Tropical Medicine andInfectious Disease

ArticleCardiovascular Manifestation of the BNT162b2 mRNA
COVID-19 Vaccine in Adolescents
Suyanee Mansanguan 1 , Prakaykaew Charunwatthana 2 , Watcharapong Piyaphanee 2 , Wilanee Dechkhajorn 3 ,Akkapon Poolcharoen 4 and Chayasin Mansanguan 2, *
1
2

3
4

*

Citation: Mansanguan, S.;
Charunwatthana, P.; Piyaphanee, W.;
Dechkhajorn, W.; Poolcharoen, A.;
Mansanguan, C. Cardiovascular
Manifestation of the BNT162b2
mRNA COVID-19 Vaccine in
Adolescents. Trop. Med. Infect. Dis.
2022, 7, 196. https://doi.org/
10.3390/tropicalmed7080196
Academic Editor: John Frean
Received: 21 July 2022

Bhumibol Adulyadej Hospital, Bangkok 10220, Thailand
Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University,
Bangkok 10400, Thailand
Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand
Samitivej Srinakarin Hospital, Bangkok 10250, Thailand
Correspondence: chayasin.man@mahidol.ac.th

Abstract: This study focuses on cardiovascular manifestation, particularly myocarditis and pericarditis events, after BNT162b2 mRNA COVID-19 vaccine injection in Thai adolescents. This prospective
cohort study enrolled students aged 13–18 years from two schools, who received the second doseof the BNT162b2 mRNA COVID-19 vaccine. Data including demographics, symptoms, vital signs,
ECG, echocardiography, and cardiac enzymes were collected at baseline, Day 3, Day 7, and Day
14 (optional) using case record forms. We enrolled 314 participants; of these, 13 participants werelost to follow-up, leaving 301 participants for analysis. The most common cardiovascular signs andsymptoms were tachycardia (7.64%), shortness of breath (6.64%), palpitation (4.32%), chest pain
(4.32%), and hypertension (3.99%). One participant could have more than one sign and/or symptom.
Seven participants (2.33%) exhibited at least one elevated cardiac biomarker or positive lab assessments. Cardiovascular manifestations were found in 29.24% of patients, ranging from tachycardia or
palpitation to myopericarditis. Myopericarditis was confirmed in one patient after vaccination. Twopatients had suspected pericarditis and four patients had suspected subclinical myocarditis. In conclusion, Cardiovascular manifestation in adolescents after BNT162b2 mRNA COVID-19 vaccination
included tachycardia, palpitation, and myopericarditis. The clinical presentation of myopericarditis
after vaccination was usually mild and temporary, with all cases fully recovering within 14 days.
Hence, adolescents receiving mRNA vaccines should be monitored for cardiovascular side effects.
Clinical Trial Registration: NCT05288231.
Keywords: BNT162b2 mRNA COVID-19 vaccine; COVID-19 vaccine; cardiovascular manifestation;
myocarditis; adolescents; Thailand

Accepted: 17 August 2022
Published: 19 August 2022
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1. Introduction
In December 2020, the US Food and Drug Administration (FDA) issued an Emergency
Use Authorization (EUA) for the Pfizer–BioNTech mRNA vaccine (BNT162b2) for theprevention of COVID-19 disease. Clinical trials have revealed that the vaccine’s efficacy is
95% and its safety profile is good, similar to that of other vaccines [1–4]. Systemic reactions
to the vaccine, which were usually mild and transient, have been reported more commonly
among the younger population and more often after the second dose [1,2,5].
Historically, post-vaccination myocarditis has been reported as a rare adverse event
after vaccinations, especially smallpox [4], influenza, and hepatitis B vaccination, among
others. In the general population, myocarditis is diagnosed in approximately 10–20 individuals per 100,000 per year [6], and occurs more commonly and at younger ages in males
than females [7]. The highest reported incidence of myocarditis from vaccination occurred
after a second dose of mRNA COVID-19 vaccine, and mostly among young men [8–10].

4.0/).

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Most of these cases developed symptoms within the first week, typically 2–4 days postvaccination. The prognosis for myocarditis patients varies according to etiology [11]. In the
pre-COVID-19 era (1990–2018), among 620,195 reports filed in the Vaccine Adverse Event
Reporting System (VAERS), 0.1% were attributable to myopericarditis; of these myopericarditis reports, 79% were in males [12]. However, the VAERS is primarily a safety signal
detection and hypothesis-generating system, and cannot be used to determine whether thevaccine caused the adverse events [13]. Cardiovascular findings from mRNA COVID-19
vaccine included myocardial injury (myocarditis), coronary heart disease, heart failure,
hypertension, and rhythm disorder [13]. The cardiovascular manifestations of COVID-19
infection include elevated cardiac biomarkers, myocarditis, cardiac arrhythmia, and venous
thromboembolism [14].
Recently, a Centers for Disease Control and Prevention (CDC) advisory committee on
immunization practices identified a likely association between the two COVID-19 mRNA
vaccines from Pfizer–BioNTech and Moderna, and cases of myocarditis and pericarditis [15–17]. For the cardiovascular system, 4863 adverse events were reported among
patients who received the BNT162b2 mRNA COVID-19 vaccine. Common findings observed with vaccines under study were tachycardia (16.41%), flushing (12.17%), increased
heart rate (9.03%), hypertension (5.82%), and hypotension (3.6%) [13]. Although cardiovascular events have been reported with the COVID-19 vaccine, causality has yet to beestablished, because such cardiovascular adverse events are also common among the general public who do not receive the intervention [13]. Conducted during the implementationof the national Thai vaccination campaigns for adolescents, this study sought to characterize, classify, and evaluate the dynamics of cardiac function and electrocardiographic(ECG) abnormalities post-BNT162b2 mRNA COVID-19 vaccination to understand andidentify cardiovascular effects that may predict cardiac complication by serial echocardiographic studies, ECG, and cardiac biomarkers for the early detection of subclinicalmyocarditis cases.
2. Materials and Methods
2.1. Study Design
This prospective cohort study focused on adolescent students from Kong Thabbok
Upatham Changkol Kho So Tho Bo School and Wachirathamsatit School who received
a second dose of the BNT162b2 mRNA COVID-19 vaccine. The study included subjects
who were: (1) aged 13–18 years; (2) male or female; and (3) had received the first doseof the BNT162b2 mRNA COVID-19 vaccine without serious adverse event. Patients who
had a history of cardiomyopathy, tuberculous pericarditis or constrictive pericarditis, andsevere allergic reaction to the COVID-19 vaccine were excluded from the study. Laboratory tests included cardiac biomarkers (troponin-T, creatine kinase-myocardial band
(CK-MB)), ECG, and echocardiography at three clinical visits (baseline, Day 3, Day 7, andDay 14 (optional for subjects with cardiac manifestation)) after receiving the second doseof the BNT162b2 mRNA COVID-19 vaccine. Participant data, including demographic
data, clinical presentation, and laboratory findings, were recorded in a pre-defined caserecord form.
Potential subjects at Kong Thabbok Upatham Changkol Kho So Tho Bo School andWachirathamsatit School were informed about the study by invitation letter, followed byan online meeting for parents and students to consider enrollment in the study. Informed
consent documents were provided for interested parents to bring to the team investigatorat enrollment. Enrollment was conducted from 3 November to 7 December 2021.
2.2. Diary Card
All participants received a diary card to record cardiac symptoms, such as chest painor palpitations. Participants who developed cardiovascular effects or side effects fromthe vaccine could telephone the principal investigator and be transferred by phone tothe medical team at the Hospital for Tropical Diseases for assessment. If the participantTrop. Med. Infect. Dis. 2022, 7, 196

