Histopathological Evaluation of the Pericardium in CABG Patients With and Without Prior SARS-CoV-2 Infection: A Prospective Observational Study

Introduction

Throughout history, epidemics have affected and changed individual health status and the sociodemographic structure of societies [1]. The COVID-19 pandemic, which occurred between 2019 and 2023, stands out as one of the most important events of the 21st century [2–4]. Many unknowns, such as what kind of histopathological changes occur in different organs and tissues during and after SARS-CoV-2 infection, the extent and persistence of these changes, and the time it takes for them to return to normal, remain as questions that have entered and remained in our lives during and after the pandemic [5].

In open-heart surgeries, pericardial fibrosis often presents as a condition secondary to a prior viral infection or a systemic disease such as tuberculosis [6,7]. This pathology can adversely affect the success of the surgical procedure and impair postoperative recovery. The most obvious indicator of pericardial damage, fibrosis, and adhesion is direct visualization and histopathological evaluation. However, obtaining pericardial samples from patients is generally neither feasible nor cost-effective. CABG offers a unique opportunity, as pericardial tissue can be collected during surgery without additional risk to the patient. We hypothesized that we could address this question by analyzing samples taken from patients who had previously undergone cardiac surgery, including those who were and were not diagnosed with a history of PCR-confirmed SARS-CoV-2 infection.

Evidence has demonstrated that SARS-CoV-2 infection can be associated with acute and subacute cardiac damage, including acute myocardial injury, acute myocarditis, acute pericarditis, peripheral arterial embolism, and venous thrombosis [8]. While acute cardiac complications of COVID-19 are well documented [8], the potential chronic effects on the pericardium such as fibrosis or persistent immune cell infiltration remains unclear. Continuing with pericarditis, although it has been thought that many patients developed pericarditis during COVID-19, pathological evaluations regarding early and late effects have been limited. For this reason, the early and late effects of exaggerated systemic inflammation due to SARS-CoV-2 infection on the pericardium are not fully known.

The aim of this study was to determine whether a history of PCR-confirmed SARS-CoV-2 infection is associated with histopathological changes in pericardial tissue. Specifically, we investigated the frequency of pericardial inflammation, fibrosis, and immune cell infiltration (CD3, CD4, CD8, CD68) in patients undergoing elective isolated CABG.

Material and Methods

STUDY DESIGN:

This prospective cohort and assessor-blinded study was conducted in the Department of Cardiovascular Surgery and Department of Pathology at Samsun Education and Research Hospital between February 2023 and February 2024. The study was designed to investigate the presence of pericardial inflammation in patients undergoing isolated coronary artery bypass grafting (CABG) and its possible association with previous PCR-confirmed SARS-CoV-2 infection. The study protocol was approved by Samsun University Non-Interventional Clinical Research Ethics Committee (SÜKAEK 2023/1–10) and conducted in accordance with the principles of the Declaration of Helsinki. The study was retrospectively registered at ClinicalTrials.gov (NCT06952179). Written informed consent was obtained from all patients for participation in the study and for the collection, analysis, and publication of tissue samples.

PATIENT SELECTION AND GROUPING:

A total of 93 patients meeting the inclusion criteria were enrolled. Eligible patients were between the ages of 20 and 75 years, metabolically stable, and scheduled to undergo elective, first-time-isolated CABG. Patients with known pericardial disease, history of pericardial incision or trauma, reoperations, active infections, unstable metabolic states, renal or hepatic failure, autoimmune diseases, or indications for urgent surgery were excluded. Patients with at least 1 documented positive SARS-CoV-2 PCR test result within the past 2 years were classified as PCR-positive, while those without any recorded positive result during this period were considered PCR-negative. Asymptomatic individuals, suspected positive cases, those with threshold-level PCR positivity, and patients without clear clinical confirmation were excluded from the study. Baseline demographic variables, comorbidities, and vaccination status (all patients had received ≥3 doses of BNT162b2) are summarized in Table 1 and detailed in the Results section.

SAMPLE COLLECTION AND EVALUATION:

After standard anesthesia and appropriate surgical field preparation, a median sternotomy was performed. During the pericardiotomy following sternotomy, pericardial biopsy samples were obtained and immediately placed in isotonic solution. These samples were delivered to the pathology laboratory on the same day for histopathological examination. All patients underwent isolated multi-vessel coronary artery bypass grafting.

Samples were fixed in 10% buffered neutral formalin for 24 hours, followed by routine tissue processing. Four-micrometer-thick sections were obtained from paraffin blocks and stained with hematoxylin-eosin. Masson’s trichrome was used for fibrosis evaluation, while immunohistochemical staining with CD3 (T lymphocytes), CD4 (helper T cells), CD8 (cytotoxic T cells), and CD68 (macrophages/monocytes) was performed to assess inflammatory cells. A pathologist (SKS), blinded to all clinical and laboratory data as well as the SARS-CoV-2 status of the patients, evaluated all slides under a light microscope (Olympus BX53). Inflammation (acute, chronic, or chronic active), fibrosis, granulation tissue, neutrophil infiltration, vascular proliferation, vasculitis, mesothelial hyperplasia, erythrocyte extravasation, hemosiderin accumulation, fibrin deposition, calcification, and granuloma formation were assessed semiquantitatively (mild, moderate, or marked). CD68-positive histiocyte density was graded similarly. CD3, CD4, and CD8-positive inflammatory cells were quantitatively assessed at ×400 magnification in areas with the most intense inflammation, and categorized as follows mild (<10 positively stained cells per high-power field [HPF]), moderate (10–30 cells per HPF), or marked (>30 cells per HPF).

