Epidemiology of gastrointestinal infections: lessons learned from syndromic testing, Region Zealand, Denmark.

The aim of this study was to investigate the value of syndromic diagnostic testing for a better understanding of the epidemiology of gastrointestinal infections in Denmark. Here we evaluated the QIAstat-Dx® Gastrointestinal (GI) Panel 1 assay on 18,610 fecal samples requested for analysis for enteric pathogens in Region Zealand, Denmark, in 1 year (October 1, 2021, to September 30, 2022). In total, 6905 (37%) samples were detected positive for one or more diarrhoeal bacteria, viruses, and protozoa. The most common bacterial, viral, and parasitic pathogens detected with the QIAstat-Dx® Gastrointestinal Panel 1 were EPEC (in patients ≥ 2 years of age) (n = 1420 (20.6%)), rotavirus (n = 948 (13.7%)), and Cryptosporidium spp. (n = 196 (2.84%)). We identified a large diversity in infections likely reflecting substantial differences in the epidemiology including origin of infections, mode of transmission, seasonality, age-dependent susceptibility to disease, severity, and travel history. All pathogens were detected as both single and coinfections. Viral infections peaked in March with a positive rate of 31.6%, and bacterial infections peaked in August with a positive rate of 35.3%. ETEC, Shigella/EIEC, EAEC, and P. shigelloides were most related to travel activity, and coinfections were frequent. The distribution of Ct values varied significantly between the pathogens, with the lowest Ct values (median 17-18) observed in astrovirus, adenovirus, and rotavirus. Our results highlight the value of providing extensive diagnostic testing on fecal samples for sufficient detection of relevant diarrhoeal pathogens for optimal clinical care.

The incidence of laboratory-confirmed cases of enteric pathogens in Denmark 2018: a national observational study.

BACKGROUND: Only a subset of enteric pathogens is under surveillance in Denmark, and knowledge on the remaining pathogens detected in acute gastroenteritis is limited. Here, we present the one-year incidence of all enteric pathogens diagnosed in Denmark, a high-income country, in 2018 and an overview of diagnostic methods used for detection. METHODS: All 10 departments of clinical microbiology completed a questionnaire on test methods and provided 2018-data of persons with positive stool samples with Salmonella species, Campylobacter jejuni/coli, Yersinia enterocolitica, Aeromonas species, diarrheagenic Escherichia coli (Enteroinvasive (EIEC), Shiga toxin-producing (STEC), Enterotoxigenic (ETEC), Enteropathogenic (EPEC), and intimin-producing/attaching and effacing (AEEC)), Shigella species., Vibrio cholerae, norovirus, rotavirus, sapovirus, adenovirus, Giardia intestinalis, Cryptosporidium species, and Entamoeba histolytica. RESULTS: Enteric bacterial infections were diagnosed with an incidence of 229.9 cases/100,000 inhabitants, virus had an incidence of 86/100,000 and enteropathogenic parasites of 12.5/100,000. Viruses constituted more than half of diagnosed enteropathogens for children below 2 years and elderly above 80 years. Diagnostic methods and algorithms differed across the country and in general PCR testing resulted in higher incidences compared to culture (bacteria), antigen-test (viruses), or microscopy (parasites) for most pathogens. CONCLUSIONS: In Denmark, the majority of detected infections are bacterial with viral agents primarily detected in the extremes of ages and with few intestinal protozoal infections. Incidence rates were affected by age, clinical setting and local test methods with PCR leading to increased detection rates. The latter needs to be taken into account when interpreting epidemiological data across the country.

Establishing a donor stool bank for faecal microbiota transplantation: methods and feasibility.

Faecal microbiota transplantation (FMT) is a promising treatment, but donor selection and implementation in clinical practice are difficult. Here, we describe the establishment of a donor stool bank based on the Tissue Act. Stool donors were recruited among blood donors and asked to donate five times in a month. A screening questionnaire, a medical interview and testing of blood and stool were conducted before and after donations. Donations were made at home and transported to the lab, where 50 g of stool was suspended and filtered in saline and 20-mL glycerol (final concentration of 10%) to a volume of 170 mL. The processed stool was assigned a batch number, frozen within 2 h after defecation and stored at - 80 °C for up to 1 year. All steps were documented and cross-checked before donor stool were released for clinical use. Thirteen donors were eligible at the first interview and started donations. Two donors were excluded due to a positive Helicobacter pylori test, two withdrew consent and one was lost to follow-up. One donor took a single dose of NSAIDs 2 days prior to a donation, which was discarded. There were no other excluding findings at the second interview or testing. Eight of the 13 donors were approved as stool donors. All donated five times with each donation yielding 1-6 portions. Eighty-four portions were released for clinical use. Recruiting stool donors among blood donors is safe and effective. The Tissue Act yields an appropriate regulative framework for FMT.

Disease-Specific Enteric Microbiome Dysbiosis in Inflammatory Bowel Disease.

