Analysis of cardiac involvement in patients recovered from Covid-19 without troponin elevation, evaluated by cardiovascular magnetic resonance.

Background The disease caused by coronavirus (COVID-19) affects the cardiovascular system, whether by direct viral aggression or indirectly through systemic inammation and multiple organ compromise. A widely used method to determine cardiac injury is troponin measurement. The aim of this study is to evaluate the prevalence of cardiac involvement (CINV) in a population recovered from COVID-19, referred to cardiac MRI (CMR), who did not present troponin elevation. There were 156 patients that recovered from COVID-19 and who did not present troponin elevation referred to CMR. CINV was considered to be the presence of: late gadolinium enhancement (LGE), edema, myocarditis, pericarditis, left ventricular systolic dysfunction (LVSD) and/or depressed right ventricular systolic dysfunction (RVSD). Prevalence of CINV was 28.8%, being more frequent in men (p = 0.002), in patients who required hospitalization (p = 0.04) and in those who experienced non-mild cases of infection (p = 0.007). RVSD (17.9%) and LVSD (13.4%) were the most frequent ndings. The rate of myocarditis was 0.6%. LGE manifested in 7.1% of patients and its presence was related to less left ventricular ejection fraction (LVEF) (p = 0.0001) and right ventricular ejection fraction (RVEF) (p = 0.04).


Introduction
Coronavirus disease 2019 , caused by the coronavirus of the severe acute respiratory syndrome (SARS-CoV-2), emerged in Wuhan, China, in December 2019 in patients who presented with pneumonia by unknown origin [1]. Since then and to this date, when this paper is being written, there have been 134,719,328 cases of COVID-19 developed in the world, with at least 2,915,972 deaths [2]. Although COVID-19 mainly presents with respiratory system compromise, due to the interaction between COVID-19 and the cardiovascular system [3][4], cardiac injury is frequently observed in these patients. The mechanism of cardiac injury is multifactorial, included oxygen supply-demand imbalance without coronary obstruction, myocardial stress, in ammation, microvascular dysfunction, pre-existing atherosclerotic plaque rupture or toxicity by direct viral injury [5]. Studies show a high prevalence of cardiovascular alterations in hospitalized patients [6][7]. Cardiac injury determination through high-sensitivity cardiac troponin shows a worse evolution in patients with increase in this biomarker [8][9].
However, impact on the heart in the mid-term published in different studies is controversial. In the initial reports, a high prevalence of cardiac disease was shown in cardiovascular magnetic resonance (CMR), with elevation in native T1 and T2 mapping being the most frequent nding.
These studies included both patients with and without troponin elevation. Late gadolinium enhancement (LGE) was around 31-32% [10][11]. On the other hand, it was recently observed in patients that were admitted and with elevated troponins at hospital discharge, that LGE prevalence was 33% of non-ischemic LGE, which may be possibly attributed to COVID-19 [12].
The aim of our study is to determine the prevalence of LGE abnormalities in a large sample of patients recovered from COVID-19, who did not present troponin elevation at the time of the study.

Methods
This is an observational, prospective, single-center study, performed at the Hospital JM Cullen of the city of Santa Fe, Argentina. Consecutive patients from October 2020 to April 2021 who were initially referred for an LGE test and who met the following inclusion criteria were included: 1) signed informed consent 2) con rmed to have infection by SARS-CoV-2 by reverse transcription polymerase chain reaction (RT-PCR); 3) more than four weeks after epidemiological discharge; 4) not presenting troponin elevation at the time of the study. The exclusion criteria were: 1) refusal to sign the informed consent; 2) chronic kidney disease with creatinine clearance ≤30 ml/min; 3) device or prosthesis not compatible with CMR; 4) claustrophobia. The protocol was analyzed and approved by the institutional bioethical review board. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as re ected in a priori approval by the institution's human research committee.
According to the severity of COVID-19 symptoms reported by the patient and considering the maximum level of healthcare required, the acute infection was categorized into: mild, moderate, severe and critical, according with the Ordinal Scale for Clinical Improvement (OSCI) proposed by the World Health Organization [13].
LGE protocol and post-process LGE was conducted following local institutional guidelines for infections control, with resonator (SIGNA, Creator, GE Healthcare, US). The protocol included: Standard long-axis cine imaging (four-, two-and three-chambers, left ventricular out ow tract) and short-axis base-to-tip sweep in expiratory apnea.
Dark blood imaging on T2-weighted STIR (short-tau inversion recovery) in long axis (four-, two-and three-chambers) and three short-axis views (basal, medial and apical).
Dark blood imaging on T1-weighted, pre-and post-contrast in the same views as in the T2-weighted sequences.
The analysis was made by Cardiac YX (GE Healthcare) for the evaluation of LV volumes and mass; while trabeculae and papillary muscles were excluded. The presence of LGE, edema, myocarditis, pericarditis, left ventricular systolic dysfunction (LVSD) and/or right ventricular systolic dysfunction (RVSD) was classi ed as cardiac involvement (CINV). Active myocarditis was de ned as the presence of non-ischemic LGE (subepicardial or mesocardial location) associated to edema criterion in T2-STIR sequences and/or hyperemia in early post-contrast T1 sequences. The criterion of myocardial edema was established through the myocardial edema index (EI), that was de ned as the relationship between myocardial signal intensity (SI) and skeletal muscle SI [14]. An EI >2.0 was considered abnormal. Pericarditis criterion was the presence of pericardial LGE or pericardial thickening (≥4 mm) associated to pericardial effusion. LVSD was determined by left ventricular ejection fraction (LVEF) when it was less than 57% and RVSD when right ventricular ejection fraction (RVEF) was less than 51% in women and 52% in men [14]. The location and pattern of LGE injuries in LGE images were evaluated by 2 observers, who reviewed all PSIR images independently.

