Zhang H, Gu X, He W, Zhao SL, Cao ZJ. Epstein-Barr virus infection is an independent risk factor for surgery in patients with moderate-to-severe ulcerative colitis. World J Gastroenterol 2025; 31(16): 104758 [DOI: 10.3748/wjg.v31.i16.104758]
Corresponding Author of This Article
Zhi-Jun Cao, MD, PhD, Professor, Chief, Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, Shanghai Institute of Digestive Disease, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, No. 145 Middle Shandong Road, Shanghai 200001, China. caozj_renji@163.com
Research Domain of This Article
Gastroenterology & Hepatology
Article-Type of This Article
Retrospective Study
Open-Access Policy of This Article
This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Hui Zhang, Shu-Liang Zhao, Zhi-Jun Cao, Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, Shanghai Institute of Digestive Disease, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200001, China
Xi Gu, Wei He, Division of Pathology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200001, China
Co-corresponding authors: Shu-Liang Zhao and Zhi-Jun Cao.
Author contributions: Zhang H and Gu X participated in the conceptualization, data acquisition and analysis, and drafted the manuscript; He W participated in the analysis of data and drafted the manuscript; Zhao SL and Cao ZJ contributed to the conceptualization, supervision; and funding, and revised the article; All authors contributed to the article and approved the final version to be submitted. Zhang H was responsible for patient screening, enrollment, collection of clinical data, performed data analysis and prepared the first draft of the manuscript. Gu X conducted EBV and CMV testing and assessment of the endoscopic biopsy or surgical specimens and contributed to the data analysis and manuscript preparation. Both Zhang H and Gu X have made crucial and indispensable contributions towards the completion of the project and thus qualified as the co-first authors of the paper. Both Zhao SL and Cao ZJ have played important and indispensable roles in the study design, data interpretation and manuscript preparation as the co-corresponding authors. Zhao SL was instrumental and responsible for comprehensive literature search, data analysis and revised the manuscript. Cao ZJ applied for and obtained the funds for this research, conceptualized, designed, and supervised the whole process of the project and revised and submitted the manuscript. This collaboration between Zhao SL and Cao ZJ is crucial for the publication of this manuscript.
Supported by General Hospital Integrated Guidance Project of Shanghai Municipal Health Commission and Municipal Administration of Traditional Chinese Medicine (Phase I), No. ZXXT-202210; and "Science and Technology Innovation Action Plan" Medical Innovation Research Project of Shanghai Municipal Science and Technology Commission, No. 22Y11907900.
Institutional review board statement: This study was approved by the Ethics Committee of Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China (Ethical Committee No. LY2024-034-B, dated March 4, 2024).
Informed consent statement: The need for patient consent was waived due to the retrospective nature of the study.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Data sharing statement: No additional data are available.
Open Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Zhi-Jun Cao, MD, PhD, Professor, Chief, Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, Shanghai Institute of Digestive Disease, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, No. 145 Middle Shandong Road, Shanghai 200001, China. caozj_renji@163.com
Received: December 31, 2024 Revised: March 18, 2025 Accepted: April 14, 2025 Published online: April 28, 2025 Processing time: 117 Days and 10.8 Hours
Abstract
BACKGROUND
Epstein-Barr virus (EBV) infection of the intestinal mucosa is associated with surgical risk in ulcerative colitis (UC); however, the exact effect remains unclear.
AIM
To determine whether EBV infection can predict the need for colectomy and to develop a surgical risk predictive model.
METHODS
This was a single-center retrospective study of 153 patients with moderate-to-severe UC between September 2012 and May 2023. EBV-encoded small RNA (EBER) in situ hybridization and immunohistochemistry (IHC) were used for EBV testing and assessment. Cytomegalovirus (CMV) was detected by IHC. Logistic regression analysis was conducted to identify risk factors for colectomy and develop a predictive risk model.
RESULTS
EBER-positivity in the intestinal mucosa was present in 40.4% (19/47) and 4.7% (5/106) of patients in the surgery and non-surgery groups, respectively, with significant differences between the groups (P < 0.01, odds ratio = 13.707). The result of multivariate logistic regression revealed that age, EBV infection in the colonic mucosa, CMV infection in the colonic mucosa, and treatment with three or more immunosuppressive agents before admission were significant independent predictors of colectomy. A nomogram incorporating these variables demonstrated good discriminative ability, and exhibited good calibration and clinical utility. IHC showed that EBV-infected cells mainly included B and T lymphocytes in patients with high EBER concentrations.
