Advances in the Diagnosis of Human Opisthorchiasis: Development of Opisthorchis viverrini Antigen Detection in Urine.
Advances in the Diagnosis of Human Opisthorchiasis: Development of Opisthorchis viverrini Antigen Detection in Urine.
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DOI：
10.1371/journal.pntd.0004157
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发表时间：
2015
影响因子：
3.8
通讯作者：
Bethony JM
中科院分区：
文献类型：
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作者：
Worasith C;Kamamia C;Yakovleva A;Duenngai K;Wangboon C;Sithithaworn J;Watwiengkam N;Namwat N;Techasen A;Loilome W;Yongvanit P;Loukas A;Sithithaworn P;Bethony JM
泰国已采用许多控制阿片吸虫病的策略，但其他邻国尚未采用。具体控制方法包括大规模药物管理（MDA）和健康教育，以减少生鱼消费。这些控制工作极大地改变了过去十年中维韦里尼后睾吸虫 (OV) 感染的流行病学，从单个村庄密集的“重度”感染转变为分布在广泛地理区域的广泛的“轻度”OV 感染。目前，OV感染的“金标准”检测方法是福尔马林乙酸乙酯浓缩技术（FECT），该技术对轻型OV感染的诊断敏感性和诊断特异性有限，OV虫卵经常与粪便中微小肠吸虫卵（MIF）混淆。在本研究中，我们开发并评估了基于单克隆抗体的酶联免疫吸附测定法的诊断性能，该测定法用于测量尿液中 OV 排泄-分泌 (ES) 抗原（尿液 OV-ES 测定），与金标准检测 FECT 方法相比，用于诊断阿片吸虫病。在测试尿液 OV-ES 测定的诊断性能之前，我们测试了几种预处理尿液样本的方法。我们使用三氯乙酸 (TCA) 预处理的尿液，在泰国东北部 OV 流行地区，比较了尿液 OV-ES 检测与 FECT 对 OV 感染的检测和定量。受试者工作特征 (ROC) 曲线用于确定使用 TCA 预处理的尿液进行尿液 OV-ES 测定的诊断敏感性和特异性，并建立诊断阳性阈值。针对已建立的诊断阳性阈值计算阳性预测值以及获得阳性测试结果 (LR+) 或阴性测试结果 (LR-) 的可能性。使用逻辑回归估计诊断风险（优势比）。当尿液样本在用于尿液 OV-ES 测定之前用 TCA 进行预处理时，分析灵敏度显着提高。使用 TCA 对尿液进行预处理，尿液 OV-ES 测定的检测限 (LoD) 为 39 ng/ml，而粪便抗原检测方法报道的 LoD 为 52 ng/mL。同样，尿液 OV-ES 测定与 FECT 测量的 OV 感染强度显着相关。尿液 OV-ES 检测还能够从之前使用 FECT 确定为阴性的 63 名 (44.4%) 个体中检测出 28 名个体呈阳性。随着 FECT 确定的 OV 感染强度的增加，通过尿液 OV-ES 检测阳性诊断 OV 感染的可能性显着增加。参考FECT，尿液OV-ES测定的敏感性和特异性分别为81%和70%。与目前检测和定量 OV 感染的“金标准”FECT 方法相比，通过尿液 OV-ES 检测检测 OV 感染显示出更高的诊断灵敏度和诊断特异性。由于其易于使用和非侵入性样本采集（尿液），尿液 OV-ES 检测有可能彻底改变肝吸虫感染的诊断，并为控制和消除这些致瘤寄生虫提供有效的工具。为了有效监测和控制这种食源性寄生虫并预防 OV 诱发的胆管癌（胆管癌或 CCA），需要改进检测人类 Viverrini 后吸虫 (OV) 感染的诊断方法。本研究建立了一种新的尿抗原检测方法，通过基于单克隆抗体的酶联免疫吸附测定（尿OV-ES测定）定量诊断阿片吸虫病。对泰国东北部孔敬省 Don Chang 分区 235 名受试者的配对粪便和尿液样本进行分析后发现，与当前的金标准诊断方法相比，尿液 OV-ES 检测的灵敏度为 81%，特异性为 70%。此外，尿液OV-ES测定检测到的抗原水平与OV感染强度显着相关（P<0001），并且抗原阳性诊断的比例与感染强度增加相关。通过金标准方法福尔马林乙酸乙酯浓缩技术确定为卵子阴性受试者的个体中有 44% 通过尿液 OV-ES 测定呈阳性。尿液样本采集的简便性和无创性以及尿液OV-ES检测的高诊断准确性为人类阿片吸虫病的诊断提供了一种替代手段，有利于东南亚资源有限地区的阿片吸虫病的预防和控制。
Many strategies to control opisthorchiasis have been employed in Thailand, but not in the other neighbouring countries. Specific control methods include mass drug administration (MDA) and health education to reduce raw fish consumption. These control efforts have greatly shifted the epidemiology of Opisthorchis viverrini (OV) infection over the last decade from presenting as densely concentrated "heavy" infections in single villages to widespread "light" OV infections distributed over wide geographical areas. Currently, the "gold standard" detection method for OV infection is formalin ethyl-acetate concentration technique (FECT), which has limited diagnostic sensitivity and diagnostic specificity for light OV infections, with OV eggs often confused with eggs of minute intestinal flukes (MIFs) in feces. In this study, we developed and evaluated the diagnostic performance of a monoclonal antibody-based enzyme-linked immunosorbent assay for the measurement of OV excretory-secretory (ES) antigens in urine (urine OV-ES assay) for the diagnosis of opisthorchiasis compared to the gold standard detection FECT method. We tested several methods for pre-treating urine samples prior to testing the diagnostic performance of the urine OV-ES assay. Using trichloroacetic acid (TCA) pre-treated urine, we