Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.

We evaluated whether provision and promotion of improved sanitation hardware (toilets and child feces management tools) reduced rotavirus and human fecal contamination of drinking water, child hands, and soil among rural Bangladeshi compounds enrolled in a cluster-randomized trial. We also measured host-associated genetic markers of ruminant and avian feces. We found evidence of widespread ruminant and avian fecal contamination in the compound environment; non-human fecal marker occurrence scaled with animal ownership. Strategies for controlling non- human fecal waste should be considered when designing interventions to reduce exposure to fecal contamination in low-income settings. Detection of a human- associated fecal marker and rotavirus was rare and unchanged by provision and promotion of improved sanitation to intervention compounds. The sanitation intervention reduced ruminant fecal contamination in drinking water and general (non-host specific) fecal contamination in soil but overall had limited effects on reducing fecal contamination in the household environment.