Introduction

American trypanosomiasis—also known as Chagas disease—is a vector-borne anthropozoonosis disease caused by the hemoflagellate parasite Trypanosoma cruzi (Trypanosomatidae). The organism infects mononuclear cells and has a predilection for the cardiac, digestive, nervous systems, skin, and soft tissues [1, 2]. The most common form of transmission is vector-borne, accounting for 70% of cases. The blood-sucking bugs belong to the genera of hematophagous Triatominae (Reduviidae family) [3–5]. Other forms of transmission include vertical transmission [6], accounting for approximately 26% of cases. Oral transmission [7], blood products [8], solid organ transplantation [9], and laboratory accidents [10] have also been reported [11]. The World Health Organization (WHO) estimates that around 75 million individuals are at risk for infection. Chagas disease is endemic in 21 Latin American countries, where approximately eight to ten million people are infected [11, 12]. Due to population mobility, the disease is not restricted to endemic areas [12]. In the USA, 300,167 individuals live with Trypanosoma cruzi infection, of whom 30,000–45,000 have cardiomyopathy; and 63–315 congenital infections occur every year [13]. The disease has two phases. An acute phase can be asymptomatic or present as an acute febrile undifferentiated syndrome. The chronic phase consists of an indeterminate form where patients are infected and asymptomatic (70% of cases) and a determinate form where the patient may present with cardiomyopathy (20–30% of cases) or a gastrointestinal mega-syndrome (megaesophagus or megacolon: 10% of cases) [14–16]. Symptomatic acute disease (~ 1%) is characterized by fever, lymphadenopathy, hepatosplenomegaly, heart failure, pericardial effusion, myocarditis, pericardial effusion, and heart failure or meningoencephalitis. Chagas disease can reactivate in the setting of immunosuppression and manifest as fulminant heart failure, panniculitis, or central nervous system rim-enhancing lesions and encephalitis [17, 18]. The annual cardiomyopathy risk reaches 2% and 5% among patients with the indeterminate form and acute infection, respectively [19••]. The parasite inoculum may influence cardiovascular disease severity and outcome [20]. Chagas disease acquired orally has been linked to a higher parasite load and is characterized by a shorter latency period and more severe cardiovascular disease than that associated with vectorial transmission.

The main burden of the disease comes from Chagas cardiomyopathy development. Once established, cardiomyopathy has an annual overall estimated mortality of 8%, of which 6.3% is of cardiovascular origin [21••]. Cardiovascular mortality mainly comes from heart failure (3.5%), followed by sudden death (2.6%) and strokes (0.4%). Cumulative heart failure mortality could reach 30% at 10 years and 50% at 20 years, assuming exponential growth (Fig. 1). The annual cardiovascular mortality is comparable to untreated AIDS [21••]. Chagas cardiomyopathy associated heart failure attributable mortality surpasses other common forms of heart failure [22].

Fig. 1
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Cumulative probability of Chagas-related heart failure mortality

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Geography influences the proportion of Chagas diseases among cohorts of patients with heart failure. In Latin American registries of heart failure patients, 0.6–20% were Chagas-related [23–25]. These numbers are expected to be substantially lower in non-endemic regions such as the USA or Europe.

People living in endemic areas are at risk of infections. Reinfections could influence the risk for congenital disease [26] or reactivation in the setting of transplantation [17]. It is unclear how repetitive exposure to the parasite through continuous vector exposure in endemic areas contributes to the development of chronic Chagas cardiomyopathy and heart failure.

This article summarizes the literature findings on Chagas disease reinfection and discusses its implications on Chagas pathogenesis and cardiomyopathy development. We explore the risk factors for reinfection, the pathological manifestations, the outcomes in experimental models, and the relevance of reinfection in diagnosis and treatment.

Trypanosoma cruzi Reinfection Definition

A standard definition for Chagas reinfection does not exist. Reinfections are defined as a new infection after recovery or a new superimposed infection. Theoretically, reinfection occurs with a new detectable blood smear parasitemia in a patient with an already established chronic infection after a recent re-exposure (vector or oral). This needs to be differentiated from reactivation disease in the immunocompromised hosts without recent exposure [27].

