Matthijs E. Grasman

Rectal Swabs for Analysis of the Intestinal Microbiota

The composition of the gut microbiota is associated with various disease states, most notably inflammatory bowel disease, obesity and malnutrition. This underlines that analysis of intestinal microbiota is potentially an interesting target for clinical diagnostics. Currently, the most commonly used sample types are feces and mucosal biopsy specimens. Because sampling method, storage and processing of samples impact microbiota analysis, each sample type has its own limitations. An ideal sample type for use in routine diagnostics should be easy to obtain in a standardized fashion without perturbation of the microbiota. Rectal swabs may satisfy these criteria, but little is known about microbiota analysis on these sample types. In this study we investigated the characteristics and applicability of rectal swabs for gut microbiota profiling in a clinical routine setting in patients presenting with various gastro-intestinal disorders. We found that rectal swabs appeared to be a convenient means of sampling the human gut microbiota. Swabs can be performed on demand, whenever a patient presents; swab-derived microbiota profiles are reproducible, whether they are gathered at home by patients or by medical professionals in an outpatient setting and may be ideally suited for clinical diagnostics and large-scale studies.

IS-pro: high-throughput molecular fingerprinting of the intestinal microbiota

The human intestinal microbiota is known to play an important role in human health and disease, and with the advent of novel molecular techniques, disease‐specific variations in its composition have been found. However, analysis of the intestinal microbiota has not yet been applicable in large‐scale clinical research or routine diagnostics because of the complex and expensive nature of the techniques needed. Here, we describe a new PCR‐based profiling technique for high‐throughput analysis of the human intestinal microbiota, which we have termed IS‐pro. This technique combines bacterial species differentiation by the length of the 16S–23S rDNA interspace region with instant taxonomic classification by phylum‐specific fluorescent labeling of PCR primers. We validated IS‐pro in silico, in vitro, and in vivo, on human colonic biopsies and feces, and introduced a standardized protocol for data analysis. IS‐pro is easy to implement in general clinical microbiological laboratories with access to capillary gel electrophoresis, and the high‐throughput nature of the test makes analysis of large numbers of samples feasible. This combination renders IS‐pro ideally suited for use in clinical research and routine diagnostics.—Budding, A. E., Grasman, M. E., Lin, F., Bogaards, J. A., Soeltan‐Kaersenhout, D. J., Vandenbroucke‐Grauls, C. M. J. E., van Bodegraven, A. A., Savelkoul, P. H. M. IS‐pro: high‐throughput molecular fingerprinting of the intestinal microbiota. FASEB J. 24, 4556–4564 (2010). www.fasebj.org

Clinical nutrition in the hepatogastroenterology curriculum

Gastroenterology (GE) used to be considered a subspecialty of internal medicine. Today, GE is generally recognized as a wide-ranging specialty incorporating capacities, such as hepatology, oncology and interventional endoscopy, necessitating GE-expert differentiation. Although the European Board of Gastroenterology and Hepatology has defined specific expertise areas in Advanced endoscopy, hepatology, digestive oncology and clinical nutrition, training for the latter topic is lacking in the current hepatogastroenterology (HGE) curriculum. Given its relevance for HGE practice, and being at the core of gastrointestinal functioning, there is an obvious need for training in nutrition and related issues including the treatment of disease-related malnutrition and obesity and its associated metabolic derangements. This document aims to be a starting point for the integration of nutritional expertise in the HGE curriculum, allowing a central role in the management of malnutrition and obesity. We suggest minimum endpoints for nutritional knowledge and expertise in the standard curriculum and recommend a focus period of training in nutrition issues in order to produce well-trained HGE specialists. This article provides a road map for the organization of such a training program. We would highly welcome the World Gastroenterology Organisation, the European Board of Gastroenterology and Hepatology, the American Gastroenterology Association and other (inter)national Gastroenterology societies support the necessary certifications for this item in the HGE-curriculum.

Tropheryma whipplei, a Potential Commensal Detected in Individuals Undergoing Routine Colonoscopy.

ABSTRACT Mucosal biopsy samples from individuals not suspected of having Whipples disease were tested for the presence of Tropheryma whipplei. A sensitive and specific real-time PCR assay targeting a sequence present seven times in the T. whipplei genome was used. T. whipplei DNA was detected in 2.0 and 3.8% of the patients undergoing gastroduodenoscopy and colonoscopy, respectively, who were tested.

Proximal esophageal cancer missed during esophagogastroduodenoscopy: should the detection of an inlet patch be added to the quality criteria for upper gastrointestinal endoscopy?

