Stool Interleukin-1ß Differentiates Clostridioides difficile Infection (CDI) From Asymptomatic Carriage and Non-CDI Diarrhea.

BACKGROUND: Despite advances in the understanding and diagnosis of Clostridioides difficile infection (CDI), clinical distinction within the colonization-infection continuum remains an unmet need. METHODS: By measuring stool cytokines and antitoxin antibodies in well-characterized cohorts of CDI (diarrhea, nucleic acid amplification test [NAAT] positive), non-CDI diarrhea (NCD; diarrhea, NAAT negative), asymptomatic carriers (ASC; no diarrhea, NAAT positive) and hospital controls (CON; no diarrhea, NAAT negative), we aim to discover novel biological markers to distinguish between these cohorts. We also explore the relationship of these stool cytokines and antitoxin antibody with stool toxin concentrations and disease severity. RESULTS: Stool interleukin (IL) 1ß, stool immunoglobulin A (IgA), and immunoglobulin G (IgG) anti-toxin A had higher (P < .0001) concentrations in CDI (n = 120) vs ASC (n = 43), whereas toxins A, B, and fecal calprotectin did not. Areas under the receiver operating characteristic curve (ROC-AUCs) for IL-1ß, IgA, and IgG anti-toxin A were 0.88, 0.83, and 0.83, respectively. A multipredictor model including IL-1ß and IgA anti-toxin A achieved an ROC-AUC of 0.93. Stool IL-1ß concentrations were higher in CDI compared to NCD (n = 75) (P < .0001) and NCD + ASC+ CON (CON, n = 75) (P < .0001), with ROC-AUCs of 0.83 and 0.86, respectively. Stool IL-1ß had positive correlations with toxins A (ρA = +0.55) and B (ρB = +0.49) in CDI (P < .0001) but not in ASC (P > .05). CONCLUSIONS: Stool concentrations of the inflammasome pathway, proinflammatory cytokine IL-1ß, can accurately differentiate CDI from asymptomatic carriage and NCD, making it a promising biomarker for CDI diagnosis. Significant positive correlations exist between stool toxins and stool IL-1ß in CDI but not in asymptomatic carriers.

Detection of High-Risk Sessile Serrated Lesions: Multitarget Stool DNA Versus CT Colonography.

BACKGROUND. The serrated pathway for colorectal cancer (CRC) development is increasingly recognized. Sessile serrated lesions (SSLs) that are large (≥ 10 mm) and/or have dysplasia (i.e., high-risk SSLs) are at higher risk of progression to CRC. Detection of SSLs is challenging given their predominantly flat and right-sided location. The yield of noninvasive screening tests for detection of high-risk SSLs is unclear. OBJECTIVE. The aim of this study was to compare noninvasive screening detection of high-risk SSLs between the multitarget stool DNA (mt-sDNA) test and CT colonography (CTC). METHODS. This retrospective study included 7974 asymptomatic adults (4705 women, 3269 men; mean age, 60.0 years) who underwent CRC screening at a single center by mt-sDNA from 2014 to 2019 (n = 3987) or by CTC from 2009 to 2019 (n = 3987). Clinical interpretations of CTC examinations were recorded. Subsequent colonoscopy findings and histology of resected polyps were also recorded. Chi-square or two-sample t tests were used to compare results between mt-sDNA and CTC using 6-mm and 10-mm thresholds for test positivity. RESULTS. The overall colonoscopy referral rate for a positive screening test was 13.1% (522/3987) for mt-sDNA versus 12.2% (487/3987; p = .23) and 6.5% (260/3987; p < .001) for CTC at 6-mm and 10-mm thresholds, respectively. The PPV for high-risk SSLs was 5.5% (26/476) for mt-sDNA versus 14.4% (66/457; p < .001) and 25.9% (63/243; p < .001) for CTC at the 6-mm and 10-mm thresholds, respectively. The overall screening yield of high-risk SSLs was 0.7% (26/3987) for mt-sDNA versus 1.7% (66/3987; p < .001) and 1.6% (63/3987; p < .001) for CTC at 6-mm and 10-mm thresholds, respectively. CONCLUSION. CTC at 6-mm and 10-mm thresholds had significantly higher yield and PPV for high-risk SSLs compared with mt-sDNA. CLINICAL IMPACT. The significantly higher detection of high-risk SSLs by CTC than by mt-sDNA should be included in discussions with patients who decline colonoscopy and opt for noninvasive screening.

