title: Nanolitre real-time PCR detection of bacterial, parasitic, and viral agents from patients with diarrhoea in Nunavut, Canada.
abstract: BACKGROUND
  Little is known about the microbiology of diarrhoeal disease in Canada's Arctic regions. There are a number of limitations of conventional microbiology testing techniques for diarrhoeal pathogens, and these may be further compromised in the Arctic, given the often long distances for specimen transport.
  
  
  OBJECTIVE
  To develop a novel multiple-target nanolitre real-time reverse transcriptase (RT)-PCR platform to simultaneously test diarrhoeal specimens collected from residents of the Qikiqtani (Baffin Island) Region of Nunavut, Canada, for a wide range of bacterial, parasitic and viral agents.
  
  
  METHODS
  Diarrhoeal stool samples submitted for bacterial culture to Qikiqtani General Hospital in Nunavut over an 18-month period were tested with a multiple-target nanolitre real-time PCR panel for major diarrhoeal pathogens including 8 bacterial, 6 viral and 2 parasitic targets.
  
  
  RESULTS
  Among 86 stool specimens tested by PCR, a total of 50 pathogens were detected with 1 or more pathogens found in 40 (46.5%) stool specimens. The organisms detected comprised 17 Cryptosporidium spp., 5 Clostridium difficile with toxin B, 6 Campylobacter spp., 6 Salmonella spp., 4 astroviruses, 3 noroviruses, 1 rotavirus, 1 Shigella spp. and 1 Giardia spp. The frequency of detection by PCR and bacterial culture was similar for Salmonella spp., but discrepant for Campylobacter spp., as Campylobacter was detected by culture from only 1/86 specimens. Similarly, Cryptosporidium spp. was detected in multiple samples by PCR but was not detected by microscopy or enzyme immunoassay.
  
  
  CONCLUSIONS
  Cryptosporidium spp., Campylobacter spp. and Clostridium difficile may be relatively common but possibly under-recognised pathogens in this region. Further study is needed to determine the regional epidemiology and clinical significance of these organisms. This method appears to be a useful tool for gastrointestinal pathogen research and may also be helpful for clinical diagnostics and outbreak investigation in remote regions where the yield of routine testing may be compromised.
authors.affiliation: Department of Pediatrics, McMaster University, Hamilton, ON, Canada.
authors.forename: David M
authors.initials: DM
authors.lastname: Goldfarb
authors.forename: Brent
authors.initials: B
authors.lastname: Dixon
authors.forename: Ioana
authors.initials: I
authors.lastname: Moldovan
authors.forename: Nicholas
authors.initials: N
authors.lastname: Barrowman
authors.forename: Kirsten
authors.initials: K
authors.lastname: Mattison
authors.forename: Chad
authors.initials: C
authors.lastname: Zentner
authors.forename: Maureen
authors.initials: M
authors.lastname: Baikie
authors.forename: Sabah
authors.initials: S
authors.lastname: Bidawid
authors.forename: Francis
authors.initials: F
authors.lastname: Chan
authors.forename: Robert
authors.initials: R
authors.lastname: Slinger
mesh_terms: D001110:Arctic Regions; D003967:Diarrhea / Q000175:diagnosis* / Q000382:microbiology*; D005243:Feces / Q000382:microbiology; D006801:Humans; D020957:Nunavut; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors
keywords: Arctic; Campylobacter spp.; Clostridium difficile; Cryptosporidium spp.; Nunavut; detection; diarrhoea; gastroenteritis; multiplex real-time PCR; pathogens; viral agents