Postmarketing active surveillance of myocarditis and pericarditis following vaccination with COVID-19 mRNA vaccines in persons aged 12 to 39 years in Italy: A multi-database, self-controlled case series study.

BACKGROUND: Myocarditis and pericarditis following the Coronavirus Disease 2019 (COVID-19) mRNA vaccines administration have been reported, but their frequency is still uncertain in the younger population. This study investigated the association between Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) mRNA vaccines, BNT162b2, and mRNA-1273 and myocarditis/pericarditis in the population of vaccinated persons aged 12 to 39 years in Italy. METHODS AND
FINDINGS: We conducted a self-controlled case series study (SCCS) using national data on COVID-19 vaccination linked to emergency care/hospital discharge databases. The outcome was the first diagnosis of myocarditis/pericarditis between 27 December 2020 and 30 September 2021. Exposure risk period (0 to 21 days from the vaccination day, subdivided in 3 equal intervals) for first and second dose was compared with baseline period. The SCCS model, adapted to event-dependent exposures, was fitted using unbiased estimating equations to estimate relative incidences (RIs) and excess of cases (EC) per 100,000 vaccinated by dose, age, sex, and vaccine product. Calendar period was included as time-varying confounder in the model. During the study period 2,861,809 persons aged 12 to 39 years received mRNA vaccines (2,405,759 BNT162b2; 456,050 mRNA-1273); 441 participants developed myocarditis/pericarditis (346 BNT162b2; 95 mRNA-1273). Within the 21-day risk interval, 114 myocarditis/pericarditis events occurred, the RI was 1.99 (1.30 to 3.05) after second dose of BNT162b2 and 2.22 (1.00 to 4.91) and 2.63 (1.21 to 5.71) after first and second dose of mRNA-1273. During the [0 to 7) days risk period, an increased risk of myocarditis/pericarditis was observed after first dose of mRNA-1273, with RI of 6.55 (2.73 to 15.72), and after second dose of BNT162b2 and mRNA-1273, with RIs of 3.39 (2.02 to 5.68) and 7.59 (3.26 to 17.65). The number of EC for second dose of mRNA-1273 was 5.5 per 100,000 vaccinated (3.0 to 7.9). The highest risk was observed in males, at [0 to 7) days after first and second dose of mRNA-1273 with RI of 12.28 (4.09 to 36.83) and RI of 11.91 (3.88 to 36.53); the number of EC after the second dose of mRNA-1273 was 8.8 (4.9 to 12.9). Among those aged 12 to 17 years, the RI was of 5.74 (1.52 to 21.72) after second dose of BNT162b2; for this age group, the number of events was insufficient for estimating RIs after mRNA-1273. Among those aged 18 to 29 years, the RIs were 7.58 (2.62 to 21.94) after first dose of mRNA-1273 and 4.02 (1.81 to 8.91) and 9.58 (3.32 to 27.58) after second dose of BNT162b2 and mRNA-1273; the numbers of EC were 3.4 (1.1 to 6.0) and 8.6 (4.4 to 12.6) after first and second dose of mRNA-1273. The main study limitations were that the outcome was not validated through review of clinical records, and there was an absence of information on the length of hospitalization and, thus, the severity of the outcome.
CONCLUSIONS: This population-based study of about 3 millions of residents in Italy suggested that mRNA vaccines were associated with myocarditis/pericarditis in the population younger than 40 years. According to our results, increased risk of myocarditis/pericarditis was associated with the second dose of BNT162b2 and both doses of mRNA-1273. The highest risks were observed in males of 12 to 39 years and in males and females 18 to 29 years vaccinated with mRNA-1273. The public health implication of these findings should be considered in the light of the proven mRNA vaccine effectiveness in preventing serious COVID-19 disease and death.

Introduction

Intensive postmarketing surveillance of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccines is ongoing worldwide to provide updated information on their effectiveness and safety, thereby supporting regulatory benefit/risk assessment. Since early phase of the global vaccination campaign, case series [1-3] and pharmacovigilance reports [4,5] on myocarditis and pericarditis following the Coronavirus Disease 2019 (COVID-19) mRNA vaccine administration were published. Both events were included as related to COVID-19 disease in the early and updated Priority List of COVID-19 Adverse events of special interest, developed by Brighton Collaboration Group and Safety Platform for Emergency vACcines (SPEAC), in order to harmonize safety assessment of COVID-19 vaccines in pre- and postmarketing setting [6]. Moreover, as per core requirements for risk management plan (RMP), they have been periodically monitored through routine pharmacovigilance activities in the Monthly Summary Safety Reports of all COVID-19 vaccines [7].

On July 2021, the COVID-19 subcommittee of WHO Global Advisory Committee on Vaccine Safety reported that very rare cases of myocarditis and pericarditis had occurred more often in adolescents or young adults and after the second dose, especially within a few days after COVID-19 mRNA vaccines, and encouraged countries to strengthen the monitoring of myocarditis/pericarditis [8]. At the same time, EMA’s Pharmacovigilance Risk Assessment Committee (PRAC) began an assessment on signals of myocarditis and pericarditis with BNT162b2 and mRNA-1273 vaccine and concluded that both cardiac conditions can occur in very rare cases following vaccination with the COVID-19 mRNA vaccines. Thus, the Committee recommended to update the product information and the RMP for these vaccines, together with a direct healthcare professional communication to raise awareness among healthcare professionals [9].

In October 2021, further data were available from the Nordic population-based register study on myocarditis and pericarditis in northern Europe that prompted some public health organizations in the Nordic countries (e.g., Sweden, Finland, Norway, Iceland) [10] either to pause the use of the mRNA-1273 or to recommend the use of the BNT162b2 rather than mRNA-1273 in younger people and/or younger males. In December 2021, the PRAC reassessed the relevant safety signal, based on the Nordic study and on a study conducted using data from the French national health system (Epi-phare) [11], concluding that the risk for both events is overall “very rare” (up to 1 in 10,000 vaccinated people) and greater in younger males. A further update of product information was recommended, while the benefit/risk was confirmed as positive for the whole indications [12].

In line with these findings, recent published data from large population-based studies from Israel, United States, United Kingdom, and Denmark documented that the risks of myocarditis/pericarditis following mRNA vaccines differ by age groups, sex, and vaccine product with a higher risk in those younger than 40 years (S1 Table) [13-18].

In Italy, SARS-CoV-2 vaccines have been administered since late December 2020 and have been offered to the population according to a priority scheme, considering profession, age, and health conditions. Vaccination in adolescent (≥12 years) started on 31 May and 28 July 2021 for BNT162b2 and mRNA-1273, respectively.

Along with the enhanced passive surveillance of the Italian PharmacoVigilance network, an active surveillance, based on regional healthcare claims databases, was set up by the Italian National Institute of Health (ISS) and the Italian Medicines Agency (AIFA) to provide real-world data on SARS-CoV-2 vaccine safety.

To our knowledge, studies examining the association between mRNA-based COVID-19 vaccines in the population resident in Italy have not been published yet. Previous published studies have been conducted in other countries [13-18] and few of them have estimated risks in younger than 40 years by sex and age [13,14,17], while none of them have used a SCCS study design, with the exception of the study of Patone and colleagues [17]. The present study, while attempting to address these gaps, has the objective to investigate the association between mRNA-based COVID-19 vaccines (BNT162b2 and mRNA-1273) and myocarditis/pericarditis in the population of vaccinated persons aged 12 to 39 years, by age and sex, in Italy, during the period 27 December 2020 and 30 September 2021.

Methods

Data source

The active surveillance is based on a dynamic multiregional observational cohort. A distributed analysis framework is applied using TheShinISS, an R-based open-source statistical tool, developed by the National Institute of Health [19], that locally processes data collected and updated periodically from regional healthcare databases according to an ad hoc, study-tailored, common data model.

