title: Validation of Bacteroidales quantitative PCR assays targeting human and animal fecal contamination in the public and domestic domains in India.
abstract: We compared host-associated Bacteroidales qPCR assays developed in the continental United States and Europe for the purpose of measuring the effect of improved sanitation on human fecal exposure in rural Indian communities where both human and animal fecal loading are high. Ten candidate Bacteroidales qPCR assays were tested against fecal samples (human, sewage, cow, buffalo, goat, sheep, dog and chicken) from a test set of 30 individual human, 5 sewage, and 60 pooled animal samples collected in coastal Odisha, India. The two universal/general Bacteroidales assays tested (BacUni, GenBac3) performed equally well, achieving 100% sensitivity on the test set. Across the five human-associated assays tested (HF183 Taqman, BacHum, HumM2, BacH, HF183 SYBR), we found low sensitivity (17 to 49%) except for HF183 SYBR (89%), and moderate to high cross-reactivity with dog (20 to 80%) and chicken fecal samples (60 to 100%). BacHum had the highest accuracy (67%), amplified all sewage samples within the range of quantification (ROQ), and did not cross-react with any fecal samples from cows, the most populous livestock animal in India. Of the ruminant- and cattle-associated assays tested (BacCow, CowM2), BacCow was more sensitive in detecting the full range of common Indian livestock animal fecal sources, while CowM2 only detected cow sources with 50% sensitivity. Neither assay cross-reacted with human sources. BacCan, the dog-associated assay tested, showed no cross-reactivity with human sources, and high sensitivity (90%) for dog fecal samples. Overall, our results indicate BacUni, BacHum, HumM2, BacCan and BacCow would be the most suitable MST assays to distinguish and quantify relative amounts of human-associated and livestock/domestic animal-associated contributions to fecal contamination in Odisha, India.
authors.affiliation: Department of Civil and Environmental Engineering, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Odagiri
authors.affiliation: Department of Civil and Environmental Engineering, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Schriewer
authors.affiliation: Department of Civil and Environmental Engineering, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Kaitlyn
authors.initials: K
authors.lastname: Hanley
authors.affiliation: Department of Civil and Environmental Engineering, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA; Singapore Centre on Environmental Life Sciences Engineering (SCELSE), School of Biological Sciences, and School of Civil and Environmental Engineering, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore. Electronic address: swuertz@ucdavis.edu.
authors.forename: Stefan
authors.initials: S
authors.lastname: Wuertz
authors.affiliation: Asian Institute of Public Health, Bhubaneswar, Odisha, India.
authors.forename: Pravas R
authors.initials: PR
authors.lastname: Misra
authors.affiliation: Departments of Epidemiology and Pediatrics, Center for Global Health and Development, College of Public Health, University of Nebraska Medical Center, Omaha, NE 68198, USA.
authors.forename: Pinaki
authors.initials: P
authors.lastname: Panigrahi
authors.affiliation: Department of Civil and Environmental Engineering, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA; Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
authors.forename: Marion W
authors.initials: MW
authors.lastname: Jenkins
mesh_terms: D000818:Animals; D041963:Bacteroidetes / Q000145:classification / Q000235:genetics*; D004784:Environmental Monitoring / Q000379:methods*; D005243:Feces / Q000382:microbiology*; D006801:Humans; D007194:India; D060888:Real-Time Polymerase Chain Reaction; D012722:Sewage / Q000382:microbiology*
chemical_list: D012722:Sewage
keywords: Bacteroidales; Fecal pollution; India; Microbial source tracking; Quantitative PCR