compared detection and quantification of OV infection using the urine OV-ES assay versus FECT in OV-endemic areas in Northeastern Thailand. Receiver operating characteristic (ROC) curves were used to determine the diagnostic sensitivity and specificity of the urine OV-ES assay using TCA pre-treated urine, and to establish diagnostic positivity thresholds. The Positive Predictive Value as well as the likelihood of obtaining a positive test result (LR+) or a negative test result (LR-) were calculated for the established diagnostic positivity threshold. Diagnostic risks (Odds Ratios) were estimated using logistic regression. When urine samples were pre-treated with TCA prior to use in the urine OV-ES assay, the analytical sensitivity was significantly improved. Using TCA pre-treatment of urine, the urine OV-ES assay had a limit of detection (LoD) of 39 ng/ml compared to the LoD of 52 ng/mL reported for coprological antigen detection methods. Similarly, the urine OV-ES assay correlated significantly with intensity of OV infection as measured by FECT. The urine OV-ES assay was also able to detect 28 individuals as positive from the 63 (44.4%) individuals previously determined to be negative using FECT. The likelihood of a positive diagnosis of OV infection by urine OV-ES assay increased significantly with the intensity of OV infection as determined by FECT. With reference to FECT, the sensitivity and specificity of the urine OV-ES assay was 81% and 70%, respectively. The detection of OV-infection by the urine OV-ES assay showed much greater diagnostic sensitivity and diagnostic specificity than the current "gold standard" FECT method for the detection and quantification of OV infection. Due to its ease-of-use, and noninvasive sample collection (urine), the urine OV-ES assay offers the potential to revolutionize the diagnosis of liver fluke infection and provide an effective tool for control and elimination of these tumorigenic parasites. Improved diagnostic methods for the detection of Opisthorchis viverrini (OV) infection in humans is required for effective surveillance and control of this food borne parasite and the prevention of OV-induced bile duct cancer (cholangiocarcinoma or CCA). In this study, a novel urinary antigen detection method was established for quantitative diagnosis of opisthorchiasis by a monoclonal antibody-based enzyme-linked immunosorbent assay (urine OV-ES assay). Analysis of paired feces and urine samples from 235 subjects in Don Chang sub-district in Khon Kaen Province, Northeast Thailand revealed 81% sensitivity and 70% specificity of the urine OV-ES assay when compared to the current gold standard diagnostic method. Moreover, levels of antigen detected by the urine OV-ES assay significantly correlated with intensity of OV infection (P< 0001), with and the proportion of antigen positive diagnosis associated with increasing intensity of infection. Forty four percent of individuals determined to be egg negative subjects by the gold standard method formalin ethyl-acetate concentration technique were positive by the urine OV-ES assay. The ease and noninvasiveness of urine sample collection and the high diagnostic accuracy of the urine OV-ES assay provide an alternative means for the diagnosis of human opisthorchiasis and facilitate the prevention and control of opisthorchiasis in resource limited setting of Southeast Asia.