Data on vaccination exposure, on hospitalization for myocarditis/pericarditis, and on participant characteristics were retrieved from several routinely collected regional healthcare claims databases:

COVID-19 vaccination registry to identify information on administered vaccines (product, date of administration, and doses for all vaccinated participants);

population registry to identify information on age, sex, and vital status (causes of death are not recorded in this registry);

hospital discharge and emergency care visit databases to identify myocarditis/pericarditis events (pre and post vaccination) and information on the comorbidities of the study participants in the period preceding the vaccination;

pharmacy claims and copayment exemptions databases to obtain information on the comorbidities of the study participants in the period preceding the vaccination;

vaccination registry to identify other vaccinations (e.g., flu and pneumococcal vaccines) administered in the period pre- and post-COVID-19 vaccination;

COVID-19 surveillance system to obtain information on SARS-Cov-2 infection and related outcomes.

Study design

We used a self-controlled case series (SCCS) design [20-24]. The SCCS design has emerged as a key methodology for studying the safety of vaccines and medicines. This approach only requires information from individuals who have experienced the event of interest and automatically controls for multiplicative time-invariant confounders, even when these are unmeasured or unknown. Originally designed to analyze the association between vaccination and specific events under the key assumption that events do not influence post-event exposures, this method has been adapted to event-dependent exposures, for example, when occurrence of an event may preclude any subsequent exposure (SCCS method for censored, perturbed, or curtailed post-event exposures) [23-25]. This is the case in observational studies of vaccines when the event of interest could be a contraindication to vaccination.

By using the adapted SCCS method for event-dependent exposures, we estimated the relative incidence (RI) of myocarditis/pericarditis following prespecified windows at risk after vaccination, in a within-person comparison of different time periods. The method allows for the control of all time-independent characteristics of participants. The SCCS method allows also for adjustment of potential time-varying confounders such as seasonal variation in risks.

Study period and population

We investigated the association between mRNA-based COVID-19 vaccines and subsequent onset of myocarditis/pericarditis in the population aged 12 to 39 years in the period 27 December 2020 to 30 September 2021 (the latest date for which outcome data were available). Regional claims data were locally transformed into a study-specific common data model and locally processed using TheShinISS.

In the end, regional pseudonymized datasets were provided to the National Institute of Health for centralized analysis, in compliance with EU General Data Protection Regulation. Over the last 2 years, TheShinISS framework has been employed in several large scale observational studies exploring the association between several exposures and COVID-19 onset/prognosis as well as other drug and vaccine-related research topics and is currently maintained by a collaborative research network [19,26-28]. The relational scheme of the study databases as well as TheShinISS flow diagram is described in S1 Fig.

Four Italian Regions (northern Italy: Lombardia, Veneto, Friuli Venezia Giulia; central Italy: Lazio), representing 36% of the population aged 12 to 39 years resident in Italy, contributed data of all vaccinated persons in this age group, in a period ranging from 27 December 2020 to the latest date for which data on outcomes were available, which varied across Regions: Lombardia up to 30 September 2021, Veneto up to 20 June 2021, Friuli Venezia Giulia up to 31 August 2021, and Lazio up to 16 June 2021). We included in the study all the persons aged 12 to 39 years who received a first dose of mRNA vaccines and were admitted to emergency care or hospital with the outcomes of interest. We excluded individuals with missing or inconsistent information on relevant variables (age, sex, vaccine product and dose, date of vaccination, of death and of event). Furthermore, we excluded individuals with a history of myocarditis or pericarditis within 365 days leading up to the start of the study period. The observation period for each case ranged from 27 December 2020 to the end of follow-up, which occurred at the time of death or at the end of Region-specific study period, whichever came first.

Definition of outcomes

The outcome of interest was the first diagnosis of myocarditis/pericarditis identified through emergency care and/or hospital admission occurring between 27 December 2020 and 30 September 2021 using International Classification of Disease, Ninth Revision, Clinical Modification (ICD-9-CM codes of myocarditis: 391.2; 398.0; 422; 429.0; ICD-9-CM codes of pericarditis: 391.0; 393; 420; 423.1; 423.2; 423.9).

Definition of exposures

The exposures of interest were the first or second dose of BNT162b2 and mRNA-1273 vaccines. The exposure risk period was not prespecified in the protocol submitted to the Ethical Committee and it was decided before data collection and analysis. It was defined as [0–21) days after first or second dose administration (vaccination date), which included day 0, the day of vaccination. The risk period was further subdivided into prespecified time periods: [0 to 7), [7 to 14), and [14 to 21) after each exposure date. The unexposed baseline period (reference period) was defined as any time of observation out of the risk periods (S2 Fig).

Statistical analysis

Characteristics of the cohort of vaccinated persons and myocarditis and pericarditis cases were described by age, sex, and comorbidities. Temporal timing of myocarditis or pericarditis events in relation to first/second dose vaccination dates was described by week.

The SCCS model was fitted using unbiased estimating equations to estimate the RIs and their 95% confidence intervals (95% CI). In the following, we will use the term “association” between vaccine exposure and the study event (overall and in a given subgroup) for an RI estimate whose CI does not include the null effect. To handle event-dependent exposures, the SCCS model was properly modified considering a counterfactual exposure history for any exposures arising after occurrence of an event [20,24]. Five 45-day calendar periods were considered as time-varying covariate controlling for the seasonal effect (adjusted model). We also estimated the excess of cases (EC) per 100,000 vaccinated with 95% CIs applying nonparametric bootstrapping (10,000 replications) [29]. We carried out subgroup analyses by age group (12 to 17, 18 to 29, 30 to 39 years), sex, and vaccine product (BNT162b2 and mRNA-1273). To assess the robustness of the primary analysis, the following sensitivity analyses regarding the modified SCCS method were conducted: (a) observation/exposure time period—we restricted the analysis to the study period from 27 December 2020 to 31 May 2021 [20,30] and to the study period from 1 June 2021 to 30 September 2021; we repeated the primary analysis excluding day 0 from the [0 to 7) day risk interval; we extended the exposure period to 28 days as well reducing it to 14 days; (b) heterologous vaccination—we carried out the primary analysis excluding individuals who received 2 different vaccine products at the first and second dose or censoring at time of second dose individuals who received a different vaccine product at the second dose (in the primary analysis, the second dose was assumed to be of the same product as the first one); (c) SARS-CoV-2 infection—we restricted the analyses to participants without a SARS-CoV-2-positive test before the occurrence of the event (any time) and within 10 days after the event. Further sensitivity analyses were performed exploring different assumptions of the standard SCCS method; (d) beginning observation at exposure; (e) beginning observation at time 0; (f) with prerisk period; (g) removing post event exposure. We conducted also an ancillary analysis reproducing the primary SCCS analysis in the vaccinated persons aged over 40 years.

The analyses were performed using R version 4.1.2 (R Core Team 2021) with SCCS package [31] and STATA version 16.1. This study is reported as per the REporting of studies Conducted using Observational Routinely-collected Data for Pharmacoepidemiology (RECORD-PE) checklist (S1 Checklist).

Ethics and permissions

This study was approved by the National Unique Ethics Committee for the evaluation of clinical trials of medicines for human use and medical devices for patients with COVID-19 of the National Institute for Infectious Diseases “Lazzaro Spallanzani” in Rome (ordinance n. 335, 17/05/2021 and n. 399, 02/09/2021).

Results

Our cohort included 13,728,174 persons older than 12 years, who received COVID-19 vaccines between 27 December 2020 to 30 September 2021, of these 10,769,025 (78.4%) received mRNA vaccines.

During the study period, 5,109,231 doses of mRNA vaccines were administered to 2,861,809 persons aged 12 to 39 years (median age 26 years, interquartile range, IQR 19 to 33; 49% females); 2,405,759 (84%) persons received BNT162b2 vaccine and 456,050 (16%) received mRNA-1273 vaccine. Among 24,469,038 doses uploaded on TheShinISS, the proportion of missing or inconsistent observations was 0.7% (n. 172,174) (S3 Fig). The vaccinated persons had a median follow-up time of 120 days (IQR 52 to 185). Characteristics of mRNA-vaccinated population aged 12 to 39 years and definition of study comorbidities are reported in S2 and S3 Tables, respectively.

