Emilie Bessède

New Methods for Detection of Campylobacters in Stool Samples in Comparison to Culture

ABSTRACT Campylobacter species, especially Campylobacter jejuni and Campylobacter coli, are a major cause of human bacterial enteritis. Current detection in stools is done essentially by culture on selective and nonselective media with filtration. These methods were compared to 2 molecular biology methods, an in-house real-time PCR and a multiplex PCR named Seeplex Diarrhea ACE Detection, and 3 immunoenzymatic methods, Premier Campy, RidaScreen Campylobacter, and ImmunoCard Stat!Campy. Out of 242 stool specimens tested, 23 (9.5%) fulfilled the positivity criteria, i.e., they were positive by one or both culture methods or, in case of a negative culture, by a positive molecular method and a positive immunoenzymatic method. The striking feature of this study is the low sensitivity of culture, in the range of 60%, in contrast to immunoenzymatic and molecular tests.

Identification of Campylobacter species and related organisms by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry.

The identification of Campylobacter species and related organisms at the species level has always been difficult using phenotypic methods because of their low metabolic activity, whereas molecular methods are more reliable but time-consuming. In this study, 1007 different strains were identified using three different methods: conventional methods, molecular biology (real-time PCR and sequencing) and matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Molecular methods were considered the gold standard. The accuracy of MALDI-TOF mass spectrometry reached 100% compared with the gold standard for all of the Campylobacter species, except Campylobacter jejuni (99.4%). The accuracy of conventional methods compared with the gold standard ranged from 0% to 100% depending on the species. However, MALDI-TOF mass spectrometry was not able to identify a mixture of two different species present in the same sample in four instances. Finally, MALDI-TOF mass spectrometry is highly recommended to identify Campylobacter spp. as only 0.4% discrepancy was found, whereas conventional methods led to 4.5% discrepancy.

Helicobacter pylori infection and gastric carcinoma

Helicobacter pylori infection is considered to be the main cause of gastric cancer and the most frequent infection-induced cancer. H. pylori is a heterogeneous species which can harbour pathogenic factors such as a cytotoxin, a pathogenicity island (cag) encoding a type 4 secretion system, and the first bacterial oncoprotein, CagA. This oncoprotein appears to be involved in the carcinogenic process in addition to the inflammation generated. This process may concern either local progenitors via an epithelial-mesenchymal transition, or recruited bone marrow-derived mesenchymal cells. There are also environmental factors such as iron deficiency or high-salt diets which interact with the bacterial factors to increase the risk of gastric cancer as well as genetic polymorphism of certain cytokines, e.g. IL-Iβ. Recent data suggest that a break in coevolution of a particular phylogeographic lineage of H. pylori and its usual host may also be a risk factor. Studies are currently being performed to assess the feasibility of organized H. pylori eradication programmes to prevent gastric cancer.

MALDI-TOF mass spectrometry for early identification of bacteria grown in blood culture bottles.

This note reports an interesting way to rapidly identify bacteria grown from blood culture bottles. Chocolate agar plates were inoculated with 1 drop of the positive blood bottle medium. After a 3-hour incubation, the growth veil was submitted to MALDI-TOF mass spectrometry: 77% of the bacteria present have been correctly identified.

Evaluation of the positive predictive value of a rapid Immunochromatographic test to detect Campylobacter in stools

The recently developed rapid immunochromatographic tests (ICT) have the potential to provide a quick and easy diagnosis of Campylobacter enteritis in comparison to culture. In a previous study we found them sensitive but lacking in specificity. The aim of the present study was to focus on the problem of specificity and determine the positive predictive value (PPV) of a positive result of the ImmunoCard Stat! Campy (Meridian Bioscience, Cincinnati, OH, USA). For this purpose, the stools positive by ICT were cultured according to 3 different protocols: Karmali agar, Preston enrichment broth subcultured on Karmali agar, and a filtration method on a blood agar without antibiotics, all incubated for 7 days at 37°C. Out of 609 stools from adults and children with community acquired enteritis, the reference methods detected 25 positive cases (4.1%) (culture: 19, specific PCR and ELISA both positive: 6) and the ICT: 31 including the 25 true positives. The PPV was 80.6%. We conclude that ICT is a good method to screen Campylobacter positive stools but because of its lack of specificity the positive stools must be tested by another method.

Is Elevated Gastric Tissue NOX2 Associated with Lymphoma of Mucosa-Associated Lymphoid Tissue?

Abstract Helicobacter pylori infection plays a crucial role in the pathogenesis of gastric extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT). However, the host response to this infection is also important in the development of the disease. In particular, NADPH oxidases (NOXs) which generate reactive oxygen species are known to induce cell damage possibly leading to carcinogenesis. We analyze for the first time NOX expression in a series of well characterized gastric MALT lymphoma (GML) patients in comparison with controls. Our observation leads to the hypothesis that NOX2 expression is significantly associated with GML.

