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Predictors of Trypanosoma cruzi PCR positivity in patients with chronic Chagas disease

Abstract

BACKGROUND

A positive Trypanosoma cruzi polymerase chain reaction (PCR) is associated with a worse prognosis in patients with chronic Chagas disease (CD).

OBJECTIVES

To study the association of clinical, electrocardiographic, and echocardiographic characteristics and biomarker blood levels with positive T. cruzi PCR in chronic CD.

METHODS

This is a single-centre observational cross-sectional study. Positive T. cruzi PCR association with clinical, electrocardiographic, and echocardiographic characteristics, and biomarker blood levels were studied by logistic regression analysis. p values < 0.05 were considered significant.

FINDINGS

Among 333 patients with chronic CD (56.4% men; 62 ± 10 years), T. cruzi PCR was positive in 41.1%. Stepwise multivariate logistic regression showed an independent association between positive T. cruzi PCR and diabetes mellitus {odds ratio (OR) 0.53 [95% confidence interval (CI) 0.30-0.93]; p = 0.03}, right bundle branch block [OR 1.78 (95% CI 1.09-2.89); p = 0.02], and history of trypanocidal treatment [OR 0.13 (95% CI 0.04-0.38); p = 0.0002]. Among patients with a history of trypanocidal treatment (n = 39), only four (10%) patients had a positive T. cruzi PCR.

MAIN CONCLUSIONS

Among several studied parameters, only diabetes mellitus, right bundle branch block, and history of trypanocidal treatment showed an independent association with positive T. cruzi PCR. History of trypanocidal treatment was a strong protective factor against a positive T. cruzi PCR.

Key words:
Chagas disease; Trypanosoma cruzi; clinical forms; biomarkers


Chagas disease (CD) is a neglected tropical disease caused by the Trypanosoma cruzi parasite and affects 5-7 million people, mainly in low-income populations in endemic areas of Latin America, where the main mode of transmission is vector-borne.11. Nunes MCP, Beaton A, Acquatella H, Bern C, Bolger AF, Echeverria LE, et al. Chagas cardiomyopathy: an update of current clinical knowledge and management. A scientific statement from the American Heart Association. Circulation. 2018; 138: e169-e209. However, migration has spread the disease to non-endemic countries where other forms of transmission, such as blood transfusion, organ donation, and vertical transmission, may occur. Those countries include but are not limited to the USA, with an estimated 288,000 infected individuals,22. Irish A, Whitman JD, Clark EH, Marcus R, Bern C. Updated estimates and mapping for prevalence of Chagas disease among adults, United States. Emerg Infect Dis. 2022; 28: 1313-20. and European countries, with an estimated 120,000 infected individuals.33. Basile L, Jansa JM, Carlier Y, Salamanca DD, Angheben A, Bartoloni A, et al. Chagas disease in European countries: the challenge of a surveillance system. Euro Surveill. 2011; 16: 19968. Therefore, CD is still a challenge for public health, causing more than 7,000 deaths per year44. WHO - World Health Organization. Chagas disease (American trypanosomiasis). 2017. Available from: http://www.who.int/mediacentre/factsheets/fs340/en/.
http://www.who.int/mediacentre/factsheet...
and a high economic and health burden.55. Lee BY, Bacon KM, Bottazzi ME, Hotez PJ. Global economic burden of Chagas disease: a computational simulation model. Lancet Infect Dis. 2013; 13: 342-8.,66. Abuhab A, Trindade E, Aulicino GB, Fujii S, Bocchi EA, Bacal F. Chagas' cardiomyopathy: the economic burden of an expensive and neglected disease. Int J Cardiol. 2013; 168: 2375-80.,77. Olivera MJ, Palencia-Sánchez F, Riaño-Casallas M. The cost of lost productivity due to premature Chagas disease-related mortality: lessons from Colombia (2010-2017). Trop Med Infect Dis. 2021; 6: 17.