During the study period, 441 persons had an emergency care and/or hospital admission related to myocarditis/pericarditis. Of these, 302 (68.5%) were males and 139 (31.5%) were females; there were 346 (78.5%) cases in those vaccinated with BNT162b2 and 95 (21.5%) in those vaccinated with mRNA-1273 (Table 1). Fig 1 describes the temporal trend of the occurrence of the events relative to vaccination date. We observed 1 death, for unknown cause, after 38 days following a pericarditis case that occurred 53 days after the second dose of BNT162b2 vaccine (unexposed period). The median follow-up time after the event was 93 days (IQR 56 to 113).

Table 1

Characteristics of cases of myocarditis/pericarditis (n. 441) among the mRNA-vaccinated population aged 12–39 years from 27 December 2020 to 30 September 2021 by vaccine product.

	mRNA [n.(%)]	BNT162b2 [n.(%)]	mRNA-1273 [n.(%)]
Number of participants	441	346	95
Sex
Males	302 (68.5%)	232 (67.1%)	70 (73.7%)
Females	139 (31.5%)	114 (32.9%)	25 (26.3%)
Charlson index
≥1	53 (12.0%)	46 (13.3%)	7 (7.4%)
Hospitalizations in the last 2 years
≥1	188 (42.6%)	154 (44.5%)	34 (35.8%)
Comorbidities
COVID-19 diagnosis before vaccination	60 (13.6%)	48 (13.9%)	12 (12.6%)
COPD/Asthma	32 (7.3%)	28 (8.1%)	4 (4.2%)
Chronic pulmonary disease	16 (3.6%)	15 (4.3%)	1 (1.1%)
Chronic kidney failure	9 (2.0%)	7 (2.0%)	2 (2.1%)
Neoplasms	16 (3.6%)	14 (4.0%)	2 (2.1%)
Diabetes mellitus	8 (1.8%)	4 (1.2%)	4 (4.2%)
Hematologic disease	50 (11.3%)	43 (12.4%)	7 (7.4%)
Cardiovascular/cerebrovascular diseases	170 (38.5%)	138 (39.9%)	32 (33.7%)
Hypertension	82 (18.6%)	66 (19.1%)	16 (16.8%)
Hepatopathy	2 (0.5%)	1 (0.3%)	1 (1.1%)
HIV	2 (0.5%)	2 (0.6%)	0
Rheumatic diseases	23 (5.2%)	18 (5.2%)	5 (5.3%)
Cystic fibrosis	0	0	0
Neurological diseases	26 (5.9%)	20 (5.8%)	6 (6.3%)
Peptic ulcer	152 (34.5%)	122 (35.3%)	30 (31.6%)
Colitis	5 (1.1%)	4 (1.2%)	1 (1.1%)
Celiac disease	1 (0.2%)	1 (0.3%)	0
Infection	155 (35.1%)	125 (36.1%)	30 (31.6%)
Prior drugs use
Prescriptions in the last 12 months (1+)	307 (69.6%)	246 (71.1%)	61 (64.2%)
Corticosteroids for systemic use	57 (12.9%)	49 (14.2%)	8 (8.4%)
NSAIDs use	48 (10.9%)	40 (11.6%)	8 (8.4%)
Estroprogestinics use	2 (0.5%)	1 (0.3%)	1 (1.1%)

COPD, chronic obstructive pulmonary disease; HIV, human immunodeficiency virus; n., number; NSAID, nonsteroidal anti-inflammatory drug; yrs, years.

Fig 1

Days from mRNA vaccination to myocarditis/pericarditis in population aged 12–39 years by vaccine product and dose.

Top (panel A-BNT162b and B-mRNA-1273): days from dose 1, for events occurring before dose 2 if present, or at any time if dose 2 not present. Bottom (panel C-BNT162b and D-mRNA-1273): days from dose 2, for events occurring after dose 1. *Each bar corresponds to 1 week starting from day 0.

Table 2 reports the results of the primary analysis from the SCCS model, with RIs adjusted by calendar period, for the 441 cases aged 12 to 39 years. The unadjusted estimates of RI are shown in S4 Table. ECs are reported for RIs with 95% CI not including the null effect. During the 21-day risk interval, there were a total of 114 cases of myocarditis/pericarditis (74 with BNT162b2 and 40 with mRNA-1273), corresponding to RIs of 1.08 (95% CI: 0.70 to 1.67) and 1.99 (95% CI: 1.30 to 3.05) after first and second dose of BNT162b2, respectively, and 2.22 (95% CI 1.00 to 4.91) and 2.63 (95% CI 1.21 to 5.71) after first and second dose of mRNA-1273, respectively. The majority of these cases occurred within the [0 to 7) day risk period after the first or second dose administration of mRNA vaccines (n. 70, 61.4%). An increased risk of myocarditis/pericarditis [0 to 7) days following a first dose of mRNA-1273 was observed (RI = 6.55, 95% CI: 2.73 to 15.72), while no association was found with BNT162b2. An increased risk of myocarditis/pericarditis [0 to 7) days was also observed following a second dose of BNT162b2 (RI = 3.39, 95% CI: 2.02 to 5.68) and mRNA-1273 (RI = 7.59, 95% CI: 3.26 to 17.65). Over the [0 to 7) days postvaccination, we estimated an additional 2.0 (95% CI: 0.8 to 3.6) myocarditis/pericarditis cases per 100,000 vaccinated persons following the first dose of mRNA-1273; following a second dose of the BNT162b2 and mRNA-1273, over the [0 to 7) days post vaccination, we estimated an additional 0.8 (95% CI: 0.4 to 1.4) and 5.5 (95% CI: 3.0 to 7.9) myocarditis/pericarditis cases per 100,000 vaccinated, respectively.

Table 2

Adjusted RI estimated by SCCS and excess cases per 100,000 vaccinated by vaccine product and risk intervals: 346 myocarditis/pericarditis events in the BNT162b2 and 95 events in the mRNA-1273 vaccinated population aged 12–39 years from 27 December 2020 to 30 September 2021.

Risk interval	Dose	BNT162b2 (n. 346)			mRNA-1273 (n. 95)
		Events in the risk interval (n)	Adjusted RI (95% CI)*	Excess cases per 100,000 vaccinated (95% CI)**	Events in the risk interval (n)	Adjusted RI (95% CI)*	Excess cases per 100,000 vaccinated (95% CI)**
[0–21)	Dose 1	35	1.08 (0.70–1.67)		15	2.22 (1.00–4.91)	1.8 (−0.2–3.7)
	Dose 2	39	1.99 (1.30–3.05)	1.0 (0.3–1.7)	25	2.63 (1.21–5.71)	4.2 (0.8–7.2)
[0–7)	Dose 1	14	1.27 (0.70–2.31)		11	6.55 (2.73–15.72)	2.0 (0.8–3.6)
	Dose 2	22	3.39 (2.02–5.68)	0.8 (0.4–1.4)	23	7.59 (3.26–17.65)	5.5 (3.0–7.9)
[7–14)	Dose 1	10	0.92 (0.46–1.82)		3	1.58 (0.45–5.58)
	Dose 2	7	1.07 (0.50–2.30)		0	—
[14–21)	Dose 1	11	1.09 (0.56–2.12)		1	0.49 (0.06–4.07)
	Dose 2	10	1.58 (0.78–3.21)		2	0.71 (0.17–3.09)
Ref.		272	1.0		55	1.0

*Adjusted by calendar period.

**Excess cases are not given when the 95% CI of RI included the null effect.

CI, confidence interval; n., number; Ref., reference period (unexposed period); RI, relative incidence; SCCS, self-controlled cases series.

Subgroup analysis by sex and age group

Table 3 and Fig 2 show the adjusted RIs (unadjusted estimates in S5 Table) and ECs by age, sex, and product in the 7 days risk period (S6–S15 Tables and S4 Fig).

Table 3

Adjusted RI and excess of cases per 100,000 vaccinated in the 7-day risk periods after mRNA vaccination in the vaccinated population aged 12–39 years from 27 December 2020 to 30 September 2021 by sex, age group, and vaccine product.

