Combined Analytical and Clinical Performance Evaluation of a Novel Dengue NS1 Rapid Test in a Real-World Endemic...

Objectives: This study evaluated the analytical and clinical performance of a novel NS1 rapid diagnostic test in a dengue-endemic setting in Thailand. Methods: The K-Dengue NS1 Ag test (K.Bio Sciences, Pathumthani, Thailand) was developed. Analytical performance included determination of LOD, reproducibility, and evaluation against potentially cross-reactive pathogens and interfering substances. Unlike conventional assays employing 40 nm colloidal gold, this test incorporates 80 nm gold nanospheres to enhance detection sensitivity. The LOD was determined by serial dilution of recombinant NS1 proteins representing all four dengue virus serotypes. Clinical performance was assessed using 185 archived plasma samples collected between January 2024 and February 2025 from two tertiary care hospitals in Thailand, with a commercial NS1 ELISA serving as the reference standard. Results: The K-Dengue NS1 test demonstrated serotype-specific limits of detection (LODs) for recombinant NS1 antigen, 2.9 ng/mL (DENV-1), 0.5 ng/mL (DENV-2), 25.2 ng/mL 27 (DENV-3), and 4.5 ng/mL (DENV-4). Cross-reactivity testing revealed no false positives against closely related arboviruses or common co-infections, and no interference was observed from frequently encountered pathogens or biochemical substances. In clinical evaluation, the assay achieved a sensitivity of 98.08% (51/52), a specificity of 100% (133/133), and an overall accuracy of 99.37%. Importantly, sensitivity was significantly higher in primary infections (100.00%) than in secondary infections (93.3%, p = 0.288). Conclusions: In this clinically oriented evaluation, the K-Dengue NS1 rapid test showed high specificity and good sensitivity, particularly in primary dengue infections. While the assay may be useful as part of early diagnostic workflows in comparable healthcare settings, reduced sensitivity in secondary infections indicates that negative NS1 results should be interpreted with caution and, where appropriate, supplemented with additional diagnostic methods.