A 75-year-old woman was investigated by the pulmonologist for pulmonary abnormalities and weight loss. In the past she had undergone repeated endoscopic rubber-band ligations for bleeding esophageal varices. A combined positron emission tomography–computed tomography (PET-CT) scan revealed intense fluorodeoxyglucose (FDG) avidity in the proximal esophagus. An esophagogastroduodenoscopy (EGD) showed a squamous cell carcinoma just distal to the upper esophageal sphincter. Remarkably, an EGD 2 months earlier had been reported to be without abnormalities. The proximal esophagus needs careful screening to detect esophageal carcinoma, especially in high risk patients [1]. A quality indicator for EGD could consist of the detection of an esophageal gastric inlet patch (GIP), which is present in 0.4%–11% of patients [2–4]. A required minimum lower limit for GIP detection rate (GIPDR) could result in a lower miss-rate for proximal abnormalities, in a manner similar to that for adenoma detection rate during colonoscopy [5]. In a retrospective cohort, we reviewed EGD reports for the presence of a GIP in both the text and images of the reports. Thereafter, consecutive patients undergoing an EGD were actively screened for the presence of a GIP. Diagnostic EGDs, performed mainly because of gastroesophageal reflux disease, dyspepsia, or anemia, were included in both groups. Exclusion criteria were therapeutic EGD or altered anatomy from mouth to duodenum. The total population consisted of 157 patients: 65 men (41%) and 92 women (59%). Retrospective review of 95 EGD reports revealed no reported GIPs (0%; 95% confidence interval [CI] 0.0%–3.8%); in the prospective group, three GIPs were observed among the 62 patients (4.8%; 95%CI 1.0%–13.5%; P=0.065). Our case report and subsequent analysis of our GIPDR underline the importance of actively focusing on the proximal esophagus during EGD. GIPDR seems to be influenced by the awareness of the endoscopist. A lower limit of GIPDR could be helpful in improving the quality of EGD. Endoscopy_UCTN_Code_CPL_1AH_2AK

P668 Interspace microbiome profiling (IS-pro) enables to differentiate IBD subclasses and disease activity by specific loss of bacterial diversity

P667 Low prevalence of Blastocystis sp. in active ulcerative colitis patients N. Rossen1 *, A. Bart2, N. Verhaar2, E. van Nood3, R. Kootte4, P. de Groot3, M. Nieuwdorp4, G. D’Haens1, C. Ponsioen1, T. van Gool2. 1Academic Medical Center, Gastroenterology & Hepatology, Amsterdam, Netherlands, 2Academic Medical Center Amsterdam, Department of Medical Microbiology, Parasitology Section, Amsterdam, Netherlands, 3Academic Medical Center Amsterdam, Department of Internal Medicine, Amsterdam, Netherlands, 4Academic Medical Center Amsterdam, Department of Vascular Medicine, Amsterdam, Netherlands

Tu2028 The Effect of Viral, Bacterial and Parasitic Infections on the Intestinal Microbiota

G A A b st ra ct s JEJ mucosa at both 6 (0.69 fold change) and 24 h (0.53 fold change) after Dox treatment, but these bacteria were increased in DI mucosa at 6 (1.05 fold change) and 24h (1.77-fold change; p,0.04 compared to jejunum). Conversely, Lactobacillus species were increased after Dox in JEJ mucosa at 6 (1.77 fold change) and 24 h (1.95 fold change), but were decreased in DI mucosa at 6 (0.59 fold change; p ,0.02) and 24 h (0.74 fold change). Faecalibacterium prausnitzii were also increased in JEJ mucosa at 6 h (1.08 fold change), but were decreased in DI mucosa (0.78 fold change). Bifidobacterium spp. were largely increased in both the JEJ and DI after Dox administration, while E. coli were increased in the lumen but decreased in the mucosa of both JEJ and DI. Conclusions: Dox induces a profound dysbiosis resulting in differences between JEJ and DI in changes in concentrations of bacterial groups. Decreases in C. coccoides, which have important immunoregulatory properties, in the JEJ may explain more severe injury induced by Dox compared to the DI. Meanwhile, proliferation of anti-inflammatory Lactobacillus spp. and F. prausnitzii in the JEJ may be compensatory responses to injury. These results can guide selective colonization experiments and translational and clinical trials aimed at preventing Dox-induced small intestinal injury by targeting specific bacterial groups with antibiotics, probiotics, or prebiotics.