Chronic CD can have three different clinical forms, ranging from a long-lasting indeterminate form, without clinical evidence of a major organ damage, to cardiac and digestive forms, which can occur simultaneously. Patients with the cardiac form may present a dismal prognosis due to arrhythmias, thromboembolic events, and heart failure (HF).88. Saraiva RM, Mediano MFF, Mendes FS, Sperandio da Silva GM, Veloso HH, Sangenis LHC, et al. Chagas heart disease: an overview of diagnosis, manifestations, treatment, and care. World J Cardiol. 2021; 13: 654-75. However, the mechanisms underlying the progression from the indeterminate to the cardiac form comprehend several possible pathways that are still under investigation. For decades, it was believed that the parasite was not present in the chronic phase and that auto-immune mechanisms would prevail and determine the progression of the disease.99. Dias JC, Ramos Jr AN, Gontijo ED, Luquetti A, Shikanai-Yasuda MA, Coura JR, et al. II Brazilian Consensus on Chagas Disease, 2015. Rev Soc Bras Med Trop. 2016; 49(Suppl. 1): 3-60. However, CD reactivation in immunocompromised patients and molecular studies proved otherwise.1010. Benatti RD, Oliveira GH, Bacal F. Heart transplantation for Chagas cardiomyopathy. J Heart Lung Transplant. 2017; 36: 597-603.,1111. Lattes R, Lasala MB. Chagas disease in the immunosuppressed patient. Clin Microbiol Infect. 2014; 20: 300-9. Today, the persistence of the parasite seems to be a key element for the persistent low grade chronic fibrosing myocarditis present in chronic CD.11. Nunes MCP, Beaton A, Acquatella H, Bern C, Bolger AF, Echeverria LE, et al. Chagas cardiomyopathy: an update of current clinical knowledge and management. A scientific statement from the American Heart Association. Circulation. 2018; 138: e169-e209.,1212. Gonzalez FB, Villar SR, Pacini MF, Bottasso OA, Perez AR. Immune-neuroendocrine and metabolic disorders in human and experimental T. cruzi infection: new clues for understanding Chagas disease pathology. Biochim Biophys Acta Mol Basis Dis. 2020; 1866: 165642.,1313. Dutra WO, Menezes CA, Magalhães LM, Gollob KJ. Immunoregulatory networks in human Chagas disease. Parasite Immunol. 2014; 36: 377-87. Moreover, an imbalance between pro- and anti-inflammatory cytokines is regarded as an important element in CD progression.1212. Gonzalez FB, Villar SR, Pacini MF, Bottasso OA, Perez AR. Immune-neuroendocrine and metabolic disorders in human and experimental T. cruzi infection: new clues for understanding Chagas disease pathology. Biochim Biophys Acta Mol Basis Dis. 2020; 1866: 165642.,1313. Dutra WO, Menezes CA, Magalhães LM, Gollob KJ. Immunoregulatory networks in human Chagas disease. Parasite Immunol. 2014; 36: 377-87.

The importance of the persistence of the parasite for CD progression was suggested by several findings, such as a more frequent identification of the parasite in the bloodstream of patients with the cardiac form than in those without the cardiac form,1414. Keating SM, Deng X, Fernandes F, Cunha-Neto E, Ribeiro AL, Adesina B, et al. Inflammatory and cardiac biomarkers are differentially expressed in clinical stages of Chagas disease. Int J Cardiol. 2015; 199: 451-9.,1515. Nielebock MAP, Miranda LFC, Brasil PEAA, Pereira TOJS, da Silva AF, Hasslocher-Moreno AM, et al. Blood culture positivity rate for Trypanosoma cruzi in patients with chronic Chagas disease differs among different clinical forms. Trans R Soc Trop Med Hyg. 2021; 115: 720-5. and a worse prognosis in patients with parasitaemia identified by haemoculture1515. Nielebock MAP, Miranda LFC, Brasil PEAA, Pereira TOJS, da Silva AF, Hasslocher-Moreno AM, et al. Blood culture positivity rate for Trypanosoma cruzi in patients with chronic Chagas disease differs among different clinical forms. Trans R Soc Trop Med Hyg. 2021; 115: 720-5. and molecular methods.1616. Echeverría LE, Rojas LZ, Rueda-Ochoa OL, Gómez-Ochoa SA, Rugeles CIG, Díaz ML, et al. Circulating Trypanosoma cruzi load and major cardiovascular outcomes in patients with chronic Chagas cardiomyopathy: a prospective cohort study. Trop Med Int Health. 2020; 25: 1534-41.,1717. Mendes VG, Rimolo L, de Lima ACB, Ferreira RR, Oliveira LS, Nisimura LM, et al. Biomarkers and echocardiographic predictors of cardiovascular outcome in patients with chronic Chagas disease. J Am Heart Assoc. 2023; 12: e028810.

We therefore aimed to investigate which clinical characteristics and immunological biomarkers would correlate with a positive T. cruzi polymerase chain reaction (PCR) in patients with chronic CD.

