Exploring Fungal Biomarkers as Predictors of Treatment Response in Ulcerative Colitis Patients Receiving Anti-TNF Therapy

Exploring Fungal Biomarkers as Predictors of Treatment Response in Ulcerative Colitis Patients Receiving Anti-TNF Therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exploring Fungal Biomarkers as Predictors of Treatment Response in Ulcerative Colitis Patients Receiving Anti-TNF Therapy Maryam Farmani, Shaghayegh Baradaran Ghavami, Nesa Kazemifard, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7749916/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease, is a chronic immune-mediated condition marked by recurrent intestinal inflammation. Although anti-TNF therapies like adalimumab (ADA) have improved disease management, predictive biomarkers for treatment response remain limited. Emerging evidence highlights fungal biomarkers as potential indicators of therapeutic outcomes, especially in UC. This study investigates fungal load dynamics and their association with clinical response in UC patients receiving ADA. Methods: We analyzed samples from 23 UC patients and 20 healthy individuals. Using quantitative PCR (qPCR), we measured the prevalence and DNA copy numbers of key fungal species, then assessed their relationships with disease activity, response to ADA, and inflammatory markers. Patients were further stratified by disease phase (flare-up vs. remission) and treatment response. Results: Our findings revealed an increase in Candida albicans levels in UC patients on ADA compared to healthy controls, while Candida glabrata was significantly reduced. Among the UC group, C. tropicalis showed the most marked increase during flare-ups compared to remission. When comparing treatment outcomes, non-responders exhibited notably higher levels of C. glabrata and C. tropicalis than responders. Further analysis suggested that disease phase may influence the relationship between fungal burden and treatment response, particularly for C. tropicalis . Conclusion: These findings reveal distinct fungal profiles associated with UC activity and response to adalimumab. C. tropicalis may indicate disease flare-ups, while elevated C. glabrata levels could reflect treatment resistance. Further studies are needed to validate fungal biomarkers as tools for guiding UC management. Ulcerative colitis Fungal biomarkers Anti-TNF therapy Candida species Treatment response Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Inflammatory Bowel Disease (IBD), which encompasses Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic and relapsing inflammatory condition of the gastrointestinal (GI) tract [ 1 , 2 ]. The exact causes of IBD remain unclear, but it is understood to result from a complex interplay between genetic predisposition, environmental triggers, immune system dysregulation, and the gut microbiome [ 3 , 4 ]. However, recent studies suggest that fungi, an integral part of the gut microbiota, may also play a crucial role in the pathogenesis and progression of IBD. This often-overlooked component of the gut microbiome, known as the mycobiome, may have both harmful and potentially beneficial effects on disease outcomes, influencing IBD activity during both active and remission [ 5 ]. Fungal species such as Candida albicans (C. albicans) , Saccharomyces cerevisiae (S. cerevisiae) , and Malassezia spp. have been identified as prevalent in the gut mycobiome, and their abundance appears to fluctuate with disease activity [ 6 ]. During active phases of IBD, specific fungi can become overrepresented, potentially exacerbating inflammation. For instance, C. albicans has been found to increase significantly during IBD flare-ups, potentially promoting inflammation by interacting with immune cells and producing metabolites that disturb the intestinal barrier [ 6 ]. These fungi may stimulate pro-inflammatory pathways, activating immune responses that can worsen tissue damage and contribute to the persistence and severity of symptoms in the active disease phase [ 7 , 8 ]. In contrast, during remission phases, a balanced and stable fungal community may support mucosal healing and help maintain intestinal homeostasis [ 8 ]. However, imbalances in fungal populations potentially triggered by factors like dietary changes, antibiotic exposure, or immune shifts could destabilize this equilibrium. Such disruptions in the mycobiome could create conditions that facilitate relapse, highlighting fungi as possible contributors to disease recurrence. One of the virulence factors of fungi is hyphae, which help form biofilms with bacteria. This interaction between bacteria and fungi leads to antibiotic resistance and complications in IBD pathogenesis [ 9 ]. Understanding the distinct roles that fungi play in the active and remission phases of IBD suggests valuable insights into the disease’s pathophysiology and underscores the potential of the mycobiome as a therapeutic target. As research into the gut mycobiome advances, it may be possible to identify specific fungal biomarkers for disease monitoring, as well as to develop novel therapies aimed at modulating the fungal microbiome to support remission and prevent relapse. Overall, fungi represent a promising frontier in the study of IBD, offering new avenues to improve management and outcomes for patients affected by this challenging and often debilitating condition. This study aimed to investigate the fungal load in two states of IBD and to compare the differences between responders and non-responders to adalimumab (ADA), providing a more in-depth setup for a detailed examination of fungi’s role in IBD. Method and Material Enrolment of Patients and Healthy Subjects 23 adult patients diagnosed with UC were enrolled at the Gastroenterology and Hepatology clinic (Taleghani Hospital, Tehran, Iran) from 2018 to 2021. Adalimumab (ADA) (CinnoRA®) therapy was initiated for patients due to active inflammation without response or with intolerance to prior conventional or biological IBD medications. Patients were classified as responders if they exhibited significant clinical and endoscopic improvement following ADA therapy, whereas non-responders were defined as those who showed minimal or no therapeutic benefit despite treatment. Individuals with other autoimmune disorders, as well as those diagnosed with cancer or viral infections (such as tuberculosis (TB), Hepatitis B virus (HBV), hepatitis C virus (HCV), and HIV), were excluded from the study. The exclusion criteria for healthy participants were consistent with those applied to the patient group. Furthermore, it is important to note that none of the participants had a history of long-term antibiotic and antifungal therapy within the last three months. The diagnosis of IBD was conducted based on established clinical parameters, including the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), fecal calprotectin (FC) levels, and the results of colonoscopy. The study design complied with the Declaration of Helsinki. All patients and healthy subjects gave written informed consent for sample collection according to the sampling protocol approved by the Ethical Review Committee of the Research Institute for Gastroenterology and Liver Diseases at Shahid Beheshti University of Medical Sciences (Project No. IR.SBMU.RIGLD.REC.1401.006). Sample Collection Fecal samples were obtained from patients diagnosed with UC at three distinct time points: prior to the initiation of ADA (CinnoRA®) therapy, as well as three- and six-months post-treatment commencement (before the second and third injection, respectively). Sample collection adhered to standardized protocols to ensure consistency and reliability [ 10 ]. Immediately after collection, the samples were stored at -80°C to preserve microbial integrity and prevent degradation. These preserved samples were subsequently utilized for fungal composition assessments to investigate potential therapeutic effects and specific fungal species alterations throughout treatment. ELISA Assay Fecal calprotectin levels were quantified using enzyme-linked immunosorbent assays (ELISA) performed on fecal supernatants. The analyses were conducted utilizing the Calprest NG ELISA Kit (Eurospital Diagnostic, Trieste, Italy), following the manufacturer’s protocol to ensure accuracy and reproducibility. The procedure involved the extraction of calprotectin from fecal samples, followed by incubation with specific antibodies to enable precise detection. Absorbance measurements were recorded to determine calprotectin concentrations, providing valuable insights into intestinal inflammation. All assays were performed under standardized laboratory conditions to minimize variability and ensure the reliability of results for subsequent statistical analyses. DNA Extraction from Fecal Samples Frozen fecal samples were thawed under controlled conditions and then completely homogenized to preserve sample integrity and minimize potential degradation of nucleic acids. DNA extraction was performed using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol to ensure optimal yield and purity. The extraction process included enzymatic and mechanical disruption to effectively lyse fungal cells and facilitate the isolation of high-quality genomic material. The purity and concentration of the extracted DNA were assessed using a NanoDrop® 192 ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), measuring absorbance at specific wavelengths (260/280 nm) to evaluate contamination levels and sample suitability for downstream molecular analyses. Fungal Culture C. albicans (ATCC 10231), C. glabrata (ATCC 90030), and C. tropicalis C. tropicalis (PTCC 5028) were cultivated on Sabouraud Dextrose Agar (SDA) (Sigma-Aldrich, USA) plates, a selective medium designed to support fungal growth by providing an optimal nutrient-rich environment. The cultures were incubated aerobically at 30°C to facilitate robust proliferation and ensure physiological consistency across samples. In parallel, S. cerevisiae (ATCC 9763) was cultured on a universal yeast medium (YM) (Sigma-Aldrich, USA), which contains essential nutrients for optimal yeast metabolism. The incubation conditions for S. cerevisiae were set at 28°C for 24 hours under aerobic conditions to promote consistent colony formation and metabolic activity. All culturing procedures were performed under sterile conditions to prevent contamination. Fungal DNA Extraction Genomic DNA was extracted from cultured Candida species and S. cerevisiae using a DNA extraction kit (ROJE TECHNOLOGIES, Iran), following the manufacturer’s protocol to ensure optimal yield and purity. The extraction process involved enzymatic lysis and purification steps designed to remove potential contaminants such as proteins and polysaccharides, thereby maximizing the integrity of fungal DNA. Quality control assessments were conducted using a NanoDrop® ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), measuring absorbance at 260/280 nm to evaluate purity and concentration. Additionally, the integrity of the extracted DNA was verified through agarose gel electrophoresis, wherein DNA fragments were visualized under ultraviolet illumination following SYBR Safe staining. Quantitative Absolute Real-time PCR For the standard curve employed in absolute quantification, genomic DNA extracted from C. albicans, C. glabrata, C. tropicalis, and S. cerevisiae cultures was prepared in a series of tenfold dilutions ranging from 10–10 5 . These serial dilutions were incorporated into each real-time PCR run to serve as reference standards for the generation of the standard curve, facilitating the precise quantification of fungal species copy numbers. Real-time PCR was performed in a final reaction volume of 10 µL, comprising 50% RealQ Plus 2x Master Mix Green (Ampliqon, Odense, Denmark). The assays were carried out utilizing species-specific primer pairs (Table 1 ), designed to target conserved genomic regions, thereby ensuring accurate and reproducible quantification of fungal DNA across all experimental replicates. All PCR procedures were executed under optimized thermocycling conditions to maximize sensitivity while minimizing potential amplification biases. Table 1 Primers used in