CONCLUSION
EBV infection of the intestinal mucosa is a significant independent risk factor for colectomy in patients with moderate-to-severe UC. The nomogram model, which includes EBV infection, effectively predicts colectomy risk.
Core Tip: This retrospective single-center observational study investigated the effect of Epstein-Barr virus (EBV) infection of the intestinal mucosa on surgical risk in patients with moderate-to-severe ulcerative colitis (UC). Surgical treatment remains necessary for patients refractory to medical therapy or with severe complications in patients with moderate-to-severe UC. Our study determined that EBV infection of the intestinal mucosa is a significant independent risk factor for colectomy in patients with moderate-to-severe UC. In addition, we developed a new nomogram model including EBV infection to help predict colectomy risk in patients with moderate-to-severe UC.
Citation: Zhang H, Gu X, He W, Zhao SL, Cao ZJ. Epstein-Barr virus infection is an independent risk factor for surgery in patients with moderate-to-severe ulcerative colitis. World J Gastroenterol 2025; 31(16): 104758
Ulcerative colitis (UC), a major form of inflammatory bowel disease (IBD), is a chronic idiopathic inflammatory condition affecting the colon characterized by relapsing and remitting mucosal inflammation. UC typically begins in the rectum and extends to the proximal segments of the colon. The pathogenesis involves genetic predisposition, epithelial barrier defects, dysregulated immune responses, and environmental factors. Symptoms include bloody diarrhea, abdominal pain, urgency, fecal incontinence, increased frequency of bowel movements, and, in severe cases, fatigue, fever, and weight loss[1,2]. A systematic review of 147 population-based studies demonstrated that UC is more prevalent in Europe and North America. Although the incidence of UC in Western countries has remained stable or decreased, it has been increasing in newly industrialized countries in Africa, Asia, and South America since 1990[3].
Although the use of biologics and small-molecule drugs has improved the outcomes and prognosis of moderate-to-severe UC, surgical treatment remains necessary for patients refractory to medical therapy or with severe complications. The current 1-, 5-, and 10-year risks of colectomy are 2.8%, 7.0%, and 9.6%, respectively[4]. In hospitalized patients with acute severe UC (ASUC), short-term colectomy rates are 25%-30%[5]. However, determining the optimal timing for surgical intervention remains challenging[6]. Given that delayed surgery is associated with an increased risk of postoperative complications and worse outcomes[7-9], identifying risk factors or predictors of colectomy is crucial for timely surgical intervention. Various clinical, hematological, biochemical, radiological, endoscopic, and pharmacological factors have been identified as predictors of colectomy, including age, clinical course, albumin, hemoglobin, C-reactive protein (CRP), fecal calprotectin, colon dilatation, endoscopic score, Clostridioides difficile infection, and exposure to immunosuppressive agent[10-14].
Recent widespread use of immunosuppressive agents and biologics has increased the incidence of opportunistic infections in patients with IBD, with a notable increase in herpes virus infections such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV)[15]. CMV colitis is an independent risk factor for poor outcomes in patients with UC, and the association between CMV infection and the need for colectomy in UC patients has been extensively studied[11,16,17]. However, the impact of EBV colitis on surgical outcomes in UC remains unknown. Some studies have indicated that high concentrations of EBV-encoded small RNA (EBER) are associated with poor prognosis in UC[18,19]. Hosomi et al[20] identified the six most common types of human herpesviruses in the colonic mucosa of patients with IBD and found that the combined infection of CMV with EBV or humanherpesvirus-6 was a significant independent predictor of subsequent colectomy in patients with UC, whereas single CMV or EBV infections were not independent risk factors for colectomy.
In this study, both EBV and CMV were identified in the colonic mucosa using endoscopic biopsy or surgical specimens from the pathology department of our center. Additionally, data on common predictive factors, including clinical characteristics, laboratory tests, endoscopic manifestations, pathological findings, and treatment regimens, were collected. We aimed to identify the impact of EBV colitis on surgical risk in patients with moderate-to-severe UC and develop a new risk predictive model for colectomy.
MATERIALS AND METHODS
Patients and study design
This single-center, retrospective study screened patients who were hospitalized for moderate-to-severe UC at the Department of Gastroenterology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, between September 2012 and May 2023. This study was approved by the Ethics Committee of Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China (Ethical Committee No. LY2024-034-B, dated March 4, 2024).