Age, years	Sex	Risk interval	Dose	BNT162b2 (n. 346)			mRNA-1273 (n. 95)
				Events in the risk interval (n)	AdjustedRI (95% CI)*	Excesscases per100,000vaccinated (95% CI)**	Events in the risk interval (n)	AdjustedRI (95% CI)*	Excesscases per100,000vaccinated (95% CI)**
12–39	Males+Females	[0–7)	Dose 1	14	1.27 (0.70–2.31)		11	6.55 (2.73–15.72)	2.0 (0.8–3.6)
			Dose 2	22	3.39 (2.02–5.68)	0.8 (0.4–1.4)	23	7.59 (3.26–17.65)	5.5 (3.0–7.9)
		Ref.		272	1		55	1
	Males	[0–7)	Dose 1	9	1.53 (0.71–3.31)		10	12.28 (4.09–36.83)	3.8 (1.5–6.3)
			Dose 2	13	3.45 (1.78–6.68)	1.0 (0.3–1.8)	19	11.91 (3.88–36.53)	8.8 (4.9–12.9)
		Ref.		184	1		38	1
	Females	[0–7)	Dose 1	5	0.88 (0.34–2.32)		1	0.69 (0.08–5.75)***
			Dose 2	9	3.38 (1.47–7.74)	0.7 (0.1–1.4)	4	2.08 (0.45–9.72)***
		Ref.		88	1		17	1
12–17	Males+Females	[0–7)	Dose 1	3	1.06 (0.17–6.59)		0	****
			Dose 2	7	5.74 (1.52–21.72)	1.7 (0.04–3.2)	3	****
		Ref.		31	1		7	1
18–29	Males+Females	[0–7)	Dose 1	7	1.76 (0.76–4.05)		9	7.58 (2.62–21.94)	3.4 (1.1–6.0)
			Dose 2	11	4.02 (1.81–8.91)	1.1 (0.2–2.0)	18	9.58 (3.32–27.58)	8.6 (4.4–12.6)
		Ref.		121	1		28	1
30–39	Males+Females	[0–7)	Dose 1	4	0.86 (0.31–2.38)		2	6.57 (1.32–32.63)	1.0 (^NE–3.3)
			Dose 2	4	1.64 (0.59–4.53)		2	3.22 (0.69–15.10)
		[7–14)	Dose 1	4	0.97 (0.34–2.80)		2	5.87 (1.34–25.74)	1.0 (^NE–3.3)
		Ref.		120	1		20	1

*Adjusted by calendar period.

**Excess cases are not given when the 95% CI of RI included the null effect.

***Unadjusted RIs due to the small number of cases.

****Considering the small number of cases, it was not possible to provide any estimates.

^Considering the small number of cases, bootstrapping produced implausible results for inferior limit of 95% CIs.

CI, confidence interval; NE, not estimable; n., number; Ref., reference period (unexposed baseline period); RI, relative incidence.

Fig 2

Excess of cases per 100,000 vaccinated in the [0–7) days risk period following BNT162b2 and mRNA-1273 vaccination in the vaccinated population aged 12–39 years from 27 December 2020 to 30 September 2021, by sex, age group, and dose (first dose blue, second dose orange).

*Considering the small number of cases in the vaccinated with mRNA-1273 of age 12–17 years, it was not possible to provide any estimates; excess cases are not given when the 95% CI of RI included the null effect over the [0–7) day risk interval post vaccination. RI, relative incidence; yrs, years.

In males, the risk of myocarditis/pericarditis increased in the [0 to 7) days following a first dose of mRNA-1273 (RI = 12.28, 95% CI: 4.09 to 36.83) and following a second dose of BNT162b2 (RI = 3.45, 95% CI: 1.78 to 6.68) and mRNA-1273 (RI = 11.91, 95% CI: 3.88 to 36.53). In females, we found an increased risk of myocarditis/pericarditis [0 to 7) days following a second dose of BNT162b2 (RI = 3.38, 95% CI: 1.47 to 7.74), while no association was observed with mRNA-1273.

In males, we estimated an additional 3.8 (95% CI: 1.5 to 6.3) EC per 100,000 in the [0 to 7) days following a first dose of mRNA-1273, and an additional 1.0 (95% CI: 0.3 to 1.8) and 8.8 (95% CI: 4.9 to 12.9) EC per 100,000 in the [0 to 7) days following a second dose of BNT162b2 and mRNA-1273, respectively. In females, we estimated an additional 0.7 (95% CI: 0.1 to 1.4) EC per 100,000 in the [0 to 7) days following a second dose of BNT162b2.

In the analyses by age group, we estimated an increased risk of myocarditis/pericarditis [0 to 7) days following the second dose of BNT162b2 (RI = 5.74, 95% CI: 1.52 to 21.72) in those aged 12 to 17 years. Number of events was insufficient to fit the SCCS model with mRNA-1273 aged 12 to 17 years. Of note, there were 3 events after the second dose in the [0 to 7) day interval compared to 7 events in the reference period. In the 18 to 29 year age group, we observed an increased risk of myocarditis/pericarditis [0 to 7) days following a first and second dose of mRNA-1273 (RI = 7.58, 95% CI: 2.62 to 21.94 and RI = 9.58, 95% CI: 3.32 to 27.58, respectively) and following the second dose of BNT162b2 (RI = 4.02, 95% CI: 1.81 to 8.91). In the age group 30 to 39 years, we found an increased risk of myocarditis/pericarditis [0 to 7) days (RI = 6.57, 95% CI: 1.32 to 32.63) and [7 to 14) days (RI = 5.87, 95% CI: 1.34 to 25.74) following the first doses of mRNA-1273, while no association was observed with BNT162b2.

In the age group 12 to 17 years, we estimated an additional 1.7 (95% CI: 0.04 to 3.2) EC per 100,000 in the [0 to 7) days following a second dose of BNT162b2. In the age group 18 to 29 years, we estimated an additional 3.4 (95% CI: 1.1 to 6.0) EC per 100,000 in the [0 to 7) days following a first dose of mRNA-1273; an additional 1.1 (95% CI: 0.2 to 2.0) and 8.6 (95% CI: 4.4 to 12.6) EC per 100,000 in the [0 to 7) days following a second dose of BNT162b2 and mRNA-1273, respectively. In the age group 30 to 39 years, we estimated an additional 1.0 EC per 100,000 (95% CI: not estimable-3.3) both in the [0 to 7) days and [7 to 14) days following the first dose of mRNA-1273, respectively.

Sensitivity and ancillary analyses

All sensitivity analyses, using the modified SCCS method for event-dependent exposure, were consistent with the main results of the study (S16 Table). The sensitivity analysis that was conducted to highlight the potential effect of notoriety bias (by restricting the observation period before and after 31 May 2021) indicated that RIs estimates and CIs are largely overlapping, even though we cannot rule out a slight inflation of the estimates in the second period. Additional sensitivity analyses, based on the standard SCCS model, showed an inflation of the estimates, with the exception of the first analysis (standard SCCS beginning observation at exposure) usually used as an alternative approach to the modified SCCS method (S17 Table).

The ancillary analysis on 2,050 cases aged over 40 years (BNT162b2 n. 1,759; mRNA-1273 n. 291) did not show an increase risk of myocarditis/pericarditis for BNT162b2 and mRNA-1273 after 7 days following the first dose (RI = 0.59, 95% CI: 0.42 to 0.82 and RI = 0.56, 95% CI: 0.23 to 1.36) and the second dose (RI = 0.84, 95% CI: 0.61 to 1.16 and RI = 1.11, 95% CI: 0.57 to 2.17) (S18 Table).

Discussion

Principal findings

This first Italian large population-based study covering about 3 million of vaccinated persons aged 12 to 39 years found an association between myocarditis/pericarditis within a week following each dose of mRNA vaccines.

The risk of myocarditis/pericarditis is particularly higher after 7 days following the first or second dose of mRNA-1273 vaccine in the overall population. Subgroup analysis by sex suggested that the increased risk was present only in males after both the first and second dose with 3.8 and 8.8 EC per 100,000 vaccinated, respectively. Stratifying by age, greater risks were found in those aged 18 to 29 years with EC of 3.4 and 8.6 per 100,000 following the first and the second doses, respectively. In the age group 12 to 17 years, the number of events was insufficient for risk estimate.

We also observed an association between BNT162b2 and myocarditis/pericarditis, but only in the 7 days following the second dose, where the risks remain similar between males and females with 1.0 and 0.7 EC per 100,000 vaccinated, respectively. In the age groups 12 to 17 years and 18 to 29 years, where the increased risks were confined, the estimated EC were 1.7 and 1.1 per 100,000 vaccinated, respectively.

