Abstract
Objectives
This study aimed to identify risk factors for postoperative recurrence in patients with perianal fistulizing Crohn’s disease and assess the predictive value of fecal calprotectin and serum miRNA6086.
Methods
From 105 patients with perianal fistulizing Crohn’s disease, blood and fecal samples as well as clinical data were collected. Analysis of blood tests, C-reactive protein, miRNA6086, and fecal calprotectin revealed postoperative recurrence risk factors. Receiver operating characteristic curve analysis assessed the predictive accuracy of miRNA6086 and fecal calprotectin for perianal fistulizing Crohn’s disease recurrence and determined their optimal cutoff values, sensitivity, and specificity.
Results
Of the 105 patients with perianal fistulizing Crohn’s disease, 33 (31.4%) experienced recurrence. Anal fistula type, preoperative miRNA6086, and fecal calprotectin levels were identified as independent risk factors for postoperative recurrence. Receiver operating characteristic curve analysis revealed that miRNA6086 had a cutoff value of 0.3195, sensitivity of 65.28%, specificity of 66.67%, and area under curve value of 0.6589 (95% confidence interval, 0.5503–0.7674). Fecal calprotectin had a cutoff value of 0.6073, sensitivity of 81.94%, specificity of 78.79%, and area under curve value of 0.8224 (95% confidence interval, 0.5503–0.7674). Combined miRNA6086 and fecal calprotectin detection had a cutoff value of 0.7121, sensitivity of 83.33%, specificity of 87.88%, and area under curve value of 0.9146 (95% confidence interval, 0.8547–0.9744).
Conclusion
Anal fistula type, preoperative miRNA6086, and fecal calprotectin levels are independent risk factors for perianal fistulizing Crohn’s disease recurrence. Combined detection of miRNA6086 and fecal calprotectin levels enhances predictive accuracy for postoperative recurrence.
Introduction
Crohn’s disease (CD) is a chronic, nonspecific inflammatory bowel disorder that affects the gastrointestinal tract. Although CD is a global health concern impacting approximately 1 in 200 individuals worldwide, its etiology and pathogenesis remain elusive. 1 Notably, the incidence of CD has been increasing in recent years. Among adult patients with CD, 20%–30% experience perianal complications, with perianal fistulizing CD (pfCD) being the predominant form. 2 This condition places a substantial burden on affected individuals and considerably impairs their quality of life.
The management of pfCD encompasses both pharmacological and surgical interventions. 3 However, pharmacotherapy is associated with certain limitations, 4 necessitating surgical treatment in numerous cases. Anal fistula surgery, although necessary, carries the risk of serious complications, including recurrence requiring reoperation and fecal incontinence. Literature has indicated that the recurrence rate following anal fistula surgery ranges from 3% to 57%, with up to 40% of patients experiencing fecal incontinence, which substantially diminishes their quality of life.5,6
Several risk factors can influence the surgical outcomes of anal fistula, particularly in the context of pfCD. Owing to the inherent nature of the disease and its high recurrence rate, approximately one-third of patients require additional surgery, and nearly a quarter of patients may continue to suffer from perianal fistula disease even a decade postoperatively. 7 Despite these challenges, there is a dearth of clear quantitative indicators for predicting recurrence following pfCD surgery. Current assessments often rely heavily on the clinical acumen of physicians and the results of invasive diagnostic procedures, such as colonoscopy. Consequently, the development of a noninvasive, straightforward diagnostic method capable of more accurately assessing and predicting postsurgical recurrence in patients with CD and anal fistulas is of paramount importance.
pfCD, a prevalent complication of CD, lacks a robust set of direct laboratory indicators for its diagnosis and assessment. Recent studies have identified fecal calprotectin (FC) as a biochemical marker that reflects intestinal inflammation. 8 Additionally, characteristic differential changes in serum microRNA (miRNA) expression profiles have been shown to mirror alterations in numerous chronic infectious diseases. 9 Specifically, miRNA6086 has been implicated in cell immunity, differentiation, and other chronic inflammatory responses within anal fistula tissue.10,11
The objective of this study was to evaluate the comparative performance of FC and serum miRNA6086 in patients with pfCD undergoing treatment. We aimed to discern differences between patients who experienced postsurgical recurrence and those who did not and investigate the correlation of these biomarkers with postoperative recurrence in patients with pfCD. This approach may enhance the understanding of pfCD pathophysiology and guide more precise therapeutic interventions.