Determinant Factors for Trypanosoma cruzi Reinfections

The population at risk for reinfection is heterogeneous and determined by factors such as the living conditions, occupation, and intra-domiciliary presence of animals. These and other factors can modulate the risk and rate of T. cruzi reinfections:

Endemic Areas

The highest risk for reinfection with T. cruzi exists in endemic areas. Chagas disease is endemic in 21 continental Latin American countries, particularly in poor, rural areas of Central and South America, where residents are continuously exposed to competent vectors. In Latin America, the prevalence of T. cruzi infection is highest in Bolivia (6.1 cases per 100 inhabitants), Argentina (3.6), Paraguay (2.1), Ecuador (1.4), El Salvador (1.3), and Guatemala (1.2). The Gran Chaco region includes eastern Bolivia, western Paraguay, Northern Argentina, and parts of Brazil. In the hyperendemic region of the Bolivian Chaco, the incidence of T. cruzi infection fluctuates between 0.1% and 4% per year [1], and the all-age prevalence in rural areas is > 50% [28]. In the USA, enzootic cycles of T. cruzi transmission exist in southern states. However, only a few autochthonous infections have been reported [1].

Tomasini et al. developed models that predict the transmission of T. cruzi discrete typing units (DTUs) that were detected in Chaco province, Argentina. The model simulated T. cruzi transmission in an environment corresponding to a rural village made of contiguous houses. A fitted model using a hybrid genetic algorithm of epidemiological data of Argentinean rural villages found a 56% rate of mixed infections by different genotypes of T. cruzi in humans. The best model predicted 0.032 (0.008–0.042) annual reinfections per individual, with 98.4% occurring in the chronic phase of the disease. Based on these low yearly rates, the risks of reinfection were predicted to be 14% (4–18%) and 60% (21–70%) after 5 and 30 years, respectively, with most individuals being reinfected 1–3 times over their lifetime [29••].

Housing

The vector usually infests the corners and fissures inside poorly constructed homes associated with poor hygiene practices [30]. Mott et al. described a high rate of seroreactivity in residents of unplastered mud-stick houses, mud-brick houses, and plastered mud-stick homes [31]. Houses near henhouses, pigsties, and animal pens increase vectorial transmission [32]. Coura et al. reported that wood, fibers (e.g., piaçava fiber), and palm leaves used as roofing material in rural areas could carry infected wild triatomines to the homes. The uncontrolled deforestation of certain areas in endemic countries with subsequent construction of urbanized areas increases the exposure of individuals to reservoirs and the vector [32, 33].

Socioeconomic Determinants

Certain subsistence activities and customs practiced in rural areas of Latin America provide an opportunity for repetitive T. cruzi infections [34]. Certain fruits and juices, such as acai, bacaba, guava, and sugar cane, have been associated with outbreaks of Chagas disease through oral transmission [35]. Due to economic and social crises, there has been an increased number of migrants from endemic areas to Europe and the USA [36]. Also, the geographic distribution widening of the disease increases the population at risk. Climate change enormously influences the rate of vector-borne infections. Tamayo et al. described the effect of increasing temperature on the vector and the parasite. They noticed a rapid appearance, an increase in the infective forms of T. cruzi, and a decreased development time in the vector, suggesting an increased probability of infection in higher temperatures [37]. Type of occupation, migrants in transit, and residence of areas affected by climate changes can be crucial socio-economical risk factors for Chagas infection and reinfection.

Vector

Transmission of T. cruzi in the domestic and peridomestic cycles is related to the density and index of intradomiciliary infection of the vector and the vector species [38]. A revision of the impact of Chagas control in Latin America showed a reduction in the morbidity and mortality of Chagas after eliminating domestic vectors and transfusion-related transmission [39]. Sustained vector exposure increased the risk of Chagas cardiomyopathy among women in Bolivia. Among 302 infected women, the risk of having ECG abnormalities consistent with Chagas cardiomyopathy was higher in older women (OR 1.06 [95% CI 1.01–1.12] per year) and those with vector exposure (OR 3.7 [95% CI 1.4–10.2]). Interestingly, vector exposure showed an inverse relationship between maternal parasitemia and mother-to-child transmission. The authors hypothesize that pathogenic and protective effects may result from frequent exposure to infected vectors and repeated superinfection by the parasite [40].