SUBJECTS AND METHODS

Design and study subjects - This is a cross-sectional secondary analysis of a previous study approved by the local ethical committee on 01/27/2014 under number 515851 whose main paper was already published.1717. Mendes VG, Rimolo L, de Lima ACB, Ferreira RR, Oliveira LS, Nisimura LM, et al. Biomarkers and echocardiographic predictors of cardiovascular outcome in patients with chronic Chagas disease. J Am Heart Assoc. 2023; 12: e028810.

Data from adult patients with chronic CD who had been included in the previous study between July 2014 and March 2017 and who had had blood samples collected for T. cruzi quantitative PCR (qPCR) were analysed in the present paper.1717. Mendes VG, Rimolo L, de Lima ACB, Ferreira RR, Oliveira LS, Nisimura LM, et al. Biomarkers and echocardiographic predictors of cardiovascular outcome in patients with chronic Chagas disease. J Am Heart Assoc. 2023; 12: e028810. Briefly, from 402 patients, 11 did not return for the index echocardiogram, 30 were excluded due to several reasons described in Figure, and other 28 patients did not have blood samples collected for T. cruzi qPCR. The final studied population was composed of 333 patients (Figure).


Flow diagram depicting recruitment, exclusion criteria, and studied groups at baseline.

CD diagnosis was based on positivity in two different serological tests (enzyme-linked immunosorbent assay and indirect immunofluorescence).1818. Nascimento CA, Gomes VA, Silva SK, Santos CR, Chambela MC, Madeira FS, et al. Left atrial and left ventricular diastolic function in chronic Chagas disease. J Am Soc Echocardiogr. 2013; 26: 1424-33.

Data regarding clinical characteristics, comorbidities, self-reported skin colour, previous trypanocidal treatment history, and use of medications were based on face-to-face interviews complemented by medical records review. Skin colour was categorised into white, brown, black, yellow, and indigenous, following the classification used by the Brazilian Official Bureau of Statistics (Instituto Brasileiro de Geografia e Estatística - IBGE), changing only brown for mixed-race.1919. Travassos C, Laguardia J, Marques PM, Mota JC, Szwarcwald CL. Comparison between two race/skin color classifications in relation to health-related outcomes in Brazil. Int J Equity Health. 2011; 10: 35. As no patient self-reported as yellow, only four categories were described in Table I.

TABLE I
Clinical and echocardiographic characteristics of studied subjects (n = 333)

Patients were classified at the time of their enrolment in the study according to the Brazilian CD consensus99. Dias JC, Ramos Jr AN, Gontijo ED, Luquetti A, Shikanai-Yasuda MA, Coura JR, et al. II Brazilian Consensus on Chagas Disease, 2015. Rev Soc Bras Med Trop. 2016; 49(Suppl. 1): 3-60. into: indeterminate (no evidence of cardiac involvement), stage A [no HF symptoms with isolated changes in the electrocardiogram (ECG)], stage B (no HF symptoms with segmental or global LV systolic dysfunction), stage C (symptomatic HF), or stage D (end-stage HF). ECGs were analysed by experienced cardiologists and classified using the Minnesota Code criteria, modified for CD.2020. Prineas RJ, Crow RS, Zhang ZM. The Minnesota code manual of electrographic findings. 2nd ed. Springer; 2010. doi: https://doi.org/10.1007/978-1-84882-778-3.
https://doi.org/10.1007/978-1-84882-778-...
The ECG changes considered compatible with CD cardiac form followed previously published criteria.99. Dias JC, Ramos Jr AN, Gontijo ED, Luquetti A, Shikanai-Yasuda MA, Coura JR, et al. II Brazilian Consensus on Chagas Disease, 2015. Rev Soc Bras Med Trop. 2016; 49(Suppl. 1): 3-60.

Echocardiography - Studies were performed using a phased-array ultrasound system (Vivid 7, GE Medical Systems, Milwaukee, WI) equipped with M4S phased-array and 1.5- to 4-MHz four-matrix-array transducers by a single experienced echocardiographer. Cardiac dimensions, measured using M-mode and 2D echocardiography, and Doppler measurements were obtained as recommended.2121. Lang RM, Badano LP, Mor-Avi V, Afilalo J, Armstrong A, Ernande L, et al. Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging. J Am Soc Echocardiogr. 2015; 28: 1-39.,2222. Nagueh SF, Appleton CP, Gillebert TC, Marino PN, Oh JK, Smiseth OA, et al. Recommendations for the evaluation of left ventricular diastolic function by echocardiography. J Am Soc Echocardiogr. 2009; 22: 107-33. 2D LV ejection fraction and 2D maximum LA volume were determined using modified Simpson’s rule with images obtained from apical 4- and 2-chamber views.

