Chagas disease and SARS-CoV-2 coinfection does not lead to worse in-hospital outcomes

Chagas disease (CD) continues to be a major public health burden in Latina America. Information on the interplay between COVID-19 and CD is lacking. Our aim was to assess clinical characteristics and in-hospital outcomes of patients with CD and COVID-19, and to compare it to non-CD patients. Consecutive patients with confirmed COVID-19 were included from March to September 2020. Genetic matching for sex, age, hypertension, diabetes mellitus and hospital was performed in a 4:1 ratio. Of the 7018 patients who had confirmed COVID-19, 31 patients with CD and 124 matched controls were included (median age 72 (64–80) years-old, 44.5% were male). At baseline, heart failure (25.8% vs. 9.7%) and atrial fibrillation (29.0% vs. 5.6%) were more frequent in CD patients than in the controls (p < 0.05). C-reactive protein levels were lower in CD patients compared with the controls (55.5 [35.7, 85.0] vs. 94.3 [50.7, 167.5] mg/dL). In-hospital management, outcomes and complications were similar between the groups. In this large Brazilian COVID-19 Registry, CD patients had a higher prevalence of atrial fibrillation and chronic heart failure compared with non-CD controls, with no differences in-hospital outcomes. The lower C-reactive protein levels in CD patients require further investigation.

Since the first case of coronavirus disease 19  described in Brazil on February 26th, 2020, SARS-CoV 2 infection has evolved as a global pandemic. The disease has a wide spectrum of clinical manifestations, ranging from asymptomatic cases to severe pneumonia and acute respiratory distress syndrome 1,2 .
Although the great majority of symptoms are unspecified, mild, flu-like or belonging to respiratory sphere, other organs could be affected, as the cardiovascular system. COVID-19 has been associated with multiple cardiac manifestations, including cardiac arrhythmias, myocardial infarction, acute heart failure and acute fulminant myocarditis. Cardiovascular involvement has shown to be associated with increased mortality 3,4 .
Underlying comorbidities have been widely associated with a worse prognosis for COVID-19 patients, since viral infections could act as triggers for worsening of chronic diseases [5][6][7] . Chagas disease (CD) is a multisystemic disorder, potentially affecting, cardiovascular, digestive, and neurological systems. It is the most common cause of infectious cardiomyopathy worldwide, and it may play a role in the clinical prognosis of COVID-19 patients 8,9 . Although CD is endemic in Latin America, it has been recognized that the disease is now a worldwide concern, as the disease spread with population movements from endemic to non-endemic countries 10 . In Brazil, CD still remains a public health challenge, being one the countries with more absolute number of patients and an annual incidence rate of approximately 0.16 per 100,000 inhabitants/year 11 .
Potential interactions between COVID-19 and Chagas cardiomyopathy could be probable, because both conditions share the same immunological pathway. SARS-CoV-2 spike proteins bind to angiotensin-converting enzyme-2 (ACE-2), which is needed to invade the host cell. On the other hand, ACE2 is involved in heart function and the development of hypertension and diabetes mellitus (DM), risk factors frequently observed in patients with chronic Chagas cardiomyopathy 12,13 . Those patients could have increased levels of ACE2 because of the chronic use of ACE inhibitors and/or angiotensin receptor blockers (ARBs).
Limited information is available regarding the characteristics and outcomes of patients with CD and COVID-19. Therefore, we aim to describe the characteristics, laboratory, and imaging findings, as well as in-hospital outcomes of CD and COVID-19 coinfected patients included in the Brazilian COVID-19 Registry.

