagas disease, known for over a hundred years, but still lists neglected diseases. We hope that the reader can turn his attention to the importance of this disease and the limitations or difficulties of diagnosis in endemic areas so that we can move toward the solution of this problem.

A long-standing challenge over the history of Chagas disease, especially in endemic areas, is its timely diagnosis for the treatment of infection and follow-up of the evolution of the clinical picture of the condition since the lack of diagnosis or incorrect diagnosis contributes to the social impacts of high morbidity and mortality [1, 2]. This challenge can be directly attributed to the need for a rapid and easy-to-use diagnostic test that can be applied in endemic areas that often have limited access to health services and contribute to the neglect of resident populations [3, 4].

Chagas disease is one of the tropical diseases that most affect neglected populations, being endemic to Latin America, where the insect vector of the Triatomine species is also found (Figure 1) [5].

First described at the beginning of the twentieth century by the researcher Carlos Justiniano Ribeiro das Chagas, the disease is caused by the protozoan Trypanosoma cruzi. This flagellate heteroxenic parasite can infect humans [6]. As a heterogenic parasite, T. cruzi requires both intermediate and definitive hosts to complete its biological cycle, where its intermediate host is the triatomine hematophagous insect. In contrast, the ideal host is the human [7].

2. Chagas disease epidemiology

We cannot rule out the importance of Chagas disease because it is endemic in Latin American countries but is becoming a global problem in other regions, attributed to infected individuals’ migratory flow [5, 8, 9]. There are an estimated 8 million infected individuals worldwide, with 6 million located in the Americas, where the annual incidence is around 30,000 new cases that lead to up to 12,000 deaths from Chagas disease [9].

From the data available in the Brazilian Information System for Notifiable Diseases [10], 3609 acute Chagas disease cases were reported between 2006 and 2021 (Figure 2). The highest incidence in Brazil (3414) was in its Northern region [10]. However, these numbers do not represent the actual situation. The underreporting rate has estimated that the reported infections may be less than 10% of all incidences, principally due to the person not seeking medical care [9].

The Sinan Net data demonstrate the high incidence of acute Chagas disease in Brazil, highlighting the need for epidemiological control measures. Considering the last 6 years of available data (2016 to 2021), the lowest incidence occurred in 2020, during the peak of the COVID-19 pandemic. This fact suggests a potential lack of demand for Chagas disease diagnosis or cases of co-infection with SARS-CoV-2, where the focus was primarily on diagnosing the viral infection.

According to the follow-up data available through the Ministry of Health (2023), 51 of these patients died of Chagas disease, suggesting that, if untreated and/or cured, 3548 of these individuals are affected by chronic Chaga disease, regardless of clinical form [10]. In 2020, the Ministry of Health included chronic Chagas disease on the list of compulsorily notifiable diseases, although data are not yet available [10].

3. Biological cycle of Trypanosoma cruzi and host immune response

The biological cycle of T. cruzi consists of four morphological stages: epimastigote, metacyclic trypomastigote, amastigote, and blood trypomastigote [6, 7]. Epimastigotes are the replicative form in the insect vector that differentiates into the non-replicative, infective metacyclic trypomastigotes. In humans, amastigotes are the intracellular replicative form, and blood trypomastigotes are the non-replicative virulent form. When a metacyclic trypomastigote leaves a triatomine and infects a person, it triggers a series of immune reactions, such as the activation of cells of the innate immune system, including macrophages, natural killer (NK) cells and dendritic cells (DC) that produce cytokines, namely interleukin 12 (IL-12), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), and effector molecules (nitrogen and oxygen reactive intermediates) that control parasite replication [11].

Cells of the innate immune system, such as dendritic cells, bridge innate and acquired immunity by producing cytokines, such as IL-12, which is necessary for the differentiation and clonal expansion of Th1 CD4+, CD8+, B, and T cells. In turn, IFN-γ produced by Th1 CD4+ cells or CD8+ T cells activates effector mechanisms in macrophages that destroy phagocytosed amastigotes and trypomastigotes forms. In contrast, the cytotoxic activity of CD8+ T cells destroys cells with internalized amastigotes [11].

The complex of immune reactions is triggered by the biochemical composition of the parasite, which presents molecules that interact with pattern recognition receptors, such as TLR (Toll-like receptor). This interaction activates the transcription of pro-inflammatory cytokines such as IFN-γ and TNF-α and interleukins such as IL-1, IL-6, IL-12, IL-18, and nitric oxide (NO). Antigen-presenting cells are activated, and phagocytes are recruited to the infected tissue [12, 13].

