Data Sheet 5_A longitudinal molecular surveillance of genetic heterogeneity of Orientia tsutsugamushi in humans, reservoir animals, and vectors in Puducherry, India.docx

BackgroundScrub typhus (ST), is a vector borne zoonotic disease, transmitted by the larva of the trombiculid mites. The enzootic cycle of the pathogen involves rodents/shrews as the animal reservoirs and humans are the accidental dead-end host. A transposon-like activity in its major antigen 56 kDa, has led to the evolution of several serotypes/strains, and more than 40 serotypes are reported globally. Puducherry, India, is endemic to scrub, but limited data exist on local serotype distribution across hosts and vectors.
MethodologyA longitudinal molecular surveillance was conducted in Puducherry to investigate the genetic diversity of Orientia tsutsugamushi among humans, animal reservoirs, and vectors. Samples from febrile patients, trapped rodents/shrews, and their infesting mites were screened using real-time and nested PCR. Serotype analysis was performed by partial amplification and sequencing of the 56 kDa gene, followed by phylogenetic, pairwise genetic distance and amino acid analysis.
ResultST infection was detected in 4.37% (95% CI: 3.05–5.71%) of human, 11.52% (95% CI: 8.6–14.4%) of rodent/shrew, and 2.36% (95% CI: 0.95–4.87%) of mite samples. Karp-like (51.72%) and Gilliam-like (41.38%) strains were predominant in both humans and animal hosts, with pairwise genetic distance (<0.1) and amino acid identity (>85%) analysis revealing a close relationship between the strains identified across the region. Notably, the only mite pool that tested positive for the 56 kDa gene, along with a shrew, was identified to belong to the TA678-like serotype (6.90%), which has not been previously reported from Puducherry.
ConclusionThis study provides molecular evidence of the enzootic maintenance and active human transmission of O. tsutsugamushi in Puducherry, with multiple co-circulating serotypes. The first detection of the TA678-like strain in the region suggests the possible introduction of new strains and underscores the need to monitor for strain-specific clinical manifestations in future studies.

背景 恙虫病（Scrub typhus, ST）是一种由恙螨（trombiculid mites）幼虫传播的媒介传播性人兽共患病。该病原体的兽疫循环以啮齿类/鼩鼱为动物储存宿主，人类为偶然的终末宿主。其主要抗原56kDa蛋白存在转座子样活性，推动了多种血清型/毒株的演化，目前全球已报道超过40种血清型。印度本地治里（Puducherry）是恙虫病流行区，但目前关于当地不同宿主与媒介中的血清型分布数据仍较为匮乏。
方法学 本研究在本地治里开展了一项纵向分子监测，以探究恙虫病东方体（Orientia tsutsugamushi）在人类、动物储存宿主以及媒介中的遗传多样性。研究采集发热患者、捕获的啮齿类/鼩鼱及其体表寄生恙螨的样本，通过实时荧光定量PCR（real-time PCR）与巢式PCR（nested PCR）进行筛查。血清型分析则通过对56kDa基因的部分片段进行扩增与测序，随后开展系统发育分析、两两遗传距离分析以及氨基酸序列分析。
结果 本研究在4.37%（95%置信区间：3.05~5.71%）的人类样本、11.52%（95%置信区间：8.6~14.4%）的啮齿类/鼩鼱样本以及2.36%（95%置信区间：0.95~4.87%）的恙螨样本中检测到恙虫病感染。在人类与动物宿主中，均以Karp样（Karp-like，51.72%）毒株与Gilliam样（Gilliam-like，41.38%）毒株为优势毒株；两两遗传距离（<0.1）与氨基酸同源性（>85%）分析显示，本区域检出的各毒株间亲缘关系密切。值得注意的是，仅有的1份56kDa基因检测阳性的恙螨混合样本，与1份鼩鼱样本一同被鉴定为TA678样（TA678-like）血清型（占比6.90%），该血清型此前从未在本地治里地区被报道过。
结论 本研究为本地治里地区恙虫病东方体的兽疫循环维持以及活跃的人间传播提供了分子生物学证据，同时证实该地区存在多种血清型共同流行。本研究首次在该地区检出TA678样（TA678-like）毒株，提示可能有新毒株传入该区域，同时强调了未来研究中开展毒株特异性临床表现监测的必要性。