Quantitative Absolute Real-time PCR Name Primer Sequence Product Size (bp) C. albicans 5´-GCTCCTGCTCCTGAAATGAC-3´ 3´-CTGGAGCAATTGGTGAGGTT-5´ 184 C. glabrata 5´-AAAGAAAGAACACACTCTGCC-3´ 3´-GGGTCTGAGTTGCTAATGTTG-5´ 153 C. tropicalis 5´-GCGGTAGGAGAATTGCGTT-3´ 3´-TCATTATGCCAACATCCTAGGTTTA-5´ 117 S. cerevisiae 5´-GAAATGCCACCGTGAATGC-3´ 3´-CTTTGGTGGTGATCCTCTATGATTG-5´ 113 [Add Table here] Statistical Analysis Quantitative real-time PCR data were obtained using Roche Light Cycler 96 Software based on standard curve and copy number calculations, with copy number data analyzed using log10 transformation (log₁₀[x + 1]) to ensure normalization. Statistical analyses were conducted in GraphPad Prism (v8.4.3) and R (v4.5.0), with Student’s t-test used for group comparisons (significance threshold: p < 0.05). Descriptive statistics summarized demographic, clinical, and fungal variables at Phase 1 of follow-up, with continuous data presented as mean ± SD or median (IQR), and categorical data as counts and percentages, without statistical comparison. Fungal quantification, including DNA copy numbers and qPCR cycle threshold (Ct) values for C. albicans, C. glabrata, C. tropicalis , and S. cerevisiae , along with fecal calprotectin levels, served as predictors in multivariable models. Generalized estimating equations (GEE) accounted for repeated measures, with two models: (1) main effects of fungal load and sampling phase, and (2) an interaction model including fungal load × phase interaction. Patient ID was the clustering unit. Appropriate statistical models were applied based on outcome type: binary (binomial link), ordinal (cumulative logit link), and continuous (Gaussian link function). Results were reported as odds ratios (ORs) or regression coefficients (β) with 95% confidence intervals (CIs). Significance was set at p < 0.05. Longitudinal trends in fungal DNA copy numbers were visualized using log₁₀-transformed line plots, stratified by clinical parameters such as disease state, treatment response, severity, clinical remission, and endoscopic remission. Results Demographic and Clinical Characteristics As shown in Table 2 , 23 patients with ulcerative colitis (UC) were included in the Phase 1 assessment. The mean age was 32.09 years (SD: 9.89), and the sex distribution was nearly equal, with 52.17% female and 47.83% male. The median duration of disease was 54.00 months (IQR: 24.00–93.00). All patients were in the flare-up phase of disease activity at the baseline. The majority of participants had severe disease (73.91%), followed by moderate to severe (13.04%) and moderate disease (8.70%) at the baseline. Only one patient (4.35%) was classified as having mild to moderate disease, while no cases of mild or no inflammation were observed. Regarding disease extension, 60.87% had pancolitis and 39.13% had left-sided colitis; no patients presented with proctosigmoiditis or no inflammation. Moreover, a cohort of 20 healthy control participants was enrolled in this study, with a mean age of 53.3 years (SD: 11.1). The sample consisted of 60% females and 40% males. A qualified physician conducted a comprehensive health assessment to confirm the absence of gastrointestinal disorders in all individuals. Table 2 Demographic and Clinical Characteristics of Patients with Ulcerative Colitis at Phase 1 of Follow-Up Variable Level Mean or Median /Frequency Demographic Variables Age ------ 32.09 ± 9.89 Gender Female 12 (52.17) Male 11 (47.83) Disease Duration (months) ------ 54.00 (24.00, 93.00) Disease Characteristics Disease state Flare-up 23 (100.00) Remission 0 (0.00) Treatment response status Non-responder 8 (34.78) Responder 15 (65.22) Severity of disease No inflammation 0 (0.00) Mild 0 (0.00) Mild to moderate 1 (4.35) Moderate 2 (8.70) Moderate to severe 3 (13.04) Severe 17 (73.91) Disease Extension No inflammation 0 (0.00) Proctosigmoiditis 0 (0.00) Left sided colitis 9 (39.13) Pancolitis 14 (60.87) Endoscopic Remission No 7 (30.43) Yes 16 (69.57) Clinical Remission No 8 (34.78) Yes 15 (65.22) [Add Table 2 here] Calprotectin Measurement Calprotectin levels in stool samples were measured using ELISA. The mean calprotectin concentration in the study population was 81.64 ± 57.86 µg/g, with a 95% confidence interval (CI) ranging from 0 to 153 µg/g. Non-responders exhibited significantly elevated fecal calprotectin levels, measuring 1.62-fold higher than those of responders (p = 0.009) (Fig. 1 A). Disease activity was classified according to the Mayo score, distinguishing remission from flare-up phases. Patients in the flare-up phase demonstrated a substantial increase in fecal calprotectin levels, with concentrations rising by 2.53-fold compared to those in remission (p < 0.0001) (Fig. 1 B). [Add Fig. 1 here] Fungal Populations in Ulcerative Colitis Patients and Healthy Subjects Quantitative analysis revealed notable differences in Log10-transformed fungal species DNA copy numbers between healthy individuals and UC patients undergoing anti-TNF therapy. The mean values for C. albicans were 1.049 (SD = 1.65) in healthy subjects and 2.38 (SD = 1.78) in UC patients, showing a 2.27-fold increase (p = 0.005). Similarly, C. glabrata exhibited a mean of 0.79 (SD = 1.41) in healthy individuals compared to 1.83 (SD = 1.52) in UC patients, reflecting a significant increase (p = 0.009). In contrast, C. tropicalis decreased from 0.4 (SD = 0.93) to 0.24 (SD = 0.59) in UC patients (p = 0.386). While S. cerevisiae showed a 1.4-fold reduction in UC patients (p = 0.082), the declines in C. tropicalis and S. cerevisiae did not reach statistical significance (Fig. 2 ). [Add Fig. 2 here] Fungal Prevalence in Patients Stratified by Disease Status Fungal species distribution varied between remission and flare-up groups. The Log10-transformed mean for C. albicans was 2.24 (SD = 1.49) in remission patients and 2.17 (SD = 1.70) during flare-ups (p = 0.879), indicating a non-significant decline. Similarly, C. glabrata measured 1.92 (SD = 1.36) in remission and 1.82 (SD = 1.78) during flare-ups (p = 0.811). In contrast, C. tropicalis showed a statistically significant 2.04-fold increase (p < 0.0001), rising from 0.97 (SD = 0.93) in remission to 1.98 (SD = 0.40) in flare-ups. S. cerevisiae displayed a modest decline from 2.45 (SD = 1.71) to 1.88 (SD = 1.98), though this difference was not statistically significant (p = 0.257). These results suggest that fluctuations occur among certain fungal species while others remain stable across disease phases (Fig. 3 ). [Add Fig. 3 here] Fungal Profiles in Responders and Non-Responders to Anti-TNF Therapy The stratification of UC patients based on their response to ADA treatment revealed notable differences in fungal burden across responder and non-responder groups. The Log10-transformed mean for C. albicans was 2.42 (SD = 1.75) in responders and 2.34 (SD = 1.95) among non-responders (p = 0.192). C. glabrata increased significantly from 1.59 (SD = 1.3) in responders to 2.7 (SD = 2.1) in non-responders (p = 0.024). In addition, C. tropicalis exhibited a noticeable 3.61-fold increase (p = 0.034), rising from 0.13 (SD = 0.44) to 0.5 (SD = 0.78). S. cerevisiae showed slight variation (2.23 [SD = 1.83] in responders vs. 1.94 [SD = 2] in non-responders), though not statistically significant (p = 0.612). These findings underscore fungal alterations associated with treatment response, particularly in C. glabrata and C. tropicalis (Fig. 4 ). [Add Fig. 4 here] Associations Between Fungal Load and Clinical Outcomes Associations between fungal DNA copy numbers, sampling phase, and various clinical outcomes are presented in Table S1 . The sampling phase (flare-up vs. remission) consistently demonstrated significant associations with multiple outcomes, including disease status, clinical and endoscopic remission, disease severity, disease duration, treatment response, and fecal calprotectin levels. For disease status, the phase variable remained significantly associated with outcome across all models (e.g., OR = 12.07 for C. albicans , p < 0.001). Similar patterns were observed for clinical remission (e.g., OR = 1.61 for C. albicans , p = 0.027), endoscopic remission (e.g., OR = 1.85 for C. albicans, p = 0.012), and disease severity (e.g., OR = 8.87 for C. albicans , p < 0.001). Calprotectin levels were significantly lower during remission phases in all fungal models (e.g., β = − 34.65 for C. albicans, p < 0.001). Treatment response also showed moderate associations with phase and limited associations with fungal load. For instance, phase was significantly associated with treatment response for C. albicans (OR = 3.21, p = 0.015) and C. tropicalis (OR = 2.48, p = 0.032) in interaction models. The interaction term for C. tropicalis reached statistical significance (p = 0.049), suggesting a potential moderating effect of disease phase. In contrast, fungal DNA copy numbers alone showed limited associations with clinical outcomes. While C. glabrata DNA copy number was significantly associated with clinical and endoscopic remission in some models (e.g., p = 0.017 and p = 0.042, respectively), the majority of fungal predictors were not statistically significant. Interaction terms between fungal load and phase were generally nonsignificant, although a few showed trends suggesting potential moderation effects (Figure S1 ). Discussion We have investigated the fecal fungal compositions in a prospective IBD cohort treated with the TNF-α blocker ADA (CinnoRA®) and followed over 6 months. While gut bacterial composition in IBD is well studied, fungal communities remain less characterized. Despite growing interest, studies exploring the fungal mycobiota in IBD remain limited particularly in UC, as most existing research has assessed the mycobiota in the stool and colon biopsies of CD patients [ 11 ]. We found differences in the frequency of some selected gut mycobiota species between UC patients and healthy controls. Additionally, we observed a slight variation in the load of these fungal species among UC patients, depending on their disease phases and response to Adalimumab treatment. It has been previously described that the fungal species belonging to the phyla Ascomycota and Basidiomycota were overrepresented in the gut mycobiota community of IBD patients [ 6 , 12 ]. Furthermore, the most common genera in the human gut mycobiota are Saccharomyces and Candida , [ 6 , 13 – 15 ] which were analyzed in this study. Our quantitative analysis revealed significant fungal shifts, particularly in Candida species, among UC patients. C. albicans and C. glabrata exhibited notable increases compared to healthy subjects (2.27-fold and 2.3-fold, respectively, p < 0.005 and p = 0.009), reinforcing their potential role in gut dysbiosis associated with IBD [ 16 ]. This aligns with the previous observations that Candida is more abundant in IBD patients compared with healthy controls [ 5 , 17 , 18 ]. In addition, it has been reported that IBD patients are characterized by elevated C. albicans levels and reduced S. cerevisiae levels, further highlighting the dysbiotic properties of C. albicans [ 6 , 15 ]. Notably, C. albicans colonizes the intestinal mucosa, inducing both protective immunity and inflammation depending on the context [ 19 ]. Furthermore, due to its pathogenic potential, C. albicans can invade epithelial cells and release cytolytic enzymes, leading to damage to the epithelial cells. It can also promote inflammatory responses by inducing IL-1, IL-36, and IL-17. Thus, C. albicans plays a significant role in regulating cytokine responses and inducing mucosal inflammation [ 11 , 20 ]. Interestingly, we found that C. tropicalis decreased in UC patients, although this reduction was not statistically significant. These findings are in agreement with those of Iliev et al. reported that the levels of C. albicans are consistently elevated in fecal samples from IBD patients, while the abundance of C. tropicalis varies across different studies [ 15 , 21 ]. Moreover, the investigation revealed a reduction in S. cerevisiae among patients with UC. However, this trend did not reach statistical significance. Further, Pascal et al. demonstrated that the abundance of Saccharomyces species is diminished in patients