Acarological investigation and detection of Orientia tsutsugamushi (Rickettsiales: Rickettsiaceae) in field-collected chiggers (Acari: Trombiculidae) and small mammals from scrub typhus positive localities in Pahang, West Malaysia.

Scrub typhus (ST) is an acute febrile illness caused by Orientia tsutsugamushi (Hayashi, 1920) that is transmitted to humans through the bite of infected chiggers. In recent years, ST has re-emerged as a public health concern in Malaysia. In 2024, more than 50 laboratory-confirmed human cases of ST were reported in Pahang. A collaborative investigation was undertaken on a subset of 26 cases to identify potential vector sources and animal hosts associated with O. tsutsugamushi transmission. Chigger-based sampling was conducted by trapping small mammals in suspected infection hotspots, including residential surroundings, frequently visited locations, and work areas of confirmed cases. A total of 95 small mammals were captured, of which 65.3% infested with chiggers (Chigger index, CI = 40.3). Overall, 3,831 on-host chiggers were collected and identified morphologically, with Leptotrombidium deliense (Walch, 1922) being the most frequently collected species (46.7%), followed by Ascoschoengastia indica (Hirst, 1915) (33.9%), Leptotrombidium spp. (18.4%), and Helenicula mutabilis (Gater, 1932) (1.0%). DNA was extracted from 188 pooled chigger samples (N = 3,450) as well as from blood and spleen tissues of captured animals, and screened for O. tsutsugamushi using nested PCR targeting the 56-kDa TSA gene. Notably, O. tsutsugamushi was detected in 13 (6.9%) chigger pools solely of L. deliense, and in 11 spleens and six blood samples. Phylogenetic analysis showed high nucleotide similarity (97-100%), with the Karp genotype predominating, followed by Gilliam, Kato, and TA763 genotypes. These findings confirm the presence of O. tsutsugamushi in vectors and animal hosts in Pahang, highlighting the region as a potential ST hotspot, and underscoring the need for continued monitoring and control efforts.