Increasing Frequency of Secondary Dengue Infections in Sequential Outbreaks (2016-2024)-Clinical Impact and Diagnostic Challenges.

Successive dengue virus (DENV) outbreaks progressively reshape population immunity, influencing disease expression and diagnostic performance. This study evaluated the impact of secondary infections across sequential outbreaks on disease severity markers, serotype dynamics, and the agreement between routinely used direct diagnostic methods. This retrospective study analyzed 976 serum samples from three outbreaks in Misiones, Argentina. Clinical and serotype analyses included 869 acute-phase dengue cases confirmed by at least one direct detection method (2016: n = 512; 2019: n = 148; 2024: n = 209). A subset of 318 samples, including 107 RT-PCR/NS1-negative samples, was used to compare NS1 antigen rapid diagnostic tests (NS1 Ag) and RT-PCR. Viral serotypes and clinical and laboratory severity markers were evaluated. Secondary infections increased from 31.1% in 2016 to 43.2% in 2019 and 53.1% in 2024 (p < 0.001). Serotype distribution shifted from DENV-1 predominance in 2016 (95.1%) to DENV-1/DENV-4 co-circulation in 2019 (60.7%/39.3%) and DENV-2 predominance in 2024 (97.6%). Secondary infections were associated with increased frequency of laboratory severity markers, particularly in 2024, including higher hematocrit, liver enzyme levels and lower leukocyte and platelet counts. Concordance between NS1 Ag and RT-PCR was lower in secondary infections (κ = 0.457 vs. 0.759, p = 0.0013). The increasing frequency of secondary infections may influence laboratory markers associated with dengue severity and reduce the diagnostic concordance between direct detection methods, highlighting the need for optimized surveillance and diagnostic strategies during dengue outbreaks.