Improved and customized dengue serodiagnostics through combined NS1/IgM testing and novel dual-cut-off IgG ELISA

BACKGROUND: Accurate diagnostics of dengue virus (DENV) infection are essential for patient management, outbreak control, and vaccine implementation. Serological testing plays a key role, especially when molecular assays are unavailable or viremia subsides; yet, cross-reactivity with other flaviviruses remains a challenge. This study examined the diagnostic accuracy of four Euroimmun ELISAs, including a newly developed dual-cut-off IgG ELISA. METHODS: The Dengue Virus NS1 ELISA, Anti-Dengue Virus Type 1-4 ELISA (IgM), Anti-Dengue Virus Type 1-4 ELISA (IgG; native antigen/gE-based), and the novel Anti-Dengue Virus NS1 ELISA 2.0 (IgG; recombinant NS1-based, with an alternative higher cut-off for flavivirus-endemic regions) were analyzed. Sensitivity was determined using sera from 22 Vietnamese patients with RT-PCR-confirmed DENV infection, collected during acute (t1, 1-6 dpo), early convalescent (t2, 4-9 dpo), and late convalescent (t3, 13-19 dpo) phases. Specificity was assessed with samples from 500 healthy German blood donors (HBD) and 40 patients each with West Nile virus (WNV) or Zika virus (ZIKV) infection. RESULTS: Sensitivities were 90.5%/70.0%/0% (t1/t2/t3) for NS1, 33.3%/85.0%/77.3% for IgM, 66.7%/100%/100% for IgG, and 33.3%/65.0%/100% vs. 19.1%/50.0%/100% for IgG 2.0 (standard vs. alternative cut-off). Combined NS1/IgM testing achieved 100% sensitivity in single acute-phase samples. Combined IgM and IgG 2.0 testing confirmed recent infection by IgM/IgG seroconversion or ≥4-fold IgG increase in 100% of paired samples. Overall specificity was 85.7% (HBD/WNV/ZIKV: 95.0%/50.0%/5.0%) for IgG, compared to 95.7% (98.2%/95.0%/65.0%) and 99.5% (99.8%/97.5%/97.5%) for IgG 2.0 using standard and alternative cut-offs, respectively. CONCLUSIONS: Euroimmun ELISAs support customized, highly accurate and versatile diagnostic strategies applicable to various dengue testing contexts. Combining NS1 and IgM ELISAs may offer a practical alternative to molecular assays during acute infection. The native antigen/gE-based IgG ELISA enables early sensitive IgG detection, although with limited specificity. With minimal cross-reactivity, the NS1-based dual-cut-off ELISA 2.0 (IgG) reliably captures DENV-specific IgG dynamics and enhances differentiation from other flaviviruses, which could provide an advantage in the use for convalescent-phase diagnostics, epidemiological surveillance, and pre-vaccination screening.