Parasite Strains

The complex domestic, peridomestic, and wild cycles of this parasite include a diverse group of mammalian reservoirs (dogs, cats, raccoons, opossums, armadillos, woodrats, and others) [3, 41]. Due to its genetic diversity, the parasite has undergone multiple nomenclature changes; two major lineages of the parasite, T. cruzi I and T. cruzi II, were initially defined [42]. There are currently six DTUs, TcI to TcVI, TcI being the most abundant DTU extending from South to North America. T. cruzi has one of the largest genomes of the Trypanosomatids [43], with considerable genetic diversity within DTUs. [44] This genetic diversity is associated with different tissue tropisms, trypanocidal agent susceptibilities, and interactions with the host immune system. Triatomines can be infected by multiple T. cruzi strains simultaneously [45]. Data from various endemic areas have shown that mixed infections with more than one T. cruzi DTU are more prevalent in humans than in triatomes [46]. It is important to note that the isolation of different T. cruzi strains or DTUs in an individual does not establish a reinfection event.

Animal Exposures

Dog ownership has been suggested as a risk factor for T. cruzi transmission [47]. Dogs have been shown to increase house infestation with species adapted to human households such as Triatoma infestans [48] and non-domiciliated species such as Triatoma dimidiata [49]. By contrast, the model of Tomasini et al. based in the Chaco province of Argentina found that one or two dogs only slightly increased the prevalence of T. cruzi infection and did not necessarily explain the observed levels of mixed infections[29••]. However, the findings suggest keeping dogs outside of dormitories to lower transmission to humans [29••] [47]. Cats are also an important reservoir in the domestic cycle. However, their role is minor compared to dogs [50, 51]. The presence of the peri domiciliary animals, like opossums, may pose a public health threat in areas where the other components of the parasite cycle are present [52]. Opossums have been linked to acute Chagas outbreaks acquired by ingesting contaminated food products. These marsupials are known to harvest trypomastigotes in their anal glands and can contaminate food products.

Pathophysiology of Reinfection

Deposition of contaminated feces with the parasites occurs in the skin or mucosal areas during or immediately after a vector’s blood meal [14]. T. cruzi will replicate at the inoculation site. The released trypomastigotes would infect multiple cellular lines once they enter the bloodstream. Despite having the capacity to infect any nucleated cell [53], the parasite has a tropism for cardiac muscle and gastrointestinal tract neurons. As soon as a cell is infected, the parasite differentiates into an amastigote form and replicates intracellularly (Fig. 2). Cell rupture ensues, releasing trypomastigotes that will infect other cells. The acute phase can last from 2 weeks to months [54]. The chronic phase can be indeterminate—characterized by a lack of symptoms and no evidence of cardiac injury based on electrocardiographic and echocardiographic evaluation—or determinate, characterized by cardiomyopathy or gastrointestinal pathology [14]. During the acute phase, diffuse myocarditis characterized by vacuolization, cytolysis, and fibrillar degeneration is typically found. The initial inflammatory response is composed of neutrophils and macrophages and is later replaced with lymphocytes, eosinophils, mast cells, and plasma cells [55]. Polyclonal activation of B cells and subsequent hypergammaglobulinemia can be seen in the acute phase of Chagas disease [56]. Cellular immunity is vital to control the parasite burden in tissues. Cytokines, chemokines, antibody production, and complement activation produce tissue-level changes characterized by interstitial fibrosis and myocardial fiber hypertrophy [57]. Chronic Chagas cardiomyopathy consists of focal areas of low-grade myocarditis with interstitial fibrosis. In these areas, parasites are scarce, and the primary immune cells are T lymphocytes [57, 58]. In the gastrointestinal tract, T. cruzi causes myenteric plexus injury due to immune cross-reactivity of the flagellar antigen of T. cruzi and myenteric neurons. [59]. Autoimmunity may play a role in the pathogenesis of the disease [60], although controversial.