3 of 13developed abnormal ECG, echocardiographic findings, or increased cardiac enzymes,
the principal investigator scheduled patients for follow-up per protocol and for Day 14
lab assessments.
2.3. Definition of Cardiovascular Manifestation
In this study, cardiovascular manifestation was defined as one or more of the following:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.

Chest pain/pericarditis
Dyspnea/orthopnea
Palpitation
Hypertension/hypotension
Tachycardia/bradycardia
Shock/cardiogenic shock
Abnormal ECG or abnormal rhythm or ECG change
Bundle branch block
Decreased ejection fraction
Diastolic dysfunction
Elevation in at least one cardiac biomarker (troponin-T, CK-MB)/myocarditis

2.4. Definition of Myocarditis [18]The diagnostic criteria for myocarditis were classified as either probable cases or
confirmed cases. Myocarditis patients were those with the presence or worsening of morethan one of the following clinical symptoms along with evidence of inflammation: (1) chest
pain, pressure, or discomfort; (2) dyspnea, shortness of breath, or pain with breathing;(3) palpitation; or (4) syncope and more than one new finding of: (a) troponin level
above upper normal limit of normal; (b) abnormal ECG or rhythm monitoring consistent
with myocarditis; (c) abnormal cardiac function or wall motion on echocardiography;(d) cardiac magnetic resonance imaging (cMRI) findings consistent with myocarditis andno identifiable cause for symptoms and findings.
2.5. Definition of Pericarditis [18]The diagnostic criteria for pericarditis included the new presence or worsening of
more than two of the following clinical features: (1) acute chest pain; (2) pericardial rub on
exam; (3) new ST-segment elevation or PR-segment depression on ECG; and (4) pericardial
effusion on echocardiography or cMRI.
2.6. Cardiac Enzymes
High-sensitivity cardiac troponin-T assay (HS-cTnT) and CK-MB isoenzyme levels
were determined for all participants at baseline, and on Day 3, Day 7, and Day 14 (optional)
after the second vaccination dose. HS-cTnT was measured using the Elecsys troponin-T hs
assay (Roche Diagnostics, Mannheim, Germany); serum levels > 14 ng/L were consideredelevated. CK-MB was also measured by electrochemiluminescence immunoassay with Elecsys CK-MB (Roche Diagnostics, Mannheim, Germany); serum CK-MB levels > 6.22 ng/mL
in males and >4.88 ng/mL in females were considered elevated. CRP levels > 5 mg/L wereconsidered elevated. ESR > 20 mm/h was considered elevated. All tests during the study
were performed by hospital technicians.
2.7. Echocardiography Protocol
Echocardiograms during the examination were recorded using the Vivid E9 ultrasound platform (GE Healthcare, Chicago, IL, USA). All echocardiographic images wererecorded and reviewed by two cardiologists. Routine two-dimensional echocardiograms
and color-flow Doppler images were obtained in the standard parasternal long axis view,
subcostal view, and apical four-chamber views. The left ventricular walls and dimensions
were measured in accordance with the guidelines of the American Society of Cardiology.

