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.

Originally, the confirmatory assays did not verify the NGS results (2 pools) of O. tsutsugamushi detected in rodents. However, since scrub typhus detection has been run in our lab as part of routine surveillance assays, three O. tsutsugamushipositive rodents were verified by a routine real-time PCR test (Table 4) . Similarly, four Bartonella species detected in fleas from domesticated mammals were also verified by a routine real-time PCR test and they were included in Table 4 . NGS seems to have less sensitivity than the conventional method. In support of this observation, a previous study has compared MiSeq and RNA-seq, and found that MiSeq cannot detect bacteria at a value lower than 4% prevalence in the population and thus RNA-seq is better in terms of sensitivity (Razzauti et al., 2015) . In all likelihood, this is due to differences in sequencing depth for each of the techniques used. The detection of B. quintana in one UFI patient and B. clarridgeiae in Ctenocephalides felis fleas provides significant evidence that Trench fever and catscratch disease-causing bacteria are present in the study area. The seroprevalence data (IgG) of B. quintana and B. henselae also confirmed previous human exposure to these bacteria. Although we detected A. phagocytophilum (anaplasmosis) in rodents and ticks, only one UFI patient was seropositive (IgG) to anaplasmosis. Further characterization of Anaplasma species detected in the UFI patient is required to identify this pathogenic species causing human infection. Given the fact that a few Anaplasma species were detected from rodents and ticks in this study area such as A. phagocytophilum, A. bovis, and A. platys, knowing what species caused infection in humans would lead to a better understanding of the transmission dynamics among the vector, host, and reservoir enable to and to better understand its transmission in the area and reservoir host and the vector involved. Other bovine and canine ehrlichiosis were also detected in ticks. Interestingly, Bor. miyamotoi and Bor. yangtzensis were detected in rodents and Ixodes ticks which marks the first detection of these human pathogenic species in Thailand. More research and surveillance is needed to further characterize their prevalence and distribution in the country. Borrelia spp. detected in chiggers could be some other unidentified bacteria since their sequence identity to most Borrelia species were quite low based on flaB gene sequences (64.0-70.2%), while the percent identity among all reference sequences used in the alignment ranges from 69.6 to100%. While the 16S rRNA sequences of two chigger pools were 97.4% identical to some unknown Borrelia species and Candidatus Borrelia africana (Accession No. KT364339), additional analysis such as multilocus sequence typing (MLST) should be performed in order to determine whether Borrelia spp. detected in chiggers are new Borrelia species or some other bacterial genus. Since some sample types included in this study were not collected across all years of sampling (2014, 2017, and 2018) such as ectoparasites collected from domesticated mammals (2014) and UFI patients (2017), the observed pathogens reported here might not represent the true picture of pathogens shared among the vectors, reservoirs, and hosts in Bo Kluea district, Nan province. In this study, co-infection between Anaplasma spp. (A. phagocytophilum, A. bovis) and Bartonella spp. was observed in Rattus and Bandicota rats (2/309, 0.65%). It is unfo