Superior performance of human wastewater-associated viral markers compared to bacterial markers in tropical environments

Identifying human sewage contamination via microbial source tracking (MST) marker genes has proven useful for effective water quality management worldwide; however, performance evaluations for these genes in tropical areas are limited. Therefore, this research assessed four human-associated MST marker genes in aquatic environments of Central Thailand: human polyomaviruses (JC and BK viruses [HPyVs]), bacteriophage crAssphage (CPQ_056), Lachnospiraceae Lachno3, and Bacteroides BacV6-21. HPyV and crAssphage assays were highly sensitive and specific to sewage (n = 19), with no cross-detection in 120 swine, cattle, chicken, duck, goat, sheep, and buffalo composite fecal samples. Lachno3 and BacV6-21 demonstrated high sensitivity but moderate specificity; however, using both markers could improve specificity to >0.80 (max value of 1.00). The most abundant markers in sewage were Lachno3 and BacV6-21 (5.42-8.02 and non-detected-8.05 log10 copies/100 mL), crAssphage (5.28-7.38 log10 copies/100 mL), and HPyVs (3.66-6.53 log10 copies/100 mL), respectively. HPyVs showed higher levels (up to 4.33 log10 copies/100 mL) and higher detection rates (92.7%) in two coastal beaches (n = 41) than crAssphage (up to 3.51 log10 copies/100 mL and 56.1%). HPyVs were also found at slightly lower levels (up to 5.10 log10 copies/100 mL), but at higher detection rates (92.6%), in a freshwater canal (n = 27) than crAssphage (up to 5.21 log10 copies/100 mL and 88.9%). Overall, both HPyVs and crAssphage are suggested as human sewage-associated MST markers in aquatic environments of Central Thailand. This study underlines the importance of characterizing and validating MST markers in host groups and environmental waters before including them in a water quality management toolbox.