Trop. Med. Infect. Dis. 2022, 7, 196

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Transmitral pulsed-wave Doppler velocities (peak E- and A-wave velocities) were measured
in the apical four-chamber view with the sample volume positioned at the mitral valve.
Pericardial effusion and other anatomical and functional findings were recorded whenpresent. Echocardiographic studies were performed at baseline, and on Day 3, Day 7, andDay 14 (optional) after the second BNT162b2 mRNA COVID-19 vaccine dose.
2.8. Sample Size Calculation
To calculate estimated sample size, we used the estimated prevalence described in aprevious study, where the incidence of cardiac manifestation among patients who received
COVID-19 vaccine was found to be 4.6% [19]. Based on a population size of 1000 students
after the first BNT162b2 mRNA COVID-19 vaccine dose, a minimum sample size of 297
was calculated to be sufficient for this study to determine cardiovascular effects, with anerror of 2% at a 95% confidence interval. We expected that 5% of patients could be lost tofollow-up or drop out before study end.
2.9. Statistical Analysis
All data were analyzed using SPSS version 18 (IBM Corp., Armonk, NY, USA). Categorical variables were summarized and expressed as frequencies and percentages. Quantitative variables were presented as mean ± SD. The chi-square test or Fisher’s exact test
was used to assess differences between groups, as appropriate. For all analyses, p < 0.05
was considered statistically significant.
3. Results
A total of 314 participants were enrolled into the study; 13 of these were excluded
from analysis, being lost to follow-up. The remaining 301 participants in the study madeup the analysis set, as shown in Figure 1.
3.1. Characteristics of the Study Population
The mean age of the participants was 15 years (standard deviation (SD) 1.6 years;
range 13–18 years). Of the 301 participants, 202 (67.1%) were male. All participants were
healthy and without any abnormal symptoms before receiving the second dose of vaccine.
The majority of the participants (257/301, 85.38%) had no underlying diseases. There were
44 participants with underlying medical conditions, including allergic rhinitis, asthma,thalassemia trait, and G6PD deficiency. There was no significant difference in clinicalcharacteristics between the 13–15-year-old group and the older adolescents in this study
cohort. The clinical characteristics of all 301 participants are shown in Table 1.
During the follow-up period, after receiving the second dose of vaccine, two patientswere hospitalized and one patient was supervised in the ICU during hospitalization,
mainly for observation of arrhythmia. The mean length of stay in the hospital was 4.5 days
(range 2–7). None of the participants died, required mechanical ventilation, or required
inotropic support.
3.2. Cardiovascular Findings
Cardiovascular adverse events observed during the study were tachycardia (7.64%),
shortness of breath (6.64%), palpitation (4.32%), chest pain (4.32%), and hypertension(3.99%). Fifty-four patients had abnormal electrocardiograms (predominantly sinus tachycardia or sinus arrhythmia) after vaccination. All 54 of these patients had normal left
ventricular ejection fraction. Three patients had minimal pericardial effusion. The cMRI
revealed findings compatible with subacute myopericarditis (no evidence of myocardial
edema with evidence of nonischemic delayed enhancement at lateral wall and pericardial
enhancement at inferolateral wall) in one patient who was diagnosed with myopericarditis, as shown in Figure 2A–C. In addition, follow-up cMRI 5 months later showed noevidence of myocardial edema, myocardial delayed enhancement, or myocardial fibrosis.
There is evidence of resolved myocarditis, as shown in Figure 2D–F. Incidental findingsTrop. Med. Infect. Dis. 2022, 7, 196

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on echocardiography included a bicuspid aortic valve and a dilated coronary sinus frompersistent left superior vena cava. Six patients had mitral valve prolapse, and six patients
had hypertension (HTN). Three patients diagnosed with myopericarditis and pericarditis
were treated with nonsteroidal anti-inflammatory drugs (NSAIDs) for 2 weeks with noresidual symptoms and complete follow-up. The patients presenting with myopericarditis,
subclinical myocarditis, and pericarditis are shown in Table 2.

Figure 1. Study flow chart. CK-MB, creatine kinase-myocardial band; ECG, electrocardiography;
Trop-T, troponin-T.

Figure1. Study
flow chart.ofCK-MB,
kinase-myocardial
ECG,vaccination.
electrocardiography;
Table
1. Clinicalcharacteristics
the 301 creatine
adolescentsafter the second band;
COVID-19
Trop-T, troponin-T.
Characteristic
Age, yBMI (kg/m2 )Male sex, n (%)
Underlying disease n (%)

Overall (n = 301)

13–15 y (n = 207)3.1. Characteristics of the Study Population
15 ± 1.6

14 ± 0.8

16–18 y (n = 94)

p-Value

17 ± 0.7-The
age of the participants
was 15 years22(standard
deviation (SD)21 ± mean
5.0
20 ± 4.8± 5.2
0.017 1.6 years;
202(67.1)years). Of the 301
110participants,(53.1)
92 (97.9)<0.0001 *
range
13–18
202 (67.1%)
were male. All participantswere
44 (14.6)
31 (15.0)(13.8) the second dose
0.795of vaccine.
healthy
and without any abnormal
symptoms before 13
receiving
The majority of the participants (257/301, 85.38%) had no underlying diseases. There were
44 participants with underlying medical conditions, including allergic rhinitis, asthma,thalassemia trait, and G6PD deficiency. There was no significant difference in clinicalTrop. Med. Infect. Dis. 2022, 7, 196

6 of 13

Table 1. Cont.
Characteristic

Overall (n = 301)

Allergic rhinitis
24 (8.0)
Asthma7 (2.3)
Thalassemia trait
5 (1.7)
G6PD deficiency
4 (1.3)
Attention deficit
1 (0.3)
Epilepsy
1 (0.3)Migraine
1 (0.3)Thyrotoxicosis
1 (0.3)Symptoms, n (%)
Fever
50 (16.6)
Palpitation
12 (4.0)
Chest pain
8 (2.7)
Shortness of breath
19 (6.3)
Headache35 (11.6)
Laboratory findings
Troponin-T, ng/L
5.6 ± 2.5
CK-MB ng/mL
1.4 ± 0.9
Treatment
andhospitalcourseTrop. Med. Infect. Dis. 2022, 7, x FOR PEER REVIEWNSAIDS, n (%)
3 (1.0)
Hospitalization, n (%)
2 (0.7)
ICU admission, n (%)
1 (0.3)

13–15 y (n = 207)

16–18 y (n = 94)

p-Value

17 (8.2)5 (2.4)3 (1.4)3 (1.4)1 (0.5)1 (0.5)1 (0.5)0

7 (7.4)2 (2.1)2 (2.1)1 (1.1)000
1 (1.1)

0.813
0.869
0.688
0.782
0.500
0.500
0.500
0.500

30 (14.5)
10 (4.8)5 (2.4)16 (7.7)
27 (13.0)

20 (21.3)
2 (2.1)3 (3.2)3 (3.2)8 (8.5)

0.093
0.268
0.699
0.134
0.257

5.4 ± 2.5
1.4 ± 0.9

5.9 ± 2.5
1.5 ± 0.9

0.112
0.473

1 (0.5)00

2 (2.1)2 (2.1)1 (1.1)

0.178
0.035
0.138

7 of 13residual
symptoms
complete
The index;
patients
presenting
with myopericardi* Statisticallysignificantand(chi-squaretest).follow-up.
BMI, body mass
CK-MB,
creatine kinase-myocardial
band;
NSAIDS,
nonsteroidal
anti-inflammatory
drugs;
Trop-T,
Troponin-T.
tis, subclinical myocarditis, and pericarditis are shown in Table 2.

