Purpose:

To find the relation between changes in resting electrocardiography (ECG) ventricular
repolarization parameters and the consequences of COVID-19. To assess these parameters
connection with arrhythmias and overall cardiovascular events and transthoracic impedance
cardiography (ICG) parameters, to evaluate the persistence of changes over a 2-year
period after the illness, and to identify prognostic ventricular repolarization and ECG
parameters in COVID-19 patients.

Goals of the study:

1. To identify the most common differences in ECG ventricular repolarization parameters
among COVID-19 patients who experienced arrhythmias and/or other cardiovascular
events within 2 years of hospital discharge, compared to those who did not have
arrhythmias and/or other cardiovascular events.

2. To assess the persistence of changes in ECG ventricular repolarization parameters 2
years after hospital discharge.

3. To identify and determine the differences in ICG parameters among individuals who
have recovered from COVID-19 2 years after their hospital discharge.

4. To find the most appropriate prognostic ECG criteria for monitoring recommendations
for patients who have recovered from COVID-19.

Duration of the study: the study is estimated to take 3 years (from 2024 11 to 2027 09).
Each enrolled participant participates in study for 2 days (a little more than 24 hours
total): one hour is allocated for the examination (taking medical history, objective
examination, ECG, echocardiography, placing the Holter monitoring device, and performing
the ECG) and the following day for about 5 minutes when participant returns for the
removal of the Holter monitoring device.

Methods. Study design: this is a prospective, nested case-control study.

Patients who had COVID-19 since January 1, 2022, and were treated in the LUHS Kaunas
Hospital 2 years ago (beginning January 1, 2022), are invited to participate in the study
if they consent to be enrolled, meet the selection criteria, and do not have any
exclusion criteria. The enrollment will last until the required sample size is collected.

The following data is going to be collected: age, anthropometric data (height, weight),
detailed history of COVID-19 (including previous illnesses, vaccination status), history
of comorbidities (chronic cardiovascular diseases, particularly detailed history of
arrhythmias and overall cardiovascular events over the last two years, including
conduction disorders, also lung diseases, diabetes, kidney and urinary system diseases,
hematological diseases, gastrointestinal diseases, connective tissue, joint, systemic
diseases, infectious diseases), history of harmful habits, and history of medications
used (including those taken during hospitalization).

Anamnestic and examination data will be assessed based on participants records, including
ECG performed during hospitalization, imaging studies (chest X-ray), and laboratory
tests. Information about medications prescribed during treatment will also be collected.
Cardiovascular events, episodes of arrhythmias, and conduction disturbances over the last
2 years after COVID-19 will also be evaluated retrospectively.

An objective examination focusing on the cardiovascular system will be performed (blood
pressure, heart rate, heart auscultation findings).

Participants will undergo a resting 12-lead ECG. The following parameters will be
assessed- left/right ventricular hypertrophy, arrhythmias (sinus, supraventricular, and
ventricular types), conduction abnormalities, ischemia/infarction signs, QRS duration.
Parameters of ventricular repolarization such as QT, QTc, Tpe, Tpec, Tpe/QT, Tpec/QTc,
and the QRS-T angle will be evaluated.

Transthoracic echocardiography will be performed to assess signs of ventricular
hypertrophy, systolic and diastolic left ventricular function, right ventricular
function, atrial abnormalities, signs of pulmonary hypertension and heart valve
pathology.

A transthoracic impedance cardiography (ICG) will be performed focusing on the thoracic
fluid capacity, thoracic fluid volume index, systemic vascular resistance, and systemic
vascular resistance index.

24-hour ECG Holter monitoring will be performed to evaluate arrhythmias (sinus,
supraventricular, ventricular), conduction disturbances, and heart rate variability
changes.

Data will be collected on an Excel spreadsheet system. A dedicated hard-copy paper copy
of the spreadsheet will be produced and kept as a backup.

Results will be analyzed using the SPSS. A p-value < 0.05 will be considered
statistically significant. Descriptive statistical methods will be used to describe key
features of groups. Parametric or non-parametric tests (depending on the distribution of
the obtained data) will be used for comparing the means and probabilities of two
independent populations (Student's t-test, Z-test, or Mann-Whitney U test). When
evaluating different diagnostic tests between each other correlation coefficients will be
used. Finally, when calculating differences between cardiovascular outcomes/complications
- odds ratio assessment (logistic regression, conditional logistic regression) will be
done.

Expected outcomes: this study will help evaluate possible long term cardiovascular
complications after COVID-19 depending on repolarization changes during the disease and
its persistence. These findings could help in the development of long-term monitoring
recommendations for patients who have recovered from COVID-19 and forecasting long-term
outcomes.

Adverse reactions: The is no expectation of any adverse outcomes or reactions.

Reasons for withdrawal or termination: A subject may be discontinued from the study at
any time if the subject feels that it is not in the subject's best interest to continue.

The following is a list of reasons for study discontinuation:

1. Subject withdrawal of consent.

2. Due to temporary or complete withdrawal of researchers or specialists conducting the
analysis from the execution of the biomedical study.

3. After collecting the required number of subjects needed to obtain statistically
reliable data.

4. If no valuable information is obtained from the primary data.

5. If potential harm to subjects is observed.

All subjects are free to withdraw from participation at any time, for any reason,
specified or unspecified, and without prejudice. Reasonable attempts will be made by the
Investigators to provide a reason for subject withdrawals.

SAMPLE SIZE JUSTIFICATION Null hypothesis: the frequency of arrhythmias and overall
cardiovascular events over two years after the disease in patients who had COVID-19 and
changes in ECG ventricular repolarization does not differ from frequency of these events
in patients who had COVID-19without changes in ECG ventricular repolarization parameters.

Type I error rate: α=0.05. The required minimum number of subjects to obtain
statistically reliable conclusions was calculated using formulas with the 'sample size
calculator' (https://sample-size.net/sample-size-means/). The formulas used for comparing
means (using the T statistical criterion) and for comparing means of repeated
measurements (paired T-test) were applied. A confidence level of 0.95 and a power of 0.8
were chosen for the calculations.

Since participants are planned to be evaluated based on several criteria, the
calculations were performed separately for each criterion, and the largest sample size
was selected (based on the mean differences in repolarization disorders between those who
had and did not have severe COVID-19; the prevalence of repolarization disorders and
arrhythmias during the post-recovery period from severe COVID-19. Since this prevalence
in the literature ranges from 10% to 27% for this study calculations the investigators
chose 15%) Based on the aforementioned criteria, the required sample size of 195
individuals (30 subjects with repolarization disorders and 165 subjects without). was
calculated. The number was increased by 10%.

The final number is 215 patients.