Fig. 2
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H&E stain, high power magnification showing parasite amastigotes (arrow), and associated myocarditis

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Heart failure in chronic Chagas cardiomyopathy may develop after repetitive cardiac injury events from subsequent reinfections. These changes can ultimately lead to myocardial abnormalities, including ventricular wall dilation, mitral and tricuspid valve regurgitations, and decreased ejection fraction [61]. Other pivotal mechanisms are the development of left ventricular apical aneurysms and cardiac arrhythmias. Autopsy findings in advanced Chagas heart failure have found evidence of cardiac denervation, specifically intramural neuronal depopulation, ganglionic injury, and reduction of subepicardial neurons [62]. In addition to dysautonomia, cardiac tissue parasite persistence has also been implicated in Chagas-related heart failure. Hypothetically, reinfection could lead to increased myocarditis and risk of advanced heart failure. Gastrointestinal manifestations of the disease—especially megacolon—shared the neural denervation component pathogenesis.

Animal Studies

During reinfection studies, multiple animal models have failed to replicate consistent results of the pathophysiological changes, likely due to the genetic diversity of the parasite, differences in immune host response, and the interaction between the parasite and the host (Table 1). Lauria-Pires and Teixeira studied superinfection in mice with low and high virulence clones. Their results showed that mice infected with low virulence organisms that were subsequently infected with highly virulent parasites did not have statistically significant differences in mortality and histopathological lesions compared with single infected mice [63]. These results suggested immune protection with reinfection. Machado exposed crossbreed dogs to 5 different types of reinfections [64]. A progressive decrease in parasitemia was noted with every reinfection, and the antibody titers during the chronic phases were higher in reinfected dogs than in the single infections. Despite multiple infections, alterations were found exclusively in the cardiac tissue. No significant increase in tissue damage was noted in reinfected dogs independent of the isolates [64].

Table 1 Summary of experimental Chagas studies related to reinfections
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In contrast, Bustamante noticed higher levels of parasitemia with reinfections with Tulahuen strain in albino Swiss mice compared with single infected mice, especially in groups receiving higher parasite loads in the reinfection event [65]. Later in 2007, Bustamante noted differences in reinfections with different parasite strains. Tulahuen strain-infected and reinfected mice had higher parasitemia levels and structural cardiac damage than SGO-Z12 infected mice. That study also showed a significant reduction in beta-adrenergic receptor affinity, concluding that reinfection can produce severe disease, disease progression, and exacerbation of the disease [66]. Andrade studied the effect and differences between single, double, and triple infections (single, mixed, and reinfections) with different strains (Colombian, 21SF, and Y strain). The study proved the coexistence of three different strains within the same mice. Reinfections resulted in the exacerbation of already-existing cardiac lesions in chronically infected mice and demonstrated that the order and strain of the reinfection influenced the outcome [67]. These results are likely explained due to the specific histiotropisms of different strains of the parasite. Restriction fragment length polymorphism (RFLP) studies revealed infection differences in single infected mice with only one strain [68].

The skeletal muscle was the largest reservoir of trypomastigote nests. Reis Machado found decreased parasitemia with reinfection, and higher levels of TNF-alpha, IFN-γ, and moderate to severe inflammatory infiltrate in reinfections with the Colombian strain compared to the Y strain [69]. Perez et al. evaluated the effect of mixed infections versus reinfections. During the study, simultaneous mixed infections did not produce acute morbidity compared to reinfections that impacted the course of the disease [70].

The mode of transmission and inoculum size may also affect the course of the infection. This has been tested by comparing intraperitoneal injections versus oral infections in mice [71]. Lewis et al. study showed that the parasites were “pan-tropic” during acute infections in mice. During the chronic infection, the gastrointestinal tract functioned as a reservoir for T. cruzi. In immunosuppression states, the infection was present in multiple organs. Differences in the immune control of T. cruzi occur between tissues. The strain differences in various tissues may also be related to tissue-related host responses and not strictly to tropism. They proposed that the cardiac damage accumulates over time according to the frequency of transient episodes of parasitism rather than persistent infection. The severity of fibrosis in the heart depends on host-parasite strain combinations, and different combinations showed heterogeneity of symptoms and disease progression rates [72]. Mortality is also affected by reinfections. Perez and Andrade et al. demonstrated sudden deaths in experimental mice models of reinfections; however, not all the deaths were related to cardiac dysfunction, suggesting that could be multiple mechanisms related to the mortality in reinfections [67, 70]. Meningoencephalitis—a clinical manifestation that can occur as part of acute Chagas disease or reactivation in immunosuppressed hosts—could also contribute to mortality in animal models of reinfection [67, 70].