Patients with moderate-to-severe UC who were either dependent on or refractory to corticosteroids, with Mayo Clinic scores of 6-12 or endoscopic subscores of 2 or 3, were analyzed[21]. The inclusion criteria were: (1) Diagnosis of moderate-to-severe UC based on clinical, endoscopic, radiological, and histological characteristics by experienced gastroenterologists; and (2) Patients who had undergone endoscopic biopsy or surgical treatment during admission with the availability of pathological specimens in our hospital. The exclusion criteria were: (1) Patients aged < 18 years; (2) Patients who refused to use immunosuppressive drugs and chose surgical treatment directly for personal reasons; (3) Patients who had undergone colectomy for intestinal adenoma or cancer; and (4) Patients in whom the diagnosis of UC was ruled out or could not be confirmed by postoperative pathology.
Clinical data collection
We retrospectively collected clinical data from the electronic medical database of our center, including general demographics (sex and age), clinical characteristics (including disease course, history of smoking, history of surgery, cardiovascular and cerebrovascular risk factors, history of malignant tumor, fever (temperature ≥ 37.3 °C), extraintestinal manifestations, complications, intestinal stenosis), laboratory tests [white blood cell count, hemoglobin, platelet count, CRP, erythrocyte sedimentation rate (ESR), albumin, prealbumin, infections (fungal infection)], endoscopic findings (UC endoscopic index of severity to determine endoscopic activity[22] and Nancy histological index to assess histological activity[23]), and treatment regimens (history of immunosuppressive therapy and antibiotic therapy with third-generation cephalosporins or more advanced antibiotics). All predictors were collected within 1 week of admission.
EBER in situ hybridization
Given its high sensitivity and precise cellular localization, EBER detection via in situ hybridization (EBER-ISH) is considered the gold standard[24]. Therefore, EBV infection of the intestinal mucosa was assessed using an EBER ISH kit (PB0589, Leica Biosystems Newcastle Ltd., Newcastle upon Tyne, United Kingdom). Paraffin-embedded histology specimens were sliced into 4 µm-thick sections. Prior to ISH, the slides were baked in a dry oven at 90 °C for 2 hours. The dried sections were processed in an automatic IHC and ISH apparatus (Leica BOND-III; Leica Biosystems Newcastle Ltd.). The sections were deparaffinized using Leica dewaxing solution three times, followed by rinsing with alcohol three times, and cleaning using concentrated solution four times. The specimens were then digested using Leica in situ hybridization protease K for 15 minutes and rinsed four times with a concentrated cleaning solution. The slides were incubated for 2 hours at room temperature with EBER probe solution. The slides were rinsed with concentrated cleaning solution 10 times, sealed with hydrogen peroxide for 5 minutes, and rinsed with concentrated cleaning solution three times. The slides were incubated for 15 minutes at room temperature with an anti-fluorescein antibody (AR0222; Leica Biosystems Newcastle Ltd.) and incubated for 8 minutes at room temperature after application of the post-primary reagent. After the application of the secondary antibody labeled with horseradish peroxidase (HRP) (BOND Polymer Refine Detection; Leica Biosystems Newcastle Ltd.) for 8 minutes at room temperature, the hybridization signals were tested using a 3,3-N-diaminobenzidine (DAB) tetrahydrochloride color development system (Leica Biosystems Newcastle Ltd.). Reliable EBER-positive staining was clearly indicated by brown-colored cell nuclei (nasopharyngeal carcinoma, which is known for its EBV-positivity, was used as the positive control).
Based on clinical experiences and previous studies[18,25], UC with superimposed EBV colitis was defined as more than two EBER-positive cells/high-power field (HPF) in the colonic mucosa. EBER-positive cases were further divided into two groups: Cases with low EBV concentrations (< 10/HPF) and high EBV concentrations (≥ 10/HPF).
