List of primers used in this study.

Background
Dengue virus (DENV), a mosquito-borne flavivirus responsible for dengue disease, has emerged as an escalating global health concern, with cases increasing sharply in recent decades. In Argentina, dengue has transitioned from a sporadic disease to a recurrent epidemic, now affecting 18 of 23 provinces and exposing gaps in diagnostic capacity.
Methodology and Principal Findings
The DENV genome encodes the non-structural protein 1 (NS1), a key biomarker for early infection detection. Given the limited access to commercially available diagnostic kits within the public health system, we developed a combined ELISA system incorporating Nanobodies designed to target NS1 across all four DENV serotypes as detection antibodies. This system demonstrates excellent discriminative performance (AUC > 0.9), with a diagnostic sensitivity of 93.6% (95% CI: 86.6–97.6%) and a specificity of 81.1% (95% CI: 70.3–89.3%). The analytical sensitivity showed strong correlation between sera pool dilutions and detected signals, with a limit of detection aligning with reported NS1 concentrations in human samples. While the system exhibits limitations in detecting NS1 from DENV-4, it successfully identified cases in patients five days post-symptom onset who were initially considered epidemiologically negative for dengue infection.
Significance
Our results underscore the urgent need for accessible, high-precision diagnostic tools in regions facing a surge in dengue outbreaks. Additionally, they highlight the necessity of revising current diagnostic algorithms to enhance the detection of late-presenting cases.