with IBD; however, this reduction is primarily observed in individuals with CD, rather than in those with UC [ 22 ]. In a separate study examining fungal dysbiosis, Imai et al. found that the genus Saccharomyces was more prevalent in healthy controls than in IBD patients [ 23 ]. Studies suggest that S. cerevisiae exerts a protective role in the inflammatory process, indicating it could function as an anti-inflammatory species. It is proposed that S. cerevisiae may regulate host responses by inducing IL-10 production [ 6 ]. Disease activity influenced fungal burden, with C. tropicalis showing a substantial increase during flare-ups (2.04-fold, p < 0.0001), while S. cerevisiae , C. albicans , and C. glabrata remained largely stable across disease phases. These findings suggest that specific fungal populations fluctuate during inflammation. This dynamic behavior warrants further investigation into their role in UC pathogenesis [ 16 ]. Intriguingly, in contrast to our findings, Sokol et al. reported a reduction in Saccharomyces during UC flares compared to remission, suggesting a potential protective role of this genus against disease activity [ 6 ]. They also confirmed a significant increase in C. albicans abundance among patients with IBD, particularly during flare-ups [ 6 ]. These contrasting findings underscore the challenge of determining whether a fungal organism has a probiotic or dysbiotic property, emphasizing the species-specific nature of their interactions with the host [ 15 ]. In addition, the small sample size, variations in investigation methods, and limitations in patient follow-up may contribute to these divergent findings. In a recent study, Hsia et al. showed that the Ascomycota phylum, along with the genera Saccharomyces and Candida, are enriched among UC patients with severe colonic inflammation compared to periods of remission. This suggests that these fungal taxa could serve as biomarkers and potential therapeutic targets for UC [ 24 ]. Only a few studies have investigated the possible markers of response to anti-TNF therapy in the gut mycobiota. In the current study, stratification of patients based on response to adalimumab revealed significant fungal variations. Non-responders exhibited higher C. glabrata (p = 0.024) and C. tropicalis (3.61-fold increase, p = 0.034), suggesting these species may affect treatment efficacy. While S. cerevisiae and C. albicans levels were similar between groups, the observed shifts in C. tropicalis and C. glabrata suggest fungal microbiota composition might influence therapeutic outcomes in UC [ 25 ]. A similar study by Ventin-Holmberg et al. demonstrated that Candida species were more abundant in non-responders to anti-TNF therapy (Infliximab) in both CD and UC patients, which is consistent with our results [ 15 ]. Additionally, a further study conducted by this group focusing on pediatric patients with IBD demonstrated that individuals who did not respond to treatment with the TNF-α antagonist infliximab exhibited higher levels of Candida spp. compared to responders to the therapy [ 26 ]. We performed a correlation analysis aiming to investigate the DNA copy numbers, sampling phase, and various clinical outcomes about ADA therapy response. Interaction models revealed that disease phase significantly influenced treatment response in relation to both C. albicans and C. tropicalis . Also, the effect of C. tropicalis may vary depending on the stage of the disease, exhibiting different behaviors across clinical states. A recent study by Catalán-Serra et al. showed that C. albicans was significantly more prevalent in patients with UC and CD. However, it was not found to be significantly overrepresented in patients experiencing active disease, and its abundance did not correlate with the inflammatory characteristics of the conditions [ 5 ]. These findings suggest that fungal behavior may not be uniform across clinical stages and could play a modulatory role in therapeutic efficacy. Although correlation analyses of the relationship between fungal load and disease activity revealed that flare-up and remission patients showed differences in correlations between C. glabrata and disease states. In such a way that C. glabrata was associated with clinical and endoscopic remission. In consistent with our results, Hsia et al. identified a correlation between high levels of Candida species and disease activity in UC patients, while a lower frequency of Candida was associated with disease quiescence [ 24 ]. Most studies conducted so far focus on patients with IBD who are not undergoing specific treatments. While much of the existing research has concentrated on patients with CD, there is a significant lack of studies investigating fungal prevalence and its relationship with clinical outcomes in patients with UC. Furthermore, these studies typically evaluate fungal load at the genus level rather than at the species level. In our study, the absolute fungal DNA copy numbers showed only limited associations with clinical outcomes, suggesting that fungal burden alone may not fully capture the complexities of host-mycobiota interactions influencing treatment response. Most fungal predictors assessed did not reach statistical significance, indicating that while C. glabrata may hold promise as a potential biomarker, fungal burden alone may not consistently predict treatment response across all species. These findings highlight the complexity of host-fungal interactions and suggest that species-specific dynamics rather than overall fungal load may be more relevant to therapeutic outcomes. The limited number of patients in our study may hinder significant findings. Additionally, variability in response assessment methods, microbiota analysis techniques, selected fungal strains, therapeutic protocols, follow-up duration, and the inclusion of control subjects further complicates meaningful comparisons across studies. Conclusion In conclusion, we found significant differences in the mentioned gut mycobiota composition between UC patients and healthy individuals. UC patients exhibited a higher abundance of C. albicans and C. glabrata species, while the load of C. tropicalis remained stable throughout the study. Our findings indicate that non-responders to ADA therapy had a higher abundance of C. glabrata and C. tropicalis . Notably, a higher abundance of C. tropicalis in patients experiencing flare-ups was observed. Throughout therapy, S. cerevisiae remained stable between UC patients in flare-ups and remission phases, as well as between responders and non-responders. These findings underscore the potential of fungal biomarkers in predicting treatment response. While calprotectin remains a reliable inflammation marker, fungal dysbiosis particularly C. glabrata and C. tropicalis elevations in non-responders require deeper investigation. Future research should focus on longitudinal, multi-center studies that incorporate fungal biomarkers into integrated omics frameworks, and to validate fungal profiling as a predictive tool, potentially guiding personalized therapeutic strategies in UC management. Abbreviations Inflammatory Bowel Disease (IBD), Ulcerative colitis (UC), Crohn’s disease (CD), Gastrointestinal (GI), Adalimumab (ADA), Tuberculosis (TB), Hepatitis B virus (HBV), Hepatitis C Virus (HCV), Human Immunodeficiency Virus (HIV), Erythrocyte Sedimentation Rate (ESR), C-reactive protein (CRP), Fecal Calprotectin (FC), Enzyme-Linked Immunosorbent Assays (ELISA), Sabouraud Dextrose Agar (SDA), Yeast Medium (YM), Interquartile Range (IQR), Standard deviation (SD), Cycle threshold (Ct), Generalized estimating equations (GEE), Odds ratios (OR), Confidence Intervals (CI), Healthy Control (HC), Tumor Necrosis Factor (TNF), Interleukin (IL). Declarations Ethics approval and consent to participate: The research was approved and performed according to the Ethical Review Committee of the Research Institute for Gastroenterology and Liver Diseases at Shahid Beheshti University of Medical Sciences (Project No. IR.SBMU.RIGLD.REC.1401.006). Consent for publication: Written informed consent was obtained from all participants. Availability of data and materials: The supporting data are available upon request to the corresponding author [Shaghayegh Baradaran Ghavami]. Competing interests: The authors declare that they have no competing interests. Funding: This work was supported by the Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Authors' contributions: Conceptualization, S.B.G., E.L., H.S.; Data curation, M.F., H.B.; Formal analysis, M.F. M.A.L.; Funding acquisition, S.S., E.L.; Investigation, M.F., M.F, A.T., N.K.; Methodology, S.B.G., H.H.; Project administration, S.B.G.; Supervision, S.B.G., H.H.; Writing original draft, M.F., S.B.G. M.A.L; Review and editing, S.B.G., H.H., M.A.L. All authors have read and agreed to the published version of the manuscript. All the authors read and approved the final manuscript. Declaration of Generative AI and AI-assisted technologies in the writing process During the preparation of this work, the authors used Grammarly and ChatGPT (GPT-4o) in order to improve readability and language. After using these tools, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication. References Saez A et al (2023) Pathophysiology of inflammatory bowel disease: innate immune system. Int J Mol Sci 24(2):1526 Ghavami SB et al (2022) Serological response to SARS-CoV-2 is attenuated in patients with inflammatory bowel disease and can affect immunization. JGH Open 6(4):266–269 Ananthakrishnan AN et al (2018) Environmental triggers in IBD: a review of progress and evidence. Nat reviews Gastroenterol Hepatol 15(1):39–49 Aghdaei HA et al (2022) Overexpression of toll-like receptors and co-stimulatory molecules on immature dendritic cells of Crohn's disease. Gene Rep 27:101579 Catalan-Serra I et al Fungal Microbiota Composition in Inflammatory Bowel Disease Patients: Characterization in Different Phenotypes and Correlation With Clinical Activity and Disease Course (dec, 10.1093/ibd/izad289 , (2023)). INFLAMMATORY BOWEL DISEASES, 2024. 30(5): pp. 876–876). INFLAMMATORY BOWEL DISEASES, 2024. 30(5): pp. 876–876 Sokol H et al (2017) Fungal microbiota dysbiosis in IBD. Gut 66(6):1039–1048 Jena A, Dutta U (2024) Role of fungus in inflammatory bowel disease: The butterfly effect? Indian J Gastroenterol 43(4):697–699 Yu M et al (2023) Fungal dysbiosis facilitates inflammatory bowel disease by enhancing CD4 + T cell glutaminolysis. Front Cell Infect Microbiol 13:1140757 Wang F, Wang Z, Tang J (2023) The interactions of Candida albicans with gut bacteria: a new strategy to prevent and treat invasive intestinal candidiasis. Gut pathogens 15(1):30 Freiberger T The Impact of DNA Extraction Methods on Stool Bacterial and Fungal Microbiota Community Recovery. Patnaik S et al (2024) Role of Candida species in pathogenesis, immune regulation, and prognostic tools for managing ulcerative colitis and Crohn's disease. World J Gastroenterol 30(48):5212 Ott SJ et al (2008) Fungi and inflammatory bowel diseases: alterations of composition and diversity. Scand J Gastroenterol 43(7):831–841 Huseyin CE et al (2017) The fungal frontier: a comparative analysis of methods used in the study of the human gut mycobiome. Front Microbiol 8:1432 Honkanen J et al (2020) Fungal dysbiosis and intestinal inflammation in children with beta-cell autoimmunity. Front Immunol 11:468 Ventin-Holmberg R et al (2021) Bacterial and fungal profiles as markers of infliximab drug response in inflammatory bowel disease. J Crohn's Colitis 15(6):1019–1031 Singh A et al (2021) Identifying anti-TNF response biomarkers in ulcerative colitis using a diffusion-based signalling model. Bioinf Adv 1(1):vbab017 Chehoud C et al (2015) Fungal signature in the gut microbiota of pediatric patients with inflammatory bowel disease. Inflamm Bowel Dis 21(8):1948–1956 Lewis JD et al (2015) Inflammation, antibiotics, and diet as environmental stressors of the gut microbiome in pediatric Crohn’s disease. Cell Host Microbe 18(4):489–500 Leonardi I et al (2020) Fungal trans-kingdom dynamics linked to responsiveness to fecal microbiota transplantation (FMT) therapy in ulcerative colitis. Cell Host