Biomarkers measurement - Biomarker blood measurements were performed by researchers blinded to the clinical classification of the patients. Tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-4, interleukin-6 (IL-6), IL-10, and IL-17 measurements were performed using commercially available enzyme-linked immunosorbent assay (ELISA) kits (TIMP-1, and TIMP-4: Picokine ELISA Human ImmunoAssay, Boster Biologics, CA, USA; IL-6, IL-10, and IL-17: Quantikine ELISA Human ImmunoAssay, R&D systems, EUA) according to the manufacturer’s instructions using SpectraMax 190 absorbance micro plate reader (Molecular Devices, CA, USA).

Trypanosoma cruzi PCR - T. cruzi PCR was performed as previously described.1717. Mendes VG, Rimolo L, de Lima ACB, Ferreira RR, Oliveira LS, Nisimura LM, et al. Biomarkers and echocardiographic predictors of cardiovascular outcome in patients with chronic Chagas disease. J Am Heart Assoc. 2023; 12: e028810. Briefly, ten millilitres of whole blood samples were obtained from each patient. The samples were processed for DNA extraction using the High Pure PCR Template Preparation kit (Roche Diagnostics Corp., Indianapolis, IN). The qPCR assays were performed by absolute quantification to estimate the parasite load, as previously described.2323. Ramírez JC, Parrado R, Sulleiro E, de la Barra A, Rodríguez M, Villarroel S, et al. First external quality assurance program for bloodstream Real-Time PCR monitoring of treatment response in clinical trials of Chagas disease. PLoS One. 2017; 12: e0188550. The qPCR assays were carried out with 5 μL of DNA, using the FastStart Universal Probe Master Mix (Roche Diagnostics, Mannheim, Germany) in a final volume of 20 μL. A sample was considered positive when the fluorescence generated by the Cruzi 3 TaqMan probe (FAM) crossed the threshold set at 0.02 for the T. cruzi satellite DNA target, at least in one of the technical replicates, during the 40 cycles of the real time PCR assay. The amplifications were performed in an ABI 7500 Fast Real Time PCR device (Applied Biosystems, USA).

Statistical analysis - Calculations were done using MedCalc version 20.113 (MedCalc Software, Mariakerke, Belgium) and Stata version 13.0 (StataCorp, College Station, TX). Continuous variables are expressed as mean ± standard deviation or median (interquartile range), and categorical variables as absolute and percentage values. Kolmogorov-Smirnov tests provided support that continuous variables were normal, as p values were > 0.10. Data between groups were compared using one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls post-hoc analysis, Kruskal-Wallis test followed by Dunn’s multiple comparison test, or contingency tables, as appropriate. Associations between studied variables and T. cruzi PCR positivity were tested using univariate logistic regression analysis. All variables with a p value under 0.20 were entered in a stepwise multivariate logistic regression analysis. Associations between continuous variables and parasite load were tested using linear regression after parasite load log transformation due to its skewed distribution. Missing data were handled by listwise deletion. The null hypothesis was rejected at p < 0.05. The data that support the findings of this study are available on request from the corresponding author.

Ethics - This study was approved by the local ethical committee on 01/27/2014 under number 515851. This study conformed to standards currently applied by the Brazilian National Committee for Research Ethics and Resolution 466/2012 of the National Health Council, of the Ministry of Health, and to the Declaration of Helsinki of 1975, as revised in 1983. All subjects gave their written informed consent before participating.

RESULTS

Patients- The studied patients presented the following CD clinical forms at the time of their enrolment: 92 (27.6%) presented the indeterminate form, 185 (55.6%) presented the cardiac form [stage A (n = 71; 21.3%), stage B (n = 70; 21.0%), stage C (n = 38; 11.4%), stage D (n = 6; 1.8%)], 11 (3.3%) presented digestive form, and 45 (13.5%) presented cardiodigestive form [stage A (n = 19; 5.7%), stage B (n = 19; 5.7%), stage C (n = 6; 1.8%), stage D (n = 1; 0.3%)].

