title: A novel isolation protocol and probe-based RT-PCR for diagnosis of gastric infections with the zoonotic pathogen Helicobacter suis.
abstract: BACKGROUND
  Helicobacter suis is a very fastidious microorganism associated with gastritis, gastric ulcers, and mucosa-associated lymphoid tissue lymphoma in humans. In vitro isolation of this agent from human patients has so far been unsuccessful.
  
  
  METHODS
  A probe-based real-time PCR (RT-PCR) for the rapid detection of H. suis in gastric biopsies was developed. Secondly, a mouse-passage-based protocol was optimized for isolation of low numbers of viable H. suis bacteria. Mice were inoculated with different numbers of viable H. suis (102-108) and kept for 4 weeks to allow multiplication of this pathogen.
  
  
  RESULTS
  The probe-based real-time PCR (RT-PCR) exhibited a high degree of diagnostic specificity and analytical sensitivity, high linear correlations (r2between 0.995 and 0.999), and high amplification efficiencies (>90%) for H. suis. No cross-reactivity was detected with human, porcine, non-human primate, and murine DNA nor with DNA from other bacteria including Helicobacter spp. and Campylobacter spp. H. suis was successfully re-isolated from the stomach of mice inoculated with at least 104viable H. suis, using a biphasic medium (pH 5), consisting of Brucella agar with Brucella broth on top, both supplemented with vitox supplement, Campylobacter-selective supplement, amphotericin (5 μg/mL), HCl (0.05%), fetal bovine serum (20%), and linezolid (5 μg/mL). Linezolid was necessary to inhibit proliferation of contaminants, including lactobacilli.
  
  
  CONCLUSIONS
  The methods described above can be implemented for detection or isolation of H. suis from human gastric biopsies.
authors.affiliation: Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
authors.forename: Caroline
authors.initials: C
authors.lastname: Blaecher
authors.affiliation: Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
authors.forename: Eva
authors.initials: E
authors.lastname: Bauwens
authors.affiliation: The Marshall Centre for Infectious Diseases Research and Training, University of Western Australia, Perth, WA, Australia.
authors.forename: Alfred
authors.initials: A
authors.lastname: Tay
authors.affiliation: The Marshall Centre for Infectious Diseases Research and Training, University of Western Australia, Perth, WA, Australia.
authors.forename: Fanny
authors.initials: F
authors.lastname: Peters
authors.affiliation: Institute of Pharmaceutical Science, King's College London, London, UK.|The Maudsley Hospital, London, UK.|Department of Gastroenterology, King's College Hospital, London, UK.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Dobbs
authors.affiliation: Institute of Pharmaceutical Science, King's College London, London, UK.|The Maudsley Hospital, London, UK.|Department of Gastroenterology, King's College Hospital, London, UK.
authors.forename: John
authors.initials: J
authors.lastname: Dobbs
authors.affiliation: Institute of Pharmaceutical Science, King's College London, London, UK.|Statistics Unit, Centre for Infectious Disease Surveillance and Control, Public Health England, London, UK.
authors.forename: André
authors.initials: A
authors.lastname: Charlett
authors.affiliation: Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
authors.forename: Richard
authors.initials: R
authors.lastname: Ducatelle
authors.affiliation: Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
authors.forename: Freddy
authors.initials: F
authors.lastname: Haesebrouck
authors.affiliation: Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
authors.forename: Annemieke
authors.initials: A
authors.lastname: Smet
mesh_terms: D000818:Animals; D001706:Biopsy; D004195:Disease Models, Animal; D005260:Female; D016481:Helicobacter Infections / Q000175:diagnosis* / Q000382:microbiology; D018247:Helicobacter heilmannii / Q000235:genetics / Q000302:isolation & purification*; D008810:Mice, Inbred C57BL; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D011323:Primates; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013272:Stomach Diseases / Q000175:diagnosis* / Q000382:microbiology
keywords: 
  Helicobacter suis
  ; diagnosis; isolation; probe-based RT-PCR