4. Trypanosoma cruzi escape of defense mechanisms

A common question surrounding an infection by T. cruzi is how the parasite escapes both the innate and adaptive immune response. Its persistence is due to the properties of its surface molecules that impart resistance and protection against immune response mechanisms to favor infection, occupation, and replication in host cells [14]. The coating molecules consist mainly of glycoproteins that allow blood trypomastigotes to adhere to the plasma membrane of host cells.

The glycoproteins gp30 and gp82 activate intracellular signaling pathways upon binding that mobilize intracellular calcium (Ca++) in the parasite, which is a primary event during host cell invasion and leads to occupying parasitophorous vacuoles [15].

Inside the cell, parasites change to the amastigote form and multiply before entering morphogenesis back to the blood trypomastigote form. The secretion of hemolysin and trans-sialidase ruptures the cell and permits its entry into the bloodstream to infect other cells [15]. The parasite also has antioxidant enzymes, such as peroxidases and superoxide dismutase, which enable it to resist lysis promoted by macrophages. In addition, the activation of NADPH oxidase allows the secretion of superoxide radicals, which contribute to its escape [16].

5. Stages of infection, clinical manifestations, and diagnoses

The T. cruzi infection is divided into two temporal stages, the acute and chronic phases [17]. The acute phase is usually asymptomatic and undetected. However, recently infected individuals can present symptoms such as fever, malaise, headache, myalgia, lymphadenopathy, hepatosplenomegaly, edema of the face or limbs, adenomegaly, hemorrhages, jaundice, tachycardia, or signs of heart failure, already detected in the first weeks after infection, manifesting dyspnea, fatigue, and other symptoms similar to myocarditis in general [18]. According to the Guidelines of the Brazilian Society of Cardiology on Analysis and Issuance of Electrocardiographic Reports, electrocardiographic examinations can be altered, revealing sinus tachycardia, decreased QRS complex voltage (corresponding to ventricular depolarization), first-degree atrioventricular block (AVB), primary alteration of ventricular repolarization, and the presence of extrasystoles [19].

In addition to the clinical signs that can sometimes be observed, suspected cases must be confirmed or ruled out by laboratory tests combined with clinical and epidemiological investigations. Among laboratory tests, the literature and guidelines recommend methods based on detecting both antigens and antibodies. Some techniques are recommended for the acute phase and others for the chronic phase of infection [20].

The diagnostic method or technique varies in sensitivity during infection due to the parasite’s behavior and the host’s immune response. While during acute infection, there is an abundance of parasites circulating in the bloodstream (parasitemia), in the chronic phase, this parasitemia decreases, leading to the consensus of the detection of priority antibodies for laboratory diagnosis (Figure 3) [5].

In this way, the diagnosis of Chagas disease relies on a combination of clinical, parasitological, and serological methods. Here are the diagnostic techniques and limitations of Chagas disease diagnosis based on the search results. Let us then understand the principle and application of each method/technique described by Junqueira et al. 2011 [20].

Parasitological testing or microscopic examination: an investigation makes the classical diagnosis during the acute phase for parasites within a blood smear on a glass slide. The direct observation of parasites can be highly specific in the acute phase when parasitemia is high. Sensitivity with fresh blood can be improved by thick drop distention, parasite concentration (micro-hematocrit, leukocyte cream, and Strout), QBC (Quantitative Buffy Coat), and Ficoll-Hypaque. All require a microscope for diagnosis; the last three methods require a centrifuge. It is not recommended for the chronic phase because parasitemia is very low [5, 20]. Microscopist training is an important element of the assay to differentiate T. cruzi from other protozoa. Early detection of infection can permit treatment and a possible cure.

Molecular testing: consists of the detection of the genetic material of the parasite in a biological sample from the infected individual. The techniques commonly used are PCR (Polymerase Chain Reaction), which requires an adequate environment to obtain and prepare the material, as well as equipment for the reaction, and Western Blotting, used as a confirmatory test, consisting of the separation of the extract of antigens of the parasite by electrophoresis and its transfer to a nitrocellulose membrane. An advantage of using molecular tests during the acute phase is the high parasitemia, which favors obtaining the genetic material of the circulating parasite. In turn, the limitation in the chronic phase is precisely the low parasitemia, which makes molecular testing difficult due to the scarcity of parasite genetic material [20].