Vaccine-associated acute myocarditis is generally attributable to allergic/hypersensitivity reactions as observed in other vaccines [32]. However, the pathophysiology of myocarditis and pericarditis associated to mRNA vaccines is not clearly understood and different mechanisms have been postulated. Molecular mimicry between the spike protein and self-antigens [33], trigger of preexisting dysregulated immune pathways, immune response to mRNA [34] or dysregulated cytokine expression [35] have been proposed.

Our results on the increased risk in the 7 days after each dose of mRNA-1273 and the second dose of BNT162b2 are consistent with the onset of viral myocarditis symptoms often reported in the first week from the infection [32,36].

Moreover, it has been postulated that a very high antibody response to mRNA vaccines in predisposed young people may elicit an uncontrolled inflammatory response similar to multisystem inflammatory syndrome observed in children (MIS-C) with SARS-CoV-2 infection [37]. To date, no clear evidence is available, and further studies are needed to clarify which is the exact mechanism of mRNA vaccines-related myocarditis and pericarditis.

Furthermore, our observation on the increased risk in young males resembles classical epidemiological features of myocarditis due to other causes [38], included COVID-19-related myocarditis [39], but the exact role of age and sex is still unclear. In a recent review, a possible effect of sex hormones in immune response is summarized, with a possible role of testosterone by a combined mechanism of inhibition of anti-inflammatory cells and commitment to a Th1-type immune response in male and of inhibitory effects of estrogen on proinflammatory T cells in female [40].

Comparison with related studies

In line with a previous US study [15], we identified an association between mRNA vaccines and myocarditis/pericarditis in individuals younger than 40 years within the 0- to 7-day period following the first and the second dose.

Our results are also consistent with observational studies that documented markedly increased risk of myocarditis in England [16] and myocarditis or myopericarditis in Denmark [18] in the population vaccinated with mRNA-1273. Specifically, in the Danish study, it was reported a strong association between mRNA-1273 and myocarditis or myopericarditis within 28 days from vaccination (hazard ratio, HR = 5.24; 2.47 to 11.12) with an estimated 5.7 EC per 1,000,000 vaccinated. The UK study also suggested a strong association within the 1 to 28 days after first and second mRNA-1273 dose (incidence rate ratio, IRR = 3.89; 1.60 to 9.44; and 20.71, 4.02 to 106.68; respectively) corresponding to 8 and 15 EC per 1,000,000 vaccinated [16]. A recent updated SCCS analysis of English data (preprint publication) [17], stratified by age and sex, also reported a higher risk in male aged less than 40 years (first dose IRR = 2.34; 1.03 to 5.34; second dose IRR = 16.52; 9.10 to 30.00) corresponding to EC of 12 and 101 per 1,000,000, respectively; a markedly increased risk was also observed in females after the second dose of mRNA-1273 (IRR = 7.55; 1.67 to 34.12) with 8 EC per 1,000,000 vaccinated [17]. Our findings are in line with a higher risk observed with mRNA-1273 vaccine in a recently published study conducted in Denmark, Finland, Norway, and Sweden [41].

Results on the association between BNT162b2 and myocarditis/pericarditis are less conclusive. We found an association in the 7 days after the second dose both in males and females. Findings from Israel [14] and England [17] confirmed an association in adolescent and adult males younger than 40 years, but not in female participants. Particularly, the English study, including data on the third dose of BNT162b2, highlighted that in males 12 to 39 years, the risk sequentially increased following each dose of vaccine (IRR = 1.66, 3.41, and 7.60, respectively) with an EC of 3, 12, and 13 per 1,000,000 vaccinated, respectively. No association was found in females and in males older than 40 years [17].

Conversely, a population-based study conducted in Denmark [18], with a more stringent case definition, did not support the association between BNT162b2 and myocarditis or myopericarditis in the 28 days after vaccination, both overall and in the 12 to 39 year group, but an association only in females (HR = 3.73; 1.82 to 7.65) was found.

Strengths and limitations

Our study strengths include the large sample size, the broad geographical distribution of the cohort and the availability of data on COVID-19 vaccination and outcomes, and comorbidities and patients’ demographic characteristics from healthcare databases. The large sample size (about 3 million vaccinated people aged 12 to 39 years) allowed us to look at fine risk intervals following vaccinations and conduct several subgroup analyses. Since data were collected from routinely collected data in the claims databases, irrespective of research question, there is no potential for recall or selection bias.

An interesting methodological point of our study is the choice of the SCCS method modified to handle event-dependent exposures. In a very recent paper on the use of the SCCS method with application to COVID-19 vaccine safety [25], the authors quantify, by simulation, the overestimation of risk in the standard SCCS method when vaccination is severely delayed or canceled after the occurrence of an event. They argue that when vaccination is only delayed by a short time, this bias may be corrected within the standard SCCS methodology by including a prevaccination risk period. Instead, the modified SCCS model for event-dependent exposures needs to be applied when vaccination may be severely delayed or canceled entirely. In addition, they discuss the usefulness of including unvaccinated cases in the analysis, this inclusion is deemed to be necessary to avoid once again overestimation of the risk estimates. This was not possible in our study since the surveillance collected data only on vaccinated cases. However, it is also shown in the paper that the presence of an appreciable proportion of vaccinated cases for whom the event occurs before the first vaccine dose, mitigated this effect. In their simulation, where the above proportion was about 50%, the overestimation of risk was only approximately 10%. In our study this proportion is 48%, and a slight inflation of the relative risk cannot be excluded. It is reassuring that the sensitivity analysis (S17 Table) in which the observation time began at first or second vaccination dose in a standard SCCS method gave similar estimates to our main analysis.

However, our study also has limitations. First, there is the possibility of notoriety bias due to overdiagnosis of cases of myocarditis/pericarditis because of the increased public and medical awareness of these potential adverse events following mRNA vaccination. The effect observed in the sensitivity analysis could be partly explained by a different age profile and characteristics of the 2 vaccinated population before and after 31 May 2021. Second, diagnoses of myocarditis and pericarditis were retrieved from hospital discharge and emergency care visit databases, and they were not validated through the review of clinical records. For this reason, a misclassification of the outcomes that biased the association cannot be excluded. Third, we did not collect further information to assess the severity of the outcomes. To date, data were collected without differentiating between emergency care admission and hospital admission, and length of the hospitalization was not available. Lastly, although the large sample size including about 3 million of vaccinated persons, due to the small number of events, it was not possible to provide robust model estimates in some subgroup analyses (i.e., mRNA-1273 in the subgroup of adolescents aged 12 to 17 years).

In conclusion, this population-based study suggests that mRNA vaccines were associated with myocarditis/pericarditis in the population younger than 40 years. According to our results, the risk increased after the second dose of BNT162b2 vaccine and after both doses of mRNA-1273 vaccine. The highest risks were observed in males of 12 to 39 years and in males and females of 18 to 29 years of age vaccinated with mRNA-1273 vaccine. However, vaccine-associated risks should always be evaluated in the light of the proven vaccine effectiveness in preventing serious COVID-19 disease and death. After the evaluation of all data available, the Italian Medicines Agency (AIFA) continued to consider a positive benefit-risk profile of mRNA COVID-19 vaccines in this population.

Further monitoring of data from this active surveillance is needed to evaluate the relationship between mRNA vaccines and myocarditis/pericarditis by age within sex, including population of children (5 to 11 years old) and the effect of the third dose (booster dose).

RECORD-PE Checklist.

(PDF)

Click here for additional data file.

Observational studies on safety of COVID-19 mRNA vaccines and myocarditis and/or pericarditis outcomes.

(DOCX)

Click here for additional data file.

Characteristics of mRNA-vaccinated population aged 12–39 years (n. 2,861,809) from 27 December 2020 to 30 September 2021, by vaccine product.

n., number; yrs, years; COPD, chronic obstructive pulmonary disease; HIV, human immunodeficiency virus; NSAIDs, nonsteroidal anti-inflammatory drugs.

(DOCX)

Click here for additional data file.

Definition of study comorbidities.