EBV immunohistochemistry
To determine the phenotype of EBV-infected cells, we performed immunohistochemistry (IHC) following EBER-ISH on paraffin sections. We used CD3 antigen (Maixin Biotech. Co.; Ltd., Fuzhou, China) as T-cell-specific marker, CD20 (Agilent Technologies Inc., China) as B lymphocyte-specific marker, CD56 (Maixin Biotech) as NK-cell-specific marker, and cytokeratin (CK, Maixin Biotech) as an epithelial-cell-specific marker. After completion of EBER-ISH, the slides were rinsed with distilled water twice, repaired with PH6 citric acid to 100 °C for 20 minutes, and rinsed with concentrated cleaning solution five times. The primary antibody (CD3/CD20/CD56/CK) was added, and the slides were incubated for 15 minutes. The slides were rinsed three times with a concentrated cleaning solution, incubated at room temperature for 20 minutes after application of the post primary reagent, and rinsed three times with a concentrated cleaning solution. The polymer alkaline phosphatase (AP) labeled with AP (BOND Polymer Red Refine Detection) was added, and the slides were incubated for 30 minutes and then washed five times with concentrated cleaning solution and once with distilled water. The red color development system was added twice to detect the immunohistochemical signals. Positive staining appeared red in the cytoplasm and cell membrane.
CMV IHC
Tissue CMV IHC is considered the gold standard with high sensitivity and specificity[26]. Therefore, the presence of CMV infection in the intestinal mucosa was assessed using a CMV IHC kit (ZSGB-BIO; Ltd., Beijing, China). The detailed steps as follows. Paraffin-embedded histology specimens were sliced into 4 µm thick sections. Before dyeing, the slides were baked in a dry oven at 90 °C for 2 hours. The dried sections were placed in an automatic IHC and ISH apparatus (Leica BOND-III). The sections were deparaffinized using Leica dewaxing solution three times, rinsed with alcohol three times, and washed with concentrated cleaning solution three times. The slides were heated to 100 °C in a pH 9 alkali solution and incubated for 20 minutes. The slides were rinsed four times with a concentrated cleaning solution, sealed with hydrogen peroxide for 5 minutes, and rinsed three times with the concentrated cleaning solution. After adding the primary antibody for CMV, the slides were incubated for 20 minutes and rinsed five times with the concentrated cleaning solution. The slides were incubated for 8 minutes at room temperature and rinsed five times with a concentrated cleaning solution. After application of a secondary antibody labeled with HRP (BOND Polymer Refine Detection; Leica Biosystems Newcastle Ltd.) for 8 minutes at room temperature, immunohistochemical signals were detected using a DAB tetrahydrochloride color development system (Leica Biosystems Newcastle Ltd.). CMV antigen expression was assessed, and the number of CMV-positive cells per section was counted. High-grade CMV infection was defined as ≥ 5 IHC-positive cells per section, and low-grade CMV infection was defined as < 5 IHC-positive cells per section, based on previous studies[27,28].
Statistical analysis
Kolmogorov-Smirnov tests were used to determine the normality of continuous variables. Normally distributed variables were expressed as mean ± SD and as median and interquartile range when the normal distribution was not followed. Depending on the normality of the distribution, Student’s t test or Mann-Whitney U test was used for comparison between groups. Categorical variables were described as frequencies with percentages and compared using the χ2 test or Fisher’s exact test. Potential predictors were sequentially screened using univariate logistic analysis and presented as odds ratios (OR) with 95%CI. A stratified analysis was performed to minimize the effects of confounding factors when evaluating the impact of EBV colitis on surgical risk. Statistically significant variables in the univariate logistic analysis (P < 0.05) were included in the multivariate logistic analysis to develop a risk model. A visualizing nomogram was developed with independent risk factors from multivariate logistic analysis using the rms package in R. Discrimination was evaluated using the concordance index (C index), which was measured by the area under the receiver operating characteristic curve (AUC). Calibration and clinical utility were evaluated using a visual calibration plot and decision-curve analysis (DCA). Internal validation was performed with 1000 bootstrap resamples to evaluate the predictive consistency of the model. Two-sided P < 0.05 indicated statistical significance. All statistical analyses were performed using SPSS (version 26.0; IBM Corp., Armonk, NY, United States) and R software (version 4.3.1; R Core Team, Vienna, Austria). The statistical methods of this study were reviewed by a biomedical statistician from Renji Hospital.
RESULTS
Study population
In total, 153 patients with moderate-to-severe UC were included in the final analysis. There were 47 patients who underwent colectomy after failure of medical rescue therapy with corticosteroids and immunomodulators during admission in the surgery group, and 106 patients who responded to medical intervention in the control group (Figure 1). All patients were Chinese (Table 1).
Figure 1 Flowchart of participant selection.
UC: Ulcerative colitis.
Table 1 Comparison of baseline demographic and clinical characteristics of patients with moderate to severe ulcerative colitis (surgery vs control group), n (%).