Methods
This manuscript adheres to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guideline 14 . All methods were performed in accordance with the relevant guidelines and regulations.
Study design and subjects. Patients were selected from the Brazilian COVID-19 Registry, a prospective multicenter cohort project with 37 participant hospitals in 17 cities from three Brazilian states (Minas Gerais, Pernambuco, Rio Grande do Sul, Santa Catarina, São Paulo). Details of the cohort were published elsewhere 5 .
COVID-19 diagnosis was confirmed through real time polymerase-chain reaction (RT-PCR) nasopharyngeal and oropharyngeal swab testing or anti-SARS-CoV-2 IgM detected in serological assay in serum or plasma sample, according to World Health Organization guidance 15 .
For the present study, patients with previous history of CD recorded in the database were selected. CD diagnosis were retrieved by their own hospital record or self-referred by the patient. Patients were admitted from March 1 to September 30, 2020. At the moment of the analysis 7018 patients were introduced in the registry, 31 of those were classified as suffering from CD. Data collection. Study data were collected by trained hospital staff or interns using Research Electronic Data Capture (REDCap) tools 16 . Medical records were reviewed to collect data on patients' demographic and clinical characteristics, including age, sex, pre-existing medical conditions and home medications; COVID-19 symptoms at hospital presentation; clinical assessment upon hospital admission, third and fifth admission days; laboratory, imaging, electrocardiographic data; inpatient medications, treatment and outcomes. Definitions were published elsewhere 5 . Statistical analysis. Genetic matching for sex, age, hypertension, DM and hospital was performed in a 4:1 ratio (MatchIt package in R). Genetic matching is a multivariate matching method that uses an evolutionary search algorithm to determine the weight each covariate is given, to maximize the balance of observed covariates across individuals of both groups 17 . Sample size of 132 controls was calculated considering and expected risk ratio for mortality 2.5 in CD-group, power of 80%, alfa-error probability of 5% for a 4:1 CD/control. Categorical data were presented as absolute numbers and proportions, and continuous variables were expressed as medians and interquartile ranges. The χ 2 and Fisher Exact test were used to compare the distribution of categorical variables, and the Wilcoxon-Mann-Whitney test for continuous variables. Results were considered statistically significant if the two-tailed p-value was < 0.05. All statistical analysis was performed with R software (version 4.0.2).
Ethics. The study was approved by the National Commission for Research Ethics (CAAE 30350820.5.1001.0008). Individual informed consent was waived by the National Commission for Research Ethics owing to the pandemic situation and the use of deidentified data, based on medical chart review only.
Transparency declaration. The lead authors (MSM, IM and MCP) affirm that the manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as originally planned (and, if relevant, registered) have been explained.
When comparing CD patients with controls (Table 1), there were no significant differences in demographic and medical characteristics, except for the prevalence of chronic heart failure (8 [25.8%] vs 12 [9.7%]; p = 0.031) and atrial fibrillation (9 [29.0%] vs 7 [5.6%]; p < 0.001), which were more prevalent in CD patients. Although the median number of comorbidities was higher in CD patients The median time since from symptom onset to hospital admission was 6 (8-4) days. Dyspnea and cough (dry or productive) were present in more than one half of patients. There were no differences in the clinical presentation between both groups (Table 2).
Laboratory and imaging findings are presented in Supplementary Table S1 and S2. Median C-reactive protein was lower in CD patients than the controls (55. 5  www.nature.com/scientificreports/ At admission, diffuse interstitial infiltrate pattern and ground glass opacities were the most prevalent findings in the chest X-ray and chest computer tomography (CT), respectively. No significant differences were found in the frequency of abnormalities and radiological progression in both groups, expect for the frequency of pleural effusion in the follow-up CT, more frequent in CD patients.
Among CD, patients 10 had an EKG performed. Of those, 4 patients had atrial fibrillation and 2 had a pacemaker rhythm, so the proportion of patients with sinus rhythm in controls were significantly higher than in CD patients (68.8% vs 40.0%, p = 0.142) ( Table 3).
During hospitalization, 72 (46.5%) of patients required admission to the intensive care unit, and among them 55 (35.4%) needed mechanical ventilation and 26 (16.8%) substitutive renal therapy. Overall, there were no differences in in terms of clinical evolution and outcomes (Table 5). www.nature.com/scientificreports/