ATC, Anatomical Therapeutic Chemical Classification System; COPD, chronic obstructive pulmonary disease; HIV, human immunodeficiency virus; ICD, International Classification of Disease; NSAID, nonsteroidal anti-inflammatory drugs.

(DOCX)

Click here for additional data file.

RI estimated by SCCS by vaccine product and risk intervals: 346 myocarditis/pericarditis events in the BNT162b2 and 95 events in the mRNA-1273 vaccinated population aged 12–39 years from 27 December 2020 to 30 September 2021.

(DOCX)

Click here for additional data file.

RI estimated by SCCS in the [0–7) risk period after mRNA vaccination in the vaccinated population aged 12–39 years from 27 December 2020 to 30 September 2021 by sex, age group, and vaccine product.

*Considering the small number of cases, it was not possible to provide any estimates. CI, confidence interval; n., number; Ref., reference period (unexposed baseline period); RI, relative incidence.

(DOCX)

Click here for additional data file.

Adjusted RI estimated by SCCS and excess cases per 100,000 vaccinated by risk intervals: 232 myocarditis and/or pericarditis events in the BNT162b2 vaccinated males aged 12–39 years from 27 December 2020 to 30 September 2021.

*Adjusted by calendar period. **Excess cases are not given when the 95% CI of RI included the null effect. CI, confidence interval; n., number; Ref., reference period (unexposed period); RI, relative incidence; SCCS, self-controlled cases series.

(DOCX)

Click here for additional data file.

Adjusted RI estimated by SCCS and excess cases per 100,000 vaccinated by risk intervals: 114 myocarditis and/or pericarditis events in the BNT162b2 vaccinated females aged 12–39 years from 27 December 2020 to 30 September 2021.

*Adjusted by calendar period. **Excess cases are not given when the 95% CI of RI included the null effect. CI, confidence interval; n., number; Ref., reference period (unexposed period); RI, relative incidence; SCCS, self-controlled cases series.

(DOCX)

Click here for additional data file.

Adjusted RI estimated by SCCS and excess cases per 100,000 vaccinated by risk intervals: 46 myocarditis and/or pericarditis events in the BNT162b2 vaccinated population aged 12–17 years from 27 December 2020 to 30 September 2021.

*Adjusted by calendar period. **Excess cases are not given when the 95% CI of RI included the null effect. CI, confidence interval; n., number; Ref., reference period (unexposed period); RI, relative incidence; SCCS, self-controlled cases series.

(DOCX)

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Adjusted RI estimated by SCCS and excess cases per 100,000 vaccinated by risk intervals: 154 myocarditis and/or pericarditis events in the BNT162b2 vaccinated population aged 18–29 years from 27 December 2020 to 30 September 2021.

*Adjusted by calendar period. **Excess cases are not given when the 95% CI of RI included the null effect. CI, confidence interval; n., number; Ref., reference period (unexposed period); RI, relative incidence; SCCS, self-controlled cases series.

(DOCX)

Click here for additional data file.

Adjusted RI estimated by SCCS and excess cases per 100,000 vaccinated by risk intervals: 146 myocarditis and/or pericarditis events in the BNT162b2 vaccinated population aged 30–39 years from 27 December 2020 to 30 September 2021.

*Adjusted by calendar period. **Excess cases are not given when the 95% CI of RI included the null effect. CI, confidence interval; n., number; Ref., reference period (unexposed period); RI, relative incidence; SCCS, self-controlled cases series.

(DOCX)

Click here for additional data file.

Adjusted RI estimated by SCCS and excess cases per 100,000 vaccinated by risk intervals: 70 myocarditis and/or pericarditis events in the mRNA-1273 vaccinated males aged 12–39 years from 27 December 2020 to 30 September 2021.

*Adjusted by calendar period. **Excess cases are not given when the 95% CI of RI included the null effect. CI, confidence interval; n., number; Ref., reference period (unexposed period); RI, relative incidence; SCCS, self-controlled cases series.

(DOCX)

Click here for additional data file.

Adjusted RI estimated by SCCS and excess cases per 100,000 vaccinated by risk intervals: 25 myocarditis and/or pericarditis events in the mRNA-1273 vaccinated females aged 12–39 years from 27 December 2020 to 30 September 2021.

*Adjusted by calendar period; **excess cases are not given when the 95% CI of RI included the null effect. CI, confidence interval; n., number; Ref., reference period (unexposed period); RI, relative incidence; SCCS, self-controlled cases series.

(DOCX)

Click here for additional data file.

Adjusted RI estimated by SCCS and excess cases per 100,000 vaccinated by risk intervals: 11 myocarditis and/or pericarditis events in the mRNA-1273 vaccinated population aged 12–17 years from 27 December 2020 to 30 September 2021.

*Adjusted by calendar period. **Excess cases are not given when the 95% CI of RI included the null effect. §Considering the small number of cases in this age group, it was not possible to provide any estimates. CI, confidence interval; n., number; Ref., reference period (unexposed period); RI, relative incidence; SCCS, self-controlled cases series.

(DOCX)

Click here for additional data file.

Adjusted RI estimated by SCCS and excess cases per 100,000 vaccinated by risk intervals: 57 myocarditis and/or pericarditis events in the mRNA-1273 vaccinated population aged 18–29 years from 27 December 2020 to 30 September 2021.

*Adjusted by calendar period. **Excess cases are not given when the 95% CI of RI included the null effect. CI, confidence interval; n., number; Ref., reference period (unexposed period); RI, relative incidence; SCCS, self-controlled cases series.

(DOCX)

Click here for additional data file.

Adjusted RI estimated by SCCS and excess cases per 100,000 vaccinated by risk intervals: 27 myocarditis and/or pericarditis events in the mRNA-1273 vaccinated population aged 30–39 years from 27 December 2020 to 30 September 2021.

*Adjusted by calendar period. **Excess cases are not given when the 95% CI of RI included the null effect. CI, confidence interval; n., number; Ref., reference period (unexposed period); RI, relative incidence; SCCS, self-controlled cases series.

(DOCX)

Click here for additional data file.

Sensitivity analyses.

*Adjusted by calendar period. **Only participants vaccinated and with event in each period were included in this analysis. ***Considering the small number of cases, it was not possible to provide any estimates. CI, confidence interval; SCCS, self-controlled cases series.

(DOCX)

Click here for additional data file.

Sensitivity analyses: standard SCCS method.

*Adjusted by calendar period. CI, confidence interval; SCCS, self-controlled cases series.

(DOCX)

Click here for additional data file.

Adjusted RI estimated by SCCS and excess cases per 100,000 vaccinated by risk intervals: 1,759 myocarditis and/or pericarditis events in the BNT162b2 and 291 events in the mRNA-1273 vaccinated population aged ≥40 years from 27 December 2020 to 30 September 2021 (ancillary analysis).

*Adjusted by calendar period. **Excess cases are not given when the 95% CI of RI included the null effect. CI, confidence interval; n., number; Ref., reference period (unexposed period); RI, relative incidence; SCCS, self-controlled cases series.

(DOCX)

Click here for additional data file.

Diagram showing the data flow when using TheShinISS to locally process healthcare data structured according to a CMD.

CDM, common data model.

(DOCX)

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Schematic presentation of the SCCS method.

SCCS, self-controlled cases series.

(DOCX)

Click here for additional data file.

Flow chart of study population.

(DOCX)

Click here for additional data file.

Adjusted RI in the [0–7) risk period after mRNA vaccination in the vaccinated population aged 12–39 years from 27 December 2020 to 30 September 2021 by vaccine product, sex, and age group.

(*) Considering the small number of cases in the vaccinated with mRNA-1273 of age 12–17 years, it was not possible to provide any estimates. CI, confidence interval; F, females; M, males; RI, relative incidence.

(DOCX)

Click here for additional data file.

8 Mar 2022

Dear Dr Morciano,

Thank you for submitting your manuscript entitled "Post-marketing active surveillance of myocarditis and pericarditis following vaccination with COVID-19 mRNA vaccines in persons aged 12-39 years in Italy: a multi-database, self-controlled case series study" for consideration by PLOS Medicine.