EBV colitis is a promising independent predictor for colectomy in patients with moderate-to-severe UC
Among the 153 patients with moderate-to-severe UC, 24 (15.7%) had EBV infection of the colonic mucosa. EBER-positivity was higher in the surgery group than in the control group (40.4% vs 4.7%; P = 0.000). The incidence of high EBER concentration (≥ 10/HPF) and low EBER concentration (2-10/HPF) was significantly higher in the colectomy group than in the control group (P = 0.000). However, no patients in the control group had high EBER concentrations. There were significant differences in the prevalence of EBV single infection, and combined infection of EBV and CMV of the colonic mucosa between the two groups (P < 0.01; Table 1).
Univariate logistic analysis revealed that EBV colitis was a significant risk factor for colectomy in patients with moderate-to-severe UC (P = 0.000, OR: 13.707, 95%CI: 4.700-39.977; Table 2). Stratified analysis confirmed that EBER positivity was associated with surgical risk in patients with moderate-to-severe disease when controlling for the effect of confounding factors such as age, fever, CMV infection, fungal infection, hemoglobin, CRP, ESR, albumin, prealbumin, treatment with three or more types of immunosuppressive drugs before admission, and therapy with third-generation cephalosporins or more advanced antibiotics during admission (Table 3). Eight significant variables (P < 0.05, AUC > 0.6) screened using univariate logistic analysis were included in the multivariate logistic analysis, and the results showed that EBV colitis was a significant independent predictor of colectomy (P = 0.003, OR: 9.634, 95%CI: 2.137-43.424; Table 4). Subgroup analysis was performed with 138 patients after excluding patients with CMV IHC positivity using the same statistical methods previously described, which yielded similar results, indicating that EBV single infection was a promising predictor of surgical risk in patients with moderate-to-severe UC (Table 5).
EBER-ISH and EBV IHC indicated that EBV-infected cells in patients with high EBER concentrations in the surgery group mainly included B and T lymphocytes, and EBV-infected intraepithelial lymphocytes were found among the intestinal epithelial cells in our cohort (Figure 2).
Figure 2 Phenotype of Epstein-Barr virus-infected cells in patients with moderate to severe ulcerative colitis determined by Epstein-Barr virus-encoded small RNA in situ hybridization and Epstein-Barr virus-encoded small RNA-immunohistochemistry (× 400).
A: Immunohistochemical colocalization with Epstein-Barr virus-encoded small RNA (EBER)-positive cells: Blue arrows indicate positive staining. Cytoplasmic and membrane signals are red for antigen CD20 positive (a specific B lymphocyte marker), and nuclear signals are brown for EBER-positive cells; B: Immunohistochemical colocalization with EBER-positive cells: Blue arrows indicate positive staining. Cytoplasmic and membrane signals are red for antigen CD3 positive (a specific T lymphocyte marker), and nuclear signals are brown for EBER-positive cells; C: Blue arrows indicate EBER-positive cells (nuclear signal is brown), and no EBER-positive cells show positive staining for antigen CD56 (a specific NK cell marker); D: Immunohistochemical colocalization with EBER-positive cells: Blue arrows indicate EBER-positive intraepithelial lymphocytes (nuclear signal is brown) among the intestinal epithelial cells (cytoplasmic and membrane signals are red in CK staining positive areas).
Construction of a surgical predictive model
Univariate logistic regression was performed, and eight variables with statistical significance (P < 0.05, AUC > 0.6) were screened, including age, fever, EBV infection, CMV infection, CRP, ESR, treatment with three or more types of immunosuppressive drugs before admission, and therapy with third-generation cephalosporins or more advanced antibiotics during admission. The eight variables were analyzed using multivariate logistic analysis, and four significant independent factors (P < 0.05), age, EBV infection, CMV infection, and treatment with three or more types of immunosuppressive drugs before admission, were included in the final predictive model (Table 3). R software was used to construct a visual nomogram based on multivariate logistic regression to predict the risk of colectomy in patients with moderate-to-severe UC (Figure 3). The risk of colectomy was estimated from the figure on the bottom axis. The detailed manipulation steps were as follows: Draw a vertical line from the value of each variate line to the “Points Line” on the figure; sum up the five figures on the “Points Line” to obtain the total figure and read the figure on the “Total Points Line”; then, draw a vertical line from the “Total Points Line” and read the figure on the bottom line, which shows the risk percentage of predicting colectomy in patients with moderate-to-severe UC.