Statistical analysis
Statistical analyses were made with IBM SPSS v23. Normality of quantitative variables were evaluated by the Shapiro-Wilk test. To describe quantitative variables, mean or median were used as central tendency measures and standard deviation (SD) or interquartile range (IQR) as corresponding dispersion measures. Qualitative variables were presented as absolute frequencies and their respective percentages. Independent samples t-tests were used to evaluate differences in means between two groups. In the case of not meeting normality criteria, the Mann-Whitney U test was used to compare distributions. Fisher's exact tests and Pearson's chi-squared tests were used to compare differences in rates between qualitative variables. Correlations between quantitative variables were evaluated by means of Pearson correlation coe cient. Con dence intervals of 95% (95% CI) were attached to the parameters when deemed necessary. α statistical signi cance was established as 0.05.

Results
Clinical characteristics.
The baseline characteristics of patients are shown in Table 1. There were 156 patients included, from whom 55.8% were men. Age average was 48.4 (SD 13.3). There was 58.9% of patients with a mild evolution in the acute phase of the disease; only 5.9% of patients were severe/critical. Patients that required admission were 35.2%. Median and IQR of hospital stay was 10 (5-15) days; while for the ICU it was 6 (4-20). Twenty-one percent of patients reported presenting atypical precordial pain, 18% palpitations and 515 shortness of breath when undergoing CMR. All these symptoms were not incapacitating and no case required admission. Mean and IQR of time from the end of symptoms until performing CMR was 58.5 (44-82) days. In 28.8% of patients, CINV was observed. Figure 1 shows the prevalence of the different alterations constituting CINV. LVSD and RVSD, with 13.5% and 17.9% respectively, were the most frequent alterations. Myocardial LGE nding was observed in 11 patients (7.1%) and was the most frequent variable found of myocardial tissue involvement.  The location of LGE was subepicardial in 45.4% of cases, in 36.4% it was mesocardial, and subendocardial in 18.2%. Patients who presented LGE had a mean LVEF of 54.8% (SD 8.7%) and those who did not have LGE had 62.8% (p = 0.0001), RVEF mean in patients with no LGE was 57.8% (SD 6.6) and in those with LGE, 53.5% (p = 0.04) (Fig. 2).

Discussion
In a cohort of 156 patients recovered from COVID-19, in whom CMR was performed and who did not present troponin elevation at the time of performing the study, 28.8% of CINV was found. RVSD and LVSD were the most frequently observed alterations, and in most of these, impairment was mild. In our study, the only preexisting condition that showed a signi cant difference in the presence of CINV was male gender, coinciding with data about a poor evolution in male patients in acute infection [16][17]. The patients who presented non-mild cases and patients who required hospitalization presented more prevalence of CINV, these data differ from the study by Puntmann et al, showing a higher prevalence of CINV (78%) and this nding is not related to previous comorbidities or the severity of acute clinical symptoms or hospitalization requirement due to COVID-19 [11].
The most frequent alterations were those in relation to functionality, and to a lesser extent, those that may re ect direct impact (in ammation, brosis, infection) on the heart. These alterations of functionality (with no tissue alterations) may be related to indirect CINV[18]; i.e. dependent on multiple organ dysfunction, with myocardial oxygen supply-demand imbalance caused by the infection, and that are generally observed in nonmild cases, a more frequent compromise than direct viral cardiac injury [19][20][21].
In our cohort, the prevalence of myocarditis was 0.65%, using the original Lake Louis diagnostic criteria [22]. These were updated in year 2018, with the introduction of T1 and T2 mapping [23]. Only T2 mapping is better than the original Lake Louis criteria in terms of diagnostic sensitivity [24], and when both criteria are compared, the update only improves sensitivity with no impact on speci city. A myocarditis prevalence of 0% was observed in a series of 16 autopsies and 4.5% in 201 cases, with the latter being selected cases, which may entail an ever lower prevalence of myocarditis in the general population [25].
In our study, the presence of LGE, a prognostic marker[26], had a prevalence of 7.1%, which is substantially less than that observed in a recent study, where it was 30%. This also included patients with troponin elevation and their values were more elevated in the patients that presented LGE [27]. Unlike this last study, in ours the patients presenting LGE had less LVEF and RVEF (p = < 0.001 and p = 0.04, respectively) than patients that do not present them.

Limitations
This study presents the limitation of not having CMR data before the COVID-19 infection. Data are shown on a single CMR test of up to 3 months after the infection, and an extended follow-up is essential to determine the progression or regression of the cardiac compromise and its prognostic impact. We do not have T1 and T2 mapping parameters and extracellular volume estimation, which are part of the Lake Louis diagnostic criteria review. However, we did use diagnostic criteria for myocarditis that present a similar speci city.

Conclusion
Almost a third of patients recovered from COVI-19 with no troponin elevation, cardiac alterations are found. These are more prevalent in patients of the male gender and who have required admission or that presented non-mild cases. Functional alterations (RVSD-LVSD) are most frequent. Myocarditis prevalence was very low. LGE is the most frequent tissue alteration and determinant for LVEF and RVEF.