The clinical characteristics, laboratory results, electrocardiogram and echocardiogram findings of the included patients are depicted in Table I. The most common ECG abnormalities were right bundle branch block, left anterior haemiblock, and primary T wave changes. Most patients presented a normal LV systolic function and grade I LV diastolic dysfunction (Table I). Forty-four patients (13.2%) had a cardiac device at baseline: 24 (7.2%) had a dual-chamber pacemaker, seven (2.1%) had a single-chamber pacemaker, eight (2.4%) had an implantable cardioverter-defibrillator (ICD) implant, 3 (0.9%) had a cardiac resynchronisation therapy pacemaker device, and 2 (0.6%) had a cardiac resynchronisation therapy defibrillator device. The frequency of use of cardiovascular medications was proportional to the number of patients with more advanced stages of the cardiac form and/or concomitant arterial hypertension.

A total of 39 patients had a previous history of trypanocidal treatment. The mean time elapsed between trypanocidal treatment and blood collection for T. cruzi PCR was 11.1 ± 7.2 years. Twenty-four patients (61.5%) received at least 300 mg a day of benznidazole. Other 14 patients were also treated with benznidazole: one patient (2.6%) received 100 mg a day, nine patients (23.1%) received 200 mg a day, and in four patients (10.3%) the dose information was missing. An additional patient was treated with ketoconazole. The trypanocidal duration of treatment was at least 60 days in 33 patients (84.6%), less than 60 days in four patients (10.3%), and unknown in one patient (2.6%).

Trypanosoma cruzi PCR positivity - PCR for T. cruzi DNA was positive in 137 out of 333 (41.1%) patients. T. cruzi PCR positivity was similar across the studied groups: 34.8% in the indeterminate group; 42.4% in stages A/B of the cardiac form; 47.1% in stages C/D of the cardiac form; and 45.4% among patients with the digestive form (p = 0.48).

Univariate analysis between T. cruzi PCR positivity and studied variables - The only clinical characteristic associated, in the univariate analysis, with a positive T. cruzi PCR was a previous history of trypanocidal treatment, which was strongly protective. The only ECG characteristic associated with T. cruzi PCR result was the presence of a right bundle branch block that increased by 65% the chance of a positive T. cruzi PCR. There was no 2D echocardiographic parameter associated with a positive T. cruzi PCR (Table II).

TABLE II
Univariate associations between clinical, echocardiographic, and biomarkers characteristics of studied subjects and Trypanosoma cruzi polymerase chain reaction (PCR) positivity

Among biomarkers, the univariate analysis did not reveal any significant association with a positive T. cruzi PCR (Table II).

Other variables with a p value under 0.20 in the univariate analysis and which were included in the multivariate analysis were CD cardiac form, sex, body mass index, diabetes mellitus, LV aneurysm, MMP-9, use of either ACE inhibitor or ARB, use of warfarin, and use of amiodarone.

Adjusted analysis between T. cruzi PCR positivity and studied variables - Multivariate logistic regression analysis revealed that diabetes mellitus, right bundle branch block (RBBB), and history of previous trypanocidal treatment remained independently associated with a positive T. cruzi PCR with a p value < 0.0001 and an area under the ROC curve (AUC) of 0.65 (95% CI 0.60-0.70) for the overall model fit (Table III).

Among patients with a previous history of trypanocidal treatment, only four patients had a positive T. cruzi PCR and none of them received benznidazole 300 mg daily for at least 60 days: one patient received benznidazole 100 mg a day for 60 days; one patient was given benznidazole 200 mg a day for 60 days; one patient was treated with ketoconazole for 60 days; and one patient used benznidazole 300 mg a day for only 11 days due to adverse events.

TABLE III
Adjusted association between clinical and biomarker characteristics of studied subjects and Trypanosoma cruzi polymerase chain reaction (PCR) positivity

Regression analysis between parasite load and studied variables - There was no significant univariate association between parasite load and age, body mass index, 2D echocardiographic parameters or biomarkers. For all analyses, residuals had a normal distribution (Table IV). A multiple regression analysis including variables with a p value under 0.20 on the univariate analysis did not retain any variable with significant association with the parasite load.

TABLE IV
Regression analysis associations between studied variables and parasite load

Biomarkers - As none of the biomarkers were associated with a positive T. cruzi PCR, we further investigated whether there were differences in the biomarker blood levels across the studied groups. For this analysis, patients with cardiac and cardiodigestive forms were grouped together, and the digestive form group was not included due to the low number of individuals in this group.