Figurea patientwithsubacute
myopericarditis
at thetimetimeof diFigure 2.
2. (A–F)(A–F)cMRI
cMRIillustrating
illustratingLGE
LGEinina patientwithsubacute
myopericarditis
at theofagnosis (A–C) and 5 months post-diagnosis (D–F). cMRI, cardiac magnetic resonance imaging; LGE,
diagnosis (A–C) and 5 months post-diagnosis (D–F). cMRI, cardiac magnetic resonance imaging;late gadolinium enhancement.
LGE, late gadolinium enhancement.
Table 2. Presentation with myopericarditis, subclinical myocarditis, and pericarditis after second
dose vaccination.

Variable
Presenting symptoms and signs—Number/total number (%)
Chest pain
Chest discomfort

Value
3/7 (42.86)
3/7 (42.86)

Trop. Med. Infect. Dis. 2022, 7, 196

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Table 2. Presentation with myopericarditis, subclinical myocarditis, and pericarditis after second
dose vaccination.
Variable

Value

Presenting symptoms and signs—Number/total number (%)
Chest pain
Chest discomfort
Pericardial effusion
Fever
Headache
Palpitation
Dyspnea

3/7 (42.86)
3/7 (42.86)
3/7 (42.86)
4/7 (57.14)
2/7 (28.57)
1/7 (14.29)
1/7 (14.29)

Vital signs on day of symptoms (Mean ± SD)
Temperature—◦ C
Blood pressure—mmHg
Systolic
DiastolicHeart rate—beats/min

114.9 ± 10.9
70.7 ± 7.8
92.71 ± 21.3

Shock-Number/total number. (%)

0/7 (0)Electrocardiographic findings—Number/total number (%)
Normal sinus rhythm
Sinus rhythm with sinus arrhythmia
Diffuse ST elevation with PR depression
Sinus arrhythmia with PAC
Sinus tachycardia
Junctional escape rhythm

1/7 (14.29)
2/7 (28.57)
1/7 (14.29)
1/7 (14.29)
1/7 (14.29)
1/7 (14.29)

Laboratory values
Elevated troponin-T

5/7 (71.43)

Clinical course
Arrhythmia
ICU admission
Need for inotrope or vasopressor
Death

4/7 (57.14)
1/7 (14.29)
0/7 (0)0/7 (0)Treatment and hospital course
Ibuprofen (NSAIDs)

3/7 (42.86)

36.4 ± 0.4

Data are reported as percentage (%) and means ± standard deviations; ◦ C, degree Celsius; NSAIDs, nonsteroidal
anti-inflammatory drugs; PAC, premature atrial contraction.

3.3. Evaluation of Patients with Elevated Biomarkers or Positive Lab Assessments
Seven patients had elevated biomarkers or positive lab assessments. The most commonly presented symptom was chest pain, followed by chest discomfort, fever, andheadache. Three patients aged 13–18 years, who presented with chest pain and biomarker
elevation, were evaluated; all three presented 24–48 h after receiving the second dose of thevaccine. Four patients had no symptoms but elevated cTnT (peak level 15.44–38.68 ng/L;
normal level < 14 ng/L). The characteristics of the patients with elevated biomarkers or
positive lab assessments are shown in Table 3. All patients were male and had abnormal electrocardiograms, particularly sinus tachycardia. The clinical course was mild in
all patients.
3.4. Evaluation of Patients Developing Abnormal ECG Post-Vaccination
After vaccination, ECG revealed that of the 301 patients, 247 (82.06%) had normal
sinus rhythm, while an abnormal ECG finding was noted in 54 patients (17.94%) (Table 4).
The most common abnormal ECG finding was sinus rhythm with sinus arrhythmia (7.31%),
followed by sinus tachycardia (6.64%) and sinus bradycardia (1.33%). Of the two patientswith abnormal rhythm, one had junctional escape rhythm, and one had ectopic atrial

Trop. Med. Infect. Dis. 2022, 7, 196

8 of 13rhythm. Arrhythmia was observed as premature ventricular contractions in two patients(0.66%), and three (1%) had premature atrial contraction. One case (0.33%) had diffused ST
elevation with PR depression.
Table 3. Characteristic of patients with elevated biomarker levels or positive lab assessments.
DemographicNo.

Age (y)Sex

Clinical Presentation
Classification

Peak
CRP
(mg/L)Peak
ESR(mm/hr)1

2

Echocardiography

CK-MB Level (ng/mL)

Troponin-T (pg/mL)

3

4

1

2

3

LVEF%
4

1

2

3

4

Pericardial
Effusion

1

16

Male

Myopericarditis

86.6

19

1.25

109.6

2.36

1.67

3.18

593

37.2

10.9

75.3

73.7

77.2

84.7

Yes

2

15

Male

Pericarditis

1.3

7

1.11

1.34

1.52

1.46

2.58

3.77

6.04

3.93

61.5

60.2

74.1

70.7

Yes

3

17

Male

Pericarditis

10.5

8

1.99

1.87

1.72

2.71

4.54

8.03

7.87

6.75

78.9

77.5

61.0

67.2

Yes

4

13

Male

Subclinicalmyocarditis

0.3

-

1.39

1.72

2.28

-

8.56

10.3

34.94

-

58.6

59.2

75.4

-

No

0.5

-

3.00

2.06

3.06

-

3.73

28.6

38.68

-

79.6

60.1

76.2

-

No

5

14

Male

Subclinicalmyocarditis

6

13

Male

Subclinicalmyocarditis

0.9

-

3.90

3.67

5.10

-

5.35

14.87

16.81

-

64.3

76.2

78.9

-

No

7

17

Male

Subclinicalmyocarditis

4.3

-

2.25

2.32

2.41

-

3.12

13.06

15.44

-

70.8

52.4

53.8

-

No

CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; CK-MB, creatine kinase-myocardial band; LVEF, left
ventricular ejection fraction.

Table 4. Electrocardiographic findings after second vaccine dose.
RhythmNumber (n = 301)

Normal sinus rhythm

247 (82.06%)

Sinus rhythm with sinus arrhythmia

22 (7.31%)

Sinus tachycardia

20 (6.64%)

Sinus bradycardia

4 (1.33%)

Premature atrial contraction (PAC)

3 (1%)

Premature ventricular contraction (PVC)

2 (0.66%)

Junctional escape rhythm

1 (0.33%)

Ectopic atrial rhythm

1 (0.33%)

Diffuse ST elevation with PR depression

1 (0.33%)

Data are reported as percentage (%). PAC, premature atrial contraction; PVC, premature ventricular contraction.