Methods
Patients and study design
This retrospective, observational, case–control study enrolled a total of 105 patients diagnosed with pfCD who were treated surgically at the Yancheng TCM Hospital Affiliated to Nanjing University of Chinese Medicine from September 2021 to June 2023. The patients were randomly categorized into two groups based on the occurrence of recurrence: recurrence of pfCD (R-pfCD) group (n = 33) and no recurrence of pfCD (NR-pfCD) group (n = 72).
The inclusion criteria were as follows: (a) a confirmed diagnosis of Crohn’s fistula-in-ano, aligning with the clinical practice guidelines for the management of anorectal abscess, fistula-in-ano, and rectovaginal fistula published by the American Society of Colon and Rectal Surgeons in 2022; 12 (b) age ≥16 years and a history of undergoing anal fistula surgery for CD; and (c) availability of complete clinical data for analysis.
The exclusion criteria were as follows: (a) presence of other infectious diseases; (b) autoimmune diseases apart from CD; and (c) concurrent large bowel tumors, other partial tumors, or hematological malignancies.
This study was conducted in accordance with the Declaration of Helsinki (revised in 2013), and written informed consent was obtained from all patients prior to treatment. The patients were informed that their medical records might be utilized in future medical research. All identifiable patient data were de-identified. Owing to the retrospective nature of the study, the Ethics Committee of Yancheng Hospital affiliated with Nanjing University of Chinese Medicine granted a waiver for the ethical review process. The reporting of this study adheres to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines. 13
Data collection and definition
The study data encompassed demographic characteristics, details of anal fistula surgery, and postoperative outcomes, including incidence rates. CD was classified using the Montreal classification. 14 Disease activity was quantified using the Crohn’s Disease Activity Index (CDAI) 15 and the Perianal Disease Activity Index (PDAI). 16 Clinical remission was defined as a CDAI score of <150 points, with ≥150 points assigned to active disease phases, which are further categorized as mild (150–220 points), moderate (221–450 points), and severe (>450 points). The PDAI, which assesses perianal secretion, pain and activity, sexual activity, perianal appearance, and induration, assigns a score of 0–4 for each item, with a maximum total score of 20 points. A PDAI score of >4 points indicates the presence of active fistulas or localized inflammation. Blood samples were collected in the fasting state in the morning, and preoperative tests included serum miRNA6086, C-reactive protein (CRP), white blood cells (WBCs), neutrophil percentage (NELT%), blood platelets (PLTs), FC, and miRNA6086. R-pfCD was defined as the occurrence of perianal abscess or fistula within 6 months of surgery; otherwise, it was classified as NR-pfCD. Fistula recurrence was confirmed through local exploration and perianal magnetic resonance imaging. Surgical approaches were categorized into two types: (a) incision and drainage with seton placement, with or without fistulotomy following seton placement, and (b) advancement flap transposition or fistulotomy with intersphincteric fistula tract ligation.
Statistical analysis
All statistical analyses were performed using SPSS version 27.0. Continuous data following a normal distribution were presented as mean ± standard deviation and compared between two groups using independent t-tests or among multiple groups using one-way analysis of variance. Non-normally distributed continuous data were presented as median (Q1, Q3) and compared between two groups using the Mann–Whitney U test or among multiple groups using the Kruskal–Wallis H test, with post-hoc pairwise comparisons adjusted by the Nemenyi test. Categorical data were expressed as numbers and percentages and compared using the chi-squared test between two groups or the Kruskal–Wallis H test among multiple groups. Logistic regression analyses were conducted to develop a model for predicting the recurrence of CD anal fistulas, and the receiver operating characteristic (ROC) curve was utilized to determine the optimal cutoff value for prediction. Statistical significance was set at P < 0.05, with P-values of <0.01 denoting a highly significant and substantial difference.
