title: Development and assessment of molecular diagnostic tests for 15 enteropathogens causing childhood diarrhoea: a multicentre study.
abstract: BACKGROUND
  Childhood diarrhoea can be caused by many pathogens that are difficult to assay in the laboratory. Molecular diagnostic techniques provide a uniform method to detect and quantify candidate enteropathogens. We aimed to develop and assess molecular tests for identification of enteropathogens and their association with disease.
  
  
  METHODS
  We developed and assessed molecular diagnostic tests for 15 enteropathogens across three platforms-PCR-Luminex, multiplex real-time PCR, and TaqMan array card-at five laboratories worldwide. We judged the analytical and clinical performance of these molecular techniques against comparator methods (bacterial culture, ELISA, and PCR) using 867 diarrhoeal and 619 non-diarrhoeal stool specimens. We also measured molecular quantities of pathogens to predict the association with diarrhoea, by univariate logistic regression analysis.
  
  
  RESULTS
  The molecular tests showed very good analytical and clinical performance at all five laboratories. Comparator methods had limited sensitivity compared with the molecular techniques (20-85% depending on the target) but good specificity (median 97·3%, IQR 96·5-98·9; mean 95·2%, SD 9·1). Positive samples by comparator methods usually had higher molecular quantities of pathogens than did negative samples, across almost all platforms and for most pathogens (p<0·05). The odds ratio for diarrhoea at a given quantity (measured by quantification cycle, Cq) showed that for most pathogens associated with diarrhoea-including Campylobacter jejuni and Campylobacter coli, Cryptosporidium spp, enteropathogenic Escherichia coli, heat-stable enterotoxigenic E coli, rotavirus, Shigella spp and enteroinvasive E coli, and Vibrio cholerae-the strength of association with diarrhoea increased at higher pathogen loads. For example, Shigella spp at a Cq range of 15-20 had an odds ratio of 8·0 (p<0·0001), but at a Cq range of 25-30 the odds ratio fell to 1·7 (p=0·043).
  
  
  CONCLUSIONS
  Molecular diagnostic tests can be implemented successfully and with fidelity across laboratories around the world. In the case of diarrhoea, these techniques can detect pathogens with high sensitivity and ascribe diarrhoeal associations based on quantification, including in mixed infections, providing rich and unprecedented measurements of infectious causes.
  
