Mote Akash Balasaheb, Dhanze Himani, Thomas Prasad, Kumar M Suman, Singh Vibha, Palavesam Azhahianambi, Singh Rajeev, Chaudhari Sandeep P, Singh K P, Ram Hira, Hegde Nagendra R, Sharma G Taru
Indian Council of Agricultural Research (ICAR)- Indian Veterinary Research Institute, Uttar Pradesh, India.
Indian Council of Agricultural Research (ICAR)- Indian Veterinary Research Institute, Uttar Pradesh, India.
J Microbiol Methods. 2025 Nov;238:107274. doi: 10.1016/j.mimet.2025.107274. Epub 2025 Sep 22.
Scrub typhus, a vector-borne zoonosis prevalent in the Asia-Pacific region, poses diagnostic challenges due to the pathogen's complex genome and diverse rodent and shrew hosts. The scarcity of reliable diagnostic tests hinders effective sentinel surveillance. This study aims to identify novel diagnostic gene targets using a bioinformatics approach to develop a highly sensitive and specific PCR assay for scrub typhus detection. Genome sequences of Orientia tsutsugamushi, the causative agent, were analyzed, leading to the selection of 11 potential diagnostic biomarkers. In-house conventional PCR assays targeting these biomarkers and published nested PCR assays, were tested on blood and tissue (spleen) samples of 150 field rodent and shrew. Among the tested genes, the tsa56 gene consistently demonstrated the highest detection rate in both conventional (55.6 %, n = 15) and nested (74 %, n = 20) assays, indicating it to be the most reliable diagnostic marker for scrub typhus. A novel nested PCR was designed targeting a unique tsa56 gene segment, which showed an analytical sensitivity of 4.7 (95 % CI: 2-7.4) copies/μL, and was able to detect O. tsutsugamushi in multiple hosts including human and mite samples. Moreover, fewer primer-template mismatches and no mismatches at the critical 3' terminus with O. tsutsugamushi strains were observed. The assay did not show any cross-reaction with available non-target organisms. Thus, the developed nested PCR assay demonstrates enhanced sensitivity, specificity, and broader strain inclusivity. Overall, the study presents a promising tool for scrub typhus detection, which will aid in improved disease surveillance, outbreak prediction, and timely implementation of control measures.
恙虫病是一种在亚太地区流行的媒介传播人畜共患病，由于病原体复杂的基因组以及多样的啮齿动物和鼩鼱宿主，给诊断带来了挑战。可靠诊断测试的匮乏阻碍了有效的哨点监测。本研究旨在通过生物信息学方法识别新的诊断基因靶点，以开发一种用于恙虫病检测的高度灵敏且特异的PCR检测方法。对病原体恙虫东方体的基因组序列进行了分析，从而筛选出11个潜在的诊断生物标志物。针对这些生物标志物的内部常规PCR检测方法以及已发表的巢式PCR检测方法，在150份野外啮齿动物和鼩鼱的血液和组织（脾脏）样本上进行了测试。在所测试的基因中，tsa56基因在常规检测（55.6%，n = 15）和巢式检测（74%，n = 20）中均始终显示出最高的检出率，表明它是恙虫病最可靠的诊断标志物。针对tsa56基因的一个独特片段设计了一种新的巢式PCR，其分析灵敏度为4.7（95%置信区间：2 - 7.4）拷贝/μL，并且能够在包括人类和螨类样本在内的多种宿主中检测到恙虫东方体。此外，观察到引物 - 模板错配较少，且与恙虫东方体菌株在关键的3'末端无错配。该检测方法与可用的非靶标生物未显示任何交叉反应。因此，所开发的巢式PCR检测方法具有更高的灵敏度、特异性和更广泛的菌株包容性。总体而言，该研究为恙虫病检测提供了一种有前景的工具，将有助于改善疾病监测、疫情预测以及及时实施控制措施。
