Performance of electrochemical aptasensor as antigen test in clinical samples for early diagnosis of leptospirosis

Abstract

Early diagnosis of leptospirosis is critical for timely treatment and effective disease management. This study evaluated the diagnostic performance of a novel electrochemical aptasensor targeting the electron transfer flavoprotein subunit beta (EtfB) of Leptospira interrogans in clinical samples collected during the acute phase of leptospirosis. The aptasensor assay was tested using plasma samples and compared to the microscopic agglutination test (MAT), the standard reference method. To assess diagnostic performance, aptasensor results were evaluated against leptospirosis status as determined by MAT. Receiver operating characteristic (ROC) analysis identified a 40% decrease in electrochemical signal relative to the blank as the optimal cut-off, yielding an area under the curve (AUC) of 0.93. The assay demonstrated a sensitivity of 100% and a specificity of 80%. For diagnostic concordance, aptasensor results were compared with those obtained from the reference quantitative PCR (qPCR) method. The aptasensor exhibited 100% positive agreement and 57.1% negative agreement with qPCR. Notably, in patients with high MAT titers, the aptasensor outperformed qPCR in detection rates (100% vs. 25%). These findings indicate that the aptasensor assay is a highly reliable and effective antigen-based diagnostic tool for early leptospirosis detection, making it suitable for both low- and high-prevalence settings.

Keywords: Antigen test; Aptamer biosensor; Aptasensor; Diagnostic performance; Early diagnosis; Leptospirosis.

Fig. 1

The calibration curve between the concentration of 6xHis-ETFΒ and the percentage of the signal decreases relative to the blank. (A): The plot fitted with 4-parameter logistic regression (R² = 0.982). (B): The plot fitted with a linear regression (R² = 0.956) with the slope values y = 4.8682x + 22.72.

Fig. 2

Receiver operating characteristic (ROC) curve analysis for estimation of cut-off value showing the diagnostic performance of the aptasensor with an area under the curve (AUC). (A): Overall, the ROC curve for the diagnostic performance with an AUC of 0.93. (B): A scatter plot showing the percentage decrease in signal relative to the blank (mean ± standard deviation) for patients with leptospirosis and those without. The mean percentage signal decrease is significantly higher in leptospirosis patients compared to non-leptospirosis patients (p < 0.0001, Mann–Whitney U test). The dashed line indicates an optimal cut-off value at the 40% signal decrease relative to the blank.

Fig. 3

The diagnostic agreement between the aptasensor using optimum cut-off at 40% signal decrease threshold and PCR methods among leptospirosis and non-leptospirosis patients. (A): ROC curves exhibit sensitivity and specificity for the aptasensor (red) and PCR (blue) in identifying leptospirosis. (B): Scatter plots show the classification of leptospirosis and non-leptospirosis patients based on the aptasensor and PCR results. Positive and negative classifications are represented by red and purple dots, respectively. The green dotted circles indicate the discordant results.

Fig. 4

Schematic diagrams of the electrochemical aptasensor for detection of the EtfB protein of Leptospira interrogans. (A): The principle of the assay and (B): The assay procedures.