Parasite Strains

In humans, the variations of tropism can also be seen within one organ, and multiple variations of one DTU can predominate in specific areas of an organ [17]. Apart from the inflammatory changes at tissue levels, the diversity of T. cruzi influences the host’s humoral immune response. Del Puerto et al. evaluated the effect of specific DTUs infection and their association with clinical forms of Chagas disease for 7 years. No lineages or sublineages were significantly associated with any clinical manifestations in Bolivian chronic Chagas patients [73]. However, this study was performed in a specific geographical area, and variations between strains from other areas were not evaluated. Burgos et al. demonstrated that blood strains in heart transplant patients with mixed infections differ from those in tissues or even within the same organ [17]. From animal studies’ findings, it is fair to hypothesize that different Trypanosoma cruzi strains may possess various patterns and severity of myocarditis [74, 75], which may correlate with heart failure risk and staging. The lack of clear human validation or an established geographic clinical variation of cardiomyopathy severity and heart failure makes this hypothesis less apparent.

Human Studies

In an experimental model, Dos Santos et al. measured the linkage between the phylogenetic divergence of T. cruzi and immunoglobulin G (IgG) levels. T. cruzi I was more efficient in over-expressing all subclasses of specific anti-parasite IgG than other T. cruzi strains. Benznidazole treatment also induced IgG alterations. These can differ depending on the stage of the disease when the medication was administered [76]. In humans, Llaguno et al. studied IgG changes in patients with Chagas disease 4 years after treatment with benznidazole. At 48 months, the total levels of IgG were decreased. In patients with cardiomyopathy and exacerbated clinical forms, increased IgG1 and decreased IgG3 were observed. Patients with the cardiac form also presented a decreased IgG3/IgG1ratio [77•]. Higher anti-T. cruzi antibodies correlated with an increased rate of cardiomyopathy and death in a long-term follow-up study suggesting higher parasite exposure can work as a surrogate for reinfections [78••].

Reinfections have also been evaluated in specific scenarios like congenital transmission. Burgos et al. assessed the genetic diversity of T. cruzi in infected mothers and infants with congenital Chagas. They found no association between a particular genotype and vertical transmission. The strains present in the mothers and the infants (including twin deliveries) were nearly identical [79].

Although there is no specifically human clinical evidence of the role of reinfections in cardiomyopathy development, some animal studies suggest that repeat infections may lead to a higher degree of cardiac injury.

Diagnosis

The diagnosis of Chagas disease is challenging and typically based on microscopic analysis of blood smears or tissue, polymerase chain reaction (PCR), and serologic studies (Fig. 3). The diagnosis of acute Chagas, reactivation, and congenital Chagas relies on PCR and microscopic analysis of blood smears. The diagnosis of chronic Chagas is based on serological tests [80].

Fig. 3
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Factors involved in diagnosis and treatment of Chagas disease during reinfections