STATISTICS AND SAMPLE SIZE:

The sample size of 93 patients (70 PCR-negative, 23 PCR-positive) was determined based on patient availability during the study period. Post hoc power analysis using a chi-square test showed that the sample size provided >80% power (α=0.05, effect size=0.25) to detect a clinically significant difference in the primary outcome – chronic pericardial inflammation – between the 2 groups, given the observed proportions (54.3% in PCR-negative vs 30.4% in PCR-positive). Thus, the study was adequately powered for its main comparison. Continuous variables are presented as medians with interquartile ranges (IQRs) due to non-normal distribution.

The data were analyzed using SPSS version 22.0. The normality of distribution for continuous variables was assessed with the Shapiro-Wilk test, and it was determined that the data did not follow a normal distribution. Therefore, comparisons between 2 groups were performed using the Mann-Whitney U test. Relationships between categorical variables were analyzed using the chi-square (χ²) test. Yates’ continuity correction was applied where appropriate in 2×2 contingency tables. A P value of less than 0.05 was considered statistically significant.

Results

A total of 93 patients were included in the study, of whom 22 (24%) were female and 71 (76%) were male. All patients had received at least 3 doses of the BNT162b2 (BioNTech) vaccine in accordance with the national vaccination program. Preoperative SARS-CoV-2 PCR tests were negative in all cases to exclude active infection or long COVID-19. The mean age was 61.6 years, mean ejection fraction (EF) was 51.7%, the average preoperative hemoglobin level was 13.5 g/dL, and the mean serum creatinine level was 0.92 mg/dL. Comorbidities included diabetes mellitus in 59% of patients, pulmonary hypertension in 58%, and an ascending aorta diameter greater than 30 mm in 58% (Table 1).

Twenty-three patients (24.7%) had a documented history of PCR-confirmed COVID-19 within the previous 2 years. These individuals had undergone home isolation and symptomatic treatment. The mean interval between the positive PCR result and the date of surgery was 14.6 months. All surgeries were completed without intraoperative complications. The study was reported in accordance with the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines.

Demographic and clinical characteristics were compared between PCR-positive and PCR-negative groups (Tables 1, 2). A statistically significant association was found between SARS-CoV-2 PCR status and sex (χ2=9.885, P=0.002), with a higher proportion of males in the PCR-positive group. No significant associations were observed for pulmonary artery pressure, presence of diabetes mellitus, or ascending aortic diameter (P>0.05 for all).

Laboratory variables, including age, ejection fraction, hemoglobin, and creatinine levels, were also compared between groups. Since none of the variables met the assumption of normality based on the Shapiro-Wilk test, the Mann-Whitney U test was used. No statistically significant differences were found in any parameter (Table 2).

Histopathological evaluation revealed a statistically significant difference in chronic inflammation between the groups (χ2=3.943, P = 0.047). Chronic inflammation was significantly higher in the PCR-negative group (54.3% vs 30.4%; χ2=3.943, P=0.047, Cramer’s V=0.206) (Table 3, Figure 1). Other parameters, such as macroscopic congestion, vascular proliferation, mesothelial hyperplasia, erythrocyte extravasation, fibrin deposition, calcification, fibrosis, and extravascular neutrophilia, showed no significant differences between groups (P>0.05 for all) (Table 3, Figure 2).

Immunohistochemical analysis showed no significant differences between groups in CD3+ (T lymphocytes), CD4+ (helper T cells), CD8+ (cytotoxic T cells), or CD68+ (macrophages/monocytes) counts per high-power field (HPF) (P>0.05) (Table 4, Figure 3). Although mean values for all 3 markers were slightly lower in the PCR-positive group, the differences were not statistically significant.

Discussion

LIMITATIONS:

Although our hospital is a regional center and our clinic is a cardiac surgery unit, we serve a geographically limited population. It has been suggested that COVID-19 manifests more severely – or at least differently – in patients with certain genotypes. Therefore, our data may not be generalizable to all populations. Multicenter and multinational studies are needed to strengthen and validate our findings. The study group consisted specifically of patients who underwent CABG. While this allowed pericardial tissue sampling, broader, more heterogeneous populations would provide a more comprehensive understanding. Importantly, patients with clinically diagnosed pericarditis were excluded from this study, which may have underestimated the full spectrum of pericardial involvement following COVID-19. However, pericardial biopsy is not a routine or risk-free procedure – it can only be performed during surgery. Considering that pericardial involvement is not currently a pressing clinical issue in the general population, alternative sampling methods were not pursued. Another limitation is the small number of patients included. This was partly due to the opportunistic nature of tissue collection during surgery. Additionally, the post-COVID-19 intervals varied among patients, and it is possible that histopathological changes may have resolved over time.

CLINICAL RELEVANCE & FUTURE DIRECTIONS:

Clinically, our findings suggest that a history of COVID-19 does not increase the risk of chronic pericardial inflammation in CABG patients. This may provide reassurance for surgeons and clinicians when assessing surgical candidates with prior COVID-19. However, these results should be interpreted with caution and confirmed in larger multicenter studies including more diverse patient populations. Future research should also include long-term follow-up of patients diagnosed with pericarditis or myocarditis and those who undergo cardiac surgery after COVID-19.

Conclusions

In summary, although COVID-19 can cause pericardial inflammation and fibrosis in the acute phase, our results did not demonstrate any lasting histopathological damage linked to prior infection in this patient group. The higher prevalence of chronic pericardial inflammation in patients without a history of COVID-19 suggests that current assumptions regarding long-term pericardial sequelae should be reconsidered. This unexpected inverse finding contradicts common assumptions and highlights the need for further research into potential protective mechanisms. However, these findings should be interpreted very carefully due to the relatively small sample size and the exclusive focus on CABG patients. Larger, multicenter studies are required to confirm these findings.