Serological testing: in addition to exams based on the presence of parasites in a biological sample, serological tests can also be performed, primarily detecting IgM (immunoglobulin M) class anti-T. cruzi antibodies [5]. Methods include:

Indirect hemagglutination assay (IHA): Indirect Hemagglutination in Chagas disease is an important auxiliary tool in diagnosing and evaluating the patient’s immune response to T. cruzi. However, it is essential to combine the results of this technique with other diagnostic approaches to obtain a complete and accurate clinical picture of the infection.

Indirect immunofluorescence assay (IFA): The indirect immunofluorescence technique is based on detecting specific antigens using antibodies labeled with fluorophores. In this method, a sample containing the antigen of interest is incubated with an unlabeled primary antibody that binds to the antigen. Next, a secondary antibody labeled with a fluorophore is added, recognizing and binding to the primary antibody. This procedure allows the visualization of the antigen through the emission of fluorescence when exposed to ultraviolet light or specific wavelengths (Figure 4) [20].

Enzyme-linked immunosorbent assay (ELISA) consists of antibody detection employing specific binding of an antigen and an antibody, followed by a colorimetric or fluorescent signal detection (Figure 5). The ELISA reader obtains the absorbance, and the determination of reagents and non-reagents is done by calculating the cut-off from positive and negative controls, which this chapter will not cover.

In acute infection, these serological tests seek to detect IgM. IgM antibodies are antigen receptor immunoglobulins in virgin B cells stimulated by the first contact with an antigen. They are the first antibody isotype produced in the acute phase because of the adaptive response. Among its functions are activation of the complement system (protein cascade) in the classical pathway, triggering of the membrane attack complex (antigen–antibody complex), and promoting cell lysis as well as acting as an antigen receptor for virgin B lymphocytes [21, 22]. In cases where direct exams do not identify the parasite, verification for the presence of anti-T. cruzi IgM antibodies in the peripheral blood are considered positive for the acute phase, particularly when associated with epidemiological history and clinical manifestations. The great advantage of the serological test is that it can be used in all stages of the infection, logically seeking to detect the antibody of each phase [23].

Contributing to the complexity of Chagas disease, when the individual is not cured in the acute phase, the infection becomes chronic, and the parasite lodges in tissues and remains latent for varying periods. During the chronic phase, an asymptomatic phase can occur, classified as the indeterminate form of the disease, which can last for decades. It can also evolve into a clinical disorder characterized by cardiac, digestive, neurological, and mixed conditions [24]. The mechanisms involved with maintaining a persistent, asymptomatic infection or its transition to a clinical state are unknown.

The cardiac clinical form of the disease, also called Chronic Chagas’ Cardiopathy (CCC), affects about 30% of patients with Chagas disease and may present with inflammatory and fibrosing characteristics. In addition, it may be accompanied by complex ventricular arrhythmias associated with disturbances in the formation and conduction of the atrioventricular and intraventricular electrical stimuli, thromboembolic phenomena, right ventricular dysfunction, and ventricular aneurysms [25, 26].

Studies show that CCC starts with an inflammatory response, destroying myocardial fibers. As a result of this destruction, fibrous tissue compromises the ventricular contractions, which become hypertrophied and characterize myocarditis. The evolution of the condition leads to a progressive loss of the ejection capacity of the heart and can be aggravated by arrhythmia, pulmonary thromboembolism, and heart valve insufficiency [27, 28].

However, epidemiological, electrocardiographic, radiological, patient history, and serological evidence should be considered for the diagnosis of CCC. Patient history should consider the time spent living in an endemic area, digestive involvement, family, and neighborhood history. Among the cardiac alterations that can be encountered are right bundle branch conduction block (RBBB), left anterior fascicular hemiblock (LAFH), left bundle branch block (LBBB), atrioventricular (AVB), and ventricular extrasystole (VES) [27, 28].

As discussed above, during the chronic phase of Chagas disease, parasitemia tends to decrease to undetectable levels in blood smears. Serologically, this phase of the disease is characterized by a predominance of the IgG class of anti-T. cruzi antibodies in patient serum. Consequently, diagnosis in the chronic phase involves investigating reactive IgG class antibodies due to the scarcity of circulating parasites [29]. IgG antibodies present in the later phases of infection mainly activate the complement system and opsonization and stimulate antibody cytotoxicity [21].

Similarly to detecting IgM, techniques include indirect hemagglutination (HAI) that displays agglutination of RBCs sensitized with anti-T. cruzi antigen in serum containing antibodies; IFA and ELISA. The most commonly used serological tests are IFA and ELISA due to their high sensitivity and specificity [5, 20]. Still, we must remember that a gold standard test for the serological diagnosis of Chagas disease has yet to be made available.