Fecal S100A12 in Takayasu arteritis predicts disease activity and intestinal involvement.

Gastrointestinal involvement in Takayasu Arteritis (TA) requires invasive or expensive diagnostic studies. Instead a fecal biomarker can be used as an initial screening test. In this context, S100A12 is promising as an established biomarker in intestinal inflammation and its role in TA pathogenesis. As such we aimed to test the feasibility of fecal S100A12 as a means of the fecal biomarker in gastrointestinal involvement in TA in this pilot study. Our study population consisted of 30 TA patients and 14 control patients with non-inflammatory arthralgia. Patients with inflammatory or infectious gastrointestinal tract diseases, or used oral antibiotics or NSAIDs for the 3 weeks were excluded. Vasculitis involvements were determined with cross-sectional radiologic studies. TA disease activity was evaluated per Indian Takayasu's Activity Score (2010) criteria and vascular involvements were classified according to Numano classification. ELISA test was used to determine fecal S100A12 levels. Fecal S100A12 levels were higher in TA patients when compared to the controls (37.9 ng/ml vs. 12.5 ng/ml p = 0.038). ESR and CRP levels were also higher in the TA group, however not correlated with fecal S100A12. Among TA patients, fecal S100A12 levels were higher inactive ones with ITAS2010 > 1 (72.9 ng/ml vs. 16.7 ng/ml p = 0.016) correlated with total ITAS2010 scores. (R = 0.52 p = 0.003). TA patients with abdominal symptoms had higher fecal S100A12 levels when compared to the remaining TA population (327.8 ng/ml vs. 28.0 ng/ml p = 0.003). However, fecal S100A12 levels in patients with or without mesenteric vessel involvement did not differ. Fecal S100A12 shows promise as a fecal biomarker to screen intestinal ischemia and inflammatory bowel disease in TA patients.

Fecal MicroRNA-Based Algorithm Increases Effectiveness of Fecal Immunochemical Test-Based Screening for Colorectal Cancer.

BACKGROUND & AIMS: An algorithm based on fecal levels of 2 microRNAs (miR-421 and miR-27a-3p), fecal hemoglobin concentration, and patient age and sex can identify patients with advanced colorectal neoplasia. We investigated whether this algorithm, called miRFec, could increase effectiveness and efficiency of fecal immunochemical test (FIT)-based colorectal cancer (CRC) screening programs. METHODS: We obtained data and fecal samples from 767 persons with a positive result from the FIT who then underwent colonoscopy examination while participating a population-based CRC screening program, from March 2011 through May 2017 in Barcelona, Spain. Fecal miRNAs were isolated from the buffer contained in the original FIT collection device and analyzed by quantitative reverse transcription PCR. Aims were to evaluate the usefulness of the miRFec algorithm in identifying persons at greatest risk for CRC who should be prioritized for colonoscopy examination and individuals at low risk for whom colonoscopy could be avoided. RESULTS: Of the 767 study subjects, 414 (54.0%) were found by colonoscopy to have advanced colorectal neoplasia (67 with CRC and 347 with advanced adenomas) and 353 (46.0%) were found to have either non-advanced adenomas (n = 136) or a normal examination (n = 217). MiRFec algorithm scores (1-4) were independently associated with the presence of advanced colorectal neoplasia (P < .001). The miRFec algorithm differentiated patients with CRC from those with non-advanced adenomas or normal colonoscopy with an area under the receiver operating characteristic curve of 90% (95% CI, 86-94). Subjects with miRFec scores in the 4th quartile (above 3.09, high-risk group) were 8-fold more likely to have advanced colorectal neoplasia than subjects with miRFec scores in the 1st quartile (below 2.14, low-risk group). Subjects in the low-risk group had a positive predictive value below 30% for detection of advanced colorectal neoplasia. When we used a 50% specificity cut-off value, the miRFec algorithm identified 97% of patients with CRC and would allow 264 subjects (34.4%) to avoid colonoscopy examination. CONCLUSIONS: An algorithm based on fecal levels of 2 miRNAs and hemoglobin, patient age and sex (miRFec) differentiated patients with CRC from those with non-advanced adenomas or normal colonoscopy with an area under the receiver operating characteristic curve value of 90% and avoided 34% of colonoscopies. Inclusion of this algorithm in FIT-based CRC screening programs could increase their effectiveness and efficiency.

