Evaluation of the applicability of clinical samples for rapid detection of Yersinia pestis using up-conversion technology-based immunochromatographic assay (UPT-ICA)

Objective To identify optimal clinical samples for rapid detection of Y. pestis using up-conversion technology-based immunochromatographic assay (UPT-ICA) and develop a standardized sample processing protocol.Methods A total of 464 clinical samples, including serum, sputum, pus, tracheal secretions, and cerebrospinal fluid were collected. After mixing with the sample diluent at a ratio of 1 : 9, these samples were used to evaluate the specificity of UPT-ICA. From the clinical samples that had been tested as negative by UPT-ICA, 141 samples of diverse types were randomly selected. These samples were mixed with the National Reference Material for Y. pestis to prepare simulated positive samples containing 1×105 CFU/mL Y. pestis, followed by evaluation of detection efficacy. Sample processing methods were optimized by adjusting the proportions of sample diluent and digestive fluid. Based on the detection results, the clinical sample processing protocol was refined. Finally, various sample types were evaluated to assess the specificity and detection efficacy of the UPT-ICA using the optimized protocol.Results The detection sensitivity of UPT-ICA to Y. pestis for the national reference stain of Y. pesits was 1 × 105 CFU / mL, significantly exceeding that of colloidal gold immunochromatographic assay. For tracheal secretions and cerebrospinal fluid, UPT-ICA showed 100% specificity. The false positive rates were 2.35% for normal serum, 8.70% for hemolytic serum, and 12.64% for lipemic serum, indicating that hemolytic and lipemic samples exhibited significant interference and were deemed unsuitable. After 1:99 dilution, the false positive rate for normal serum samples decreased to 0.58%. When sputum and sputum digestate were mixed at a 1:1 ratio, one false positive result was generated (with a false positive rate of 1.20%), however, no false positive result was observed at a 1:2 ratio. Among 42 pus samples, 3 false positive results were detected; however, when all samples were collected using the cotton swab sampling method, no further false positives were observed. Mouse liver and spleen homogenates of 10 mg/mL and 100 mg/mL, respectively, did not affect UPT-ICA specificity. UPT-ICA successfully detected all spiked simulated positive samples with Y. pestis at a final concentration of 1×105 CFU/mL. Following optimization and validation of the improved sample processing protocol, the false positive rate of UPT-ICA for various clinical samples declined significantly from 7.14% (pre-optimization) to 0.59% (post-optimization).Conclusions The UPT-ICA reagent for detection of Y. pestis demonstrated rapid and accurate detection performance for all tested clinical samples following standardized preprocessing.