title: Optimization of Quantitative PCR Methods for Enteropathogen Detection.
abstract: Detection and quantification of enteropathogens in stool specimens is useful for diagnosing the cause of diarrhea but is technically challenging. Here we evaluate several important determinants of quantification: specimen collection, nucleic acid extraction, and extraction and amplification efficiency. First, we evaluate the molecular detection and quantification of pathogens in rectal swabs versus stool, using paired flocked rectal swabs and whole stool collected from 129 children hospitalized with diarrhea in Tanzania. Swabs generally yielded a higher quantification cycle (Cq) (average 29.7, standard deviation 3.5 vs. 25.3 ± 2.9 from stool, P<0.001) but were still able to detect 80% of pathogens with a Cq < 30 in stool. Second, a simplified total nucleic acid (TNA) extraction procedure was compared to separate DNA and RNA extractions and showed 92% (318/344) sensitivity and 98% (951/968) specificity, with no difference in Cq value for the positive results (ΔCq(DNA+RNA-TNA) = -0.01 ± 1.17, P = 0.972, N = 318). Third, we devised a quantification scheme that adjusts pathogen quantity to the specimen's extraction and amplification efficiency, and show that this better estimates the quantity of spiked specimens than the raw target Cq. In sum, these methods for enteropathogen quantification, stool sample collection, and nucleic acid extraction will be useful for laboratories studying enteric disease.
authors.affiliation: Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia, United States of America.
authors.forename: Jie
authors.initials: J
authors.lastname: Liu
authors.affiliation: Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia, United States of America.
authors.forename: Jean
authors.initials: J
authors.lastname: Gratz
authors.affiliation: Haydom Global Health Institute, Haydom, Tanzania.
authors.forename: Caroline
authors.initials: C
authors.lastname: Amour
authors.affiliation: Haydom Global Health Institute, Haydom, Tanzania.
authors.forename: Rosemary
authors.initials: R
authors.lastname: Nshama
authors.affiliation: Haydom Global Health Institute, Haydom, Tanzania.
authors.forename: Thomas
authors.initials: T
authors.lastname: Walongo
authors.affiliation: Kilimanjaro Clinical Research Institute, Moshi, Tanzania.
authors.forename: Athanasia
authors.initials: A
authors.lastname: Maro
authors.affiliation: Haydom Global Health Institute, Haydom, Tanzania.
authors.forename: Esto
authors.initials: E
authors.lastname: Mduma
authors.affiliation: Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia, United States of America.
authors.forename: James
authors.initials: J
authors.lastname: Platts-Mills
authors.affiliation: Department of Pediatrics, University of Virginia, Charlottesville, Virginia, United States of America.
authors.forename: Nadia
authors.initials: N
authors.lastname: Boisen
authors.affiliation: Department of Pediatrics, University of Virginia, Charlottesville, Virginia, United States of America.
authors.forename: James
authors.initials: J
authors.lastname: Nataro
authors.affiliation: Department of Pathology, University of Virginia, Charlottesville, Virginia, United States of America.
authors.forename: Doris M
authors.initials: DM
authors.lastname: Haverstick
authors.affiliation: Department of Pediatrics and Child Health, Aga Khan University, Karachi, Pakistan.
authors.forename: Furqan
authors.initials: F
authors.lastname: Kabir
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: 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: Pimmada
authors.initials: P
authors.lastname: Jeamwattanalert
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 Enteric Diseases, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand.
authors.forename: Carl
authors.initials: C
authors.lastname: Mason
authors.affiliation: International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), Dhaka, Bangladesh.
authors.forename: Sharmin
authors.initials: S
authors.lastname: Begum
authors.affiliation: International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), Dhaka, Bangladesh.
authors.forename: Rashidul
authors.initials: R
authors.lastname: Haque
authors.affiliation: Christian Medical College, Vellore, Tamil Nadu, India.
authors.forename: Ira
authors.initials: I
authors.lastname: Praharaj
authors.affiliation: Christian Medical College, Vellore, Tamil Nadu, India.
authors.forename: Gagandeep
authors.initials: G
authors.lastname: Kang
authors.affiliation: Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia, United States of America.
authors.forename: Eric R
authors.initials: ER
authors.lastname: Houpt
mesh_terms: D003967:Diarrhea / Q000175:diagnosis* / Q000209:etiology*; D005243:Feces / Q000382:microbiology* / Q000469:parasitology* / Q000821:virology*; D018628:Gene Dosage; D006801:Humans; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling
chemical_list: D011933:Reagent Kits, Diagnostic