Comparison of Fecal MicroRNA Isolation Using Various Total RNA Isolation Kits.

Fecal specimens have long been regarded as promising sources for gastrointestinal cancer screening and have, thus, been extensively investigated in biomarker research. MicroRNAs (miRNAs) are small, non-coding RNA molecules involved in regulating various biological processes. They are commonly dysregulated during tumor development and exhibit differential expression in feces. To assess the preanalytical feasibility of fecal miRNA analysis, we systematically compared the performance of commonly used total RNA extraction methods. Fecal samples from healthy subjects were utilized for this evaluation. Various methods, including miRNeasy, Universal, Trizol, RNeasy, and mirVana kits, were employed to isolate total RNA. MiRNA expression analyses were conducted using TaqMan or SYBR Green qRT-PCR for a subset of miRNAs, with externally spiked-in cel-miR-39 used for normalization. Most methods demonstrated similar performance in terms of the total RNA concentration and purity. Externally spiked cel-miR-39 and endogenous miRNAs (RNU6b, miR-16, and miR-21) exhibited comparable concentrations across the different RNA isolation methods, whereas the RNeasy mini kit consistently yielded lower values. Our findings suggest that various isolation methods produce reproducible and comparable miRNA expression results, supporting the potential comparability and translational applicability of miRNA-based biomarker research in the future.

The fecal biomarker ovotransferrin associates with broiler performance under field conditions.

Broilers often suffer from subclinical intestinal health problems of ill-defined etiology, which have a negative impact on performance. Macroscopic and microscopic evaluations can be used to monitor intestinal health, but because these are subjective and time-consuming, respectively, objective and easy-to-measure biomarkers are urgently needed. Fecal biomarkers can potentially be used as noninvasive, objective measures to evaluate gut health in broilers. The aim of the current study was to evaluate ovotransferrin (OVT) as a biomarker in fecal/colonic samples derived from broilers from 27 industrial farms by investigating associations between OVT, broiler performance and gut histology parameters. Eight chickens per farm were randomly selected, weighed and euthanized on d 28 of the production round. A duodenal section was collected to measure the intestinal villus structure (villus length, crypt depth) and the inflammatory status of the gut (CD3+ T-lymphocytes area percentage). The coefficient of variation for the OVT (between farms; 83.45%, within farms; 95.13%) was high compared to the villus length (between farms; 10.91%, within farms; 15.48%), crypt depth (between farms; 15.91%, within farms; 14.10%), villus-to-crypt ratio (between farms; 22.08%, within farms; 20.53%), and CD3+ (between farms; 36.38%, within farms; 26.13%). At farm level, colonic OVT was significantly associated with the average slaughter weight (P = 0.005), daily weight gain (P = 0.007) and the European production index (EPI) (P = 0.009). At broiler level, significant associations were found between colonic OVT and the villus length (P = 0.044) and between the colonic OVT and villus-to-crypt ratio (P = 0.050). These results thus show that quantifying OVT in colon can have merit for evaluation of intestinal health in broilers under field conditions.

Fecal miR-223 is a noninvasive biomarker for estimating Crohn's disease activity.