3.5. Evaluation of Patients with Serial Echocardiographic Findings
All participants underwent a follow-up echocardiography examination at baseline,
Day 3, Day 7, and Day 14 (optional). There were no significant differences between theechocardiographic findings by study day, as shown in Table 5.
Table 5. Comparison between cardiac function on day of baseline (D0), Day 3 after the second vaccine
dose (D3), and Day 7 after the second vaccine dose (D7).
Cardiac Function

Day0

Day3

Day7

p-Value
D0 vs. D3

p-Value
D0 vs. D7

p-Value
D3 vs. D7

IVSD,mean ± SD

1.18 ± 4.83

0.99 ± 1.51

0.93 ± 1.32

0.508

0.383

0.596

LVIDd,
mean ± SD

4.43 ± 3.22

4.34 ± 3.02

4.81 ± 5.02

0.735

0.209

0.168

LVPWd,
mean ± SD

0.96 ± 0.67

0.91 ± 0.81

1.28 ± 5.16

0.431

0.299

0.234

LVIDs,
mean ± SD

2.64 ± 1.65

2.85 ± 2.82

2.60 ± 0.5

0.250

0.730

0.128

Trop. Med. Infect. Dis. 2022, 7, 196

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Table 5. Cont.
Cardiac Function

Day0

Day3

Day7

p-Value
D0 vs. D3

p-Value
D0 vs. D7

p-Value
D3 vs. D7

LVEF,
mean ± SD

68.68 ± 9.27

68.21 ± 9.18

68.30 ± 8.56

0.490

0.585

0.878

MV flow E-wave
velocity mean ± SD

99.32 ± 18.77

98.98 ± 20.47

99.93 ± 21.05

0.791

0.773

0.391

MV flow A-wave
velocity mean ± SD

53.37 ± 16.10

52.39 ± 15.39

51.23 ± 16.18

0.432

0.046

0.217

MV annulus E-wave
velocity, mean ± SD

2.11 ± 1.28

2.03 ± 0.63

2.06 ± 1.18

0.281

0.632

0.653

e’,
mean ± SD

0.12 ± 0.05

0.13 ± 0.10

0.18 ± 0.55

0.227

0.097

0.065

E/e’,
mean ± SD

8.33 ± 2.63

8.22 ± 2.03

9.29 ± 8.27

0.551

0.277

0.125

a’,
mean ± SD

0.08 ± 0.06

0.08 ± 0.04

0.16 ± 0.79

0.393

0.201

0.091

Data are presented as means ± standard deviations. p-values correspond to paired t-tests. a’, late (atrial) diastolicmitral annular velocity; e’, early diastolic mitral annular velocity; E/e’, ratio of peak early mitral inflow velocity
to early diastolic mitral annular velocity; IVSD, interventricular septal end diastole; LVEF, left ventricular ejection
fraction; LVIDS, left ventricular internal diameter end systole; LVIDd, left ventricular internal diameter end
diastole; LVPWd, left ventricular posterior wall end diastole; MV, mitral valve.

4. Discussion
This prospective cohort study focuses on cardiovascular manifestations after BNT162b2
mRNA vaccination. Immunization against COVID-19 infection using mRNA-based vaccines is a new technology [20]. On 10 May 2021, the US Food and Drug Administration(USFDA) expanded the use of the Pfizer–BioNTech vaccine to include adolescents aged
12–15 years [21]. In the COVID-19 era, the risks of myocarditis after an mRNA vaccine injection, especially in male adolescents, have raised particular concerns. In July
2021, the CDC reported an association between COVID-19 mRNA vaccines and suspectedcases of myocarditis and pericarditis. The incidence rate of myocarditis/pericarditis aftermRNA COVID-19 vaccine was reported to be as low as 12.6 cases per million second
dose mRNA vaccines among those aged 12–39 years [8,15]. In contrast, our study found
one case of myopericarditis, four cases of subclinical myocarditis, and two cases of pericarditis among 301 participants, and each case had mild symptoms. The incidence of
myocarditis/pericarditis found in our study may be higher than the other studies due tothe study protocol, which required determining baseline troponin-T, CK-MB, ECG, andechocardiography before vaccination. Two retrospective studies from Israel [8,9] showeda slightly different incidence compared with CDC data, possibly resulting from different
data collection methods and different criteria for diagnosing myocarditis. Montgomery
and colleagues reported on 23 male military personnel diagnosed with myocarditis after
presenting with acute sudden onset of chest pain within 4 days after mRNA COVID-19
vaccine [22]. Another prospective study reported six males who were hospitalized withsuspected myocarditis, all shortly after a second dose of BNT162b2 mRNA COVID-19
vaccine [23].
The potential mechanism of mRNA COVID-19 vaccine-induced myocarditis remains
unknown. It has been suggested that excessive innate immune activation by both lipid
nanoparticle and RNA components of COVID-19 vaccines can cause myocarditis [24,25].
Endosomal toll-like receptor (TLR) TLR3, TLR7, and TLR8 in immune cells and RIG-I andMDA5 in nonimmune cells act as a natural defense against foreign RNA but can crossreact with in vitro transcribed (IVT) RNA [26]. The activation of these receptors triggers an
inflammatory cascade, assembly of inflammasome platform production of type I interferons,
and nuclear translocation of NK-kB [24,25,27]. Inflammasomes are large multiproteins that

