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, all samples present in each positive NGS pool were individually tested by confirmatory assays using either real-time PCR or conventional PCR (Supplementary Table S1 ). All amplified products were sequenced by the Sanger method. The correlation between the number of NGSpositive pools and the number of positive pools verified by confirmatory assays was determined using linear regression analysis (Figure 4) . The results from both assays were positively correlated with R 2 = 0.8968 (95% confidence interval = 0.7440-0.9368) or R 2 = 0.6004 (95% confidence interval = 0.3950-0.7024) when the far point was removed. Prevalence rates for each pathogen were calculated based on NGS results verified by confirmatory assays or a combination of both NGS results and routine screening tests as mentioned earlier. Table 3 shows the prevalence rate for each pathogen detected among samples. A high prevalence of Bartonella spp. was FIGURE 4 | Correlation of metagenomic sequencing with confirmatory assays using qPCR and PCR methods for detection of bacterial pathogens in a variety of samples. seen in rodents, rodent fleas, and rodent lice populations (41.1, 65.2, and 75.0%, respectively). The prevalence of Rickettsia spp. was highest in fleas collected from domesticated mammals, mostly from dogs (84.1%), followed by rodent lice (25.0%), and rodent ticks (6.8%). Coxiella spp. was detected at highest prevalence in ticks collected from rodents and domesticated mammals (32.2 and 71.4%), but later was identified as a Coxiella endosymbiont (Table 4 ). Other highly pathogenic species known to cause disease in humans and animals were detected among vectors, rodents, and UFI patient samples, although at low prevalence. These included O. tsutsugamushi, Anaplasma spp., Borrelia spp., and Leptospira spp.