Microbe 27(5):823–829e3 Moyes DL et al (2016) Candidalysin is a fungal peptide toxin critical for mucosal infection. Nature 532(7597):64–68 Iliev ID, Cadwell K (2021) Effects of intestinal fungi and viruses on immune responses and inflammatory bowel diseases. Gastroenterology 160(4):1050–1066 Pascal V et al (2017) A microbial signature for Crohn's disease. Gut 66(5):813–822 Imai T et al (2019) Characterization of fungal dysbiosis in Japanese patients with inflammatory bowel disease. J Gastroenterol 54(2):149–159 Hsia K et al (2023) Alterations in the fungal microbiome in ulcerative colitis. Inflamm Bowel Dis 29(10):1613–1621 Cui G, Florholmen J, Goll R (2022) Could Mucosal TNF Transcript as a Biomarker Candidate Help Optimize Anti-TNF Biological Therapy in Patients With Ulcerative Colitis? Front Immunol 13:881112 Ventin-Holmberg R et al (2022) The gut fungal and bacterial microbiota in pediatric patients with inflammatory bowel disease introduced to treatment with anti-tumor necrosis factor-α. Sci Rep 12(1):6654 Additional Declarations No competing interests reported. Supplementary Files SupplementaryTableS1.docx SupplementaryFigureS1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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13:41:19","extension":"png","order_by":43,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":35229,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7749916/v1/829208fb56725da55413dd4a.png"},{"id":94672722,"identity":"f766aab8-995e-4184-9ebd-4d2f92faf121","added_by":"auto","created_at":"2025-10-29 13:40:52","extension":"png","order_by":44,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":29275,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7749916/v1/ec91c536aa716dddaf240cd9.png"},{"id":94673472,"identity":"0e7280b2-4fef-4b1c-9021-2ca4b8f30a1e","added_by":"auto","created_at":"2025-10-29 13:41:25","extension":"png","order_by":45,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":29893,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7749916/v1/fe2cc6c6e96443685604bad7.png"},{"id":94672539,"identity":"457f4471-133b-4c91-97cc-76f56201ed25","added_by":"auto","created_at":"2025-10-29 13:40:42","extension":"png","order_by":46,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":21658,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7749916/v1/a50476dd5f7c4097c18b2676.png"},{"id":94665453,"identity":"b28c4875-d1bc-4541-91b6-574637775597","added_by":"auto","created_at":"2025-10-29 12:28:14","extension":"xml","order_by":47,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":110699,"visible":true,"origin":"","legend":"","description":"","filename":"babb71c8f6bc4b0cad42782325d293711structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7749916/v1/abb94e5a88ad1a63767321ff.xml"},{"id":94665455,"identity":"0361df7d-e3d4-4584-98d3-706ba994f1b5","added_by":"auto","created_at":"2025-10-29 12:28:14","extension":"html","order_by":48,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":124753,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7749916/v1/a3d1985fc768198d15b30199.html"},{"id":94672889,"identity":"76d4a371-65d1-43ac-8fdf-76ea43103d5d","added_by":"auto","created_at":"2025-10-29 13:41:03","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":38930,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of fecal calprotectin levels between study groups:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eNon-responders exhibited significantly higher calprotectin concentrations, showing a 1.62-fold increase compared to responders to Adalimumab therapy (p = 0.009).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eComparing different disease states indicated that patients in the flare-up phase demonstrated a 2.53-fold increase in calprotectin levels compared to those in remission (p \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003e(The asterisks indicate levels of statistical significance: *, p \u0026lt;0.05; **, p \u0026lt;0.001; ***, p \u0026lt;0.0001)\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7749916/v1/9b897493b7ceff1296b5ae72.jpg"},{"id":94665375,"identity":"c32e1ef3-a454-4cfa-944e-2a096240a49f","added_by":"auto","created_at":"2025-10-29 12:28:13","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":56826,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFungal abundance in UC patients undergoing anti-TNF therapy compared to healthy controls\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003e\u003cem\u003eC. albicans\u003c/em\u003e showed a significant 2.27-fold increased abundance in UC patients (p = 0.005), and also, \u003cstrong\u003e(B) \u003c/strong\u003e\u003cem\u003eC. glabrata \u003c/em\u003eincreased by 2.31-fold in UC patients compared to HC (p = 0.009), though. In contrast, (\u003cstrong\u003eC) \u003c/strong\u003e\u003cem\u003eC. tropicalis\u003c/em\u003e displayed a significant 1.66-fold decline (p = 0.386), while\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e(D) \u003c/strong\u003e\u003cem\u003eS. cerevisiae\u003c/em\u003e exhibited a 1.4-fold reduction in UC patients (p = 0.082). (ns: non-significant, HC: Healthy control, UC: Ulcerative colitis) (The asterisks indicate levels of statistical significance: *, P\u0026lt;0.05; **, P\u0026lt;0.001)\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7749916/v1/a911fdfbea458a3c8cbf2d1b.jpg"},{"id":94665376,"identity":"e7bb5eb2-5438-4292-bbf7-b62be031c78f","added_by":"auto","created_at":"2025-10-29 12:28:13","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":63667,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in fungal prevalence between UC patients in flare-up and remission phases.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e The prevalence of \u003cem\u003eC. albicans\u003c/em\u003e increased by 1.03-fold in flare-ups compared to remission; however, this difference was not statistically significant (p = 0.879). \u003cstrong\u003e(B)\u003c/strong\u003e \u003cem\u003eC. glabrata\u003c/em\u003e exhibited a 1.05-fold increase in flare-up patients relative to those in remission, though the difference did not reach statistical significance (p = 0.811). \u003cstrong\u003e(C)\u003c/strong\u003e Among UC patients in the flare-up phase, \u003cem\u003eC. tropicalis\u003c/em\u003e showed the highest observed elevation, with a significant 2.04-fold increase compared to remission patients (p \u0026lt; 0.0001). \u003cstrong\u003e(D)\u003c/strong\u003e The prevalence of \u003cem\u003eS. cerevisiae\u003c/em\u003eshowed a 1.3-fold increase in the remission phase whereas these differences did not reach statistical significance (p = 0.257). (ns: non-significant) (The asterisks indicate levels of statistical significance: *, P\u0026lt;0.05; **, P\u0026lt;0.001; ***, p \u0026lt;0.0001)\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7749916/v1/eb4148301a63658ae79e4bbf.jpg"},{"id":94665381,"identity":"c32f250c-0ce7-4704-a237-4ea84deef83a","added_by":"auto","created_at":"2025-10-29 12:28:13","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":56966,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAbsolute copy number differences in fungal species between non-responders and responders to anti-TNF therapy.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003e\u003cem\u003eC. albicans\u003c/em\u003e displayed a 1.03-fold increase in non-responders compared to responders; however, this difference was not statistically significant (p = 0.891). \u003cstrong\u003e(B) \u003c/strong\u003eA significant 1.69-fold elevation in \u003cem\u003eC. glabrata\u003c/em\u003e was observed in non-responders relative to responders (p = 0.024). \u003cstrong\u003e(C) \u003c/strong\u003eThe abundance of \u003cem\u003eC. tropicalis\u003c/em\u003e was notably higher in non-responders, showing a 3.61-fold increase compared to responders (p = 0.034). \u003cstrong\u003e(D) \u003c/strong\u003eAlthough \u003cem\u003eS. cerevisiae\u003c/em\u003e levels were 1.14-fold higher in responders, this difference did not reach statistical significance (p = 0.612). (ns: non-significant) (The asterisks indicate levels of statistical significance: *, P\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7749916/v1/1853d0cc4ba3850d86f16422.jpg"},{"id":103687256,"identity":"87ac68fe-d66e-488b-8398-5331aa522ad8","added_by":"auto","created_at":"2026-03-01 11:54:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1310948,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7749916/v1/7361a5cb-f190-4401-9baa-c4300df777df.pdf"},{"id":94665379,"identity":"e927b5a2-8056-4eb7-97f3-6c9a917f8c5f","added_by":"auto","created_at":"2025-10-29 12:28:13","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":26854,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7749916/v1/02b2549d8982dab36a12c5ac.docx"},{"id":94672758,"identity":"81797cd0-78df-477c-962e-f006dae35f7d","added_by":"auto","created_at":"2025-10-29 13:40:55","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":829009,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7749916/v1/0ee8883de20c9aae3bb39994.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring Fungal Biomarkers as Predictors of Treatment Response in Ulcerative Colitis Patients Receiving Anti-TNF Therapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInflammatory Bowel Disease (IBD), which encompasses Crohn\u0026rsquo;s disease (CD) and ulcerative colitis (UC), is a chronic and relapsing inflammatory condition of the gastrointestinal (GI) tract [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The exact causes of IBD remain unclear, but it is understood to result from a complex interplay between genetic predisposition, environmental triggers, immune system dysregulation, and the gut microbiome [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, recent studies suggest that fungi, an integral part of the gut microbiota, may also play a crucial role in the pathogenesis and progression of IBD. This often-overlooked component of the gut microbiome, known as the mycobiome, may have both harmful and potentially beneficial effects on disease outcomes, influencing IBD activity during both active and remission [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFungal species such as \u003cem\u003eCandida albicans (C. albicans)\u003c/em\u003e, \u003cem\u003eSaccharomyces cerevisiae (S. cerevisiae)\u003c/em\u003e, and \u003cem\u003eMalassezia\u003c/em\u003e spp. have been identified as prevalent in the gut mycobiome, and their abundance appears to fluctuate with disease activity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. During active phases of IBD, specific fungi can become overrepresented, potentially exacerbating inflammation. For instance, \u003cem\u003eC. albicans\u003c/em\u003e has been found to increase significantly during IBD flare-ups, potentially promoting inflammation by interacting with immune cells and producing metabolites that disturb the intestinal barrier [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These fungi may stimulate pro-inflammatory pathways, activating immune responses that can worsen tissue damage and contribute to the persistence and severity of symptoms in the active disease phase [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn contrast, during remission phases, a balanced and stable fungal community may support mucosal healing and help maintain intestinal homeostasis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, imbalances in fungal populations potentially triggered by factors like dietary changes, antibiotic exposure, or immune shifts could destabilize this equilibrium. Such disruptions in the mycobiome could create conditions that facilitate relapse, highlighting fungi as possible contributors to disease recurrence. One of the virulence factors of fungi is hyphae, which help form biofilms with bacteria. This interaction between bacteria and fungi leads to antibiotic resistance and complications in IBD pathogenesis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eUnderstanding the distinct roles that fungi play in the active and remission phases of IBD suggests valuable insights into the disease\u0026rsquo;s pathophysiology and underscores the potential of the mycobiome as a therapeutic target. As research into the gut mycobiome advances, it may be possible to identify specific fungal biomarkers for disease monitoring, as well as to develop novel therapies aimed at modulating the fungal microbiome to support remission and prevent relapse. Overall, fungi represent a promising frontier in the study of IBD, offering new avenues to improve management and outcomes for patients affected by this challenging and often debilitating condition.