Inflammatory Bowel disease (IBD) is traditionally divided into Crohn's disease (CD) and ulcerative colitis (UC). UC is a relapsing non-transmural inflammatory disease that is restricted to the colon and is characterized by flare-ups of bloody diarrhea. CD is a chronic, segmental localized granulomatous disease that can affect any part of the entire gastrointestinal tract. Ileo-anal pouch is a procedure restoring functionality of the rectum after a colectomy. IBD is a multifactorial disease and flares of IBD are probably triggered by changes in the intestinal microbiota followed by an abnormal immune response. In this study, we aim to analyze the intestinal bacterial diversity in IBD patients during various stages of disease compared with healthy controls. Permission for human experiments and recruitment of participants was obtained from the Ethic Committee for Copenhagen County hospitals (Permission no. KA-03019, Permission no. KA-20060159). Stools from 26 healthy controls, 42 CD, 38 UC and 18 pouch patients were collected. Stool DNA extraction was performed using Qiagen, DNA mini stool kit Denmark. DGGE-PCR amplifying the V2-V3 region of 16S-rDNA gene of the bacteria was amplified by universal primers HDA1 and HDA2. Analysis of DGGE was performed blinded using BioNumerics version 7.5. After normalization, a DGGE gel band matching was performed. The similarities between profiles were calculated with a ranked Pearson correlation coefficient based on the band matching results using band intensities. Simpson's index of diversity and Pielou's species evenness were calculated. Based on the Shannon Diversity Index, UC patients had lower species diversity and bacterial evenness in comparison to healthy persons, p < 0.05. However, only CD and disease pouch patients had lower species diversity compared to those with inactive disease and healthy controls. Well-functioning pouch patients had decreased species evenness in comparison to diseased pouch patients and control group. During the active disease stage in CD and pouch, the patients have a low bacterial diversity in their gut when compared to the inactive stage. In UC patients, a generally low diversity was observed at all stages of the disease compared to healthy controls.

Seroreactivity to E. coli outer membrane protein C antibodies in active inflammatory bowel disease; diagnostic value and correlation with phylogroup B2 E. coli infection.

BACKGROUND: Several serologic tests, including anti-outer membrane porin C antibody (Omp C), are used for screening and as marker of disease course in inflammatory bowel diseases (IBD). Our aim was to investigate possible differences in Omp C level in patients with active and inactive IBD compared to controls. METHODS: All blood samples were tested for Omp C. Disease activity was evaluated by Harvey Bradshaw Index, Simple Clinical Activity Index and Modified Pouchitis Disease Activity Index. RESULTS: Blood samples were collected from 113 patients and 60 controls. Patients with active IBD did not have a higher level of Omp C than patients in remission. Surprisingly, in patients with active Crohn's disease a significantly lower level of Omp C was found compared with patients with inactive Crohn's disease (p < 0.05). All other groups among patients with IBD did have a significantly higher level of Omp C, compared with controls, including patients with acute gastroenteritis (p < 0.05). Although IBD patients with phylogroup B2 E. coli cultured from their fecal samples, were more likely to have a positive Omp C test (p < 0.05), this could not explain the low Omp C level in the subgroup of patients with active Crohn's disease. CONCLUSIONS: Omp C titer was not raised in patients with active IBD compared with patients in remission. In addition, there was no difference in Omp C level in patients with active Crohn's disease compared with controls. These observations do not support the use of Omp C serology testing, either in disease activity assessment, or in screening for active Crohn's disease.

Oral and fecal Campylobacter concisus strains perturb barrier function by apoptosis induction in HT-29/B6 intestinal epithelial cells.

Campylobacter concisus infections of the gastrointestinal tract can be accompanied by diarrhea and inflammation, whereas colonization of the human oral cavity might have a commensal nature. We focus on the pathophysiology of C. concisus and the effects of different clinical oral and fecal C. concisus strains on human HT-29/B6 colon cells. Six oral and eight fecal strains of C. concisus were isolated. Mucus-producing HT-29/B6 epithelial monolayers were infected with the C. concisus strains. Transepithelial electrical resistance (R(t)) and tracer fluxes of different molecule size were measured in Ussing chambers. Tight junction (TJ) protein expression was determined by Western blotting, and subcellular TJ distribution was analyzed by confocal laser-scanning microscopy. Apoptosis induction was examined by TUNEL-staining and Western blot of caspase-3 activation. All strains invaded confluent HT-29/B6 cells and impaired epithelial barrier function, characterized by a time- and dose-dependent decrease in R(t) either after infection from the apical side but even more from the basolateral compartment. TJ protein expression changes were sparse, only in apoptotic areas of infected monolayers TJ proteins were redistributed. Solely the barrier-forming TJ protein claudin-5 showed a reduced expression level to 66±8% (P<0.05), by expression regulation from the gene. Concomitantly, Lactate dehydrogenase release was elevated to 3.1±0.3% versus 0.7±0.1% in control (P<0.001), suggesting cytotoxic effects. Furthermore, oral and fecal C. concisus strains elevated apoptotic events to 5-fold. C. concisus-infected monolayers revealed an increased permeability for 332 Da fluorescein (1.74±0.13 vs. 0.56±0.17 10(-6) cm/s in control, P<0.05) but showed no difference in permeability for 4 kDa FITC-dextran (FD-4). The same was true in camptothecin-exposed monolayers, where camptothecin was used for apoptosis induction.In conclusion, epithelial barrier dysfunction by oral and fecal C. concisus strains could mainly be assigned to apoptotic leaks together with moderate TJ changes, demonstrating a leak-flux mechanism that parallels the clinical manifestation of diarrhea.