Fungal and Bacterial Loads: Noninvasive Inflammatory Bowel Disease Biomarkers for the Clinical Setting.

Microbiome sequence data have been used to characterize Crohn's disease (CD) and ulcerative colitis (UC). Based on these data, we have previously identified microbiomarkers at the genus level to predict CD and CD relapse. However, microbial load was underexplored as a potential biomarker in inflammatory bowel disease (IBD). Here, we sought to study the use of fungal and bacterial loads as biomarkers to detect both CD and UC and CD and UC relapse. We analyzed the fecal fungal and bacterial loads of 294 stool samples obtained from 206 participants using real-time PCR amplification of the ITS2 region and the 16S rRNA gene, respectively. We combined the microbial data with demographic and standard laboratory data to diagnose ileal or ileocolonic CD and UC and predict disease relapse using the random forest algorithm. Fungal and bacterial loads were significantly different between healthy relatives of IBD patients and nonrelated healthy controls, between CD and UC patients in endoscopic remission, and between UC patients in relapse and non-UC individuals. Microbial load data combined with demographic and standard laboratory data improved the performance of the random forest models by 18%, reaching an average area under the receiver operating characteristic curve (AUC) of 0.842 (95% confidence interval [CI], 0.65 to 0.98), for IBD diagnosis and enhanced CD and UC discrimination and CD and UC relapse prediction. Our findings show that fecal fungal and bacterial loads could provide physicians with a noninvasive tool to discriminate disease subtypes or to predict disease flare in the clinical setting.IMPORTANCE Next-generation sequence data analysis has allowed a better understanding of the pathophysiology of IBD, relating microbiome composition and functions to the disease. Microbiome composition profiling may provide efficient diagnosis and prognosis tools in IBD. However, the bacterial and fungal loads of the fecal microbiota are underexplored as potential biomarkers of IBD. Ulcerative colitis (UC) patients have higher fecal fungal and bacterial loads than patients with ileal or ileocolonic CD. CD patients who relapsed harbor more-unstable fungal and bacterial loads than those of relapsed UC patients. Fecal fungal and bacterial load data improved prediction performance by 18% for IBD diagnosis based solely on clinical data and enhanced CD and UC discrimination and prediction of CD and UC relapse. Combined with existing laboratory biomarkers such as fecal calprotectin and C-reactive protein (CRP), microbial loads may improve the diagnostic accuracy of IBD and of ileal CD and UC disease activity and prediction of UC and ileal CD clinical relapse.

Colonisation by Faecalibacterium prausnitzii and maintenance of clinical remission in patients with ulcerative colitis.

BACKGROUND: Although incrimination of the intestinal microbiota in the pathogenesis of IBD is widely accepted, few data are available about the role of specific bacteria. Potentially, Faecalibacterium prausnitzii, bacteria with anti-inflammatory properties, might be deficient in ulcerative colitis (UC). AIM: To quantify F. prausnitzii in the faecal microbiota of UC patients in remission and determine its relationship with relapse. METHODS: A cross-sectional study included 116 UC patients in remission, 29 first-degree relatives and 31 healthy controls. A subset of eighteen patients, recruited during the first month of remission, underwent a 1-year follow-up. Total bacteria and F. prausnitzii were measured by quantitative Real Time PCR (qPCR, copies/g). Calprotectin was determined as inflammatory index (µg/g). RESULTS: We found that F. prausnitzii was reduced in patients (median, IQR: 1.4 × 108 , 5.1 × 107-4.5 × 108) and relatives (1.7 × 108, 9.3 × 107-5.1 × 108) vs. controls (6.5 × 108, 3.7 × 108-1.6 × 109, P < 0.0001). Moreover, low counts of F. prausnitzii were associated with less than 12 months of remission (8.0 × 107, 2.0 × 107-3.5 × 108 vs. 2.1 × 108, 1.0 × 108-7.9 × 108, P < 0.001) and more than 1 relapse/year (8.0 × 107, 3.2 × 107-3.8 × 108 vs. 1.9 × 108, 6.8 × 107-6.0 × 108, P < 0.01). When patients were followed up, F. prausnitzii increased steadily until reaching similar levels to those of controls if remission persisted (2.9 × 108, 9.3 × 106-1.2 × 109; calprotectin: 76, 19-212), whereas it remained low if patients relapsed (2.2 × 108, 1.4 × 106-3.3 × 108; calprotectin: 1760, 844-3662 P < 0.05 vs. controls). CONCLUSIONS: Defective gut colonisation by F. prausnitzii occurred in UC patients during remission and in their unaffected relatives. The recovery of the F. prausnitzii population after relapse is associated with maintenance of clinical remission.