  
  BACKGROUND
  Bill & Melinda Gates Foundation Next Generation Molecular Diagnostics Project.
authors.affiliation: Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Pediatrics and Child Health, Aga Khan University, Karachi, Pakistan.
authors.forename: Furqan
authors.initials: F
authors.lastname: Kabir
authors.affiliation: Medical Research Council Unit, Banjul, The Gambia.
authors.forename: Jainaba
authors.initials: J
authors.lastname: Manneh
authors.affiliation: Department of Enteric Diseases, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand.
authors.forename: Paphavee
authors.initials: P
authors.lastname: Lertsethtakarn
authors.affiliation: International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), Dhaka, Bangladesh.
authors.forename: Sharmin
authors.initials: S
authors.lastname: Begum
authors.affiliation: Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA, USA; Kilimanjaro Clinical Research Institute, Moshi, Tanzania.
authors.forename: Jean
authors.initials: J
authors.lastname: Gratz
authors.affiliation: Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA, USA.
authors.forename: Steve M
authors.initials: SM
authors.lastname: Becker
authors.affiliation: Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA, USA.
authors.forename: Darwin J
authors.initials: DJ
authors.lastname: Operario
authors.affiliation: Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA, USA.
authors.forename: Mami
authors.initials: M
authors.lastname: Taniuchi
authors.affiliation: Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA, USA.
authors.forename: Lalitha
authors.initials: L
authors.lastname: Janaki
authors.affiliation: Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Platts-Mills
authors.affiliation: Department of Pathology, University of Virginia, Charlottesville, VA, USA.
authors.forename: Doris M
authors.initials: DM
authors.lastname: Haverstick
authors.affiliation: International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), Dhaka, Bangladesh.
authors.forename: Mamun
authors.initials: M
authors.lastname: Kabir
authors.affiliation: International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), Dhaka, Bangladesh.
authors.forename: Shihab U
authors.initials: SU
authors.lastname: Sobuz
authors.affiliation: Department of Enteric Diseases, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand.
authors.forename: Kaewkanya
authors.initials: K
authors.lastname: Nakjarung
authors.affiliation: Department of Enteric Diseases, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand.
authors.forename: Pimmada
authors.initials: P
authors.lastname: Sakpaisal
authors.affiliation: Department of Enteric Diseases, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand.
authors.forename: Sasikorn
authors.initials: S
authors.lastname: Silapong
authors.affiliation: Department of Enteric Diseases, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand.
authors.forename: Ladaporn
authors.initials: L
authors.lastname: Bodhidatta
authors.affiliation: Department of Pediatrics and Child Health, Aga Khan University, Karachi, Pakistan.
authors.forename: Shahida
authors.initials: S
authors.lastname: Qureshi
authors.affiliation: Department of Pediatrics and Child Health, Aga Khan University, Karachi, Pakistan.
authors.forename: Adil
authors.initials: A
authors.lastname: Kalam
authors.affiliation: Kilimanjaro Clinical Research Institute, Moshi, Tanzania.
authors.forename: Queen
authors.initials: Q
authors.lastname: Saidi
authors.affiliation: Kilimanjaro Clinical Research Institute, Moshi, Tanzania.
authors.forename: Ndealilia
authors.initials: N
authors.lastname: Swai
authors.affiliation: Haydom Lutheran Hospital, Haydom, Tanzania.
authors.forename: Buliga
authors.initials: B
authors.lastname: Mujaga
authors.affiliation: Kilimanjaro Clinical Research Institute, Moshi, Tanzania.
authors.forename: Athanasia
authors.initials: A
authors.lastname: Maro
authors.affiliation: Medical Research Council Unit, Banjul, The Gambia.
authors.forename: Brenda
authors.initials: B
authors.lastname: Kwambana
authors.affiliation: Medical Research Council Unit, Banjul, The Gambia.
authors.forename: Michel
authors.initials: M
authors.lastname: Dione
authors.affiliation: Medical Research Council Unit, Banjul, The Gambia.
authors.forename: Martin
authors.initials: M
authors.lastname: Antonio
authors.affiliation: Kilimanjaro Clinical Research Institute, Moshi, Tanzania.
authors.forename: Gibson
authors.initials: G
authors.lastname: Kibiki
authors.affiliation: Department of Enteric Diseases, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand.
authors.forename: Carl J
authors.initials: CJ
authors.lastname: Mason
authors.affiliation: International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), Dhaka, Bangladesh.
authors.forename: Rashidul
authors.initials: R
authors.lastname: Haque
authors.affiliation: Department of Pediatrics and Child Health, Aga Khan University, Karachi, Pakistan.
authors.forename: Najeeha
authors.initials: N
authors.lastname: Iqbal
authors.affiliation: Department of Pediatrics and Child Health, Aga Khan University, Karachi, Pakistan.
authors.forename: Anita K M
authors.initials: AKM
authors.lastname: Zaidi
authors.affiliation: Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA, USA. Electronic address: erh6k@virginia.edu.
authors.forename: Eric R
authors.initials: ER
authors.lastname: Houpt
mesh_terms: D001424:Bacterial Infections / Q000175:diagnosis* / Q000382:microbiology; D016022:Case-Control Studies; D002675:Child, Preschool; D015331:Cohort Studies; D003955:Diagnostic Tests, Routine / Q000379:methods*; D003967:Diarrhea / Q000175:diagnosis* / Q000209:etiology; D005260:Female; D006801:Humans; D007223:Infant; D008297:Male; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D014777:Virus Diseases / Q000175:diagnosis* / Q000821:virology