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are responsive to pathogen- and stress-associated cellular insults. Inflammasomes lead tothe secretion of the pleiotropic IL-1 family (IL-1β and IL-18), and pyroptosis [28]. In one
case report, patients with vaccine-induced myocarditis had increased levels of interleukin 1(IL-1) receptor antagonist, interleukin 5 (IL-5), and interleukin 16 (IL-16) [5]. Myocarditis
associated with COVID-19 mRNA vaccination mainly occurs in male teenagers within
1 week post-vaccination, typically the second dose vaccination, with recovery of cardiac
function within 1–5 weeks after hospitalization [8,12]. The mechanism is unknown, but
may be related to the mRNA sequence that encodes for the spike protein of SARS-CoV-2, orthe immune response following vaccination [18,29]. By contrast, the incidence of COVID19-associated cardiac injury or myocarditis is much higher, estimated to be 100 times higher
than mRNA COVID-19-related myocarditis [30,31]. Moreover, mRNA vaccine-related
myocarditis is characterized by overall mild presentation and favorable outcomes. In ourstudy, chest pain was considered an alarming side effect after BNT162b2 mRNA vaccine
injection. Although clinical symptoms spontaneously resolved rapidly in all patients, thepotential for cardiac fibrosis vaccine-related myocarditis remains unknown. The long-term
outcomes of COVID-19 vaccine have not been described, but in our study nearly 100%of patients with symptoms had recovered within 1–2 weeks, concordant with anotherstudy [18]. Long-term surveillance with follow-up cardiac imaging, especially cardiac MRI
in patients with vaccine-related myocarditis, is required.
In this study, one patient who was admitted to the ICU Department with negative
PCR for COVID-19, was diagnosed with myopericarditis after mRNA COVID-19 vaccine,and showed abnormalities in cardiac enzymes (troponin-T, CK-MB), ECG, echocardiography, and cardiac MRI. As our patient remained clinically stable, endomyocardial biopsy
was not indicated during hospitalization. He was treated with ibuprofen for 2 weeks
without guideline-directed medical therapy for heart failure because his imaging showednormal LVEF. Treatment of vaccine-induced myocarditis includes corticosteroids, NSAIDs,
colchicine, and, in severe cases, IVIG [32,33]. Corticosteroids have been proposed for thetreatment of vaccine-induced myocarditis [33]. Nonpharmacological strategies to prevent
cardiovascular side effects of COVID-19 vaccination include oral and systemic administration of ascorbic acid. An observational study has found that low serum ascorbic acidcan increase cardiovascular disease in humans [34]. Ascorbic acid can prevent cardiotoxic
events and can reduce the relative risk for cardiovascular events that could reduce inflammation related to cardiovascular events after COVID-19 vaccination [35]. All patients with
subclinical myocarditis had elevated troponin-T without CK-MB elevation; troponin-T level
was also highly sensitive for post-vaccination screening for myocarditis. Two patients diagnosed with pericarditis had normal troponin-T levels, but generally raised c-reactive protein(CRP) or erythrocyte sedimentation rate (ESR). All seven patients with elevated biomarker
levels or positive lab assessments showed normal left ventricular ejection fraction (LVEF),
and only three patients had minimal pericardial effusion. Conventional echocardiography
may not be the ideal diagnostic tool in suspected vaccine-induced myocarditis becauseof its mild clinical symptoms; conventional echocardiography appeared normal in thesepatients. Speckle tracking echocardiography (STE) strain and strain-rate parameters areuseful diagnostic measures with high sensitivity for the early detection of subclinical ventricular dysfunction [36], and cardiac MRI may be the best way to confirm a diagnosis of
myocarditis in the majority of cases. In this study, the disease course was mild in all of ourpatients with cardiac symptoms; they were treated with ibuprofen for 2 weeks and cardiac
enzymes returned to normal after 1–2 weeks of outpatient treatment. Myocarditis may
have more severe clinical manifestations requiring inotropic drug or mechanical support;
however, one patient with myopericarditis in our study follow-up with cMRI at 5 months
post-vaccination showed complete recovery and no scar. If an adolescent presents with
myopericarditis after COVID-19 mRNA vaccination, a booster shot is contraindicated.
This study had some limitations. Due to the national Thai vaccination campaign
against the COVID-19 pandemic, the government declared the timing for the first mRNA
COVID-19 vaccine dose urgently, so there was limited time for the Ethics Committee (EC)

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to process the first dose; however, myocarditis/pericarditis was more common with thesecond mRNA COVID-19 vaccination dose. Our study included some participants with
subclinical myocarditis presenting without symptom other than elevated troponin-T. It
was necessary to request parental permission for blood testing on Day 14, which may have
limited participation.
Strengths: this is the first prospective study in Thailand during the national campaign of vaccination against the COVID-19 pandemic for adolescents. The strengths of
our study include its prospective design employing serial cardiac enzyme, ECG, andechocardiographic measurements at three separate visits (baseline, Day 3, and Day 7). Two
cardiologists at different institutions worked together to confirm the diagnoses of myocarditis, myopericarditis, and pericarditis. Another strength of this study is that participants
and parents were able to contact the principal investigators directly online or by telephone
for consultation and immediate treatment.
5. Conclusions
In this observational study, clinically suspected myopericarditis was temporarily
associated with the BNT162b2 mRNA COVID-19 vaccine in a small proportion of adolescentpatients. Chest pain is an alarming symptom in patients receiving BNT162b2 mRNA
COVID-19 vaccination, especially a second dose of BNT162b2. The risk for these symptoms
was found to be higher than reported elsewhere. The adverse cardiovascular manifestations
observed in this adolescent cohort were both mild and transient.
Author Contributions: Conceptualization, C.M., S.M., P.C., A.P. and W.P.; Data curation, C.M., S.M.,
P.C. and A.P.; Formal analysis, C.M., S.M. and W.D.; Funding acquisition, C.M.; Investigation, C.M.;
Methodology, C.M., S.M., W.P. and A.P.; Project administration, C.M.; Validation, C.M., S.M. andW.D.; Writing—original draft, C.M. and S.M.; Writing—review and editing, C.M., S.M., P.C., A.P. and
W.P. All authors have read and agreed to the published version of the manuscript.
Funding: This study received partial funding from the Department of Clinical Tropical Medicine,
Mahidol University. The funders had no role in the study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Institutional Review Board Statement: The study design was approved by the Ethics Committee ofthe Faculty of Tropical Medicine, Mahidol University (Certificate No. MUTM 2021-068-01).
Informed Consent Statement: Written informed consent was obtained from patients before enrolment into the study.
Data Availability Statement: The dataset can be requested from the corresponding author.
Acknowledgments: We thank all participants who participated in this study. We would like to thankKong Thabbok Upatham Changkol Kho So Tho Bo School and Wachirathamsatit School for theenrollment process team and support. We thank Roche for providing reagents. We give special thanks
for partial funding provided by the Department of Clinical Tropical Medicine, Mahidol University.
Special thanks to Punnee Pitisuttithum who provided valuable advice. We also offer sincere thanks
to Benjaluck Phonrat for much-valued advice. We give special thanks to Paul Adams for his valuableadvice. Special thanks to Arun Huntrap for her team’s contributions and support.
Conflicts of Interest: The authors declare no conflict of interest.