Currently, the Second Brazilian Consensus on Chagas Disease recommends that individuals suspected of being infected by T. cruzi be submitted to serological, parasitological, and/or molecular tests according to clinical suspicion, infection route, and stage of infection. The fear must be promptly clarified because the delay in diagnosis may compromise the treatment and favor the evolution to more severe cases of the disease [5]. The World Health Organization recommends that to confirm or reject T. cruzi infection, the investigation of suspected cases should be done by performing two tests with different principles, methods, or preparations. The most used conventional tests are IFI, ELISA, and HAI. Two concordant tests reach the diagnostic conclusion. In disagreement, a new sample should be tested, and a third method should be employed [5].

We must not forget that, in addition to vector transmission, Chagas disease can also be transmitted orally (through the ingestion of contaminated food, such as undercooked meat and juices), congenitally (from the infected mother to the baby), or by blood transfusion (blood donation from an infected donor to a healthy recipient) [5].

Laboratory diagnoses are the same for the different forms of infection, but we will close this section except for diagnostic management in cases of congenital infection. According to the Brazilian Consensus on Chagas Disease, 2015, for symptomatic children or newborns of mothers with acute T. cruzi infection, these parasitological tests must be performed repeatedly, and if they are negative, parasitological enrichment and/or molecular methods have been used. However, they are not accessible and standardized for use in health services. Reactive conventional serology in children in the first months strongly indicates congenital transmission, especially when the possibilities of vectorial and transfusional transmission are excluded. In Brazil, including the serological test with IgG for T. cruzi in the National Neonatal Screening Program (“heel prick test”) is recommended, especially in endemic regions for T. cruzi infection. Anti-T. cruzi of the IgM class has low sensitivity, with difficulties in standardizing techniques and obtaining controls. Serological methods that employ recombinant antigens, such as the shed acute-phase antigen (SAPA), may be indicated if available. Reports show maternal anti-SAPA antibodies disappear earlier than conventional antibodies – in approximately three months [5].

6. Diagnostic coverage of chagas disease: a problem to be solved

So far, it has been possible to see that the diagnosis of Chagas disease involves a range of complexities, such as clinical signs that must be observed, epidemiological factors, and laboratory tests that require infrastructure with a certain degree of robustness, which is not always available in endemic areas and not accessible to neglected populations. But these are not the only limitations.

Even today, the serological tests available on the market are fragile due to being insufficiently sensitive and specific to make the diagnosis, which prevents a single test from being enough, requiring the application of two or more tests that use different techniques and detect antibodies to various antigens. Commonly used techniques include ELISA and IFA. However, serological tests may have limitations in terms of specificity and sensitivity [30].

Another problem that needs to be addressed to improve screening for Chagas disease is the observation that currently employed conventional tests show variable performance profiles across diverse geographical areas. Initially demonstrated in comparative studies between commercial serological tests (ELISA, chemiluminescence, and western blotting) from different countries that utilized other antigens (total lysate, recombinant protein, and trypomastigote protein) to capture anti-T. cruzi antibodies [30]. The analysis employed the WHO Biological References for the serological diagnosis of Chagas disease generated from two broad areas predicted to circulate principally either TcI strains or TcII strains of T. cruzi. The availability of the reference pools of sera normalized the quantity of anti-T. cruzi antibodies could illuminate differences in clinical sensitivity. Furthermore, the reactivity index at each concentration of the reference samples over a serial dilution was important to determine test sensitivity.

These studies were extended to analyze commercial kit performance in four different geographical areas of Brazil, the States of Amazonas, Piauí, Mato Grosso do Sul, and Minas Gerais, previously tested for anti-T. cruzi, IgG antibodies [31]. The initial diagnoses based on two or more assays showed a level of divergence that reached 9.5% overall and as high as 29% in samples from the State of Amazonas. Between the commercial tests employed, results varied from 5–51% in Amazonas, 6–12% in Piauí, and 5–32% in Mato Grosso do Sul. Based on the recommendations, a third test should be employed, and a new blood sample should be obtained from up to nearly half of the individuals examined. Other studies highlight the impact of divergent results on the tested individual’s quality of life and public health. Out of caution, blood banks discard bags with divergent results without any certainty that they are positive for T. cruzi [32]. A contributing problem to geographical performance in areas also endemic for visceral leishmaniasis [33], which can display cross-reactivity and generate false positive results.