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26 Apr 2022

Dear Dr. Morciano,

Thank you very much for submitting your manuscript "Post-marketing active surveillance of myocarditis and pericarditis following vaccination with COVID-19 mRNA vaccines in persons aged 12-39 years in Italy: a multi-database, self-controlled case series study" (PMEDICINE-D-22-00715R1) for consideration at PLOS Medicine.

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Requests from the editors:

The Data Availability Statement (DAS) requires revision. If the data are not freely available, please describe briefly the ethical, legal, or contractual restriction that prevents you from sharing it. Please also include an appropriate contact (web or email address) for inquiries (this cannot be a study author).

Throughout, please consider using an alternative term instead of ‘brand’ to reflect the non-proprietary presentation of the vaccines e.g. product/type.

Abstract Methods and Findings:

* Please ensure that all numbers presented in the abstract are present and identical to numbers presented in the main manuscript text.

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I could not locate the RECORD-PE checklist? Please check this was included and cite in your Methods (S1 Checklist).

When completing the checklist, please use section and paragraph numbers, rather than page numbers.

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Comments from the reviewers:

Reviewer #1: See attachment

Michael Dewey

Reviewer #2: Myocarditis/pericarditis in relation to mRNA vaccinations has been a topic of interest lately due to ongoing concerns of an association. This study, along with others based in different countries, found significant associations between mRNA vaccinations and this event. This paper in particular finds an association [0-7) days after vaccination for mRNA-1273 following both doses and BNT162b2 following the second dose. This study is of interest as it supports findings found in other countries but uses self-controlled case series adapted to handle event-dependent exposures. This is a well-written study, but I have a few suggestions that I think would improve the paper and that I ask the authors to consider:

1. An important assumption in this study is that there are event-dependent exposures. The authors mention that they conduct a subanalysis using SCCS standard, starting the observation time at the first and second dose. I assume that this means that all time before vaccination is not considered in the SCCS analysis, and the days following the 21-day risk period are the reference period. While I agree that there could be bias introduced due to a delayed vaccination following an event, I do not think that removing time prior to vaccination in the standard SCCS method and comparing it to your main results is a sufficient example in itself of why the modified SCCS model should be used. I would recommend doing a plot similar to Figure 1, but include time before the first vaccination as well so that it can be investigated whether there is a visible "dip" in events just prior to vaccination. If so, introducing a pre-risk period is justified. Long event delays or preclusions of the exposure will not be visible on this plot, but doing the standard SCCS analysis without the exclusion of this time as a sort of sensitivity analysis to see whether the results are heavily biased by the event-dependent exposure assumption could aid in this investigation. If the event delays or precludes the exposure, then the relative incidence will be biased upwards. Alternatively, (Farrington CP, Whitaker H, Weldeselassie YG. Self-Controlled Case Series Studies. A Modelling Guide with R. CRC Press, 2018) recommends removing post-event exposures in a standard SCCS analysis in order to display this bias (pg. 237-239).

2. I do not follow the conclusion "According to our results the risk increased after the second dose and in the youngest for both vaccines, remained moderate following vaccination with BNT162b2, while was higher in males following vaccination with mRNA-1273."

a. The youngest age group (12-17) did not have any estimates for mRNA-1273, so how can the risk be increased in the youngest for both vaccines?

b. I would also use the phrase "was not statistically significant" instead of "moderate" when discussing RI.

c. In general, this sentence is a bit hard to follow. Consider breaking it up into two.

3. As you reference later in the paper, different countries found varying results per dose when receiving the different vaccine brands. Therefore, I would be cautious assuming that heterologous vaccinations essentially do not exist in the primary analysis. Is this a reasonable assumption to make following the Italian vaccination plan that many do not get cross-vaccinated? I would report the results of the analysis where these individuals have been censored when they received a different vaccine brand as the main analysis to avoid the effect mixing the brands would have.

4. This comment is regarding Table S12: Sensitivity Analysis: c) Heterologous vaccination (II). If it is correct that only one person's time is censored here (and it is a person who got an mRNA-1273 to BNT162b2 combination) and the rest of the analysis remains the same, why does this change the number of cases in BNT162b2 such that it is no longer estimable? Should it not be the same number of cases? Please recheck this analysis or explain the analysis in more detail.

5. Using the time after the exposure period for a reference period allows for more cases and power to your study instead of just using the time prior to vaccination as the reference period. However, performing a sensitivity analysis on the length of the exposure period is, therefore, more important as it affects your reference period. For that I have two comments:

a. Have you preformed any sensitivity analyses using this risk period (either shortening or extending the length) justifying your choice of risk period? Other countries listed in your discussion have a risk period of 28-days.

b. I would recommend adding a sensitivity analysis either where you increase the number of days in your risk period or exclude some time shortly following your risk period so that you can investigate whether your unexposed period remains unbiased from potential delayed reported events related to your exposure.

6. A schematic illustration of the time windows used in the SCCS analysis would benefit the reader. This could potentially be put in as a supplementary plot and referenced to in "Statistical analysis" or "Definition of exposures".

Minor comments:

* Line 186: SCSS -> SCCS

* Table 1: Please format the table so that the groupings of characteristics are more visible and the group-wise percentages are easier to see.

* Fig 1: Please change the heading of the figure to BNT162b2 and mRNA-1273 to match the rest of the paper.

* Lines 317-318: The statement in these lines is correct for mRNA-1273, but only after the second dose for Pfizer. Consider rephrasing.

* Line 338: "also reported an higher risk in male" -> "also reported a higher risk in males"

* Lines 355-356: This sentence does not make sense. Consider rephrasing. "The large sample size (about 3 million vaccinated people aged 12-39 years) allowed us to look at fine risk intervals following vaccination and conduct several subgroup analyses."

* Lines 380-383: This sentence does not make sense. Consider rephrasing.

* Line 389: Remove "s" in "conclusions".

Reviewer #3: Massari et al. estimate the increased risk for myocarditis and pericarditis following receipt of mRNA Covid-19 vaccines. The study is interesting, but I do have concerns that merit addressing.

Note: The system describes this as a revision. I was not involved in the original submission and know nothing of previous comments. I therefore review this as I would review the original submission.

== Major ==

More needs to be said about the quality of the data, particularly as it synthesizes various sources. Are all sources guaranteed to be complete? If not, why and how incomplete? How long is the history for each person? How complete is the follow-up? Etc.

In the introduction, the authors should explicitly state the scientific gap they aim to feel. From what I understand, the relative risk has already been quantified for these two vaccines, including by age group and sex. What then is the goal of the study?

I do not have intimate knowledge of the SCCS methodology, and particularly not of its extension to situations in which exposure depends on past events (like it does here, as past myocarditis lowers the probability of vaccination). This is a critical aspect of this study, and others more knowledgeable should be sought to review it, as many questions arise including issues of confounding by calendar time, inclusion only of vaccinated (e.g., why isn't information on the unvaccinated used? Does this not result in bias?), etc.

In the same context, While I hesitate to suggest a different analysis and I understand the within-person advantage of SCCS, but considering that other than previous myocarditis, there aren't known strong confounders for the vaccination => myocarditis effect, why not use a standard cohort design with a modified Poisson model? This is even more warranted when considering that the authors acknowledge that not including unvaccinated persons could lead to bias.

The authors should avoid language that stems from statistical significance. E.g., saying that no association was found between the first 7 days after the first dose of the Pfizer vaccine and the outcome or that "increased risk was present only in…". The reason is simple - if statistical significance is evoked, then all its tenets must be adhered to, including adjusting for multiple comparisons, which was not performed here. A simple reporting of the CI, then, would suffice.

== Minor ==

The abstract result section is overly detailed. Should only be overall effects per each vaccine and maybe some specific interesting subgroup results.

The conclusion in the abstract "This population-based study suggested that mRNA vaccines were associated with myocarditis/pericarditis in the population younger than 40 years, whereas no association was observed in older subjects" is weird, considering that this study is limited to ages 12-39. Later I see that this was a secondary analysis. Its results then, should not appear in the abstract.

Persons with missing data were excluded. Why? How many were there? A population flow chart should be included and the number of missing mentioned.