Figure 3 Nomogram for predicting surgery in patients with moderate to severe ulcerative colitis.
EBER: Epstein-Barr virus-encoded small RNA; CMV: Cytomegalovirus.
Performance of the predictive model
The Hosmer-Lemeshow test results (χ2 = 8.576 and P = 0.379) indicated that the model had good forecasting accuracy. Receiver operating characteristic (ROC) curve analysis indicated that this predictive model possessed good discriminative ability, with an AUC of 0.906 (95%CI: 0.856-0.955) (Figure 4A). The calibration curve (Figure 4B) and DCA (Figure 4C) demonstrated good calibration and clinical utility. Bootstrap internal validation of 1000 times resampling yielded an average AUC of 0.904 (95%CI: 0.903-0.906), indicating that the model had good internal consistency.
Figure 4 Analysis of the predictive model.
A: Receiver operating characteristic curve analysis of the predictive model used in this study; B: Calibration curve of the predictive model; C: Decision curve analysis of the predictive model. AUC: The area under the receiver operating characteristic curve.
DISCUSSION
Although a recent meta-analysis[4] reported a decline in the overall surgical rate for UC, the surgical risk remains high, and surgery is unavoidable for some patients, particularly those with recurrent cases, drug dependence, refractory disease, and particularly ASUC. Determining the optimal timing for surgical intervention remains a major challenge in clinical practice[9]. Exploring the predictive risk factors for colectomy can assist clinicians in identifying critically ill patients who are likely to require surgical treatment, thereby preventing treatment delays and reducing postoperative complications.
Recent use of immunosuppressive agents and biologics in patients with IBD has led to an increased incidence of opportunistic infections, including infection with herpes viruses such as CMV and EBV[15]. CMV and EBV infections of the intestinal mucosa are associated with poor prognosis in UC[16-20].
Based on the results of the multivariate logistic regression analysis, we developed a nomogram model to predict surgical risk by incorporating four predictors: Age; EBER positivity in the intestinal mucosa; CMV positivity in the intestinal mucosa; and treatment with three or more types of immunosuppressive drugs prior to admission. We used ROC curve, calibration curve, and decision curve analyses to evaluate the performance of the model. The model demonstrated strong discriminative ability, calibration accuracy, and clinical applicability. Additionally, the bootstrap method was used for internal validation to confirm the internal consistency of the model.
Age and exposure to immunosuppressive agents or biologics are associated with the risk of colectomy in patients with UC[12-14,29,30]. Jain et al[11] demonstrated that positive CMV DNA in the intestinal mucosa was an independent risk factor for surgery in UC. Several studies[18-20] have indicated that EBV infection is associated with poor prognosis in UC; however, the specific role of EBV infection as a surgical risk factor for UC has not yet been investigated. A recent study included both EBV and CMV infections when screening for risk factors for colectomy in UC; however, the final results showed that positive EBV and CMV hematological or mucosal tests were not associated with treatment failure[13]. Our findings confirmed that EBV infection of the colonic mucosa is a significant independent predictor for colectomy in patients with moderate-to-severe UC and it was incorporated into the final predictive model for the first time. The prevalence of EBV single infection, CMV single infection, and combined EBV and CMV infection differed significantly between the surgery and control groups. Subgroup analysis further demonstrated that EBV single infection was an independent risk factor for colectomy in UC even after excluding patients with CMV positivity in the intestinal mucosa. These findings highlight the importance of detecting EBV in the colonic mucosa using the technique of EBER-ISH in patients with refractory IBD who have no response to medical therapy. The nomogram model constructed in our study could help surgeons assess the risk of surgery in patients with moderate-to-severe UC, thereby making better treatment decisions and avoiding the many harms associated with delayed surgery.
The phenotypes of EBV-infected cells in all seven patients with high EBER concentrations in the surgery group were determined using EBER ISH and IHC, and preliminary investigations were conducted to explore how EBV infection contributes to mucosal injury and poor prognosis. The EBV-infected cells primarily included B and T lymphocytes, with EBV-infected intraepithelial lymphocytes identified among the intestinal epithelial cells at our center.
