College of Medicine and Public Health, Ubon Ratchathani University, Warinchamrap, 34190, Ubon Ratchathani, Thailand.
Research Group for Biomedical Research and Innovative Development (RG-BRID), College of Medicine and Public Health, Ubon Ratchathani University, Warinchamrap, 34190, Ubon Ratchathani, Thailand.
Parasit Vectors. 2024 May 7;17(1):206. doi: 10.1186/s13071-024-06285-7.
Opisthorchiasis and cholangiocarcinoma (CCA) continue to be public health concerns in many Southeast Asian countries. Although the prevalence of opisthorchiasis is declining, reported cases tend to have a light-intensity infection. Therefore, early detection by using sensitive methods is necessary. Several sensitive methods have been developed to detect opisthorchiasis. The immunological detection of antigenic proteins has been proposed as a sensitive method for examining opisthorchiasis.
The Opisthorchis viverrini antigenic proteins, including cathepsin B (OvCB), asparaginyl endopeptidase (OvAEP), and cathepsin F (OvCF), were used to construct multi-antigenic proteins. The protein sequences of OvCB, OvAEP, and OvCF, with a high probability of B cell epitopes, were selected using BepiPred 1.0 and the IEDB Analysis Resource. These protein fragments were combined to form OvCB_OvAEP_OvCF recombinant DNA, which was then used to produce a recombinant protein in Escherichia coli strain BL21(DE3). The potency of the recombinant protein as a diagnostic target for opisthorchiasis was assessed using immunoblotting and compared with that of the gold standard method, the modified formalin-ether concentration technique.
The recombinant OvCB_OvAEP_OvCF protein showed strong reactivity with total immunoglobulin G (IgG) antibodies against light-intensity O. viverrini infections in the endemic areas. Consequently, a high sensitivity (100%) for diagnosing opisthorchiasis was reported. However, cross-reactivity with sera from other helminth and protozoan infections (including taeniasis, strongyloidiasis, giardiasis, E. coli infection, enterobiasis, and mixed infection of Echinostome spp. and Taenia spp.) and no reactivity with sera from patients with non-parasitic infections led to a reduced specificity of 78.4%. In addition, the false negative rate (FNR), false positive rate (FPR), positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy were 0%, 21.6%, 81.4%, 100%, and 88.9%, respectively.
The high sensitivity of the recombinant OvCB_OvAEP_OvCF protein in detecting opisthorchiasis demonstrates its potential as an opisthorchiasis screening target. Nonetheless, research on reducing cross-reactivity should be undertaken by detecting other antibodies in other sample types, such as saliva, urine, and feces.
华支睾吸虫病和胆管癌（CCA）在许多东南亚国家仍然是公共卫生关注的问题。尽管华支睾吸虫病的流行率正在下降，但报告的病例往往感染程度较轻。因此，有必要使用敏感方法进行早期检测。已经开发了几种敏感方法来检测华支睾吸虫病。免疫检测抗原蛋白已被提议作为一种敏感的华支睾吸虫病检查方法。
使用华支睾吸虫抗原蛋白，包括组织蛋白酶 B（OvCB）、天冬酰胺内肽酶（OvAEP）和组织蛋白酶 F（OvCF），构建多抗原蛋白。使用 BepiPred 1.0 和 IEDB 分析资源选择 OvCB、OvAEP 和 OvCF 的蛋白序列，这些蛋白序列具有高 B 细胞表位的可能性。这些蛋白片段被组合成 OvCB_OvAEP_OvCF 重组 DNA，然后在大肠杆菌 BL21（DE3）菌株中产生重组蛋白。使用免疫印迹法评估重组蛋白作为华支睾吸虫病诊断靶标的效力，并与金标准方法（改良的甲醛乙醚浓缩技术）进行比较。
重组 OvCB_OvAEP_OvCF 蛋白与来自流行地区轻度华支睾吸虫感染的总免疫球蛋白 G（IgG）抗体强烈反应。因此，报告了 100%的高灵敏度用于诊断华支睾吸虫病。然而，与其他寄生虫和原生动物感染（包括带绦虫病、旋毛虫病、贾第虫病、大肠杆菌感染、蛲虫病和 Echinostome spp.和 Taenia spp.混合感染）的血清发生交叉反应，与非寄生虫感染患者的血清无反应，导致特异性降低至 78.4%。此外，假阴性率（FNR）、假阳性率（FPR）、阳性预测值（PPV）、阴性预测值（NPV）和诊断准确性分别为 0%、21.6%、81.4%、100%和 88.9%。
重组 OvCB_OvAEP_OvCF 蛋白在检测华支睾吸虫病方面的高灵敏度表明其作为华支睾吸虫病筛查靶标的潜力。然而，应该通过检测其他样本类型（如唾液、尿液和粪便）中的其他抗体来进行降低交叉反应的研究。