Brain natriuretic peptide (BNP), cardiac troponin I, matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9), transforming growth factor β1 (TGF-β1), and tumour necrosis factor (TNF) blood levels were previously investigated in this project.1717. Mendes VG, Rimolo L, de Lima ACB, Ferreira RR, Oliveira LS, Nisimura LM, et al. Biomarkers and echocardiographic predictors of cardiovascular outcome in patients with chronic Chagas disease. J Am Heart Assoc. 2023; 12: e028810. IL-17 blood levels were higher in all cardiac form groups than in patients with the indeterminate form. IL-6 presented a tendency for higher blood levels in both initial stages of the cardiac form (p = 0.06) and advanced stages of the cardiac form (p = 0.05) than in patients with the indeterminate form. IL-10 and TIMP-1 blood levels were similar across the studied groups. TIMP-4 blood levels were higher in the initial stages of the cardiac form than in patients with the indeterminate form (Table V).

TABLE V
Biomarkers blood levels of studied subjects

DISCUSSION

Parasite persistence in T. cruzi chronic infection plays a central role in CD progression and reactivation. However, the mechanisms associated with the development of CD pathology are still not fully clarified and it has been suggested that alterations in the immune response profile are involved in the pathogenesis of the disease. Previous data have already reported a possible association between cytokine levels and individuals in the indeterminate and cardiac CD clinical forms.2424. Gomes JA, Bahia-Oliveira LM, Rocha MO, Martins-Filho OA, Gazzinelli G, Correa-Oliveira R. Evidence that development of severe cardiomyopathy in human Chagas disease is due to a Th1-specific immune response. Infect Immun. 2003; 71: 1185-93.,2525. Vitelli-Avelar DM, Sathler-Avelar R, Teixeira-Carvalho A, Pinto Dias JC, Gontijo ED, Faria AM, et al. Strategy to assess the overall cytokine profile of circulating leukocytes and its association with distinct clinical forms of human Chagas disease. Scand J Immunol. 2008; 68: 516-25. An imbalance between pro-inflammatory and anti-inflammatory networks has been suggested as being important in CD progression towards Chagas heart disease.1212. Gonzalez FB, Villar SR, Pacini MF, Bottasso OA, Perez AR. Immune-neuroendocrine and metabolic disorders in human and experimental T. cruzi infection: new clues for understanding Chagas disease pathology. Biochim Biophys Acta Mol Basis Dis. 2020; 1866: 165642.,1313. Dutra WO, Menezes CA, Magalhães LM, Gollob KJ. Immunoregulatory networks in human Chagas disease. Parasite Immunol. 2014; 36: 377-87.

However, we could not demonstrate an association between biomarker blood levels and persistent parasitaemia. The strongest association with a negative T. cruzi PCR was previous history of trypanocidal treatment indicating the high anti-parasite efficiency of benznidazole, as patients remained negative even decades after treatment. The other two factors associated with T. cruzi PCR were the presence of RBBB and diabetes mellitus.

Cure criteria after anti-parasite treatment are currently limited to seroconversion. However, the change in serological status (seronegativity using conventional serological tests) may take decades to happen.2626. Fabbro DL, Streiger ML, Arias ED, Bizai ML, del Barco M, Amicone NA. Trypanocide treatment among adults with chronic Chagas disease living in Santa Fe city (Argentina), over a mean follow-up of 21 years: parasitological, serological and clinical evolution. Rev Soc Bras Med Trop. 2007; 40: 1-10. Therefore, new cure criteria are needed in order to follow patients properly and test new treatment strategies. Thus, biomarkers before and after trypanocidal treatment have been studied and some have been shown to present significant changes after trypanocidal treatment. Those include the sera reactivity for a set of T. cruzi antigens (KMP11, PFR2, HSP70, and 3973d)2727. Egui A, Thomas MC, FernándezVillegas A, Pérez-Antón E, Gómez I, Carrilero B, et al. A parasite biomarker set for evaluating benznidazole treatment efficacy in patients with chronic asymptomatic Trypanosoma cruzi infection. Antimicrob Agents Chemother. 2019; 63: e02436-18. and a specific antibody response (antibody 3, Ab3)2828. Zrein M, Granjon E, Gueyffier L, Caillaudeau J, Liehl P, Pottel H, et al. A novel antibody surrogate biomarker to monitor parasite persistence in Trypanosoma cruzi-infected patients. PLoS Negl Trop Dis. 2018; 12: e0006226. that declined significantly in BZN-treated patients, and a change in antigen-specific response of CD8 T cells with an improved response after trypanocidal treatment.2929. Mateus J, Perez-Anton E, Lasso P, Egui A, Roa N, Carrilero B, et al. Antiparasitic treatment induces an improved CD8 T cell response in chronic chagasic patients. J Immunol. 2017; 198: 3170-80. This may be relevant, as dysfunction of CD8 T cells may contribute to an immunological imbalance that compromises pathogen control in CD.3030. Lasso P, Mateus J, Pavia P, Rosas F, Roa N, Thomas MC, et al. Inhibitory receptor expression on CD8 T cells is linked to functional responses against Trypanosoma cruzi antigens in chronic chagasic patients. J Immunol. 2015; 195: 3748-58.

