title: A Field-Expedient Method for Direct Detection of Enterotoxigenic E Coli and Shigella from Stool.
abstract: We describe a field-expedient analytic system that fills a unique and critical public health role and potentially provides a valuable aid in diagnostics. Dual-fluorigenic, hydrolysis probe (TaqMan), PCR assays for detection of causative agents of enterotoxigenic Escherichia coli (ETEC) disease and shigellosis/bacillary dysentery were prepared in a thermal-stable, hydrolytic enzyme resistant format. The assays were packaged as a kit for use with a portable, ruggedized, qRT-PCR thermocycler. The analytical limit of detection of each q
  ETEC-STIa, ETEC-STIb, and ETEC-LT assay was 30 colony forming units (CFU) and Shigella/enteroinvasive E coli assay was 3 CFU. During field evaluation, testing was conducted using a blind-panel of 138 stored stool samples previously obtained from enterotoxigenic E coli disease (n=91) and shigellosis (n=47) patients. Sample processing and analyses were completed in 3 days. Test results of the qRT-PCR assays showed promise as aid in pathogen identification when compared to culture, digoxigenin-labeled probe (ETEC), and serotyping (Shigella) the qRT-PCR. The sensitivity of each of the 4 qRT-PCR assays was 100% and specificity was ETEC-STIa (92.4%), ETEC-STIb (92.6%), ETEC-LT (79.6%), and Shigella/enteroinvasive E coli (81.6%). Sequencing of qRT-PCR amplicon indicated that the sensitivity and specificity of each qRT-PCR assay exceeded the comparator methods. The system shows promise as a rapid method for direct detection of ETEC and Shigella from stool and is applicable for use in clinical diagnostics and biosurveillance as an extension of temporary field laboratories or as a part of fixed reference laboratory facilities.
authors.affiliation: Department of Enteric Diseases, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.
authors.forename: Sasikorn
authors.initials: S
authors.lastname: Silapong
authors.forename: Pimmnapar
authors.initials: P
authors.lastname: Neesanant
authors.forename: Orntipa
authors.initials: O
authors.lastname: Sethabutr
authors.forename: Paphavee
authors.initials: P
authors.lastname: Lertsethtakarn
authors.forename: Ladaporn
authors.initials: L
authors.lastname: Bodhidatta
authors.forename: James C
authors.initials: JC
authors.lastname: McAvin
authors.forename: Carl J
authors.initials: CJ
authors.lastname: Mason
mesh_terms: D004405:Dysentery, Bacillary / Q000175:diagnosis* / Q000382:microbiology; D054307:Enterotoxigenic Escherichia coli / Q000302:isolation & purification*; D004927:Escherichia coli Infections / Q000175:diagnosis* / Q000382:microbiology; D005243:Feces / Q000382:microbiology; D006801:Humans; D009390:Nepal; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012760:Shigella / Q000302:isolation & purification*