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Multiple strains can be involved in the pathogenesis of the disease [17]. During reinfections, the difference in the tropism of T. cruzi strains can cause the parasite identified in the blood to be different from the one causing tissue disease. Animal models have demonstrated a lower parasitemia with repetitive infections, making the diagnosis more difficult by microscopic analysis and PCR[64]. The differences in humoral response based on the T. cruzi strain raised concern about the sensitivity and specificity of serological tests [76, 77•]. There is a need for studies profiling the Th2 responses based on different strains and the effect of reinfections and mixed infections. Wide use of PCR in endemic areas and rural fields may aid in understanding detectable parasitemia as a marker for Chagas disease progression related to reinfections, intrinsic parasite features, host immune control, or a combination of them. Reinfections events remain very challenging to detect and diagnose. During Chagas cardiomyopathy and heart failure diagnosis, we recommend using the standardized AHA classification of 4 stages: A, B1, B2, C, and D [61]. AHA classification uses electrocardiographic or echocardiographic changes and the presence of heart failure symptoms. Key clinical features include cardiac arrhythmias (commonly left anterior fascicular block (LAFB), incomplete or complete right bundle branch block (RBBB), ST-T changes, or monomorphic PVCs), dilated cardiomyopathy, ventricular aneurysms, and wall motion abnormalities. Symptoms are attributed to pulmonary congestion and hypervolemia. Although unclear, reinfection can present with possible heart failure exacerbation or an acute on chronic heart failure due to additional myocarditis. One of the main prognostic predictors used in clinical practice is the Rassi score. As in other forms of heart failure, NHYA class III/IV, ejection fraction, age, and cardiomegaly are the main mortality prognostic risk factors [21••, 61].

Congenital Chagas transmission occurred among 5% of infected mothers [81]. PAHO guidelines recommend screening women of childbearing age or with chronic Chagas disease, including heart failure, and their newborns.

Treatment

The challenge in the treatment of latent infection includes a lack of immune protection after initial infection, decreased efficacy of the treatment during the indeterminate and chronic phases [82], and the human immune system’s inability to eradicate the parasite in most cases. Chronic Chagas disease carries a staggering high annual mortality of 8% [21••], and any efforts to decrease mortality and progression are urgently needed. Currently, two drugs are available to treat Chagas disease, benznidazole and nifurtimox. Benznidazole is approved in the USA for the treatment of Chagas disease [83]. A limitation in the treatment trials of Chagas is that they do not routinely test for the strains that cause the disease in trial participants.

Bustamante and Tarleton demonstrated in an animal model that previous infections, active infections, and previously treated infections do not protect against reinfection with T. cruzi. Mice previously treated with benznidazole challenged with cyclophosphamide have an increased reactivation risk [84]. A T-cell-mediated inflammatory response is usually induced after the initial T. cruzi replication cycle. However, parasites remain capable of infecting cells initially with minimal immune system stimulation [84]. Teston et al. evaluated the benznidazole susceptibility of natural populations of T. cruzi DTUs (I, II, IV) from the states of Amazonas, Parana, and Minas Gerais. The overall cure rate was 62.5%. However, cure rates differ between DTUs and even within the same DTUs from multiple locations (27 to 100%). In the study, 9/23 strains were sensitive, 9/23 were intermediate, and 5/23 were resistant to benznidazole. There was no predominance of resistance patterns between the strains. Differences in cures rates between TcI strains from Amazonas and Parana were noted, highlighting the genetic diversity within DTUs [85]. A natural infection cure without medication administration is a rare phenomenon and usually takes a considerable amount of time [86–88]. The presence of reinfection phenomena may suggest that some infected patients may benefit from repeated treatment courses, along with strict vector control. The BENEFIT trial failed to show any benefit of anti-trypanosomal therapy to reduce cardiovascular events or death at 5 years in patients with already established Chagas cardiomyopathy [89]. In endemic regions with poor vector control, reinfections may account for the observed lack of antiparasitic therapy benefits. However, therapy with benznidazole or nifurtimox is largely ineffective once heart failure is clinically manifested. Also, any reinfection episode may precipitate an exacerbation in a stable heart failure patient on cardiac pharmacotherapy.

Conclusion

The literature reviewed in this article exposes the challenges in studying T. cruzi reinfections. The differences in host response between multiple animal models, the lack of a standardized model, the enormous genetic diversity of the parasite, and the subsequent variations in the individual host immune responses make the study of this area extremely complex. These data uncover the need to develop tools to study human reinfections that consider the DTUs and geographic locations of the specific strain. Other essential study variables to consider are host-specific genotypes and immune responses.

The development of new treatment modalities against this multisystemic disease is required to overcome the diverse tissue tropism and possible reservoirs of this disease in the human body. Based on the existing literature, reinfections may play a role in developing chronic Chagas cardiomyopathy and heart failure and may be linked to worse outcomes. This stresses the importance of implementing public health measures to continue strict vector control and improve house conditions of populations at risk.