Molecular Epidemiology of Orientia tsutsugamushi in Chiggers collected from Sundargarh district Odisha, Indi
Introduction Scrub Typhus, a rickettsial zoonotic infection, caused by Orientia tsutsugamushi has an estimated one million new cases occurring annually worldwide and about 18,000 cases reported in India in 2022. Infection occurs by bite of larval chigger-mites, during exposure to soil, vegetations, forests, or rodents which are maintenance hosts. During January-September 2023, Odisha, an eastern-Indian state, reported 1,212 cases with Sundargarh district reporting highest cases. We investigated a scrub typhus cluster in block-X in Sundargarh district to describe epidemiology and assess risk factors. Methodology: A case was defined as Scrub Typhus IgM-ELISA positive resident of, or hospital admission in block-X between July 1 and October 30, 2023. A line-list of IgM-ELISA positives was collected from the district Laboratory. We interviewed cases for socio-demography, symptoms, treatment-seeking behavior, and exposure to mite-habitats, by a semi-structured questionnaire. We conducted a 1:1 unmatched case-control study. Controls were test negatives in the same time frame enrolled from district-hospital in block-X.  We present odds ratio-OR with 95 percent confidence intervals-CI. We conducted an entomological survey in block-X around domestic and peri-domestic areas of positive-cases, to trap rodents and calculated entomological indices. We collected rodent-sera for confirming infection by Weil-Felix test and collected mites from captured rodents to identify the pathogen by 47kDa Real Time PCR. The chigger pools were also subjected to 56kDa Nested PCR and Sanger sequencing. Results- We identified 140 cases, with median age of 29 years, 55 percent males, in block-X with case-fatality rate of 2.8 percent. Among cases, 70 percent were adults and 63 percent were rural-residents. Symptoms included fever 100 percent, headache 65 percent, cough 37 percent, myalgia 35 percent and eschar 12 percent. Among cases, 22 percent presented to the health-facility and 45 percent were tested in second week of symptom-onset. Among 61 cases and 61 controls, recent forest or farm visit OR equals to 4.27, 95 percent and CI range from 1.9-9.1, house-proximity 10 m to bushes OR equals to 3.03, 95 percent, CI range from 1.2-7.3, dampness OR equals to 2.74, 95 percent CI range from 2-6.0, proximity 100m to forest or farm or fields OR equals to 2.30, 95 percent CI range from 1.0-5.2, open drainage OR equals to 2.23. 95 percent CI range from 1.0-4.7 were significantly associated with illness. We laid 100 rodent-traps and captured 20 rodents: trap-positivity equals to 20 percent. Of the 17 live rodents captured, 16 were infested with 322 ectoparasites and Chigger-index of 18.94 with 33 percent rodent-sera samples positive for Scrub typhus antigen. Among 11 mite-pools, four were found positive for Orientia tsutsugamushi using 47kDa Real-time PCR. Three chigger pools were positive for 56kDa gene and showed similarity with MMR2018-68 56-kDa strain from Myanmar and previously submitted PGI sequences. The phylogenetic tree reveals that these isolates from chiggers are categorized under the Karp-like strain. Conclusions This was a laboratory-confirmed case-cluster of scrub typhus in block-X of Sundargarh district, Odisha, predominantly among adults and rural-residents.  Delays in treatment seeking and testing were observed. Higher odds of illness were associated in cases with mite-habitat proximity and environment attracting rodents. We confirmed presence of maintenance hosts, vector, and agent entomologically by high trap-positivity, chigger index and microbiologically by the presence of pathogen in mites. We sensitized frontline-healthcare workers on early identification and timely referral and recommended community education on limiting mite exposure, using protective measures, rodent-proofing their residence, and seeking timely medical attention. Keywords-Infectious diseases, Scrub typhus, exposure risk factors, mites, Rodents