**3. Discussion**

Herein, we described the first report of triple infection (SARS-CoV-2 infection, *T. cruzi* infection, and CMV dissemination) in HT recipients. Patients received HT as a form of treatment for Chagas cardiomyopathy.

We described the laboratory data from the first day of hospitalization in the ICU due to COVID-19 until the time of death. Both patients were admitted to a referral center for treatment for COVID-19 in the metropolitan region of São Paulo, a city in southeastern Brazil. We hypothesize that the triple infection by SARS-CoV-2 and CMV may have been an important cause of death and of the worsening in CD patients with HT. To date, there have been no similar reports of patients presenting these three concomitant infectious diseases and HT receptors.

COVID-19 infection among transplant recipients increases the potential for developing severe illness [13] and may vary between different organ transplants [14,15].

SARS-CoV-2 entry´s receptor angiotensin converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) are expressed in different tissues, such as the lungs, heart, liver, kidneys, testicles, thyroid, and adipose tissue [16]. Some patients with COVID-19 develop severe disease characterized by respiratory distress syndrome and systemic manifestations. This condition has been associated with the dysregulated release of pro-inflammatory cytokines, termed "cytokine storm," which may induce multi-organ failure [1,17].

The use of immunosuppressants and post-surgical opportunistic infections can also lead to damage of multisystem organs or even death [18]. There is a therapeutic paradox here, because while insufficient immunosuppression results in graft loss due to rejection, excessive immunosuppression can result in serious infection, including SARS-COV-2 [13], besides contributing to the reactivation of pathogens [19].

Long-term administration of immunosuppressants to solid organ transplant (SOT) receptors to reduce the risk of graft rejection may increase the risk of respiratory infections [20], although there is no clear clinical evidence of increased morbidity/mortality in SARS-CoV-2 infection [21].

Fernandez-Ruiz and collaborators [22] described a cohort of SOT receptors affected by COVID-19, 44% of whom were kidney transplant recipients, 33.3% liver transplant recipients, and 22.2% heart transplant recipients. The lethality rate was 27.8%, suggesting that SARS-CoV-2 infection had a severe course in SOT recipients.

Recently, we described the first patients with CD affected by SARS-CoV-2. The CD patients presented an increase in COVID-19 laboratory hallmarks and a rapid disease progression. Despite the efforts of the health staff, both patients died [3].

HT may be associated with the reactivation of pathogens, as reported in CD [10]. In a Brazilian cohort, the reactivation rate of Chagas disease after heart transplantation was reported to be 38.8% [23]. This fact made HT as a treatment for Chagas cardiomyopathy initially controversial. However, currently, especially in endemic countries, it is the most viable therapeutic option for patients with end-stage heart failure [24]. We hypothesize that, in the cases presented here, HT followed by reactivation of CD conferred an additional risk factor for the worsening of COVID-19.

Transplant recipients are also commonly affected by CMV reactivation, being associated with significant morbidity and mortality [12] that may worsen the infectious condition of COVID-19 [25]. In SOT, risk factors include the use of immunosuppressants for transplantation, advanced age, acute rejection, and other concomitant infections [11], with all these characteristics being present in both patients described herein. In the present report, we observed that immunosuppression may have contributed to the susceptibility to superinfections and more severe clinical manifestations in individuals undergoing HT.

Since overactivated immune responses can be one of the causes of organ damage, the anti-inflammatory effects of immunosuppression can be protective, reducing the cytokine storm related to complications in COVID-19 [26]. In this context, it has been described that immunosuppressive therapy with calcineurin inhibitors in patients with solid organ transplantation or systemic rheumatic diseases promotes a clinical course in SARS-CoV-2 infection, which is generally mild, and with an apparently low risk of superinfection [27]. In addition, immunosuppression has not been evaluated as a risk factor for SARS or MERS [28].

However, in the present case report, we observed that immunosuppression may have contributed to the susceptibility to SARS-CoV-2 infection, the reactivation of pathogens, and more serious clinical manifestations in individuals undergoing HT. Corroborating our findings, it was shown that patients with COVID-19 and cancer, due to their systemic immunosuppressive condition caused by malignancy and anticancer treatments, such as chemotherapy, had an increased risk of SARS-CoV-2 infection and a worse prognosis [29]. It has also been reported that chronic use of corticosteroids prior to SARS-CoV-2 contaminationisassociatedwithcriticaldiseaseoutcomes,includingahighriskofdeath[30].

These contradictory observations show that knowledge about the relationship between COVID-19 and the patient's immune condition is limited. Further studies are needed to elucidate the immune responses and prognosis of COVID-19.

In general, HT in patient 1 proved to be successful for the treatment of Chagas cardiomyopathy, with no reactivation of the pathogen. However, after SARS-CoV-2 infection, she presented graft rejection, treated with methylprednisolone, thymoglobulin, and plasmapheresis. There is a description in eye transplantation that COVID-19 infection can compromise the balance of immunoregulatory responses that allow graft survival, contributing to rejection in individuals infected with SARS-CoV-2 [31]. Thus, this change in the balance of attenuation of the immune response, such as a reduction in the number of regulatory T cells (Tregs) [32], may favor direct cardiotoxic action and multiple organ dysfunction.

During postoperative hospitalization, patient 2 presented Chagas reactivation, evidenced by skin biopsy, indicating that the CD was not completely controlled. In addition, it is important to consider that immunosuppressive therapy can contribute to the reacti-

vation of CD [33]. After HT, the patient presented graft rejection and was treated with plasmapheresis and methylprednisolone. With this, before the SARS-CoV-2 infection, the patient was already weakened and died 21 days after the infection, in relation to patient 1 who died after 47 days of infection.