Cutoff values of the Inflammatory Bowel Disease Questionnaire to predict a normal health related quality of life.

UNLABELLED: One of the objectives in the treatment of the inflammatory bowel disease (IBD) is improving the patient's quality of life. However, we do not dispose of validated criteria to determine the questionnaire's scoring threshold that has to be reached in order to be able to assert that the patients' quality of life has normalized. OBJECTIVE: To determine the normality punctuation cutoff in the IBD specific quality of life questionnaire IBDQ-36. METHOD: Cross-sectional study in a random sample of IBD patients, who have completed the questionnaires IBDQ-36 and EuroQol-5D. The IBDQ-36 normality was calculated according to its equivalence with the EuroQol-5D tariff ≥ 0.90, which corresponds to the 95% CI of the average obtained in a Spanish general population. RESULTS: 218 patients were included. According to the EuroQol-5D tariff, 70 patients were considered to have a normal quality of life and 148 a quality of life poorer than the general population. The IBDQ-36 scoring was significantly higher in the normal quality of life group (222.9 ± 22.8 vs. 171.4 ± 44.8 in the bad quality of life group, p<0.001). According to the linear regression between IBDQ-36 and EuroQol-5D, the cutoff point is 209, with a sensitivity and specificity to predict normality of 0.74 and 0.71 respectively. CONCLUSIONS: Scores of the IBDQ-36 equal or superior to 209 suggest quality of life comparable to that perceived by the general population. This study allowed to set a threshold of normality in the management of the inflammatory bowel diseases.

Oral oligofructose-enriched inulin supplementation in acute ulcerative colitis is well tolerated and associated with lowered faecal calprotectin.

BACKGROUND: Inulin and oligofructose promote selective growth of saccharolytic bacteria with low inflammatory potential. OBJECTIVE: To test the effect of oligofructose-enriched inulin in patients with active ulcerative colitis. DESIGN: Prospective, randomized, placebo controlled pilot trial. Eligible patients had been previously in remission with mesalazine as maintenance therapy or no drug, and presented with a relapse of mild to moderate activity. They were treated with mesalazine (3 g/day) and randomly allocated to receive either oligofructose-enriched inulin (12 g/day, p.o., n = 10) or placebo (12 g/day of maltodextrin, p.o., n = 9) for 2 week. Primary endpoint was the anti-inflammatory effect as determined by reduction of calprotectin and human DNA in faeces. RESULTS: Rachmilewitz score decreased in both groups, reaching statistical significance at day 14 (P < 0.05). Oligofructose-enriched inulin was well-tolerated and dyspeptic symptoms scale decreased significantly with active treatment but not with placebo. At day 7, an early significant reduction of calprotectin was observed in the group receiving oligofructose-enriched inulin (day 0: 4377 +/- 659 microg/g; day 7: 1033 +/- 393 microg/g, P < 0.05) but not in the placebo group (day 0: 5834 +/- 1563 microg/g; day 7: 4084 +/- 1395 microg/g, n.s.). Changes in faecal concentration of human DNA were not significant. CONCLUSION: In active ulcerative colitis, dietary supplementation with oligofructose-enriched inulin is well tolerated and is associated with early reduction in faecal calprotectin.

The opinion of patients with inflammatory bowel disease on healthcare received.

BACKGROUND: an item to consider in analyzing a healthcare model for a population group suffering from chronic disease is necessary health-care resources, their use, and their rating by end-users. Regarding inflammatory bowel disease (IBD), healthcare resources used by patients are numerous and varied, and yet they have been never assessed. DESIGN: an anonymous self-rated questionnaire has been developed with 24 basic questions on overall disease, who is monitoring the patient, how are visits scheduled, need for urgent care, patient view on how control may be improved, etc. This questionnaire was sent to 393 patients who were asked to fill it out and then return it by mail. RESULTS: two hundred and thirty-seven patients returned a filled-out questionnaire. Most patients were followed up in a hospital, and only 8.8% were being monitored by a general practitioner or area specialist. Ninety-two percent of patients reported visits were routinely scheduled irrespective of clinical status, and 79.6% of patients reported having occasionally presented to an emergency department, because of not knowing what to do or due to having no other resources available in 25.2% of times. This entails that 38% of visits to an emergency unit may be prevented with a better understanding of disease or by means of a phone call. Thirty percent of patients reported that current healthcare is inadequate in terms of contents, form, or waiting time. In all, 97.8% of patients feel that information and knowledge on their disease would help in its control, and 69.6% consider that adequate information would allow them to initiate a proper treatment before visiting their doctor. Family care is another poorly looked-after aspect that 74.6% of subjects believe would be of help in controlling their disease. CONCLUSIONS: overall, the opinion of patients with IBD on healthcare received is good; however, a number of deficiencies were detected, as is the case with insufficient information, care of family members, and healthcare resources accessibility/agility.

