A new study from Euroimmun, published in PLoS Neglected Tropical Diseases, demonstrates how customised testing for different dengue serological markers can improve diagnostic accuracy across different stages of infection.
Diagnostic challenges
Accurate diagnosis of dengue virus (DENV) infection is challenging due to its non-specific early symptoms and potential for rapid clinical deterioration. While RT-PCR is highly effective for laboratory confirmation during the early phase of infection, its utility is constrained by the short duration of viraemia and limited availability in many dengue-endemic regions.
The diagnostic window can be extended by detection of DENV non-structural protein (NS1) and detection of anti-DENV IgM and IgG antibodies. However, interpretation of serological results may be complicated by changing biomarker profiles over the disease course and cross-reactivity resulting from prior flavivirus infection or vaccination.
Evaluating a comprehensive ELISA panel
The study assessed the diagnostic accuracy of four Euroimmun ELISAs, examining both individual assays and combined testing approaches. The panel included the Dengue Virus NS1 ELISA, the Anti-Dengue Virus Type 1-4 ELISA (IgM), the Anti-Dengue Virus Type 1-4 ELISA (IgG), which is based on native viral antigens and recombinant glycoprotein E (gE), and the newly developed Anti-Dengue Virus NS1 ELISA 2.0 (IgG), which is based on recombinant NS1 and features a novel dual cut-off.
The sample cohorts included 22 Vietnamese patients with PCR-confirmed dengue infection sampled at multiple time points, as well as healthy blood donors and individuals with acute West Nile virus (WNV) or previous Zika virus (ZIKV) infection.
Combined NS1 and IgM testing improves early detection
A key finding was that combining NS1 antigen detection with IgM antibody testing achieved 100% sensitivity for identifying acute dengue infections from a single sample.
NS1 antigen detection was highly effective during the earliest stage of infection, while IgM antibodies became increasingly detectable as the immune response developed. Due to these temporal profiles, combined testing can close diagnostic gaps that may occur when either marker is used alone.
IgM testing may be especially valuable in secondary DENV infections, where a rapid anamnestic immune response may mask or clear circulating NS1 antigen, reducing its detectability. The combined NS1/IgM approach may offer a practical alternative for identifying acute infections in settings where molecular diagnostics are unavailable.
Tailored IgG testing enhances specificity
The study also examined different IgG testing approaches in the context of flavivirus cross-reactivity. The Anti-Dengue Virus Type 1-4 ELISA (IgG) enabled sensitive DENV IgG detection from the early convalescent phase onwards. However, this high sensitivity came with the drawback of substantial cross-reactivity with antibodies generated by infections with related flaviviruses such as ZIKV and WNV.
In contrast, the Anti-DENV NS1 ELISA 2.0 (IgG) demonstrated higher specificity, although with delayed detection dynamics, reflecting later production of anti-NS1 IgG antibodies. The assay includes two interpretation thresholds: a standard cut-off and a more stringent alternative cut-off designed for flavivirus-endemic regions. Applying the higher threshold reduced cross-reactivity in both the ZIKV and WNV cohorts and boosted the overall specificity to 99.5%. This approach could therefore improve differentiation of DENV-specific IgG from cross-reactive flavivirus antibodies in areas where multiple flaviviruses co-circulate.
A flexible serodiagnostic strategy
The Euroimmun ELISA panel provides a flexible and standardised framework for customising dengue diagnostic strategies to different clinical and epidemiological settings. By combining serological biomarkers with complementary diagnostic windows, the panel offers high diagnostic accuracy across all stages of DENV infection.
The novel NS1-based IgG assay is particularly promising for identifying convalescent-stage DENV infections in flavivirus-endemic regions, where minimising cross-reactivity is critical for reliable serodiagnostics. Its enhanced specificity may improve the accuracy of DENV IgG determination in epidemiological research studies.
Read the full study here:
Saschenbrecker S, Muigg N, Klemens O, Klemens JM. Improved and customized dengue serodiagnostics through combined NS1/IgM testing and novel dual-cut-off IgG ELISA. PLoS Negl Trop Dis. 2026 Apr 27;20(4):e0014295. https://doi: 10.1371/journal.pntd.0014295