6 - Temporal stability of faecal microbiota by Inter Spacer rDNA bacterial profiling (IS-pro)

Gnotobiotic rodents facilitate the study of commensal bacteria and their roles in human physiology and pathophysiology. To ensure sterility, animals must be screened frequently for contamination by the traditional approach of culturing and Gram staining of feces. Yet many bacteria are uncultivable, fecal Gram stains can be difficult to interpret, and these methods are labor-intensive and time-consuming. Aims: To develop molecular methods of detecting contamination in gnotobiotic units.Methods:We collected fresh fecal pellets from mice housed in germ free (GF) isolators (N=8), a contaminated ex-GF isolator, an isolator colonized with 2 bacterial species, and a specific pathogen free (SPF) animal room (N=4). DNA from fecal samples was extracted using a phenol/chloroform extraction method combined with physical disruption of bacterial cells. Polymerase chain reactions (PCR) were carried out using templates of fecal DNA and primers that amplify the genes encoding the 16S rDNA from all bacterial groups. The numbers of 16S rDNA gene copies in each DNA sample were then quantified using quantitative Real-Time PCR (qPCR) with the same templates and primers and the appropriate set of standards. Finally, the 16S rDNA gene was amplified from fecal DNA from contaminated ex-GF mice, sequenced, and crossreferenced with a computerized data base to identify the bacterial species. Results: PCR yielded amplicons from isolated fecal DNA from, in descending order of intensity, all SPF mice, dual associated mice, and contaminated ex-GF mice, as well as from mice from one presumed GF isolator. Fecal DNA from mice from the other 7 GF isolators did not yield amplicons. qPCR confirmed that the level of bacteria in themouse feces from the questionable isolator, (4.0 X 108 copies 16S rDNA gene/μg feces), was 210-fold greater than the levels in the other 7 GF isolators, (1.9 X 106 copies/μg, p < 0.0001), and was comparable to the level in the known contaminated isolator, (3.0 X 108 copies/μg.) The source of DNA in mouse feces from GF isolators detected by qPCR was dead bacteria in autoclaved food. Upon review, both cultures and Gram stains from the isolator that we determined to be contaminated based on PCR and qPCR results had been deemed indeterminate by gnotobiotic facility staff. Sequencing the 16S rDNA gene revealed the contaminating bacterial species in the ex-GF isolator to be Bacillus simplex. Conclusions: We developed molecular methods to screen for contamination in gnotobiotic units that are more reliable and less timeconsuming than current traditional methods and can be used to identify the contaminating species.

W1640 Detection of Tropheryma Whipplei DNA in Intestinal Biopsy Specimens with a Novel Real-Time PCR

Introduction Whipples disease (WD) is a rare, multisystemic infectious disease caused by the bacterium Tropheryma whipplei (Tw). Periodic acid Schiff (PAS) stain is usually performed to diagnose the disease. PCR is currently used to confirm the presence of Tw DNA. However, conventional PCR is not specific, whereas real-time PCR (rtPCR) based on the 16S rRNA gene is insensitive since only one copy of the gene is present in Tw genome. Recently, we developed a novel, more sensitive and specific rtPCR to detect the presence of Tw DNA. Aims To determine prevalence of Tw in duodenal and colonic biopsy specimens with rtPCR in a random population of patients undergoing routine gastroscopy or colonoscopy. Materials and methods Mucosal biopsy specimens from the duodenum or sigmoid were harvested from subsequent patients undergoing upper or lower endoscopy. Most frequent indications for gastroscopy were dyspepsia, pyrosis, and follow-up of celiac disease. Common indications for colonoscopy were colorectal cancer screening and surveillance, and abdominal pain. Specimens were analyzed by rtPCR targeting an exclusive, repetitive sequence which is seven times present in the Tw genome. Results In total, 48 duodenal and 90 colonic mucosal biopsy specimens of 118 patients were analyzed. Of these patients, 40% were men, mean age 54y (range 18-84y). Normal appearance of duodenal and colonic mucosa was found in 87% and 83%, respectively. Main abnormalities found in the duodenum were signs of celiac disease (CD). Abnormalities of colonic specimens included signs of IBD and pseudomelanosis coli. Of the duodenal and colonic specimens 2.1% and 6.7% were positive in the Tw rtPCR, respectively (mean age 43y, male/female: 2/5). One showed CD at microscopy, whereas all others were normal. In another patient, signs and symptoms of WD were recorded namely arthralgia, weight loss and peripheral neuropathy. The Cycle threshold-value of the PCR was considerably lower in this patient, consistent with a high load of Tw DNA. Discussion/ Conclusion With a novel rtPCR, Tw DNA is present in duodenal or colonic biopsy specimens in a proportion of patients without signs or symptoms of WD. Therefore, additional factors, other than merely the presence of Tw, play a role in development of disease. Initiation of antibiotic therapy against Tw may not exclusively depend on positive PCR findings, but should additionally be based on PAS stain and signs and symptoms ofWD. Clinical significant WD appears to be associated with a higher Tw load.