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April 18, 2026Tropical Medicine and Infectious Disease0 citationsOpen Access

Systematic Evaluation of Four Cysteine Proteases (CsCP1–4) from Clonorchis sinensis for Serodiagnosis: From Single-Antigen Screening to Multi-Antigen Modeling

SWShuai Wei XCXinyan Chen SCShangkun Cai

Key Points

The aim is to evaluate the diagnostic potential of four recombinant cysteine proteases for human clonorchiasis.
Developed an indirect ELISA to measure serum reactivity against rCsCP1–4.
Validated the assay using 180 positive and 148 negative sera.
Used logistic regression to assess diagnostic performance and compared it to commercial kits.
Analyzed metrics such as area under the curve (AUC), sensitivity, specificity, accuracy, F1-score, and Kappa coefficient.
rCsCP1-IgG4, rCsCP2-IgA, rCsCP3-IgG1, and rCsCP4-IgG4 demonstrated strong performance with AUC values ranging from 0.863 to 0.958.
rCsCP1-IgG4, rCsCP3-IgG1, and rCsCP4-IgG4 offered higher sensitivity, specificity, and accuracy compared to a commercial kit.
Multi-antigen combinations significantly improved diagnostic metrics with sensitivity and specificity over 98% and AUC approaching 1.0.

Abstract

Background: Cysteine proteases of Clonorchis sinensis are potential diagnostic antigens, yet the performance of individual members within this diverse enzyme family requires systematic evaluation. This study aimed to assess the diagnostic potential of four recombinant cysteine proteases (rCsCP1–4) for human clonorchiasis. Methods: An indirect ELISA was developed to measure serum reactivity (IgG, IgG subclasses, IgA) against rCsCP1–4. The assay was validated using 180 microscopy-confirmed positive and 148 negative control sera. Samples were randomly split into training and validation sets (7.5:2.5). Diagnostic performance of single antigens and their combinations was evaluated using univariate and multivariate logistic regression and compared with a commercial kit. Key metrics included the area under the curve (AUC), sensitivity, specificity, accuracy, F1-score, and Kappa coefficient. Results: Four single antigen–antibody pairs showed high performance: rCsCP1-IgG4 (AUC = 0.928), rCsCP2-IgA (AUC = 0.863), rCsCP3-IgG1 (AUC = 0.920), and rCsCP4-IgG4 (AUC = 0.958). Among these, rCsCP1-IgG4, rCsCP3-IgG1, and rCsCP4-IgG4 outperformed the commercial kit, achieving higher sensitivity (92.0%, 96.0%, 96.0% vs. 86.0%), specificity (87.5%, 81.3%, 90.6% vs. 78.1%), accuracy (92.0%, 88.9%, 94.1% vs. 86.0%), and F1-scores (0.902, 0.902, 0.939 vs. 0.829). The Kappa values for rCsCP1-IgG4 (0.768) and rCsCP4-IgG4 (0.773) indicated substantial agreement with the microscopic standard. Multi-antigen combinations (triple or quadruple) further enhanced performance, achieving sensitivity and specificity > 98% with an AUC approaching 1.0. Conclusions: This study identifies rCsCP1 and rCsCP4, particularly in combination with IgG4 detection, as highly promising diagnostic targets for clonorchiasis. Multi-antigen combinations significantly improved diagnostic performance compared to single-antigen assays, offering a strategy for high-precision diagnosis. Furthermore, the efficacy of the rCsCP2-IgA pair suggests that detecting fecal secretory IgA could be a novel avenue for non-invasive, self-testing applications.

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