The authors dedicate Table 1 to all those vaccinated with mRNA vaccines. This is not warranted. The study population in a SCCS design are those that experienced an event and were included. The complete population should only be mentioned in the context of the population flow chart (which I do not see in this submission). Similarly, mentioning the full population as a strength does not seem to make sense.

The structure of Table 2 is not optimal. The stratification by age makes the table hard to understand and does not seem helpful.

The effect estimate of interest is called "relative incidence" throughout. It would be preferable to use the more informative and accurate "incidence rate ratio".

The strengths section is better removed. The merit of a study should be evident from the reporting and not specifically mentioned. Moreover, most of this section is devoted to what is actually a limitation - the exclusion of unvaccinated individuals.

Reviewer #4: See attached file

Any attachments provided with reviews can be seen via the following link:

[LINK]

Submitted filename: massari.pdf

Click here for additional data file.

Submitted filename: Italian paper review.docx

Click here for additional data file.

26 May 2022

Submitted filename: Massari_ResponseReviewer PLoSM (26.05.2022).docx

Click here for additional data file.

15 Jun 2022

Dear Dr. Morciano,

Thank you very much for re-submitting your manuscript "Post-marketing active surveillance of myocarditis and pericarditis following vaccination with COVID-19 mRNA vaccines in persons aged 12-39 years in Italy: a multi-database, self-controlled case series study" (PMEDICINE-D-22-00715R2) for review by PLOS Medicine.

I have discussed the paper with my colleagues and the academic editor and it was also seen again by three reviewers. I am pleased to say that provided the remaining editorial and production issues are dealt with we are planning to accept the paper for publication in the journal.

The remaining issues that need to be addressed are listed at the end of this email. Please take these into account before resubmitting your manuscript:

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We look forward to receiving the revised manuscript by Jun 22 2022 11:59PM.

Sincerely,

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PLOS Medicine

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------------------------------------------------------------

Requests from Editors:

Lines 60-61: Please update to ‘The main study limitations were that the outcome was not validated through review of clinical records, and there was an absence of information on the length of hospitalization and, thus, the severity of the outcome.’

Line 163: Please specify the variables that were considered relevant.

Line 195: Please update to ‘we extended the exposure period to 28 days as well reducing it to 14 days.’

Line 200: Please describe these further sensitivity analyses.

Line 204: Please update to ‘This study is reported as per the REporting of studies Conducted using Observational Routinely-collected Data for Pharmacoepidemiology (RECORD-PE) checklist (S1 Checklist).’

Please update your RECORD-PE checklist to use section names and paragraph numbers rather than page/line numbers (these will either change or not be present in the final published version).

Line 215: Please include the actual number of missing observations as well as the proportion.

Line 269: Please update to ‘Of note, there were…’

Line 280-281: Please include the 95% CIs.

Line 386: Please update to ‘It is reassuring that the sensitivity analysis (S17 Table) in which the observation time began at first or second vaccination dose in a standard SCCS method gave similar estimates to our main analysis.’

Line 391: ‘Such bias is probably minimal’ – please either provide a reference to support this claim or remove it.

For Internet references (e.g. reference 8), please include the data accessed.

Please ensure all Supporting Information items (e.g. Supporting Tables/Figures) are cited in the main text.

To help us extend the reach of your research, please provide any Twitter handle(s) that would be appropriate to tag, including your own, your coauthors’, your institution, funder, or lab. Please respond to this email with any handles you wish to be included when we tweet this paper.

Comments from Reviewers:

Reviewer #1: The authors have addressed all my points.

Michael Dewey

Reviewer #2: Thank you for including my suggested changes and analyses to the article.

I have no further comments. I recommend this article be accepted.

Reviewer #3: The bulk of my comments were answered in a satisfactory manner.

My most important comment was regarding the need for expert evaluation of the SCCS methodology. It seems that this was now done by a key developer of said methodology.

I have nothing further to add.

Because in my opinion, the decision for this paper should hinge mostly on methodological considerations, and because I am not an expert in said methodology, I defer to the recommendation of more knowledgeable reviewers.

20 Jun 2022

Submitted filename: Massari_ResponsEditor PLoSM (20.06.2022).pdf

Click here for additional data file.

21 Jun 2022

Dear Dr. Morciano,

Thank you very much for re-submitting your manuscript "Post-marketing active surveillance of myocarditis and pericarditis following vaccination with COVID-19 mRNA vaccines in persons aged 12-39 years in Italy: a multi-database, self-controlled case series study" (PMEDICINE-D-22-00715R3) for review by PLOS Medicine.

There are a few minor remaining issues that need to be addressed before we can proceed to acceptance - these are listed at the end of this email. Please take these into account before resubmitting your manuscript.

As before, when revising the manuscript, please ensure you address the specific points made by each reviewer and the editors. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments and the changes you have made in the manuscript. Please submit a clean version of the paper as the main article file. A version with changes marked must also be uploaded as a marked up manuscript file.

Given the relatively minimal comments, we hope to receive your revised manuscript within 2 days. Please email me(cdavidson@plos.org) if you foresee any problems with this deadline. Otherwise, we look forward to receiving the revised manuscript by Jun 23 2022 11:59PM.

Sincerely,

Callam Davidson,

Associate Editor

PLOS Medicine

plosmedicine.org

------------------------------------------------------------

Requests from Editors:

Thank you for providing your Author Summary. I have reviewed and suggested updated wording below. Please carefully review this wording and confirm that it is a) scientifically accurate and b) presents the key findings in a manner that you deem appropriate. Feel free to edit if you feel anything could be clearer.

Author summary

Why was this study done?

* Pharmacovigilance reports and observational studies have suggested an increased risk of myocarditis/pericarditis following the COVID-19 mRNA vaccine administration in people younger than 40 years.

* More information on the safety of COVID-19 mRNA vaccines is needed to further explore the relationship between mRNA vaccines and myocarditis/pericarditis in this population.

What did the researchers do and find?

* We conducted a multiregional self-controlled case series study in Italy between 27 December 2020 - 30 September 2021 to investigate the association between myocarditis/pericarditis and COVID-19 mRNA vaccines in the population aged 12-39 years (n=2,861,809).

* We found 441 myocarditis/pericarditis cases, 114 of which occurred within the 21-day risk interval after vaccination.

* Within the 21-day risk interval, the relative incidence (RI) was 1.99 (95% confidence interval [CI] 1.30-3.05) after the second dose of BNT162b2 and 2.22 (1.00-4.91) and 2.63 (1.21-5.71) after the first and second doses of mRNA-1273, respectively. Within the 0–7-day risk interval, the RI was 6.55 (2.73-15.72) after first dose of mRNA-1273, and 3.39 (2.02-5.68) and 7.59 (3.26-17.65) after the second doses of BNT162b2 and mRNA-1273, respectively.

* The highest risk was seen in males, 0-7 days after the first and second dose of mRNA-1273 (RIs of 12.28 (4.09-36.83) and 11.91 (3.88-36.53), respectively). After the second dose of mRNA-1273 in males, the excess of cases (EC) was 8.8 (4.9-12.9) per 100,000 vaccinated individuals.

What do these findings mean?

* Consistent with previous studies, the findings suggest that COVID-19 mRNA vaccines were associated with myocarditis/pericarditis in the population younger than 40 years.

* The results provide information that could be helpful for the continuous assessment of the post-marketing benefit/risk profile of the COVID-19 mRNA vaccines and should be considered within the context of the proven mRNA vaccine effectiveness in reducing COVID-19 morbidity and mortality.

Line 64: Please update 'According to our results the risk increased after second dose of BNT162b2 and after both doses of mRNA-1273' to 'According to our results, increased risk of myocarditis/pericarditis was associated with the second dose of BNT162b2 and both doses of mRNA-1273'.

Line 67 (abstract conclusions): Please change 'weighed' to 'considered'.

22 Jun 2022

Dear Dr Morciano,

On behalf of my colleagues and the Academic Editor, Dr James Beeson, I am pleased to inform you that we have agreed to publish your manuscript "Post-marketing active surveillance of myocarditis and pericarditis following vaccination with COVID-19 mRNA vaccines in persons aged 12-39 years in Italy: a multi-database, self-controlled case series study" (PMEDICINE-D-22-00715R4) in PLOS Medicine.

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To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

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