Biopsy-proven viral myocarditis: clinical and histological diagnostic and prognostic correlates from a large monocentric prospective cohort

Background: Myocarditis is an inflammatory disease of the myocardium diagnosed by established histological, immunological and immunohistochemical criteria. Molecular biology techniques, particularly quantitative real-time polymerase chain reaction (qPCR) on endomyocardial biopsy (EMB), are essential for identifying the infectious aetiology of the disease. Aims: Primary aims: 1. To characterize a large cohort of patients with biopsy-proven myocarditis and to determine the prevalence of viral genomes in EMB specimens; 2. To assess the prognostic value of a positive viral PCR result; 3. To investigate the correlation between PCR results on EMB, clinical presentation, and non-invasive diagnostic tools – with a particular focus on viral serology – in identifying patients with viral myocarditis. Secondary aims: 1. To evaluate the use of specific antiviral therapies in patients with positive viral PCR; 2. To explore the association between specific viral agents and clinical outcomes; 3. To examine the role of PVB19 in the pathogenesis of myocarditis. Materials and methods: This prospective longitudinal study includes consecutive patients with biopsy-proven viral myocarditis, diagnosed and followed up at the Cardioimmunology Outpatient Clinic of the University Hospital of Padua, between 1993 and 2025. All patients underwent qPCR analysis for viral genome detection. Patient data at diagnosis and follow-up were systematically collected using the REDCap database and compared using the Wilcoxon rank-sum test for continuous variables and the Chi-square or Fisher’s exact test for categorical variables, as appropriate. Results: The study included 343 patients (66% male, median age at diagnosis of 41 years) with biopsy-proven myocarditis, with a median follow up of 62.6 months (27.1-114.7 months). PCR testing on EMB specimens detected viral genomes in 74 patients (22%). The most frequently detected virus was PVB19 (42%, 31/74). Among patients with positive viral PCR, 11 received specific antiviral therapy (22%). No statistically significant differences in clinical presentation and non-invasive diagnostic tools were observed when comparing patients with positive versus negative viral PCR, those positive for PVB19 versus other viruses, or those with high versus low PVB19 viral loads. The findings did not demonstrate a statistically significant correlation between viral serology and the results of PCR test. PCR-positive patients had significantly lower probability of survival compared to PCR-negative patients (75% at 5 years vs 91%%, p = 0,0042). Finally, patients positive for enterovirus had a more favourable clinical course compared to those positive for other viruses. Conclusions: Within our prospective longitudinal cohort of biopsy-proven myocarditis patients, those with a positive viral PCR accounted for a substantial proportion of the study population (22%). The study showed that patients with a positive viral PCR exhibit higher mortality compared to those with a negative viral PCR. Furthermore, there were no distinctive clinical phenotypes, specific biomarker profiles, or characteristic imaging patterns capable of distinguishing the patients with viral myocarditis from those with virus-negative myocarditis. In addition, viral serology showed no correlation with PCR results on EMB, thus reinforcing the role of PCR on EMB as the gold standard for definitive diagnosis. The more favorable clinical course observed in patients positive for enterovirus highlights the importance of further investigating the pathogenetic mechanisms of individual viruses and their prognostic impact, in order to support the development of increasingly effective aetiological therapies. Furthermore, our data did not reveal any statistically significant differences between patients positive for PVB19 and those positive for other viruses. Finally, they did not provide evidence that a high PVB19 viral load is associated with a poorer clinical outcome.