Regarding T. cruzi PCR, a persistent positive test after trypanocidal treatment is recognised as a treatment failure. There is also an association between a drop of serological reactivity and a decrease of T. cruzi PCR positivity.3131. Buss LF, Oliveira-da-Silva LC, Moreira CHV, Manuli ER, Sales FC, Morales I, et al. Declining antibody levels to Trypanosoma cruzi correlate with polymerase chain reaction positivity and electrocardiographic changes in a retrospective cohort of untreated Brazilian blood donors. PLoS Negl Trop Dis. 2020; 14: e0008787. Yet another important aspect is that T. cruzi PCR positivity is associated with Chagas heart disease3232. Sabino EC, Ribeiro AL, Lee TH, Oliveira CL, Carneiro-Proietti AB, Antunes AP, et al. Detection of Trypanosoma cruzi DNA in blood by PCR is associated with Chagas cardiomyopathy and disease severity. Eur J Heart Fail. 2015; 17: 416-23. and worse clinical prognosis.1717. Mendes VG, Rimolo L, de Lima ACB, Ferreira RR, Oliveira LS, Nisimura LM, et al. Biomarkers and echocardiographic predictors of cardiovascular outcome in patients with chronic Chagas disease. J Am Heart Assoc. 2023; 12: e028810. However, a negative test cannot be used as cure criterion, as parasitaemia is low and intermittent in chronic CD. Nonetheless, it is the best parameter to be followed in clinical trials testing new treatment strategies against CD.3333. Pinazo MJ, Thomas MC, Bua J, Perrone A, Schijman AG, Viotti RJ, et al. Biological markers for evaluating therapeutic efficacy in Chagas disease, a systematic review. Expert Rev Anti Infect Ther. 2014; 12: 479-96. Therefore, we evaluated the association between a positive T. cruzi PCR and several biomarkers described to have changes in their blood levels in patients with CD. However, we could not find such a correlation even though we have shown changes in cytokine blood levels in CD clinical forms, similar to the literature. We showed that IL-17 blood levels were higher in all cardiac form groups than in patients with the indeterminate form, as shown by others.3434. Perez AR, Silva-Barbosa SD, Berbert LR, Revelli S, Beloscar J, Savino W, et al. Immunoneuroendocrine alterations in patients with progressive forms of chronic Chagas disease. J Neuroimmunol. 2011; 235: 84-90. IL-6 presented a tendency for higher blood levels in both the initial stages of the cardiac form and advanced stages of the cardiac form than in patients with the indeterminate form, as shown by others.1414. Keating SM, Deng X, Fernandes F, Cunha-Neto E, Ribeiro AL, Adesina B, et al. Inflammatory and cardiac biomarkers are differentially expressed in clinical stages of Chagas disease. Int J Cardiol. 2015; 199: 451-9.,3434. Perez AR, Silva-Barbosa SD, Berbert LR, Revelli S, Beloscar J, Savino W, et al. Immunoneuroendocrine alterations in patients with progressive forms of chronic Chagas disease. J Neuroimmunol. 2011; 235: 84-90.,3535. Sousa GR, Gomes JA, Fares RC, Damasio MP, Chaves AT, Ferreira KS, et al. Plasma cytokine expression is associated with cardiac morbidity in Chagas disease. PLoS One. 2014; 9: e87082. Additionally, a specific IL6 genotype lowered the risk of positive parasitaemia.3636. Gomes dos Santos A, Watanabe EH, Ferreira DT, Oliveira J, Nakanishi ES, Oliveira CS, et al. A Specific IL6 polymorphic genotype modulates the risk of Trypanosoma cruzi parasitemia while IL18, IL17A, and IL1B variant profiles and HIV infection protect against cardiomyopathy in Chagas disease. Front Immunol. 2020; 11: 521409. We have also shown previously that the group of patients studied in this article had changes in BNP, cardiac troponin, TNF and TGF-β1 in patients with the cardiac form like what is described in the literature, and discussed these findings.1717. Mendes VG, Rimolo L, de Lima ACB, Ferreira RR, Oliveira LS, Nisimura LM, et al. Biomarkers and echocardiographic predictors of cardiovascular outcome in patients with chronic Chagas disease. J Am Heart Assoc. 2023; 12: e028810.