EBV, also known as herpesvirus type 4, is a member of the herpesvirus family that infects > 90% of the global population[31]. Primary EBV infections typically occur during childhood and are often asymptomatic. However, some individuals may experience primary infections during adolescence or adulthood, presenting as infectious mononucleosis. Following initial infection, EBV persists in memory B cells for life. In immunocompromised populations, such as patients with primary immunodeficiencies affecting the NK and/or T cell systems, and immunosuppressed transplant recipients, EBV can reactivate and lead to an increased risk of various virus-driven lymphoproliferative diseases[31,32]. Current research has demonstrated that EBV replication is associated with the proliferation and increased infiltration of B lymphocytes in the inflammatory colonic mucosa of individuals with IBD, resulting in severe mucosal inflammation[33]. Consistent with previous studies[19,34], our findings confirmed that EBV-infected cells in patients with severe IBD were predominantly B lymphocytes, which may contribute to disease exacerbation and poor prognosis. Additionally, we found that EBV infects not only B lymphocytes but also a substantial number of T lymphocytes in Asian patients with UC and concurrent intestinal EBV infection. This suggests that the immune surveillance and clearance functions of T lymphocytes in these patients may be impaired, further promoting the reactivation and replication of B lymphocytes, exacerbating mucosal inflammation and leading to a poor response to drug therapy.
Our findings indicated the presence of EBV-infected intraepithelial lymphocytes among intestinal epithelial cells, although no EBV-infected intestinal epithelial cells were identified. Epithelial cells are involved in the replication and transmission of EBV within the host. The lytic phase of the EBV cycle is activated in these cells to establish a major productive infection, resulting in a direct cytopathic effects[35,36]. Ciccocioppo et al[36] reported that the levels of EBV-DNA in the intestinal epithelial cells were significantly higher in patients with refractory IBD than in the responsive group, utilizing cell isolation from the colon mucosa and PCR technology for the first time. A significant positive correlation was observed between the EBV load in the mucosal cell population of IBD lesions and clinical and endoscopic indicators of disease activity, supporting the critical role of EBV in causing mucosal damage in IBD when infecting intestinal epithelial cells. Additionally, they identified ZEBRA (an immediate early lytic gene of EBV) positive intraepithelial lymphocytes among the intestinal epithelial cells in a small subset of patients with refractory IBD, suggesting that close contact with intestinal epithelial cells may facilitate the transfer of EBV particles. Although no epithelial cells infected by EBV were identified in this study, EBER-positive intraepithelial lymphocytes were observed among the intestinal epithelial cells, indicating that EBV infection has penetrated the epithelium, where lytic infection may occur and facilitate the transfer of viral particles.
Our study had several limitations. First, this was a retrospective observational study that precluded the establishment of a causal relationship between EBV infection and other predictors of surgical outcomes. Second, the sample size was small, which limits generalization of the results. Additionally, the variables included were restricted, and many confounding factors were not controlled, which might have contributed to potential bias. Third, we did not ascertain the infective stage of the virus, nor could we determine whether EBV merely acts as a bystander or exacerbates mucosal damage, which needs to be addressed in future studies. Finally, this was a single-center study, and the final prediction model lacked external validation, limiting its applicability to other populations. More large-scale, multi-center and multi-ethnic prospective clinical studies and basic research are needed to detect the exact role of EBV infection on the surgical risk in patients with IBD and the potential biological mechanisms in the future.
CONCLUSION
EBV infection in the intestinal mucosa is an independent and significant risk factor for colectomy in patients with moderate-to-severe UC. The nomogram model, which included age, EBV infection in the colonic mucosa, CMV infection in the colonic mucosa, and treatment with three or more immunosuppressive agents before admission, exhibited good predictive performance for colectomy in moderate to severe UC. This model provides a basis for the clinical assessment of surgical risk and decision-making. The EBV-infected cells were predominantly B- and T-lymphocytes. Additionally, EBER-positive intraepithelial lymphocytes were observed among the intestinal epithelial cells, which supports the association between active EBV infection, disease exacerbation, and poor prognosis in UC.
ACKNOWLEDGEMENTS
We are particularly grateful to our patients, and the professionals who collectively support our work.
Footnotes
Provenance and peer review: Unsolicited article; Externally peer reviewed.
Peer-review model: Single blind
Specialty type: Gastroenterology and hepatology
Country of origin: China
Peer-review report’s classification
Scientific Quality: Grade A, Grade B, Grade B, Grade B, Grade C
Novelty: Grade A, Grade B, Grade B, Grade B, Grade D
Creativity or Innovation: Grade A, Grade B, Grade B, Grade B, Grade C
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