\u003c/p\u003e\u003cp\u003eThis study aimed to investigate the fungal load in two states of IBD and to compare the differences between responders and non-responders to adalimumab (ADA), providing a more in-depth setup for a detailed examination of fungi\u0026rsquo;s role in IBD.\u003c/p\u003e"},{"header":"Method and Material","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eEnrolment of Patients and Healthy Subjects\u003c/h2\u003e\u003cp\u003e23 adult patients diagnosed with UC were enrolled at the Gastroenterology and Hepatology clinic (Taleghani Hospital, Tehran, Iran) from 2018 to 2021. Adalimumab (ADA) (CinnoRA\u0026reg;) therapy was initiated for patients due to active inflammation without response or with intolerance to prior conventional or biological IBD medications. Patients were classified as responders if they exhibited significant clinical and endoscopic improvement following ADA therapy, whereas non-responders were defined as those who showed minimal or no therapeutic benefit despite treatment. Individuals with other autoimmune disorders, as well as those diagnosed with cancer or viral infections (such as tuberculosis (TB), Hepatitis B virus (HBV), hepatitis C virus (HCV), and HIV), were excluded from the study. The exclusion criteria for healthy participants were consistent with those applied to the patient group. Furthermore, it is important to note that none of the participants had a history of long-term antibiotic and antifungal therapy within the last three months. The diagnosis of IBD was conducted based on established clinical parameters, including the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), fecal calprotectin (FC) levels, and the results of colonoscopy. The study design complied with the Declaration of Helsinki. All patients and healthy subjects gave written informed consent for sample collection according to the sampling protocol approved by the Ethical Review Committee of the Research Institute for Gastroenterology and Liver Diseases at Shahid Beheshti University of Medical Sciences (Project No. IR.SBMU.RIGLD.REC.1401.006).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSample Collection\u003c/h3\u003e\n\u003cp\u003eFecal samples were obtained from patients diagnosed with UC at three distinct time points: prior to the initiation of ADA (CinnoRA\u0026reg;) therapy, as well as three- and six-months post-treatment commencement (before the second and third injection, respectively). Sample collection adhered to standardized protocols to ensure consistency and reliability [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Immediately after collection, the samples were stored at -80\u0026deg;C to preserve microbial integrity and prevent degradation. These preserved samples were subsequently utilized for fungal composition assessments to investigate potential therapeutic effects and specific fungal species alterations throughout treatment.\u003c/p\u003e\n\u003ch3\u003eELISA Assay\u003c/h3\u003e\n\u003cp\u003eFecal calprotectin levels were quantified using enzyme-linked immunosorbent assays (ELISA) performed on fecal supernatants. The analyses were conducted utilizing the Calprest NG ELISA Kit (Eurospital Diagnostic, Trieste, Italy), following the manufacturer\u0026rsquo;s protocol to ensure accuracy and reproducibility. The procedure involved the extraction of calprotectin from fecal samples, followed by incubation with specific antibodies to enable precise detection. Absorbance measurements were recorded to determine calprotectin concentrations, providing valuable insights into intestinal inflammation. All assays were performed under standardized laboratory conditions to minimize variability and ensure the reliability of results for subsequent statistical analyses.\u003c/p\u003e\n\u003ch3\u003eDNA Extraction from Fecal Samples\u003c/h3\u003e\n\u003cp\u003eFrozen fecal samples were thawed under controlled conditions and then completely homogenized to preserve sample integrity and minimize potential degradation of nucleic acids. DNA extraction was performed using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) following the manufacturer\u0026rsquo;s protocol to ensure optimal yield and purity. The extraction process included enzymatic and mechanical disruption to effectively lyse fungal cells and facilitate the isolation of high-quality genomic material. The purity and concentration of the extracted DNA were assessed using a NanoDrop\u0026reg; 192 ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), measuring absorbance at specific wavelengths (260/280 nm) to evaluate contamination levels and sample suitability for downstream molecular analyses.\u003c/p\u003e\n\u003ch3\u003eFungal Culture\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eC. albicans\u003c/em\u003e (ATCC 10231), \u003cem\u003eC. glabrata\u003c/em\u003e (ATCC 90030), and \u003cem\u003eC. tropicalis\u003c/em\u003e C. tropicalis (PTCC 5028) were cultivated on Sabouraud Dextrose Agar (SDA) (Sigma-Aldrich, USA) plates, a selective medium designed to support fungal growth by providing an optimal nutrient-rich environment. The cultures were incubated aerobically at 30\u0026deg;C to facilitate robust proliferation and ensure physiological consistency across samples. In parallel, \u003cem\u003eS. cerevisiae\u003c/em\u003e (ATCC 9763) was cultured on a universal yeast medium (YM) (Sigma-Aldrich, USA), which contains essential nutrients for optimal yeast metabolism. The incubation conditions for \u003cem\u003eS. cerevisiae\u003c/em\u003e were set at 28\u0026deg;C for 24 hours under aerobic conditions to promote consistent colony formation and metabolic activity. All culturing procedures were performed under sterile conditions to prevent contamination.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eFungal DNA Extraction\u003c/h2\u003e\u003cp\u003eGenomic DNA was extracted from cultured Candida species and \u003cem\u003eS. cerevisiae\u003c/em\u003e using a DNA extraction kit (ROJE TECHNOLOGIES, Iran), following the manufacturer\u0026rsquo;s protocol to ensure optimal yield and purity. The extraction process involved enzymatic lysis and purification steps designed to remove potential contaminants such as proteins and polysaccharides, thereby maximizing the integrity of fungal DNA. Quality control assessments were conducted using a NanoDrop\u0026reg; ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), measuring absorbance at 260/280 nm to evaluate purity and concentration. Additionally, the integrity of the extracted DNA was verified through agarose gel electrophoresis, wherein DNA fragments were visualized under ultraviolet illumination following SYBR Safe staining.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eQuantitative Absolute Real-time PCR\u003c/h3\u003e\n\u003cp\u003eFor the standard curve employed in absolute quantification, genomic DNA extracted from \u003cem\u003eC. albicans, C. glabrata, C. tropicalis, and S. cerevisiae\u003c/em\u003e cultures was prepared in a series of tenfold dilutions ranging from 10\u0026ndash;10\u003csup\u003e5\u003c/sup\u003e. These serial dilutions were incorporated into each real-time PCR run to serve as reference standards for the generation of the standard curve, facilitating the precise quantification of fungal species copy numbers. Real-time PCR was performed in a final reaction volume of 10 \u0026micro;L, comprising 50% RealQ Plus 2x Master Mix Green (Ampliqon, Odense, Denmark). The assays were carried out utilizing species-specific primer pairs (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), designed to target conserved genomic regions, thereby ensuring accurate and reproducible quantification of fungal DNA across all experimental replicates. All PCR procedures were executed under optimized thermocycling conditions to maximize sensitivity while minimizing potential amplification biases.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimers used in Quantitative Absolute Real-time PCR\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eName\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer Sequence\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eProduct Size (bp)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eC. albicans\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026acute;-GCTCCTGCTCCTGAAATGAC-3\u0026acute;\u003c/p\u003e\u003cp\u003e3\u0026acute;-CTGGAGCAATTGGTGAGGTT-5\u0026acute;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e184\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eC. glabrata\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026acute;-AAAGAAAGAACACACTCTGCC-3\u0026acute;\u003c/p\u003e\u003cp\u003e3\u0026acute;-GGGTCTGAGTTGCTAATGTTG-5\u0026acute;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e153\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eC. tropicalis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026acute;-GCGGTAGGAGAATTGCGTT-3\u0026acute;\u003c/p\u003e\u003cp\u003e3\u0026acute;-TCATTATGCCAACATCCTAGGTTTA-5\u0026acute;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e117\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eS. cerevisiae\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026acute;-GAAATGCCACCGTGAATGC-3\u0026acute;\u003c/p\u003e\u003cp\u003e3\u0026acute;-CTTTGGTGGTGATCCTCTATGATTG-5\u0026acute;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e113\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003e[Add Table here]\u003c/h3\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003e Quantitative real-time PCR data were obtained using Roche Light Cycler 96 Software based on standard curve and copy number calculations, with copy number data analyzed using log10 transformation (log₁₀[x\u0026thinsp;+\u0026thinsp;1]) to ensure normalization. Statistical analyses were conducted in GraphPad Prism (v8.4.3) and R (v4.5.0), with Student\u0026rsquo;s t-test used for group comparisons (significance threshold: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Descriptive statistics summarized demographic, clinical, and fungal variables at Phase 1 of follow-up, with continuous data presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD or median (IQR), and categorical data as counts and percentages, without statistical comparison.\u003c/p\u003e\u003cp\u003eFungal quantification, including DNA copy numbers and qPCR cycle threshold (Ct) values for \u003cem\u003eC. albicans, C. glabrata, C. tropicalis\u003c/em\u003e, and \u003cem\u003eS. cerevisiae\u003c/em\u003e, along with fecal calprotectin levels, served as predictors in multivariable models. Generalized estimating equations (GEE) accounted for repeated measures, with two models: (1) main effects of fungal load and sampling phase, and (2) an interaction model including fungal load \u0026times; phase interaction. Patient ID was the clustering unit. Appropriate statistical models were applied based on outcome type: binary (binomial link), ordinal (cumulative logit link), and continuous (Gaussian link function). Results were reported as odds ratios (ORs) or regression coefficients (β) with 95% confidence intervals (CIs). Significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Longitudinal trends in fungal DNA copy numbers were visualized using log₁₀-transformed line plots, stratified by clinical parameters such as disease state, treatment response, severity, clinical remission, and endoscopic remission.