Background & objectives: Scrub typhus (ST), an important zoonosis caused by Orientia tsutsugamushi, is now prevalent throughout India. While demonstration of IgM antibody by Indirect Immunofluorescence Assay (IFA) is the gold standard serological test, IgM ELISA is an alternative. Demonstration of O. tsutsugamushi DNA in the blood or eschar confirms infection in the early febrile period.
Methods: Scrub typhus nested PCR (n-PCR) for 56 kDa, 47 kDa and groEL genes and ST IgM ELISA were performed for 210 clinically suspected ST patients. As healthy controls, 70 voluntary blood donors were included. Statistical analysis was performed for laboratory parameters using Fisher exact test/chi-square test. Ninety-five PCR products of n-PCR positive samples were purified and submitted for gene sequencing.
Results: PCR was positive for one or more gene targets in 75.71% of IgM ELISA positive patients and 10% of antibody negative patients. All voluntary blood donors were negative for both antibodies and DNA. Gene sequences of 95 n-PCR positive products confirmed the presence of Orientia tsutsugamushi DNA in the samples and NCBI database accession numbers MG601875 to MG601969 were obtained.
Interpretation & conclusion: Compared to IgM ELISA, sensitivity of three PCRs was 30, 51.43 and 61.43% for 56 kDa, 47 kDa and groEL targets, respectively. Since IgM ELISA positivity can persist up to one year, PCR confirms ST diagnosis in the acute phase of the illness, in the presence of IgM and even before IgM appears. Inclusion of all three genes – 56 kDa, 47 kDa and groEL, instead of a single 56 kDa target, identifies and confirms maximum number of ST patients.

中文翻译：

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印度 Puducherry 的斑疹伤寒：针对三种不同基因的巢式 PCR 的应用 - 56 kDa、47 kDa 和 恙虫病东方体的 groEL 以及与 ST IgM ELISA 的比较

背景与目的：恙虫病 (ST) 是一种由恙虫病东方体引起的重要人畜共患病，目前在整个印度流行。虽然通过间接免疫荧光分析 (IFA) 证明 IgM 抗体是金标准血清学测试，但 IgM ELISA 是另一种选择。血液或焦痂中的O. tsutsugamushi DNA 的证明证实了早期发热期的感染。
方法：对 210 名临床疑似 ST 患者进行了 56 kDa、47 kDa 和 groEL 基因的 Scrub typhus 巢式 PCR (n-PCR) 和 ST IgM ELISA。作为健康对照，包括 70 名自愿献血者。使用Fisher精确检验/卡方检验对实验室参数进行统计分析。纯化n-PCR阳性样品的95个PCR产物并提交基因测序。
结果：在 75.71% 的 IgM ELISA 阳性患者和 10% 的抗体阴性患者中，PCR 对一个或多个基因靶标呈阳性。所有自愿献血者的抗体和 DNA 均呈阴性。95个n-PCR阳性产物的基因序列证实了样本中存在恙虫病DNA，并获得NCBI数据库登录号MG601875至MG601969。
解释与结论：与 IgM ELISA 相比，三种 PCR 对 56 kDa、47 kDa 和 groEL 靶标的灵敏度分别为 30、51.43 和 61.43%。由于 IgM ELISA 阳性可持续长达一年，因此 PCR 可在疾病的急性期、IgM 存在时甚至在 IgM 出现之前确认 ST 诊断。包含所有三个基因——56 kDa、47 kDa 和 groEL，而不是单个 56 kDa 目标，识别和确认 ST 患者的最大数量。