Discussion
We described a cohort of CD patients infected with SARS-COV-2 and admitted in hospitals belonging to a large Brazilian COVID-19 Registry project. Overall, CD patients had similar clinical characteristics and outcomes to non-CD controls, matched by age, sex, hypertension, DM and hospital, except from a higher prevalence of atrial fibrillation and chronic heart failure, and lower C-reactive protein levels. Due to the potential cardiac involvement, and the higher procoagulant state, T.cruzi and SARS-COV-2 coinfection has been postulated as condition for myocardial damage, depression of ventricular function, increased arrhythmogenic state, thromboembolism risk, and ultimately a worst prognosis [18][19][20] . However, it was only a hypothesis and no previous study has tested it using patient data. Despite the limited number of patients with CD (31) our study refuted did not confirm the hypothesis. We did not find any significant difference or even a trend of worse clinical outcomes in CD patients, even with a higher frequency of atrial fibrillation and heart failure in the CD group.
Current data demonstrates that SARS-CoV-2 infection induces immune dysfunction, widespread endothelial injury, complement-associated coagulopathy and systemic microangiopathy 21 . By the other hand, T. cruzi infection is associated with an upregulated procoagulant activity in plasma. Therefore, it could be expected a greater risk of thromboembolic manifestations. In our cohort the overall thrombosis event was 4.5% (7 out of 155), all of them were in the control group. Noteworthy that, the great majority of patients (91%) were treated with oral anticoagulants because its underlying disease or received any kind of prophylactic heparin when admitted to the hospital, as recommended by national and international guidelines for the management of in-hospital COVID-19 patients 22,23 .
The lower median C-reactive level in CD patients was an unexpected finding. We hypothesize that CD patients, as they already have an active chronic inflammatory and immune response triggered by T. cruzi infection, might have a lower risk of unregulated inflammatory response to COVID-19 24 . Therefore, what could have been a factor for worse prognosis, due to a higher frequency of associated heart failure and atrial fibrillation and the CD itself, could be equilibrated by a controlled inflammatory response. This is only a hypothesis, that merits www.nature.com/scientificreports/ consideration for future studies. If proved correct, it may add to the knowledge of understating how to prevent the unregulated inflammatory response in COVID-19. It is also interesting to discuss the influence that the use of anticoagulants in full doses may have had on the outcomes of patients with CD and COVID-19. The higher prevalence of atrial fibrillation in those patients may had led to a higher frequency of use of therapeutic dosage anticoagulants (19.3% vs. 10.5%), which did not reach statistical significance due to the sample size. The best strategy to be used-prophylactic or therapeutic heparin doses-in patients with moderate to severe COVID-19 is not yet defined, and it has been hypothesized that therapeutic anticoagulation (full dose heparin) is associated with decreased in-hospital mortality in patients with moderate COVID-19, but not in patients with severe COVID-19.
It is known the effect of immunosuppressant drugs and the risk of reactivation of CD. In the case of corticosteroids, immunosuppressive doses have not been associated with higher rates of reactivation of CD, although is controversial due to the lack of supporting evidence 25,26 . Tocilizumab, a cytokine inhibitor (recombinant humanized monoclonal antibody with an antagonist effect on the IL-6 receptor), combined with another immunosuppressant agents have been suggested to be associated with the reactivation of latent infections, including parasites.
Two published case reports of Strongyloides Hyperinfection Syndrome in COVID-19 patients immunosuppressed with dexamethasone and tocilizumab, have been recently published 27,28 . To date, no cases of CD reactivation have been published, but at least, there is a concern that COVID-19 disease therapeutics could potentially Table 3. Electrocardiographic characteristics of the study population at baseline and new abnormalities at follow-up. *New electrocardiographic abnormalities through in-hospital follow-up, and number of patients in which this outcome was assessed. CD Chagas disease, ECG electrocardiogram, QTc corrected QT interval.  www.nature.com/scientificreports/ trigger reactivation of CD. This merits further investigation and until definitive evidence is published, it should be a cause of concern in decision making, when prescribing immunosuppressors in these patients. The fact that the majority of CD patients were admitted to public hospitals (81.8%) is an indicator that CD disproportionally affects people from lower income background. In a previous multivariate analysis, we demonstrated that despite being admitted to public hospitals patients do not have worse prognosis than patients admitted to private ones 5 .
This study has limitations. In addition to the retrospective design, subject to the drawbacks of a patient records review, the number of CD was low. However, it is the largest series published to date. Due to the pragmatic study design, laboratory and imaging tests were performed at the discretion of the treating physician. In that sense, Chagas disease diagnosis was based on medical records or by self-reporting, in these cases no extra serology was performed. Despite the limited representativity of radiologic, tomographic and electrocardiographic analysis, no patient performed echocardiogram during hospital admission.

Conclusions
Although coinfection by Trypanosoma cruzi and SARS-COV-2 may pose a risk of complications and therefore a worse prognosis, in our series we did not find significant differences in terms of clinical presentation and outcomes of patients with CD compared to controls, despite a higher frequency of chronic heart failure and atrial fibrillation at baseline. We observed lower C-reactive protein levels in CD when compared to controls, and this merits further investigation.

Data availability
Data are available upon reasonable request.