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eDemographic and Clinical Characteristics\u003c/h2\u003e\n \u003cp\u003eAs shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, 23 patients with ulcerative colitis (UC) were included in the Phase 1 assessment. The mean age was 32.09 years (SD: 9.89), and the sex distribution was nearly equal, with 52.17% female and 47.83% male. The median duration of disease was 54.00 months (IQR: 24.00\u0026ndash;93.00). All patients were in the flare-up phase of disease activity at the baseline. The majority of participants had severe disease (73.91%), followed by moderate to severe (13.04%) and moderate disease (8.70%) at the baseline. Only one patient (4.35%) was classified as having mild to moderate disease, while no cases of mild or no inflammation were observed. Regarding disease extension, 60.87% had pancolitis and 39.13% had left-sided colitis; no patients presented with proctosigmoiditis or no inflammation. Moreover, a cohort of 20 healthy control participants was enrolled in this study, with a mean age of 53.3 years (SD: 11.1). The sample consisted of 60% females and 40% males. A qualified physician conducted a comprehensive health assessment to confirm the absence of gastrointestinal disorders in all individuals.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDemographic and Clinical Characteristics of Patients with Ulcerative Colitis at Phase 1 of Follow-Up\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVariable\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLevel\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean or Median /Frequency\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eDemographic Variables\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e------\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.09\u0026thinsp;\u0026plusmn;\u0026thinsp;9.89\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12 (52.17)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11 (47.83)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDisease Duration (months)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e------\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54.00 (24.00, 93.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eDisease Characteristics\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eDisease state\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFlare-up\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23 (100.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRemission\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eTreatment response status\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNon-responder\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 (34.78)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eResponder\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15 (65.22)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003eSeverity of disease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo inflammation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMild\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMild to moderate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (4.35)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModerate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (8.70)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModerate to severe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 (13.04)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSevere\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17 (73.91)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eDisease Extension\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo inflammation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eProctosigmoiditis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLeft sided colitis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9 (39.13)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePancolitis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14 (60.87)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eEndoscopic Remission\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7 (30.43)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16 (69.57)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eClinical Remission\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 (34.78)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15 (65.22)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e[Add Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e here]\u003c/h2\u003e\n \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\n \u003ch2\u003eCalprotectin Measurement\u003c/h2\u003e\n \u003cp\u003eCalprotectin levels in stool samples were measured using ELISA. The mean calprotectin concentration in the study population was 81.64\u0026thinsp;\u0026plusmn;\u0026thinsp;57.86 \u0026micro;g/g, with a 95% confidence interval (CI) ranging from 0 to 153 \u0026micro;g/g. Non-responders exhibited significantly elevated fecal calprotectin levels, measuring 1.62-fold higher than those of responders (p\u0026thinsp;=\u0026thinsp;0.009) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Disease activity was classified according to the Mayo score, distinguishing remission from flare-up phases. Patients in the flare-up phase demonstrated a substantial increase in fecal calprotectin levels, with concentrations rising by 2.53-fold compared to those in remission (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e[Add Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e here]\u003c/h2\u003e\n \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\n \u003ch2\u003eFungal Populations in Ulcerative Colitis Patients and Healthy Subjects\u003c/h2\u003e\n \u003cp\u003eQuantitative analysis revealed notable differences in Log10-transformed fungal species DNA copy numbers between healthy individuals and UC patients undergoing anti-TNF therapy. The mean values for \u003cem\u003eC. albicans\u003c/em\u003e were 1.049 (SD\u0026thinsp;=\u0026thinsp;1.65) in healthy subjects and 2.38 (SD\u0026thinsp;=\u0026thinsp;1.78) in UC patients, showing a 2.27-fold increase (p\u0026thinsp;=\u0026thinsp;0.005). Similarly, \u003cem\u003eC. glabrata\u003c/em\u003e exhibited a mean of 0.79 (SD\u0026thinsp;=\u0026thinsp;1.41) in healthy individuals compared to 1.83 (SD\u0026thinsp;=\u0026thinsp;1.52) in UC patients, reflecting a significant increase (p\u0026thinsp;=\u0026thinsp;0.009). In contrast, \u003cem\u003eC. tropicalis\u003c/em\u003e decreased from 0.4 (SD\u0026thinsp;=\u0026thinsp;0.93) to 0.24 (SD\u0026thinsp;=\u0026thinsp;0.59) in UC patients (p\u0026thinsp;=\u0026thinsp;0.386). While \u003cem\u003eS. cerevisiae\u003c/em\u003e showed a 1.4-fold reduction in UC patients (p\u0026thinsp;=\u0026thinsp;0.082), the declines in \u003cem\u003eC. tropicalis\u003c/em\u003e and \u003cem\u003eS. cerevisiae\u003c/em\u003e did not reach statistical significance (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e[Add Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e here]\u003c/h2\u003e\n \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\n \u003ch2\u003eFungal Prevalence in Patients Stratified by Disease Status\u003c/h2\u003e\n \u003cp\u003eFungal species distribution varied between remission and flare-up groups. The Log10-transformed mean for \u003cem\u003eC. albicans\u003c/em\u003e was 2.24 (SD\u0026thinsp;=\u0026thinsp;1.49) in remission patients and 2.17 (SD\u0026thinsp;=\u0026thinsp;1.70) during flare-ups (p\u0026thinsp;=\u0026thinsp;0.879), indicating a non-significant decline. Similarly, \u003cem\u003eC. glabrata\u003c/em\u003e measured 1.92 (SD\u0026thinsp;=\u0026thinsp;1.36) in remission and 1.82 (SD\u0026thinsp;=\u0026thinsp;1.78) during flare-ups (p\u0026thinsp;=\u0026thinsp;0.811). In contrast, \u003cem\u003eC. tropicalis\u003c/em\u003e showed a statistically significant 2.04-fold increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), rising from 0.97 (SD\u0026thinsp;=\u0026thinsp;0.93) in remission to 1.98 (SD\u0026thinsp;=\u0026thinsp;0.40) in flare-ups. \u003cem\u003eS. cerevisiae\u003c/em\u003e displayed a modest decline from 2.45 (SD\u0026thinsp;=\u0026thinsp;1.71) to 1.88 (SD\u0026thinsp;=\u0026thinsp;1.98), though this difference was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.257). These results suggest that fluctuations occur among certain fungal species while others remain stable across disease phases (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e[Add Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e here]\u003c/h2\u003e\n \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\n \u003ch2\u003eFungal Profiles in Responders and Non-Responders to Anti-TNF Therapy\u003c/h2\u003e\n \u003cp\u003eThe stratification of UC patients based on their response to ADA treatment revealed notable differences in fungal burden across responder and non-responder groups. The Log10-transformed mean for \u003cem\u003eC. albicans\u003c/em\u003e was 2.42 (SD\u0026thinsp;=\u0026thinsp;1.75) in responders and 2.34 (SD\u0026thinsp;=\u0026thinsp;1.95) among non-responders (p\u0026thinsp;=\u0026thinsp;0.192). \u003cem\u003eC. glabrata\u003c/em\u003e increased significantly from 1.59 (SD\u0026thinsp;=\u0026thinsp;1.3) in responders to 2.7 (SD\u0026thinsp;=\u0026thinsp;2.1) in non-responders (p\u0026thinsp;=\u0026thinsp;0.024). In addition, \u003cem\u003eC. tropicalis\u003c/em\u003e exhibited a noticeable 3.61-fold increase (p\u0026thinsp;=\u0026thinsp;0.034), rising from 0.13 (SD\u0026thinsp;=\u0026thinsp;0.44) to 0.5 (SD\u0026thinsp;=\u0026thinsp;0.78). S. cerevisiae showed slight variation (2.23 [SD\u0026thinsp;=\u0026thinsp;1.83] in responders vs. 1.94 [SD\u0026thinsp;=\u0026thinsp;2] in non-responders), though not statistically significant (p\u0026thinsp;=\u0026thinsp;0.612). These findings underscore fungal alterations associated with treatment response, particularly in \u003cem\u003eC. glabrata\u003c/em\u003e and \u003cem\u003eC. tropicalis\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003e[Add Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e here]\u003c/h2\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003eAssociations Between Fungal Load and Clinical Outcomes\u003c/h2\u003e\n \u003cp\u003eAssociations between fungal DNA copy numbers, sampling phase, and various clinical outcomes are presented in Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e. The sampling phase (flare-up vs. remission) consistently demonstrated significant associations with multiple outcomes, including disease status, clinical and endoscopic remission, disease severity, disease duration, treatment response, and fecal calprotectin levels. For disease status, the phase variable remained significantly associated with outcome across all models (e.g., OR\u0026thinsp;=\u0026thinsp;12.07 for \u003cem\u003eC. albicans\u003c/em\u003e, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similar patterns were observed for clinical remission (e.g., OR\u0026thinsp;=\u0026thinsp;1.61 for \u003cem\u003eC. albicans\u003c/em\u003e, p\u0026thinsp;=\u0026thinsp;0.027), endoscopic remission (e.g., OR\u0026thinsp;=\u0026thinsp;1.85 for C. albicans, p\u0026thinsp;=\u0026thinsp;0.012), and disease severity (e.g., OR\u0026thinsp;=\u0026thinsp;8.87 for \u003cem\u003eC. albicans\u003c/em\u003e, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Calprotectin levels were significantly lower during remission phases in all fungal models (e.g., \u0026beta; = \u0026minus;\u0026thinsp;34.65 for C. albicans, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Treatment response also showed moderate associations with phase and limited associations with fungal load. For instance, phase was significantly associated with treatment response for \u003cem\u003eC. albicans\u003c/em\u003e (OR\u0026thinsp;=\u0026thinsp;3.21, p\u0026thinsp;=\u0026thinsp;0.015) and \u003cem\u003eC. tropicalis\u003c/em\u003e (OR\u0026thinsp;=\u0026thinsp;2.48, p\u0026thinsp;=\u0026thinsp;0.032) in interaction models. The interaction term for \u003cem\u003eC. tropicalis\u003c/em\u003e reached statistical significance (p\u0026thinsp;=\u0026thinsp;0.049), suggesting a potential moderating effect of disease phase. In contrast, fungal DNA copy numbers alone showed limited associations with clinical outcomes. While \u003cem\u003eC. glabrata\u003c/em\u003e DNA copy number was significantly associated with clinical and endoscopic remission in some models (e.g., p\u0026thinsp;=\u0026thinsp;0.017 and p\u0026thinsp;=\u0026thinsp;0.042, respectively), the majority of fungal predictors were not statistically significant. Interaction terms between fungal load and phase were generally nonsignificant, although a few showed trends suggesting potential moderation effects (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe have investigated the fecal fungal compositions in a prospective IBD cohort treated with the TNF-α blocker ADA (CinnoRA\u0026reg;) and followed over 6 months. While gut bacterial composition in IBD is well studied, fungal communities remain less characterized. Despite growing interest, studies exploring the fungal mycobiota in IBD remain limited particularly in UC, as most existing research has assessed the mycobiota in the stool and colon biopsies of CD patients [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. We found differences in the frequency of some selected gut mycobiota species between UC patients and healthy controls. Additionally, we observed a slight variation in the load of these fungal species among UC patients, depending on their disease phases and response to Adalimumab treatment. It has been previously described that the fungal species belonging to the phyla \u003cem\u003eAscomycota\u003c/em\u003e and \u003cem\u003eBasidiomycota\u003c/em\u003e were overrepresented in the gut mycobiota community of IBD patients [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Furthermore, the most common genera in the human gut mycobiota are \u003cem\u003eSaccharomyces\u003c/em\u003e and \u003cem\u003eCandida\u003c/em\u003e, [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] which were analyzed in this study.