INTRODUCTION: MicroRNA-223 (miR-223) has emerged as a promising noninvasive biomarker for Crohn's disease (CD). However, it is unclear which tissue derived miRNA-223 can more accurately estimate CD disease activity. MATERIALS AND METHODS: To collect serum, terminal ileal mucosa biopsy and fecal samples from CD patients and healthy controls. The CD Activity Index (CDAI) score, Montreal classification, maintenance medicines, peripheral blood inflammatory markers, fecal calprotectin (FC) and the Simple Endoscopic Score for CD (SES-CD) were recorded. To compare the expression of miR-223 in the serum, intestinal tissue, and feces. RESULTS: MiR-223 expression levels in the serum, intestinal tissue and feces of CD patients were significantly higher than those of controls. The level of miR-223 in the serum, intestinal tissue and feces increased significantly in active CD patients compared with that in inactive CD patients. The levels of serum, intestinal tissue and fecal miR-223 were correlated with the CDAI. Serum miR-223 was also correlated with C-reactive protein (CRP) and IL-6, tissue miR-223 correlated with IL-6 and FC, and fecal miR-223 correlated with FC. In terms of the association with FC, fecal miR-223 had a higher Spearman r value than tissue miR-223. The area under the curve (AUC) values of serum, tissue and fecal miR-223 to diagnose CD were similar to those of CRP and FC (AUC > 0.8). The AUC values of tissue and fecal miR-223 to evaluate CD disease activity were 0.832 and 0.818, respectively, and were higher than serum miR-223, CRP and FC. Fecal miR-223 had a higher specificity of 92.3%. CONCLUSIONS: Fecal miR-223 might be a novel, noninvasive biomarker for estimating the disease activity of CD patients.

Fecal Volatile Metabolomics Predict Gram-Negative Late-Onset Sepsis in Preterm Infants: A Nationwide Case-Control Study.

Early detection of late-onset sepsis (LOS) in preterm infants is crucial since timely treatment initiation is a key prognostic factor. We hypothesized that fecal volatile organic compounds (VOCs), reflecting microbiota composition and function, could serve as a non-invasive biomarker for preclinical pathogen-specific LOS detection. Fecal samples and clinical data of all preterm infants (≤30 weeks' gestation) admitted at nine neonatal intensive care units in the Netherlands and Belgium were collected daily. Samples from one to three days before LOS onset were analyzed by gas chromatography-ion mobility spectrometry (GC-IMS), a technique based on pattern recognition, and gas chromatography-time of flight-mass spectrometry (GC-TOF-MS), to identify unique metabolites. Fecal VOC profiles and metabolites from infants with LOS were compared with matched controls. Samples from 121 LOS infants and 121 matched controls were analyzed using GC-IMS, and from 34 LOS infants and 34 matched controls using GC-TOF-MS. Differences in fecal VOCs were most profound one and two days preceding Escherichia coli LOS (Area Under Curve; p-value: 0.73; p = 0.02, 0.83; p < 0.002, respectively) and two and three days before gram-negative LOS (0.81; p < 0.001, 0.85; p < 0.001, respectively). GC-TOF-MS identified pathogen-specific discriminative metabolites for LOS. This study underlines the potential for VOCs as a non-invasive preclinical diagnostic LOS biomarker.

Fecal Multidimensional Assay for Non-Invasive Detection of Colorectal Cancer: Fecal Immunochemical Test, Stool DNA Mutation, Methylation, and Intestinal Bacteria Analysis.

BACKGROUND: Fecal immunochemical test (FIT), DNA mutation, DNA methylation, and microbial dysbiosis all showed promising in colorectal cancer (CRC) non-invasive detection. We assessed CRC detection with an assay combining all these strategies and investigated the effect of clinical features on the performance of this comprehensive test. METHODS: We performed a multidimensional analysis study using stool samples collected from 108 patients with CRC, 18 patients with colorectal adenoma, and 36 individuals with no evidence of colorectal disease. The multidimensional analysis of stool samples including FIT, stool DNA (sDNA) tests for three methylated genes (Septin9, NDRG4, BMP3) and three mutated genes (KRAS, BRAF, PI3KCA) using next generation sequencing as well as detection of stool bacteria level of Fusobacterium nucleatum and Parvimonas micra using qPCR method. We used a linear support vector classification model to analyze the data. RESULTS: The sensitivity of FIT alone was 69.4% for CRC and 11.1% for adenoma. Separately, the sensitivity of the detection of intestinal bacteria, DNA mutation, and DNA methylation for CRC was 58.3, 50.0, and 51.9%, respectively. The combination of FIT and sDNA tests had a sensitivity of 81.5% for CRC (AUC: 0.93, better than FIT alone, P = 0.017) and 27.8% for adenoma with 94.4% specificity. Sensitivity of the multidimensional test to detect CRC with stage II (84.6%) and III (91.9%) CRC was relatively higher (88.2%) than that of patients with stage I (60.0%) and stage IV (75.0%) (P = 0.024). The rate of CRC detection increased with tumor size (P = 0.008) and age (P = 0.04). Interestingly, the rate of CRC detection was higher in smoking persons than non-smokers with marginal significance (P = 0.08). CONCLUSIONS: The multidimensional assay of stool samples combining FIT and stool DNA tests further improved the diagnostic sensitivity for CRC. This could provide new approach for improvement of CRC screening and further demonstrations are warranted.