Results
Patient baseline characteristics
In this study, a total of 105 eligible patients were analyzed. The cohort had an average age of 31.93 ± 10.96 years, with 61 males comprising 46.61% of the sample. Within a 6-month postoperative period, 33 patients with pfCD developed R-pfCD, indicating a recurrence rate of 31.4%. The average CDAI score was 188.80 ± 74.13, with 33 patients (33.3%) in clinical remission, 33 (33.3%) exhibiting mild activity, and 39 (37.1%) presenting with moderate-to-severe activity. Preoperative medication usage rates were as follows: mesalazine was used by 14.41% of the patients, immunosuppressants by 9.32%, and biologics by 6.78%, collectively accounting for 6.78% of the total patient population. The prevalence of simple anal fistulas was 54.3% (57/105), whereas complex anal fistulas accounted for 45.7% (48/105) of the cases. The baseline characteristics of the study population are detailed in Table 1.
Clinical characteristics of the study population.
pfCD: perianal fistulizing Crohn’s disease; BMI: body mass index; CDAI: Crohn’s Disease Activity Index; PDAI: Perianal Disease Activity Index; 5-ASA: 5 aminosalicylic acid.
Preoperative laboratory examination indicators for patients with pfCD
Preoperative levels of CRP, WBC, NELT%, PLT, FC, and miRNA6086 were assessed in all patients. Comparative analysis of preoperative CRP, WBC, NELT%, and PLT between the R-pfCD and NR-pfCD groups revealed no significant differences. However, the preoperative FC levels were significantly higher in the R-pfCD group (mean value: 308.60 ± 184.78) than in the NR-pfCD group (mean value: 115.52 ± 68.16) (P < 0.001). Similarly, preoperative miRNA6086 levels were markedly elevated in the R-pfCD group, averaging 76.92 ± 45.00, compared with 53.56 ± 12.91 in the NR-pfCD group, indicating a statistically significant difference (P < 0.01). Detailed values are presented in Table 2.
Preoperative laboratory examination indicators of pfCD.
pfCD: perianal fistulizing Crohn’s disease; CRP: C-reactive protein; WBC: white blood cell; NELT: neutrophil percentage; PLT: blood platelet; FC: fecal calprotectin.
Univariate and multivariate logistic regression analysis of factors influencing R-pfCD
Both univariate and multivariate logistic regression analyses identified preoperative miRNA6086 levels, FC levels, and complexity of anal fistulas as independent risk factors for R-pfCD. In contrast, R-pfCD was not significantly correlated with other laboratory parameters such as CRP, WBC count, PLT count, or NELT% (Table 3).
Univariate and multivariate logistic regression analysis of pfCD recurrence.
pfCD: perianal fistulizing Crohn’s disease; OR: odds ratio; CI: confidence interval; CDAI: Crohn’s Disease Activity Index; PDAI: Perianal Disease Activity Index; CRP: C-reactive protein; PLT: blood platelet; NELT%: neutrophil percentage; FC: fecal calprotectin; WBC: white blood cell; CDAIC: CDAI classification; PDAIC: PDAI classification; PF: perianal fistula classification.
Evaluating the predictive accuracy of miRNA6086, FC, and their combination for surgical recurrence
ROC curve for miRNA6086 (Figure 1(a)) revealed a cutoff value of 0.3195 for predicting R-pfCD, with a sensitivity of 65.28% and specificity of 66.67%. The area under the curve (AUC) value was 0.6589 (95% confidence interval (CI), 0.5503–0.7674). For FC (Figure 1(b)), the ROC curve indicated a cutoff value of 0.6073, sensitivity of 81.94%, specificity of 78.79%, and AUC of 0.8224 (95% CI, 0.5503–0.7674). The combined detection of serum miRNA6086 and FC (Figure 1(c)) demonstrated superior efficacy in predicting postoperative R-pfCD, with a cutoff value of 0.7121, sensitivity of 83.33%, specificity of 87.88%, and AUC of 0.9146 (95% CI: 0.8547–0.9744). These results highlight the potential of combined biomarker assessment for enhanced prediction of surgical R-pfCD.

