title: Development of a real-time PCR for identification of brachyspira species in human colonic biopsies.
abstract: BACKGROUND
  Brachyspira species are fastidious anaerobic microorganisms, that infect the colon of various animals. The genus contains both important pathogens of livestock as well as commensals. Two species are known to infect humans: B. aalborgi and B. pilosicoli. There is some evidence suggesting that the veterinary pathogenic B. pilosicoli is a potential zoonotic agent, however, since diagnosis in humans is based on histopathology of colon biopsies, species identification is not routinely performed in human materials.
  
  
  METHODS
  The study population comprised 57 patients with microscopic evidence of Brachyspira infection and 26 patients with no histopathological evidence of Brachyspira infection. Concomitant faecal samples were available from three infected patients. Based on publically available 16S rDNA gene sequences of all Brachyspira species, species-specific primer sets were designed. DNA was extracted and tested by real-time PCR and 16S rDNA was sequenced.
  
  
  RESULTS
  Sensitivity and specificity for identification of Brachyspira species in colon biopsies was 100% and 87.7% respectively. Sequencing revealed B. pilosicoli in 15.4% of patients, B. aalborgi in 76.9% and a third species, tentatively named "Brachyspira hominis", in 26.2%. Ten patients (12.3%) had a double and two (3.1%) a triple infection. The presence of Brachyspira pilosicoli was significantly associated with inflammatory changes in the colon-biopsy (p=0.028).
  
  
  CONCLUSIONS
  This newly designed PCR allows for sub-differentiation of Brachyspira species in patient material and thus allows large-scaled surveillance studies to elucidate the pathogenicity of human Brachyspira infections. One-third of affected patients appeared to be infected with a novel species.
authors.affiliation: Department of Medical Microbiology, University Medical Centre Utrecht, Utrecht, The Netherlands.
authors.forename: Laurens J
authors.initials: LJ
authors.lastname: Westerman
authors.forename: Herbert V
authors.initials: HV
authors.lastname: Stel
authors.forename: Marguerite E I
authors.initials: ME
authors.lastname: Schipper
authors.forename: Leendert J
authors.initials: LJ
authors.lastname: Bakker
authors.forename: Eskelina A
authors.initials: EA
authors.lastname: Neefjes-Borst
authors.forename: Jan H M
authors.initials: JH
authors.lastname: van den Brande
authors.forename: Edwin C H
authors.initials: EC
authors.lastname: Boel
authors.forename: Kees A
authors.initials: KA
authors.lastname: Seldenrijk
authors.forename: Peter D
authors.initials: PD
authors.lastname: Siersema
authors.forename: Marc J M
authors.initials: MJ
authors.lastname: Bonten
authors.forename: Johannes G
authors.initials: JG
authors.lastname: Kusters
mesh_terms: D001706:Biopsy; D017400:Brachyspira / Q000145:classification* / Q000235:genetics*; D003106:Colon / Q000382:microbiology*; D006801:Humans; D010802:Phylogeny; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*