Novel Testing Enhances Irritable Bowel Syndrome Medical Management: The IMMINENT Study.

PRIMARY STUDY OBJECTIVE: To evaluate the economic utility of a fecal biomarker panel structured to suggest alternative, treatable diagnoses in patients with symptoms of irritable bowel syndrome (IBS) by quantifying, comparing, and contrasting health service costs between tested and non-tested patients. STUDY DESIGN: Retrospective, matched cohort study comparing direct medical costs for IBS patients undergoing fecal biomarker testing with those of matched control subjects. METHODS: We examined de-identified medical and pharmacy claims of a large American pharmacy benefit manager to identify plan members who underwent panel testing, were eligible for covered benefits for at least 180 days prior to the test date, and had data available for 30, 90, and 365 days after that date. We used propensity score matching to develop population-based control cohorts for each tested cohort, comprised of records with IBS-related diagnoses but for which panel testing was not performed. Primary outcome measures were diagnostic and medical services costs as determined from claims data. RESULTS: Two hundred nine records from tested subjects met inclusion criteria. The only significant baseline differences between groups were laboratory costs, which were significantly higher in each tested cohort. At each follow-up time point, total medical and gastrointestinal procedural costs were significantly higher in non-tested cohorts. Within tested cohorts, costs declined significantly from baseline, while costs rose significantly in non-tested control cohorts; these differences were also significant between groups at each time point. CONCLUSIONS: Structured fecal biomarker panel testing was associated with significantly lower medical and gastrointestinal procedural costs in this study of patients with IBS symptoms.

Quantitative Fecal Lactoferrin as a Biomarker for Severe Clostridium difficile Infection in Hospitalized Patients.

BACKGROUND: The incidence and severity of Clostridium difficile infection (CDI) have increased over the past decade, especially among hospitalized patients. In this study, we determined the value of published criteria for severe CDI in predicting 3 month mortality, as well as the utility of fecal lactoferrin as a biomarker for severe CDI. METHODS: Pilot Year 1 of IRB approved (HSR-IRB# 13630) prospective cohort study of hospitalized patients with CDI at US academic medical center (10/08-4/10). Medical records of hospitalized patients with clinically diagnosed CDI, via toxin assay, were evaluated to objectively define severe CDI based on current guidelines. A stool sample from CDI diagnosis was analyzed for amount of fecal lactoferrin (IBD-SCAN, TechLab, Inc.). Data was analyzed using SPSS for student's t-test and chi-squared, significance p ≤ 0.05. RESULTS: 79 subjects consented and enrolled, mean age was 64 years (standard deviation, sd, 17.2), 48 (61%) female, and average Charlson co-morbidity score was 5.8 (sd 3.8). Subjects with severe CDI were 5 times more likely to die within 3 months of diagnosis (Odds Ratio 5.66 (95% Confidence Interval 2.03-15.79), p=0.001) and had significantly more fecal lactoferrin (580.0 (sd 989.0) vs. 181.7 (sd 244.2) µg/mL, p=0.018), compared to those that did not meet severe CDI criteria. CONCLUSION: In this pilot study, subjects who meet defined criteria for severe CDI had higher mortality and more intestinal inflammation. These preliminary results were, however, underpowered to show a direct association of lactoferrin with mortality. Larger cohort studies are needed to optimize a criterion for severe CDI and evaluate a direct association of lactoferrin and mortality in hospitalized patients with CDI.