Deletion of IQGAP1 promotes Helicobacter pylori -induced gastric dysplasia in mice and acquisition of cancer stem cell properties in vitro

Helicobacter pylori infection is responsible for gastric carcinogenesis but host factors are also implicated. IQGAP1, a scaffolding protein of the adherens junctions interacting with E-cadherin, regulates cellular plasticity and proliferation. In mice, IQGAP1 deficiency leads to gastric hyperplasia. The aim of this study was to elucidate the consequences of IQGAP1 deletion on H. pylori-induced gastric carcinogenesis. Transgenic mice deleted for iqgap1 and WT littermates were infected with Helicobacter sp., and histopathological analyses of the gastric mucosa were performed. IQGAP1 and E-cadherin expression was evaluated in gastric tissues and in gastric epithelial cell lines in response to H. pylori infection. The consequences of IQGAP1 deletion on gastric epithelial cell behaviour and on the acquisition of cancer stem cell (CSC)-like properties were evaluated. After one year of infection, iqgap1+/- mice developed more preneoplastic lesions and up to 8 times more gastro-intestinal neoplasia (GIN) than WT littermates. H. pylori infection induced IQGAP1 and E-cadherin delocalization from cell-cell junctions. In vitro, knock-down of IQGAP1 favoured the acquisition of a mesenchymal phenotype and CSC-like properties induced by H. pylori infection. Our results indicate that alterations in IQGAP1 signalling promote the emergence of CSCs and gastric adenocarcinoma development in the context of an H. pylori infection.

Evaluation of the Diagnostic Accuracy of Two Immunochromatographic Tests Detecting Campylobacter in Stools and Their Role in Campylobacter Infection Diagnosis

ABSTRACT The detection of campylobacters in stools is performed essentially by culture, but this technique has a low sensitivity. New detection methods are now available. Among them, immunochromatography tests (ICTs) are very attractive in that they offer a result within 15 min. However, previous studies suggest that these tests have a relatively low specificity. The objective of this study was to evaluate the performance of these tests. During the study period, all patients who consulted the emergency units and had a stool culture were included. Their stool samples were tested with two ICTs, Ridaquick Campylobacter and ImmunoCard STAT! Campy. Stools were also tested by a home-made PCR and two commercially available enzyme-linked immunosorbent assays (ELISAs) when one of the ICTs was positive. The composite reference standard (CRS) was defined as positive if the culture was positive or, in case of a negative culture, if the PCR and one of the ELISAs were positive simultaneously. Three hundred and five patients were included. Among the 50 positive specimens with Ridaquick Campylobacter, 47 were considered true positives by the CRS, corresponding to a positive predictive value (PPV) of 94.0%. Among the 52 positive specimens with ImmunoCard STAT! Campy, 44 were considered true positives by the CRS, corresponding to a PPV of 84.6%. The negative predictive values were estimated at 94.9 and 92.4% for the Ridaquick Campylobacter and ImmunoCard STAT! Campy tests, respectively. ICTs appear to be very efficient and allow a very rapid detection of campylobacters, which is important for treating early campylobacter infections with an adapted antibiotherapy.

A new kit to detect Campylobacter species in stool specimens: the Orion GenRead Campylobacter®

Campylobacter enteritis is the most frequent bacterial enteritis including in children. Its diagnosis suffers from the lack of sensitivity and delayed result of culture. Our aim was to test a new PCR-derived method for Campylobacter diagnosis in comparison to a composite reference. Patients presenting to the emergency ward of our hospital with enteric symptoms during the 2016 summer season were included. In addition to culture, an ELISA and an in-house real-time PCR were performed, as well as the new method (Orion GenRead Campylobacter) on all stool specimens. The composite reference used to consider a case positive for Campylobacter was either culture positive and in case of negative culture both the ELISA and real-time PCR positive. One hundred fifty patients were included, 64 being infants or children. There were 29 cases positive by the composite reference, with 19 of the 64 children (29.7%) and 10 of the 86 adults (11.6%). If performed alone, culture would have missed six cases. The Orion GenRead Campylobacter detected all the positives by the composite reference but also 12 cases negative by the composite reference (sensitivity 100%, specificity 90.1%). Given the characteristics of the new method, it can be used as a screening method for Campylobacter detection.

Epidemiology of Helicobacter pylori and Mechanisms of Carcinogenesis

Helicobacter pylori is a Gram-negative, microaerophilic bacterium which colonises human gastric mucosa. Initially, B. Marshall and W. Warren discovered the etiological role of H. pylori in gastritis and peptic ulcers and received the Nobel Prize in Medicine in 2005. Indeed, since 1994, H. pylori has been also recognised by the WHO to be associated with gastric cancer, being the first cause of gastric adenocarcinoma based on epidemiological data and Hill’s criteria for causality. H. pylori infection is responsible for at least 90 % of non-cardia gastric cancers and is the third cause of mortality by cancer in the world with nearly one million new cases per year.