Chao Chien-Chung, Belinskaya Tatyana, Zhang Zhiwen, Jiang Le, Ching Wei-Mei
Viral and Rickettsial Diseases Department, Infectious Diseases Directorate, Naval Medical Research Center, Silver Spring, MD 20910, USA.
Department of Preventive Medicine and Biostatistics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA.
Trop Med Infect Dis. 2019 Jul 31;4(3):113. doi: 10.3390/tropicalmed4030113.
Scrub typhus is caused by an obligated intracellular organism, (). The disease was traditionally thought to be limited in the tsutsugamushi triangle. Recently, scrub typhus has been confirmed in areas outside the triangle. Serological diagnosis of scrub typhus relies on indirect immunofluorescence assay (IFA). Molecular assays such as PCR, qPCR, loop-mediated isothermal amplification, and recombinase polymerase amplification are often targeting a single copy gene. These assays are sensitive and specific, yet they are not broadly used in clinical settings possibly due to low circulating in blood. In this study, we compared qPCR results using a multiple copy (traD) gene with those using a single copy (47 kDa) gene to assess the improvement of sensitivity and limit of detection. Our results demonstrate that the qPCR using the traD gene provides superior sensitivity in 15 strains. The limit of detection is below single genome equivalent and the assay retains specificity with excessive DNA from mouse, chiggers and human. The clinical utility was evaluated using confirmed scrub typhus positive and negative samples. The results show 100% sensitivity and specificity in these samples suggesting that the traD gene qPCR may be useful for clinical diagnosis of infection.
恙虫病由一种专性细胞内病原体（）引起。传统上认为该疾病局限于恙虫病东方体三角区。最近，在该三角区以外的地区也确诊了恙虫病。恙虫病的血清学诊断依赖于间接免疫荧光法（IFA）。诸如PCR、qPCR、环介导等温扩增和重组酶聚合酶扩增等分子检测方法通常针对单个拷贝基因。这些检测方法灵敏且特异，但可能由于血液中循环量低而未在临床环境中广泛应用。在本研究中，我们比较了使用多拷贝（traD）基因的qPCR结果与使用单拷贝（47 kDa）基因的qPCR结果，以评估敏感性的提高和检测限。我们的结果表明，使用traD基因的qPCR在15株菌株中具有更高的敏感性。检测限低于单个基因组当量，并且该检测方法对来自小鼠、恙螨和人类的过量DNA仍保持特异性。使用确诊的恙虫病阳性和阴性样本评估了临床实用性。结果显示这些样本的敏感性和特异性均为100%，表明traD基因qPCR可能对恙虫病感染的临床诊断有用。