References
1.
2.

3.

Skowronski, D.M.; De Serres, G. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N. Engl. J. Med. 2021, 384,
1576–1577. [PubMed]
Walsh, E.E.; Frenck, R.W., Jr.; Falsey, A.R.; Kitchin, N.; Absalon, J.; Gurtman, A.; Lockhart, S.; Neuzil, K.; Mulligan, M.J.;
Bailey, R.; et al. Safety and Immunogenicity of two RNA-based COVID-19 Vaccine Candidates. N. Engl. J. Med. 2020, 383,
2439–2450. [CrossRef] [PubMed]
Oliver, S.E.; Gargano, J.W.; Marin, M.; Wallace, M.; Curran, K.G.; Chamberland, M.; McClung, N.; Campos-Outcalt, D.;
Morgan, R.L.; Mbaeyi, S.; et al. The Advisory Committee on Immunization Practice’s Interim Recommendation for use PfizerBioNTech COVID-19 vaccine–United States, December 2020. Morb. Mortal. Wkly. Rep. 2020, 69, 1922–1924. [CrossRef]Trop. Med. Infect. Dis. 2022, 7, 196

4.

5.

6.

7.
8.
9.
10.

11.

12.
13.
14.
15.

16.
17.
18.
19.
20.
21.

22.

23.
24.
25.
26.
27.
28.

12 of 13Faix, D.J.; Gordon, D.M.; Perry, L.N.; Raymond-Loher, I.; Tati, N.; Lin, G.; DiPietro, G.; Selmani, A.; Decker, M.D. Prospective
safety surveillance study of ACAM2000 smallpox vaccine in deploying military personnel. Vaccine 2020, 38, 7323–7330. [CrossRef][PubMed]Muthukumar, A.; Narasimhan, M.; Li, Q.-Z.; Mahimainathan, L.; Hitto, I.; Fuda, F.; Batra, K.; Jiang, X.; Zhu, C.; Schoggins, J.; et al.
In-Depth Evaluation of a Case of Presumed Myocarditis After the Second Dose of COVID-19 mRNA Vaccine. Circulation 2021,
144, 487–498. [CrossRef] [PubMed]
Tschöpe, C.; Ammirati, E.; Bozkurt, B.; Caforio, A.L.P.; Cooper, L.T.; Felix, S.B.; Hare, J.M.; Heidecker, B.; Heymans, S.;
Hübner, N.; et al. Myocarditis and inflammatory cardiomyopathy: Current evidence and future directions. Nat. Rev. Cardiol.
2021, 18, 169–193. [CrossRef] [PubMed]
Fairweather, D.; Cooper, L.T.; Blauwet, L.A. Sex and Gender Differences in Myocarditis and Dilated Cardiomyopathy. Curr. Probl.
Cardiol. 2013, 38, 7–46. [CrossRef] [PubMed]
Witberg, G.; Barda, N.; Hoss, S.; Richter, I.; Wiessman, M.; Aviv, Y.; Grinberg, T.; Auster, O.; Dagan, N.; Balicer, R.D.; et al.
Myocarditis after Covid-19 Vaccination in a Large Health Care Organization. N. Engl. J. Med. 2021, 385, 2132–2139. [CrossRef]
Mevorach, D.; Anis, E.; Cedar, N.; Bromberg, M.; Haas, E.J.; Nadir, E.; Olsha-Castell, S.; Arad, D.; Hasin, T.; Levi, N.; et al.
Myocarditis after BNT162b2 mRNA Vaccine against Covid-19 in Israel. N. Engl. J. Med. 2021, 385, 2140–2149. [CrossRef]
Gargano, J.W.; Wallace, M.; Hadler, S.C.; Langley, G.; Su, J.R.; Oster, M.E.; Broder, K.R.; Gee, J.; Weintraub, E.; Shimabukuro, T.; et al.
Use of mRNA COVID-19 Vaccine After Reports of Myocarditis Among Vaccine Recipients: Update from the Advisory Committee
on Immunization Practices—United States, June 2021. MMWR. Morb. Mortal. Wkly. Rep. 2021, 70, 977–982. [CrossRef]
Caforio, A.L.; Pankuweit, S.; Arbustini, E.; Basso, C.; Gimeno-Blanes, J.; Felix, S.B.; Fu, M.; Heliö, T.; Heymans, S.; Jahns, R.; et al.
European Society of Cardiology Working Group on Myocardial and Pericardial Diseases. Current state of knowledge on aetiology,
diagnosis, management, and therapy of myocarditis: A position statement of the European Society of Cardiology Working Groupon Myocardial and Pericardial Diseases. Eur. Heart J. 2013, 34, 2636–2648. [PubMed]
Su, J.R.; McNeil, M.M.; Welsh, K.J.; Marquez, P.L.; Ng, C.; Yan, M.; Cano, M.V. Myopericarditis after vaccination, Vaccine Adverse
Event Reporting System (VAERS), 1990–2018. Vaccine 2021, 39, 839–845. [CrossRef] [PubMed]
Shimabukuro, T.T.; Nguyen, M.; Martin, D.; DeStefano, F. Safety monitoring in the Vaccine Adverse Event Reporting System(VAERS). Vaccine 2015, 33, 4398–4405. [CrossRef]
Guo, T.; Fan, Y.; Chen, M.; Wu, X.; Zhang, L.; He, T.; Wang, H.; Wan, J.; Wang, X.; Lu, Z. Cardiovascular Implications of Fatal
Outcomes of Patients With Coronavirus Disease 2019 (COVID-19). JAMA Cardiol. 2019, 5, 811–818. [CrossRef]
Centers for Disease Control and Prevention (CDC). Advisory Committee on Immunization Practice (ACIP). Coronavirus Disease2019 (COVID-19) Vaccines. Available online: https://www.cdc.gov/vaccines/acip/meetings/slides-2021-10.html (accessed on
23 January 2022).
Kuntz, J.; Crane, B.; Weinmann, S.; Naleway, A.L. Myocarditis and pericarditis are rare following live viral vaccinations in adults.
Vaccine 2018, 36, 1524–1527. [CrossRef]
García, J.B.; Ortega, P.P.; Fernández, J.A.B.; León, A.C.; Burgos, L.R.; Dorta, E.C. Acute myocarditis after administration of the
BNT162b2 vaccine against COVID-19. Rev. Esp. Cardiol. 2021, 74, 812–814. [CrossRef]
Bozkurt, B.; Kamat, I.; Hotez, P.J. Myocarditis With COVID-19 mRNA Vaccines. Circulation 2021, 144, 471–484. [CrossRef][PubMed]Kaur, R.J.; Dutta, S.; Charan, J.; Bhardwaj, P.; Tandon, A.; Yadav, D.; Islam, S.; Haque, M. Cardiovascular Adverse Events Reported
from COVID-19 Vaccines: A Study Based on WHO Database. Int. J. Gen. Med. 2021, 14, 3909–3927. [CrossRef] [PubMed]
Pardi, N.; Hogan, M.J.; Porter, F.W.; Weissman, D. mRNA vaccines-a new era in vaccinology. Nat. Rev. Drug Discov. 2018, 17,
261–279. [CrossRef]
US Food and Drug Administration. Coronavirus (COVID-19) Update: FDA Authorizes Pfizer-BioNTech COVID-19 Vaccine for
Emergency Use in Adolescents in Another Importance Action in Flight Against Pandemic; US Food and Drug Administration: Silver
Spring, MD, USA, 2021.
Montgomery, J.; Ryan, M.; Engler, R.; Hoffman, D.; McClenathan, B.; Collins, L.; Loran, D.; Hrncir, D.; Herring, K.; Platzer, M.; et al.
Myocarditis Following Immunization With mRNA COVID-19 Vaccines in Members of the US Military. JAMA Cardiol. 2021, 6,
1202–1206. [CrossRef]
Mouch, S.A.; Roguin, A.; Hellou, E.; Ishai, A.; Shoshan, U.; Mahamid, L.; Zoabi, M.; Aisman, M.; Goldschmid, N.; Yanay, N.B.
Myocarditis following COVID-19 mRNA vaccination. Vaccine. 2021, 39, 3790–3793. [CrossRef] [PubMed]
Power, J.R.; Keyt, L.K.; Adler, E.D. Myocarditis following COVID-19 vaccination: Incidence, mechanisms, and clinical considerations. Expert Rev. Cardiovasc. Ther. 2022, 20, 241–251. [CrossRef]
Hajjo, R.; Sabbah, D.A.; Bardaweel, S.K.; Tropsha, A. Shedding the Light on Post-Vaccine Myocarditis and Pericarditis in
COVID-19 and Non-COVID-19 Vaccine Recipients. Vaccines 2021, 9, 1186. [CrossRef] [PubMed]
Wolff, J.A.; Malone, R.W.; Williams, P.; Chong, W.; Acsadi, G.; Jani, A.; Felgner, P.L. Direct gene transfer into mouse muscle in vivo.
Science 1990, 247 Pt 1, 1465–1468. [CrossRef]
Ahin, U.; Karikó, K.; Türeci, Ö. mRNA-based therapeutics—developing a new class of drugs. Nat. Rev. Drug Discov. 2014, 13,
759–780. [CrossRef] [PubMed]
Vora, S.M.; Lieberman, J.; Wu, H. Inflammasome activation at the crux of severe COVID-19. Nat. Rev. Immunol. 2021, 21, 694–703.
[CrossRef]Trop. Med. Infect. Dis. 2022, 7, 196