\u003c/p\u003e\u003cp\u003eOur quantitative analysis revealed significant fungal shifts, particularly in \u003cem\u003eCandida\u003c/em\u003e species, among UC patients. \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eC. glabrata\u003c/em\u003e exhibited notable increases compared to healthy subjects (2.27-fold and 2.3-fold, respectively, p\u0026thinsp;\u0026lt;\u0026thinsp;0.005 and p\u0026thinsp;=\u0026thinsp;0.009), reinforcing their potential role in gut dysbiosis associated with IBD [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This aligns with the previous observations that \u003cem\u003eCandida\u003c/em\u003e is more abundant in IBD patients compared with healthy controls [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition, it has been reported that IBD patients are characterized by elevated \u003cem\u003eC. albicans\u003c/em\u003e levels and reduced \u003cem\u003eS. cerevisiae\u003c/em\u003e levels, further highlighting the dysbiotic properties of \u003cem\u003eC. albicans\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Notably, \u003cem\u003eC. albicans\u003c/em\u003e colonizes the intestinal mucosa, inducing both protective immunity and inflammation depending on the context [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Furthermore, due to its pathogenic potential, \u003cem\u003eC. albicans\u003c/em\u003e can invade epithelial cells and release cytolytic enzymes, leading to damage to the epithelial cells. It can also promote inflammatory responses by inducing IL-1, IL-36, and IL-17. Thus, \u003cem\u003eC. albicans\u003c/em\u003e plays a significant role in regulating cytokine responses and inducing mucosal inflammation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Interestingly, we found that \u003cem\u003eC. tropicalis\u003c/em\u003e decreased in UC patients, although this reduction was not statistically significant. These findings are in agreement with those of Iliev et al. reported that the levels of \u003cem\u003eC. albicans\u003c/em\u003e are consistently elevated in fecal samples from IBD patients, while the abundance of \u003cem\u003eC. tropicalis\u003c/em\u003e varies across different studies [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Moreover, the investigation revealed a reduction in \u003cem\u003eS. cerevisiae\u003c/em\u003e among patients with UC. However, this trend did not reach statistical significance. Further, Pascal et al. demonstrated that the abundance of \u003cem\u003eSaccharomyces\u003c/em\u003e species is diminished in patients with IBD; however, this reduction is primarily observed in individuals with CD, rather than in those with UC [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In a separate study examining fungal dysbiosis, Imai et al. found that the genus \u003cem\u003eSaccharomyces\u003c/em\u003e was more prevalent in healthy controls than in IBD patients [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Studies suggest that \u003cem\u003eS. cerevisiae\u003c/em\u003e exerts a protective role in the inflammatory process, indicating it could function as an anti-inflammatory species. It is proposed that \u003cem\u003eS. cerevisiae\u003c/em\u003e may regulate host responses by inducing IL-10 production [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDisease activity influenced fungal burden, with \u003cem\u003eC. tropicalis\u003c/em\u003e showing a substantial increase during flare-ups (2.04-fold, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), while \u003cem\u003eS. cerevisiae\u003c/em\u003e, \u003cem\u003eC. albicans\u003c/em\u003e, and \u003cem\u003eC. glabrata\u003c/em\u003e remained largely stable across disease phases. These findings suggest that specific fungal populations fluctuate during inflammation. This dynamic behavior warrants further investigation into their role in UC pathogenesis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Intriguingly, in contrast to our findings, Sokol et al. reported a reduction in \u003cem\u003eSaccharomyces\u003c/em\u003e during UC flares compared to remission, suggesting a potential protective role of this genus against disease activity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. They also confirmed a significant increase in \u003cem\u003eC. albicans\u003c/em\u003e abundance among patients with IBD, particularly during flare-ups [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These contrasting findings underscore the challenge of determining whether a fungal organism has a probiotic or dysbiotic property, emphasizing the species-specific nature of their interactions with the host [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition, the small sample size, variations in investigation methods, and limitations in patient follow-up may contribute to these divergent findings. In a recent study, Hsia et al. showed that the \u003cem\u003eAscomycota\u003c/em\u003e phylum, along with the genera \u003cem\u003eSaccharomyces\u003c/em\u003e and Candida, are enriched among UC patients with severe colonic inflammation compared to periods of remission. This suggests that these fungal taxa could serve as biomarkers and potential therapeutic targets for UC [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOnly a few studies have investigated the possible markers of response to anti-TNF therapy in the gut mycobiota. In the current study, stratification of patients based on response to adalimumab revealed significant fungal variations. Non-responders exhibited higher \u003cem\u003eC. glabrata\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.024) and \u003cem\u003eC. tropicalis\u003c/em\u003e (3.61-fold increase, p\u0026thinsp;=\u0026thinsp;0.034), suggesting these species may affect treatment efficacy. While \u003cem\u003eS. cerevisiae\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e levels were similar between groups, the observed shifts in \u003cem\u003eC. tropicalis\u003c/em\u003e and \u003cem\u003eC. glabrata\u003c/em\u003e suggest fungal microbiota composition might influence therapeutic outcomes in UC [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A similar study by Ventin-Holmberg et al. demonstrated that \u003cem\u003eCandida\u003c/em\u003e species were more abundant in non-responders to anti-TNF therapy (Infliximab) in both CD and UC patients, which is consistent with our results [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Additionally, a further study conducted by this group focusing on pediatric patients with IBD demonstrated that individuals who did not respond to treatment with the TNF-α antagonist infliximab exhibited higher levels of \u003cem\u003eCandida\u003c/em\u003e spp. compared to responders to the therapy [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe performed a correlation analysis aiming to investigate the DNA copy numbers, sampling phase, and various clinical outcomes about ADA therapy response. Interaction models revealed that disease phase significantly influenced treatment response in relation to both \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eC. tropicalis\u003c/em\u003e. Also, the effect of \u003cem\u003eC. tropicalis\u003c/em\u003e may vary depending on the stage of the disease, exhibiting different behaviors across clinical states. A recent study by Catal\u0026aacute;n-Serra et al. showed that \u003cem\u003eC. albicans\u003c/em\u003e was significantly more prevalent in patients with UC and CD. However, it was not found to be significantly overrepresented in patients experiencing active disease, and its abundance did not correlate with the inflammatory characteristics of the conditions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThese findings suggest that fungal behavior may not be uniform across clinical stages and could play a modulatory role in therapeutic efficacy. Although correlation analyses of the relationship between fungal load and disease activity revealed that flare-up and remission patients showed differences in correlations between \u003cem\u003eC. glabrata\u003c/em\u003e and disease states. In such a way that \u003cem\u003eC. glabrata\u003c/em\u003e was associated with clinical and endoscopic remission. In consistent with our results, Hsia et al. identified a correlation between high levels of \u003cem\u003eCandida\u003c/em\u003e species and disease activity in UC patients, while a lower frequency of \u003cem\u003eCandida\u003c/em\u003e was associated with disease quiescence [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMost studies conducted so far focus on patients with IBD who are not undergoing specific treatments. While much of the existing research has concentrated on patients with CD, there is a significant lack of studies investigating fungal prevalence and its relationship with clinical outcomes in patients with UC. Furthermore, these studies typically evaluate fungal load at the genus level rather than at the species level.\u003c/p\u003e\u003cp\u003eIn our study, the absolute fungal DNA copy numbers showed only limited associations with clinical outcomes, suggesting that fungal burden alone may not fully capture the complexities of host-mycobiota interactions influencing treatment response. Most fungal predictors assessed did not reach statistical significance, indicating that while \u003cem\u003eC. glabrata\u003c/em\u003e may hold promise as a potential biomarker, fungal burden alone may not consistently predict treatment response across all species. These findings highlight the complexity of host-fungal interactions and suggest that species-specific dynamics rather than overall fungal load may be more relevant to therapeutic outcomes.\u003c/p\u003e\u003cp\u003eThe limited number of patients in our study may hinder significant findings. Additionally, variability in response assessment methods, microbiota analysis techniques, selected fungal strains, therapeutic protocols, follow-up duration, and the inclusion of control subjects further complicates meaningful comparisons across studies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, we found significant differences in the mentioned gut mycobiota composition between UC patients and healthy individuals. UC patients exhibited a higher abundance of \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eC. glabrata\u003c/em\u003e species, while the load of C. tropicalis remained stable throughout the study. Our findings indicate that non-responders to ADA therapy had a higher abundance of \u003cem\u003eC. glabrata\u003c/em\u003e and \u003cem\u003eC. tropicalis\u003c/em\u003e. Notably, a higher abundance of \u003cem\u003eC. tropicalis\u003c/em\u003e in patients experiencing flare-ups was observed. Throughout therapy, \u003cem\u003eS. cerevisiae\u003c/em\u003e remained stable between UC patients in flare-ups and remission phases, as well as between responders and non-responders.