Metagenomic Approach to Characterizing Disease Epidemiology in a Disease-Endemic Environment in Northern Thailand

Takhampunya, Ratree; Korkusol, Achareeya; Pongpichit, Chalermpol; Yodin, Komsan; Rungrojn, Artharee; Chanarat, Nitima; Promsathaporn, Sommai; Monkanna, Taweesak; Thaloengsok, Sasikanya; Tippayachai, Bousaraporn; Kumfao, Naruemon; Richards, Allen L.; Davidson, Silas A.

In this study, a high prevalence from NGS results was observed for few bacterial genera; however, some genera could not be verified with conventional assays such as real-time PCR, PCR, or DNA sequencing. The main reason was likely that the number of reads was too low and below the limit of detection for the conventional test, or they could have been non-pathogenic strains or species and were not picked up by confirmatory assays. For example, Leptospira spp. comprise both pathogenic, intermediate, and saprophytic (non-pathogenic) species that can be introduced as contaminants from the environment into samples. Here Leptospira spp. were only detected in the rodent population, although NGS analysis showed reads were also detected in UFI patients, chiggers, and ticks at lower levels. However, testing with confirmatory assay resulted in no signal or PCR product using a genus-based assay (Ahmed et al., 2009) with primer sets targeting house-keeping genes such as gyrB or SecY (Slack et al., 2006; Victoria et al., 2008) . In some cases, PCR assays targeting Leptospira 16S rRNA genes showed some positive bands for UFI patients but the product size was shorter than expected and DNA sequences from these products matched only human DNA (100%). Rickettsia spp. and Francisella spp. could not be verified in some sample types such as ticks, chiggers, and rodents. These could be endosymbiont bacteria which our assays could not detect (Wright et al., 2011; Takhampunya et al., 2017) .