29.
30.
31.
32.
33.

34.
35.

36.

13 of 13

Chilamahuri, R.; Agarwal, S. COVID-19: Characteristics and Therapeutics. Cells 2021, 10, 206. [CrossRef]
Aikawa, T.; Takagi, H.; Ishikawa, K.; Kuno, T. Myocardial injury characterized by elevated cardiac troponin and in-hospitalmortality of COVID-19: An insight from a meta-analysis. J. Med. Virol. 2021, 93, 51–55. [CrossRef]
Maiese, A.; Frati, P.; Del Duca, F.; Santoro, P.; Manetti, A.C.; La Russa, R.; Di Paolo, M.; Turillazzi, E.; Fineschi, V. Myocardial
Pathology in COVID-19-Associated Cardiac Injury: A Systematic Review. Diagnostics 2021, 11, 1647. [CrossRef]
Tano, E.; San Martin, S.; Girgis, S.; Martinez-Fernandez, Y.; Sanchez Vegas, C. Perimyocarditis in adolescents after Pfizer-BioNTech
COVID-19 vaccine. J. Pediatr. Infect. Dis. Soc. 2021, 10, 962–966. [CrossRef]
Marshall, M.; Ferguson, I.D.; Lewis, P.; Jaggi, P.; Gagliardo, C.; Collins, J.S.; Shaughnessy, R.; Caron, R.; Fuss, C.;
Corbin, K.J.E.; et al. Symptomatic acute myocarditis in 7 adolescents after Pfizer-BioNTech COVID-19 vaccination. Pediatrics 2021, 148, e2021052478. [CrossRef] [PubMed]
Ashor, A.W.; Lara, J.; Mathers, J.C.; Siervo, M. Effect of vitamin C on endothelial function in health and disease: A systematic
review and meta-analysis of randomized controlled trials. Athersclerosis 2014, 235, 9–20. [CrossRef] [PubMed]
Berretta, M.; Quagliariello, V.; Maurea, N.; Di Francia, R.; Sharifi, S.; Facchini, G.; Rinaldi, L.; Piezzo, M.; Manuela, C.;
Nunnari, G.; et al. Multiple Effects of Ascorbic Acid against Chronic Diseases: Updated Evidence from Preclinical and ClinicalStudies. Antioxidants 2020, 9, 1182. [CrossRef] [PubMed]
Hsiao, J.F.; Koshino, Y.; Bonnichsen, C.R.; Yu, Y.; Miller FAJr Pellikka, P.A.; Cooper, L.T., Jr. Villarraga HR. Speckle tracking
echocardiography in acute myocarditis. Int. J. Cardiovasc. Imaging 2013, 29, 275–284. [CrossRef] [PubMed]