Increased mucosal tumour necrosis factor alpha production in Crohn's disease can be downregulated ex vivo by probiotic bacteria.

BACKGROUND AND AIMS: Tumour necrosis factor alpha (TNF-alpha) plays a key role in the pathogenesis of intestinal inflammation in Crohn's disease. The effect of bacteria on TNF-alpha release by intestinal mucosa was investigated. METHODS: Ileal specimens were obtained at surgery from 10 patients with Crohn's disease (ileal stricture) and five disease controls undergoing right hemicolectomy (caecal cancer). Mucosal explants from each specimen were cultured for 24 hours with either non-pathogenic Escherichia coli, Lactobacillus casei DN-114001, L bulgaricus LB10, or L crispatus (each study contained blank wells with no bacteria). Tissue and bacterial viability was confirmed by lactate dehydrogenase (LDH) release and culture. Concentrations of TNF-alpha were measured in supernatants and the phenotype of the intestinal lymphocytes was analysed by flow cytometry. RESULTS: Coculture of mucosa with bacteria did not modify LDH release. Release of TNF-alpha by inflamed Crohn's disease mucosa was significantly reduced by coculture with L casei or L bulgaricus; changes induced by L crispatus or E coli were not significant. The effect of L casei and L bulgaricus was not prevented by protease inhibitors. Coculture with L casei and L bulgaricus reduced the number of CD4 cells as well as TNF-alpha expression among intraepithelial lymphocytes from Crohn's disease mucosa. None of the bacteria induced changes in non-inflamed mucosa. CONCLUSIONS: Probiotics interact with immunocompetent cells using the mucosal interface and modulate locally the production of proinflammatory cytokines.

Role of microecology in chronic inflammatory bowel diseases.

Inflammatory bowel diseases (IBD) are chronic conditions of unknown etiology. Current therapy mitigates the severity of acute bouts of mucosal inflammation but an eradication therapy is lacking. Growing incidence of IBD is associated with social development. Epidemiology suggests a relationship between the establishment of the individual gut flora and the risk of developing IBD. Patients show an impaired tolerance towards commensal bacteria of the resident flora. Unrestrained activation of the intestinal immune system against some commensal bacteria appears to be responsible for the characteristic relapsing course of these diseases. Wide-spectrum antibiotic therapy reduces bacterial load and mitigates intestinal inflammation in human IBD and in animal models. Current research aims at the identification of probiotics for bacterial antagonism therapies. Probiotics are living microorganisms which upon ingestion in certain numbers exert health benefits beyond inherent basic nutrition. Colonization with a Lactobacillus reuteri strain can prevent the development of colitis in genetically susceptible mice. Other studies have used a bacterium genetically engineered to secrete the antiinflammatory cytokine IL-10 and demonstrated a therapeutic effect in animal models of colitis. Moreover, some probiotics may naturally exhibit antiinflammatory properties when interacting with the human gut mucosa. Prebiotics such as inulin have also been shown to prevent colonic inflammation in animal models. Preliminary clinical trials with probiotics in IBD are encouraging. Probiotics offer a valuable tool for the prevention and control of inflammatory bowel diseases.

Helicobacter pylori eradication prevents recurrence from peptic ulcer haemorrhage.

BACKGROUND: Successful eradication of Helicobacter pylori infection clearly modifies the natural history of peptic ulcer disease and prevents further recurrences of duodenal and gastric ulcers. However, there are few prospective studies about actual rates of rebleeding after H. pylori eradication, a highly relevant aspect of management as re-infection, relapse of ulcer disease for other reasons (i.e. anti-inflammatory agents) or idiopathic ulcers unrelated to H. pylori may develop and cause further bleeding episodes. OBJECTIVE: To determine the incidence of bleeding episodes after eradication of H. pylori infection in patients who had bled from an H. pylori-positive peptic ulcer. PARTICIPANTS AND INTERVENTIONS: H. pylori-positive patients who bled from a gastric or duodenal ulcer were treated with appropriate triple and/or quadruple therapy. H. pylori eradication was confirmed by urea breath test 4 weeks after treatment. Patients received no further treatment but were followed clinically and additional urea breath tests were performed every 6 months. Endoscopy with antral and corpus biopsies and urea breath test were repeated as soon as patients manifested any dyspeptic symptoms that might signal recurrence. RESULTS: A total of 103 patients with bleeding duodenal ulcer were included in the study; H. pylori was successfully eradicated in 93 of these patients, who were followed for a median interval of 27 months. The yearly re-infection rate was calculated to be 0.6%. There were no instances of rebleeding in any patients during the follow-up period. CONCLUSIONS: Even after prolonged follow-up, successful H. pylori eradication prevents rebleeding.