We found that the presence of diabetes mellitus was a protective factor regarding T. cruzi PCR positivity. This could be interpreted as a contradictory finding, as hyperglycaemia was shown to increase parasitaemia and mortality in an animal model of acute CD3737. Tanowitz HB, Amole B, Hewlett D, Wittner M. Trypanosoma cruzi infection in diabetic mice. Trans R Soc Trop Med Hyg. 1988; 82: 90-3. and patients with diabetes mellitus are known to have increased susceptibility to infection, which is linked to immune dysfunction.3838. Berbudi A, Rahmadika N, Tjahjadi AI, Ruslami R. Type 2 diabetes and its impact on the immune system. Curr Diabetes Rev. 2020; 16: 442-9. However, the main drug used to treat diabetes patients included in this study (metformin) was recently shown to have an in vitro parasite-killing activity against T. cruzi thought to be mediated by the blocking of the mitochondrial respiratory-chain complex I.3939. Martínez-Flórez A, Galizzi M, Izquierdo L, Bustamante JM, Rodriguez A, Rodriguez F, et al. Repurposing bioenergetic modulators against protozoan parasites responsible for tropical diseases. Int J Parasitol Drugs Drug Resist. 2020; 14: 17-27. In fact, biguanides were first developed to be used as an antimalarial drug, but their effect on lowering blood glucose levels resulted in their use as a drug to treat diabetes mellitus.4040. Sweeney D, Raymer ML, Lockwood TD. Antidiabetic and antimalarial biguanide drugs are metal-interactive antiproteolytic agents. Biochem Pharmacol. 2003; 66: 663-77. Therefore, it is possible that the chronic use of metformin, among other factors, could have contributed to a decreased risk of T. cruzi PCR positivity present by patients with diabetes mellitus.

The presence of RBBB was the only characteristic evaluated by cardiac exams found to be associated with a positive T. cruzi PCR. In fact, the frequency of a positive T. cruzi PCR or the parasite load was similar across patients with different forms of the chronic CD. We can speculate that the predilection of the T. cruzi for the heart conduction system4141. Andrade ZA, Andrade SG, Oliveria GB, Alonso DR. Histopathology of the conducting tissue of the heart in Chagas' myocarditis. Am Heart J. 1978; 95: 316-24. may be linked to the higher risk of a positive T. cruzi PCR among those with a RBBB. Previous work has described that patients with low parasitaemia had a significantly higher risk of having a reduced LV ejection fraction than patients with undetectable parasitaemia, although those with a positive T. cruzi PCR had a lower ejection fraction.1616. Echeverría LE, Rojas LZ, Rueda-Ochoa OL, Gómez-Ochoa SA, Rugeles CIG, Díaz ML, et al. Circulating Trypanosoma cruzi load and major cardiovascular outcomes in patients with chronic Chagas cardiomyopathy: a prospective cohort study. Trop Med Int Health. 2020; 25: 1534-41.

Clinical implications - Although there are many biomarkers being evaluated as cure criteria, none of the biomarkers studied here, including some with prognostic association in previous studies, were found to be associated with the presence of a positive or negative T. cruzi PCR. On the other hand, the strongest factor associated with a negative T. cruzi PCR was previous history of trypanocidal treatment with benznidazole, especially in those patients who completed treatment with a dose of 5 mg/kg/day. It is noteworthy that patients within this article were treated for an average of 11 years before the PCR evaluation, which shows a long-term efficacy of the benznidazole treatment. This reinforces the importance of the trypanocidal treatment.

Limitations - This work is limited by the retrospective nature of the data collected on previous history of trypanocidal treatment. Also, parasite load is low and has a skewed distribution, which limits the study of its association with clinical characteristics.

In conclusion - Among several clinical and biomarkers parameters, only diabetes mellitus, right bundle branch block, and history of trypanocidal treatment showed an independent association with T. cruzi PCR. History of trypanocidal treatment was a strong protective factor against a positive T. cruzi PCR even more than 10 years after treatment.

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Publication Dates

  • Publication in this collection
    15 Dec 2023
  • Date of issue
    2023

History

  • Received
    30 June 2023
  • Accepted
    10 Nov 2023
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