Predictive accuracy of miRNA6086, FC, and their combination for surgical recurrence. (a) ROC curve of miRNA6086; (b) ROC curve of FC; (c) ROC curve of miRNA6086 and FC. FC: fecal calprotectin; ROC: receiver operating characteristic.
Discussion
pfCD is a complex and debilitating complication of CD, characterized by a diminished quality of life and increased healthcare costs, with a substantial proportion of patients necessitating surgical intervention.17,18 A study reported a relapse rate of approximately 30% following treatment for pfCD. 19 The intricate nature and high recurrence rate of pfCD underscore the need for thorough pretreatment evaluation to identify high-risk patients who are likely to experience recurrent pfCD early in their preoperative management. Despite this, treatment evaluation remains a formidable challenge for colorectal surgeons, who primarily rely on clinical presentation, examination under anesthesia, and radiological assessments. 20 In this study, we analyzed the fundamental clinical features and commonly utilized laboratory markers of CD to identify independent risk factors associated with postoperative R-pfCD. Subsequently, we employed these factors to predict the likelihood of recurrence following pfCD surgery. Notably, the concurrent detection of serum miRNA6086 and FC demonstrated promising potential in predicting postoperative recurrence in patients with pfCD.
A study by Vuyyuru et al., which analyzed 65 patients with pfCD, reported complete remission rates of 25% for patients who received immunomodulators, 42.8% for those who underwent fistula surgery, 39.5% for those who received biological agents, and 45.4% for those without concomitant perianal abscess. 21 The recurrence rate following fistula surgery was 52.6%, with a significantly higher likelihood of complete fistula closure in patients without perianal abscess. Surgery is a crucial component in the management of pfCD, providing effective drainage and optimal immunosuppressive effects. Contemporary surgical approaches for pfCD include fibrin glue injection, anal fistula plug, and ligation of the intersphincteric fistula tract; however, surgery alone is associated with high recurrence rates.22,23 These findings underscore the complexity of and propensity for recurrence in pfCD treatment, emphasizing the need for rigorous pretreatment assessment to identify patients at high risk of recurrent pfCD early in the preoperative phase.
In clinical practice, diagnostic and therapeutic procedures such as clinical activity indices are often cumbersome and subjective, whereas endoscopy, an invasive procedure, entails bowel preparation and carries risks of complications such as bleeding and perforation. Imaging examinations, although valuable, may involve radiation exposure and are costly. These modalities, with their inherent limitations, leave the prediction of postoperative recurrence in pfCD largely dependent on clinical experience due to the absence of definitive quantitative markers. Hence, there is a critical need for a noninvasive and straightforward method to accurately predict recurrence following pfCD surgery. FC, a neutrophil-derived protein involved in intestinal defense mechanisms, is stable, easily detectable, and serves as a reliable biochemical marker of intestinal inflammation. It is particularly useful in the differential diagnosis of functional bowel disorders such as irritable bowel syndrome 24 and is highly recommended in the 2023 Chinese guidelines for evaluating CD. However, its application in pfCD diagnosis remains underexplored. miRNAs, a class of endogenous noncoding small RNAs, play a role in cellular processes such as proliferation, differentiation, and inflammation, making them valuable for disease diagnosis and assessment. miRNA6086, in particular, has been implicated in immune responses and chronic inflammation within anal fistula tissues 11 ; however, its relationship with pfCD has not yet been explored.