\u003c/p\u003e\u003cp\u003eThese findings underscore the potential of fungal biomarkers in predicting treatment response. While calprotectin remains a reliable inflammation marker, fungal dysbiosis particularly \u003cem\u003eC. glabrata\u003c/em\u003e and \u003cem\u003eC. tropicalis\u003c/em\u003e elevations in non-responders require deeper investigation. Future research should focus on longitudinal, multi-center studies that incorporate fungal biomarkers into integrated omics frameworks, and to validate fungal profiling as a predictive tool, potentially guiding personalized therapeutic strategies in UC management.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eInflammatory Bowel Disease (IBD), Ulcerative colitis (UC), Crohn\u0026rsquo;s disease (CD), Gastrointestinal (GI), Adalimumab (ADA), Tuberculosis (TB), Hepatitis B virus (HBV), Hepatitis C Virus (HCV), Human Immunodeficiency Virus (HIV), Erythrocyte Sedimentation Rate (ESR), C-reactive protein (CRP), Fecal Calprotectin (FC), Enzyme-Linked Immunosorbent Assays (ELISA), Sabouraud Dextrose Agar (SDA), Yeast Medium (YM), Interquartile Range (IQR), Standard deviation (SD), Cycle threshold (Ct), Generalized estimating equations (GEE), Odds ratios (OR), Confidence Intervals (CI), Healthy Control (HC), Tumor Necrosis Factor (TNF), Interleukin (IL).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cul\u003e\n \u003cli\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThe research was approved and performed according to the Ethical Review Committee of the Research Institute for Gastroenterology and Liver Diseases at Shahid Beheshti University of Medical Sciences (Project No. IR.SBMU.RIGLD.REC.1401.006).\u0026nbsp;\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eConsent for publication:\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eWritten informed consent was obtained from all participants.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eAvailability of data and materials:\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThe supporting data are available upon request to the corresponding author [Shaghayegh Baradaran Ghavami].\u0026nbsp;\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eCompeting interests:\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eFunding:\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThis work was supported by the Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eAuthors' contributions:\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eConceptualization, S.B.G., E.L., H.S.; Data curation, M.F., H.B.; Formal analysis, M.F. M.A.L.; Funding acquisition, S.S., E.L.; Investigation, M.F., M.F, A.T., N.K.; Methodology, S.B.G., H.H.; Project administration, S.B.G.; Supervision, S.B.G., H.H.; Writing original draft, M.F., S.B.G. M.A.L; Review and editing, S.B.G., H.H., M.A.L. All authors have read and agreed to the published version of the manuscript. All the authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Generative AI and AI-assisted technologies in the writing process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this work, the authors used Grammarly and ChatGPT (GPT-4o) in order to improve readability and language. After using these tools, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSaez A et al (2023) Pathophysiology of inflammatory bowel disease: innate immune system. Int J Mol Sci 24(2):1526\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGhavami SB et al (2022) Serological response to SARS-CoV-2 is attenuated in patients with inflammatory bowel disease and can affect immunization. JGH Open 6(4):266\u0026ndash;269\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnanthakrishnan AN et al (2018) Environmental triggers in IBD: a review of progress and evidence. Nat reviews Gastroenterol Hepatol 15(1):39\u0026ndash;49\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAghdaei HA et al (2022) Overexpression of toll-like receptors and co-stimulatory molecules on immature dendritic cells of Crohn's disease. Gene Rep 27:101579\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCatalan-Serra I et al \u003cem\u003eFungal Microbiota Composition in Inflammatory Bowel Disease Patients: Characterization in Different Phenotypes and Correlation With Clinical Activity and Disease Course (dec, 10.1093/ibd/izad289\u003c/em\u003e, (2023)). INFLAMMATORY BOWEL DISEASES, 2024. 30(5): pp. 876\u0026ndash;876). INFLAMMATORY BOWEL DISEASES, 2024. 30(5): pp. 876\u0026ndash;876\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSokol H et al (2017) Fungal microbiota dysbiosis in IBD. Gut 66(6):1039\u0026ndash;1048\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJena A, Dutta U (2024) Role of fungus in inflammatory bowel disease: The butterfly effect? Indian J Gastroenterol 43(4):697\u0026ndash;699\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu M et al (2023) Fungal dysbiosis facilitates inflammatory bowel disease by enhancing CD4\u0026thinsp;+\u0026thinsp;T cell glutaminolysis. Front Cell Infect Microbiol 13:1140757\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang F, Wang Z, Tang J (2023) The interactions of Candida albicans with gut bacteria: a new strategy to prevent and treat invasive intestinal candidiasis. Gut pathogens 15(1):30\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFreiberger T \u003cem\u003eThe Impact of DNA Extraction Methods on Stool Bacterial and Fungal Microbiota Community Recovery.\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePatnaik S et al (2024) Role of Candida species in pathogenesis, immune regulation, and prognostic tools for managing ulcerative colitis and Crohn's disease. World J Gastroenterol 30(48):5212\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOtt SJ et al (2008) Fungi and inflammatory bowel diseases: alterations of composition and diversity. Scand J Gastroenterol 43(7):831\u0026ndash;841\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuseyin CE et al (2017) The fungal frontier: a comparative analysis of methods used in the study of the human gut mycobiome. Front Microbiol 8:1432\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHonkanen J et al (2020) Fungal dysbiosis and intestinal inflammation in children with beta-cell autoimmunity. Front Immunol 11:468\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVentin-Holmberg R et al (2021) Bacterial and fungal profiles as markers of infliximab drug response in inflammatory bowel disease. J Crohn's Colitis 15(6):1019\u0026ndash;1031\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSingh A et al (2021) Identifying anti-TNF response biomarkers in ulcerative colitis using a diffusion-based signalling model. Bioinf Adv 1(1):vbab017\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChehoud C et al (2015) Fungal signature in the gut microbiota of pediatric patients with inflammatory bowel disease. Inflamm Bowel Dis 21(8):1948\u0026ndash;1956\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLewis JD et al (2015) Inflammation, antibiotics, and diet as environmental stressors of the gut microbiome in pediatric Crohn\u0026rsquo;s disease. Cell Host Microbe 18(4):489\u0026ndash;500\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLeonardi I et al (2020) Fungal trans-kingdom dynamics linked to responsiveness to fecal microbiota transplantation (FMT) therapy in ulcerative colitis. Cell Host Microbe 27(5):823\u0026ndash;829e3\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoyes DL et al (2016) Candidalysin is a fungal peptide toxin critical for mucosal infection. Nature 532(7597):64\u0026ndash;68\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIliev ID, Cadwell K (2021) Effects of intestinal fungi and viruses on immune responses and inflammatory bowel diseases. Gastroenterology 160(4):1050\u0026ndash;1066\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePascal V et al (2017) A microbial signature for Crohn's disease. Gut 66(5):813\u0026ndash;822\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eImai T et al (2019) Characterization of fungal dysbiosis in Japanese patients with inflammatory bowel disease. J Gastroenterol 54(2):149\u0026ndash;159\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHsia K et al (2023) Alterations in the fungal microbiome in ulcerative colitis. Inflamm Bowel Dis 29(10):1613\u0026ndash;1621\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCui G, Florholmen J, Goll R (2022) Could Mucosal TNF Transcript as a Biomarker Candidate Help Optimize Anti-TNF Biological Therapy in Patients With Ulcerative Colitis? Front Immunol 13:881112\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVentin-Holmberg R et al (2022) The gut fungal and bacterial microbiota in pediatric patients with inflammatory bowel disease introduced to treatment with anti-tumor necrosis factor-α. Sci Rep 12(1):6654\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ulcerative colitis, Fungal biomarkers, Anti-TNF therapy, Candida species, Treatment response","lastPublishedDoi":"10.21203/rs.3.rs-7749916/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7749916/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e\u003cp\u003eInflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease, is a chronic immune-mediated condition marked by recurrent intestinal inflammation. Although anti-TNF therapies like adalimumab (ADA) have improved disease management, predictive biomarkers for treatment response remain limited. Emerging evidence highlights fungal biomarkers as potential indicators of therapeutic outcomes, especially in UC. This study investigates fungal load dynamics and their association with clinical response in UC patients receiving ADA.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e\u003cp\u003eWe analyzed samples from 23 UC patients and 20 healthy individuals. Using quantitative PCR (qPCR), we measured the prevalence and DNA copy numbers of key fungal species, then assessed their relationships with disease activity, response to ADA, and inflammatory markers. Patients were further stratified by disease phase (flare-up vs. remission) and treatment response.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e\u003cp\u003eOur findings revealed an increase in \u003cem\u003eCandida albicans\u003c/em\u003e levels in UC patients on ADA compared to healthy controls, while \u003cem\u003eCandida glabrata\u003c/em\u003e was significantly reduced. Among the UC group, \u003cem\u003eC. tropicalis\u003c/em\u003e showed the most marked increase during flare-ups compared to remission. When comparing treatment outcomes, non-responders exhibited notably higher levels of \u003cem\u003eC. glabrata\u003c/em\u003e and \u003cem\u003eC. tropicalis\u003c/em\u003e than responders. Further analysis suggested that disease phase may influence the relationship between fungal burden and treatment response, particularly for \u003cem\u003eC. tropicalis\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusion:\u003c/h2\u003e\u003cp\u003eThese findings reveal distinct fungal profiles associated with UC activity and response to adalimumab. \u003cem\u003eC. tropicalis\u003c/em\u003e may indicate disease flare-ups, while elevated \u003cem\u003eC. glabrata\u003c/em\u003e levels could reflect treatment resistance. Further studies are needed to validate fungal biomarkers as tools for guiding UC management.\u003c/p\u003e","manuscriptTitle":"Exploring Fungal Biomarkers as Predictors of Treatment Response in Ulcerative Colitis Patients Receiving Anti-TNF Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-29 12:28:08","doi":"10.21203/rs.3.rs-7749916/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5938bee7-58ce-46b3-9b1b-748186d2c872","owner":[],"postedDate":"October 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-01T11:54:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-29 12:28:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7749916","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7749916","identity":"rs-7749916","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}