Department of Infectious Diseases, Christian Medical College, Vellore, Tamil Nadu, India.
Department of Emergency Medicine, Christian Medical College, Vellore, Tamil Nadu, India.
PLoS Negl Trop Dis. 2020 Nov 12;14(11):e0008747. doi: 10.1371/journal.pntd.0008747. eCollection 2020 Nov.
Diagnosis of scrub typhus, caused by the bacterium Orientia tsutsugamushi, is challenging because of the overlap of its non-specific symptoms with other infections coupled with the lack of sufficient data on the performance of diagnostic tests. Early diagnosis of scrub typhus is crucial to improve outcomes and this study evaluates the diagnostic performance of various tests. The present study aims at assessing the accuracy of various rapid diagnostic tests, serologic tests, and nucleic acid amplification methods on well-characterized patient samples. Adult patients with acute febrile illness and manifestations suggestive of scrub typhus confirmed by positive PCR in the blood, eschar or tissue were characterized as cases. Patients with acute febrile illness and a confirmed alternate etiology such as culture-confirmed typhoid, smear/PCR positive for malaria, PCR/NS1 antigen positive for dengue, PCR positive for influenza, PCR/MAT positive for leptospirosis, PCR positive for spotted fever were characterized as controls with other infections. The healthy controls consisted of subjects from the same geographic region. We performed the following tests on blood samples for scrub typhus and calculated the sensitivity, specificity, positive predictive value, and negative predictive value: (1) Quantitative real time PCR using 47kDa gene (qPCR); (2) Conventional PCR using 56kDa gene (cPCR); (3) Loop-mediated isothermal amplification assay (LAMP assay); (4) Immunofluorescence assay (IFA); (5) Enzyme-linked immunosorbent assay (ELISA); (6) Weil-Felix test(WF test); and (7) Immunochromatographic Rapid Diagnostic Test (RDT).Among the 316 participants, 158 had confirmed scrub typhus (cases) and 158 were controls. ELISA and RDT detecting Orientia tsutsugamushi specific IgM antibodies had excellent discriminative potential with sensitivities and specificities of 92%, 94% and 92%, 92% respectively. The sensitivity and specificity of IFA were found to be 95% and 74% respectively. IgM serology had a false positivity rate of 8% with other acute febrile illnesses such as dengue, leptospirosis and spotted fever due to the nonspecific binding of the pentavalent IgM. LAMP assay had 91.7% sensitivity and 77.2% specificity while qPCR provided excellent sensitivity (97%) and perfect specificity. In conclusion, ELISA and RDT detecting Orientia tsutsugamushi specific IgM antibodies have excellent sensitivity and specificity while the accuracy of IFA is suboptimal for the diagnosis of scrub typhus. Given its perfect specificity and superior sensitivity, qPCR is preferred for diagnostic confirmation in reference laboratories particularly for diagnosis of early disease with less than 7 days duration. This study provides a comprehensive evaluation of all currently available diagnostic tests for scrub typhus.
恙虫病由东方立克次体引起，其诊断具有挑战性，因为其非特异性症状与其他感染重叠，并且缺乏有关诊断测试性能的充分数据。早期诊断恙虫病对于改善预后至关重要，本研究评估了各种检测方法的诊断性能。本研究旨在评估各种快速诊断检测、血清学检测和核酸扩增方法在特征明确的患者样本中的准确性。具有急性发热性疾病和恙虫病表现并通过血液、焦痂或组织中的阳性 PCR 证实的成年患者被确定为病例。具有急性发热性疾病且具有明确替代病因的患者，例如培养证实的伤寒、疟疾涂片/PCR 阳性、登革热 PCR/NS1 抗原阳性、流感 PCR 阳性、钩端螺旋体病 PCR/MAT 阳性、斑点热 PCR 阳性被确定为具有其他感染的对照组。健康对照组由来自同一地理区域的受试者组成。我们对血液样本进行了以下恙虫病检测，并计算了灵敏度、特异性、阳性预测值和阴性预测值：(1)使用 47kDa 基因的实时定量 PCR(qPCR)；(2)使用 56kDa 基因的常规 PCR(cPCR)；(3)环介导等温扩增检测(LAMP 检测)；(4)免疫荧光检测(IFA)；(5)酶联免疫吸附试验(ELISA)；(6)魏尔斐尔试验(WF 试验)；和 (7)免疫层析快速诊断检测(RDT)。在 316 名参与者中，有 158 名确诊为恙虫病(病例)，158 名为对照组。ELISA 和 RDT 检测东方立克次体特异性 IgM 抗体具有出色的鉴别能力，其灵敏度和特异性分别为 92%、94%和 92%、92%。IFA 的灵敏度和特异性分别为 95%和 74%。由于五价 IgM 的非特异性结合，IgM 血清学的假阳性率为 8%，与其他急性发热性疾病如登革热、钩端螺旋体病和斑点热有关。LAMP 检测的灵敏度为 91.7%，特异性为 77.2%，而 qPCR 提供了出色的灵敏度(97%)和完美的特异性。总之，ELISA 和 RDT 检测东方立克次体特异性 IgM 抗体具有出色的灵敏度和特异性，而 IFA 用于诊断恙虫病的准确性并不理想。鉴于其完美的特异性和优越的灵敏度，qPCR 是参考实验室诊断确认的首选方法，特别是对于病程少于 7 天的早期疾病的诊断。本研究对所有现有的恙虫病诊断检测方法进行了全面评估。