In formulating our research plan, we initially accumulated clinical data from 105 patients with pfCD who underwent surgery. These data encompassed demographic details, medical history, disease characteristics, and clinical and laboratory parameters. After scrutinizing these variables, we identified differential clinical indicators, such as anal fistula type, CDAI scores, PDAI scores, and case counts, and performed univariate and multivariate logistic regression analyses to assess R-pfCD. We determined that anal fistula type is an independent risk factor for postoperative recurrence, corroborating previous studies. In our analysis of laboratory indicators, elevated levels of miRNA6086 and FC emerged as significant independent risk factors, demonstrating greater diagnostic value in predicting postoperative R-pfCD than other inflammatory markers such as CRP, WBC count, and NELT%. We hypothesized that preoperative FC and serum miRNA6086 assessments can effectively stratify patients into low- and high-risk groups for postoperative R-pfCD.
Building on these findings, we conducted ROC curve analyses of FC and serum miRNA6086 as individual and combined predictors. The results indicated that FC and miRNA6086 can independently predict R-pfCD with high AUC values, along with high sensitivity and specificity. Although FC showed superior predictive efficiency compared with miRNA6086, the combined prediction of these markers significantly enhanced the predictive power, yielding the largest AUC and optimal predictive outcomes. Existing literature supports the efficacy of FC in accurately predicting the disease activity of CD and recurrence under endoscopic evaluation. We posit that as pfCD is a localized manifestation of CD, FC’s ability to assess and predict CD recurrence extends to pfCD. miRNA6086, although indicative of fistula recurrence, lacks precision in predicting overall CD. Hence, its standalone predictive efficacy for R-pfCD is inferior to FC. The synergistic use of both markers likely amplifies their predictive accuracy by accounting for the systemic aspects of CD and the localized characteristics of the fistula.
Our study acknowledges certain limitations. Specifically, variables such as disease classification characteristics of CD, preoperative medication use, and surgical techniques did not attain statistical significance in our clinical data analysis. This may be attributed to the limited sample size within the R-pfCD group, which potentially constrained the power of our statistical findings. Additionally, this study was designed to explore the development of a clinical prediction model, aiming to identify potential predictors and assess their predictive capacity. However, due to difficulties in data acquisition across different medical institutions or regions, external validation as required by the Transparent Reporting of a multivariable prediction model for Individual Prognosis or Diagnosis statement was not performed. The present study is limited to the initial phase of model development and internal validation. We acknowledge this limitation and plan to conduct external validation in our subsequent research.
Conclusion
Our study identified anal fistula type, preoperative miRNA6086 levels, and FC as independent risk factors for R-pfCD. The combined preoperative assessment of miRNA6086 and FC levels offers a valuable reference for predicting postoperative R-pfCD and guiding timely clinical interventions aimed at prevention. This approach holds promise as an improved preoperative predictive method for R-pfCD.
Footnotes
Acknowledgement
None.
Author contributions
Shengnan Jin and Maozhen Zhang contributed equally to this work. They were involved in the study design, data collection, and manuscript drafting.
Dongdong Gu and Xingping Zhu assisted with data analysis and interpretation.
Zuolei Wang and Xuerong Shi contributed to clinical data collection and patient management.
Yanping Hao provided statistical support and helped with the validation of results.
Su Xu, the corresponding author, oversaw the study design, coordinated with the research team, and revised the manuscript for submission.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Consent for publication
All authors have read and approved the final manuscript for submission and agree with its publication in the journal.
Declaration of conflicting interests
The authors declare that they have no competing interests.
Ethics approval and consent to participate
This study was conducted in accordance with the Declaration of Helsinki (revised in 2013), and written informed consent was obtained from all patients prior to treatment. Due to the retrospective nature of the study, the Ethics Committee of the Yancheng TCM Hospital Affiliated to Nanjing University of Chinese Medicine granted a waiver for the ethical review process.
Funding
This research was supported by Jiangsu Provincial Chinese Medicine Science and Technology Development Program Project (No. MS2021071).