The impact of delaying gastric emptying by either meal substrate or drug on the [13C]-urea breath test.

OBJECTIVE: Optimal [13C]-urea breath test (UBT) conditions for diagnosis of Helicobacter pylori infection are still being fine-tuned. In the present study we investigated the impact of delaying gastric emptying by different meal substrates or L-DOPA, a drug known to induce gastric stasis, on the performance of the [13C]-UBT. METHODS: A total of 115 patients participated in the study. On two consecutive days, participants ingested [13C]-urea (100 mg) 10 min after either 270 ml of a mixed formula meal (1 Kcal/ml) or an equivalent amount of tap water. In 11 participants two additional tests were performed with or without oral 500 mg L-DOPA given 30 min before [13C]-urea load. The 13C/12C ratio in a basal breath sample was compared with ratios in samples collected 30 and 60 min after [13C]-urea. Histological assessment of H. pylori presence in antral biopsy served as reference standard. RESULTS: Formula UBT showed excellent specificity (100% at 30 and 60 min) and good sensitivity (97% at both time intervals), whereas water UBT had the same specificity but slightly lower sensitivity (94% at 30 min and 73% at 60 min). In formula UBT, 13C/12C ratios were higher at 60 min than at 30 min (21.7+/-2 vs 17.7+/-1.8 per thousand respectively, p < 0.01, whereas in water UBT 13C/12C ratios were higher at 30 min than at 60 min (13.9+/-1.5 vs 8.4+/-0.09 per thousand respectively, p < 0.01). Pretreatment with L-DOPA did not modify either the sensitivity or the specificity of the UBT. CONCLUSIONS: The performance of the [13C]-urea with a formula meal may not be improved by pharmacologically delaying gastric emptying. A short, water-based test may be a sensible approach to worldwide standardization of the [13C]-UBT for H. pylori infection.

Antiinflammatory effects of enterically coated amoxicillin-clavulanic acid in active ulcerative colitis.

The inflammatory activity of colonic mucosal lesions may be stimulated by intraluminal bacteria. Our aim was to investigate whether administration of broad-spectrum antibiotics decreases inflammatory activity in ulcerative colitis. To this end, we performed a randomized, 5-day study with either oral enterically coated amoxicillin-clavulanic acid (1 g + 250 mg, t.i.d.); i.v. methylprednisolone (40 mg/day) and oral placebo (t.i.d.); or both i.v. methylprednisolone and oral amoxicillin-clavulanic acid as above, in 30 patients with clinically active ulcerative colitis. Before and after 5 days of treatment, intestinal inflammation was assessed by the quantification of mucosal release of eicosanoids and interleukin-8 by rectal dialysis in each patient. Breath H2 excretion after oral lactulose was determined as an index of metabolic activity of colonic flora. The total release of (IL-8) interleukin-8 and eicosanoids significantly decreased in patients treated with antibiotic or steroids and antibiotic. Antibiotic treatment, but not steroids, markedly inhibited breath H2 excretion. In conclusion, short-term treatment with enteric-coated amoxicillin-clavulanic acid decreases the intraluminal release of IL-8 and other inflammatory mediators.

Usefulness of rectal dialysis to determine intrarectal eicosanoids release in ulcerative colitis.

The quantification of the local production of eicosanoids is of interest because it has been implicated in the mucosal damage of ulcerative colitis. In situ production of eicosanoids is not reflected by its urinary or seric levels, requiring invasive examinations. Thus, new non-invasive techniques such as rectal dialysis have been investigated. The purpose of this study was to assess whether the determination of the intrarectal eicosanoid levels measured by rectal dialysis is useful in detecting the presence of rectal inflammation in patients with ulcerative colitis. Thirty one patients with clinically active colitis and 7 controls with irritable bowel syndrome have been studied. A 10 cm long dialysis bag was placed in the rectum for 1 hour. To determine the variability of the technique, the dialysis was repeated the next day in 6 controls. To detect intrarectal eicosanoids release in inactive colitis, rectal dialysis was performed in another group of 15 patients with clinical and endoscopically inactive colitis and compared with 9 patients with active colitis. PGE2, TXB2, and LTB4 were measured in rectal dialysates by immunospecific RIA. Dialysis was well tolerated by all participants. Intrarectal level of every eicosanoid was much higher in active colitis than in controls (p < 0.05) and in inactive colitis (p < 0.001). The mean coefficient of variation of duplicated dialysis ranged from 15 to 28%. In conclusion, rectal dialysis is a non-invasive technique that allows to prove the presence of active inflammation in ulcerative colitis patients.