As you can see, a single test is not sensitive and specific enough to make the diagnosis. For this reason, the standard approach is to apply two or more tests that use different techniques and detect antibodies to various antigens. Commonly used techniques include ELISA and IFA. However, serological tests may have limitations in terms of specificity and sensitivity [30, 31]. This fact is worrying, as only a correct and timely diagnosis enables the treatment and follow-up of patients.

7. Rapid diagnostic test for chronic chagas disease

By now, you must have realized that the diagnosis of Chagas disease is more complex than it seems. Due to the absence of a gold standard test, the diagnosis is made by a set of two or more methods or techniques, which generally require qualified technical labor and infrastructure, making the diagnosis expensive and inaccessible to many endemic areas [34].

We approach that classical diagnostic tests require laboratory infrastructure and personnel that are only sometimes available in endemic regions. Their distance from hard-to-reach collection sites can make it impractical to diagnose neglected populations, especially for confirmatory tests that require a new sample. A rapid screening test lateral flow (Figure 6) is the starting point for solving this problem. Lateral flow immunochromatography tests offer a method that is feasible in any location. Often called rapid tests, they are easy to perform and return results in minutes from blood obtained by digital puncture, which is much less invasive than venipuncture collections [34].

The principle of the lateral flow rapid test is the detection of antibodies. The serological sample can be obtained from the finger prick, where a small drop of blood can be obtained, which will be applied to the test device, previously sensitized with antigen (usually recombinant antigen is used, which offers greater sensitivity and specificity), enabling antigen-antibody interaction. The Brazilian Ministry of Health, through its Clinical Protocol and Therapeutic Guidelines (CPTG) published in 2018, emphasized using rapid tests to diagnose chronic Chagas disease in difficult-to-access areas and pregnant women [35]. The CPTG was drafted through consensus after evaluating the sensitivity and specificity indicated by studies but recommended that it be an alternative test whose negative result rules out the disease.

In contrast, a positive result still requires confirmation by conventional methods [35]. The rapid test is qualitative; that is, it confirms or rejects, during a screening, the infection by T. cruzi [36]. The main purpose of a rapid test is that it be highly sensitive, specific, easy to perform, accessible in remote areas, and provides the result quickly [37].

According to Suescún-Carrero et al. (2022), who conducted a systematic review and meta-analysis on rapid tests and ELISA for the diagnosis of Chagas disease, concluded that ELISA and rapid diagnostic tests “have a high validity for diagnosing chronic Chagas disease” [38]. It was also noted that some variables influencing performance require further study [39]. Yet, implementing protocols based on rapid diagnostic tests for Chagas disease could “reduce the current social, geographical, economic and techno-scientific gaps in the diagnosis and treatment of Chagas disease” [40]. Studies have shown that social and housing conditions such as residing in rural areas, lack of health services, low primary health care coverage, and poor and remote regions are determinants for poor prognosis for the disease. Thus, implementing a rapid and simple management method for diagnosing Chagas disease could help support the treatment of the disease even before the clinical manifestations appear. Studies indicate that the quick test could replace conventional serological tools for the conclusive diagnosis of chronic T. cruzi infections in endemic regions. Still, there is a need to validate the difficulties in different geographic areas since the current studies show the performance of the tests is directly associated with the antigenic profile of the predominant strain of the parasite [41].

8. Conclusion

Chagas disease is a significant health concern for persons living in endemic regions, spreading worldwide. Since the identification of T. cruzi as its causative agent at the turn of the twentieth century, a full spectrum of diagnostic tests has been developed to detect infected individuals, from direct visualization of parasites to a range of indirect assays that capture antibodies made in response to an infection. To date, no single test has been developed that is sufficient to diagnose Chagas disease confidently. The absence of a gold standard has illuminated the challenges of providing for neglected populations living in remote areas endemic to multiple diseases. Requirements for numerous tests, different patient samples, infrastructure, and qualified personnel contribute to higher costs and delays in treatments that limit access to healthcare that disproportionately impact lower social classes. Developing high-performance rapid tests can solve many of these issues and be an example for other neglected tropical diseases.

Acknowledgments

We thank Ms. Laura C. Santos and Maria J. Soares for their support and access to the Hematophagous Triatomines Collection sector from the Oswaldo Cruz Institute/FIOCRUZ. Thanks to Dr. Patrícia Zauza and Julio C. Miguel (LDP) for their support in obtaining the image of the indirect immunofluorescence slide.