Unmasking Cross-Reactivity: A Comparative Evaluation of Serological and Molecular Diagnostics in Dengue and Chikungunya Co-infections

Background: Dengue and Chikungunya are major arboviral diseases that frequently co-circulate in tropical regions like India, causing overlapping clinical symptoms and complicating diagnosis. This study aimed to evaluate the diagnostic performance of serological and molecular tests in differentiating these infections and to assess ELISA-based cross-reactivity, with a focus on Dengue Virus (DENV) serotype distribution. Methods: A total of 566 dengue IgM-positive cases were screened, of which 132 samples collected within seven days of symptom onset were selected. These were tested for Chikungunya IgM, dengue NS1 antigen, and subjected to multiplex RT-PCR for dengue and Chikungunya viruses. Dengue-positive samples by Real-Time Reverse Transcription PCR (RT-PCR) were further serotyped. Concordance between assays was analyzed using Cohen’s Kappa statistic. Results: Among 566 cases, 132 were collected within seven days of illness onset and included for further analysis. Among these, 26 tested positives for Chikungunya IgM and were subsequently screened for dengue NS1 antigen using ELISA, of which 22 were found positive indicating concurrent positivity for CHIKV IgM and DENV IgM/NS1 by serological assays. Multiplex RT-PCR confirmed 12 dengue-only cases, 7 Chikungunya-only cases, and 2 co-infections. RT-PCR serotyping showed DENV-2 (41.67%) as the predominant serotype, followed by DENV-1, -3, and -4. Concordance between dengue NS1 and IgM ELISA was 84.6% (κ=0.69), while RT-PCR and NS1 showed lower agreement (77.3%, κ=0.33). Chikungunya IgM ELISA and RT-PCR showed poor concordance (31.8%, κ=−0.36), suggesting false-positives and timing-related discrepancies. Conclusion: The study highlighted the limitations of serological assays in differentiating dengue and Chikungunya infections due to cross-reactivity and timing of sample collection. While combined IgM and NS1 testing is valuable for dengue diagnosis, reliance on Chikungunya IgM ELISA alone may be misleadingand RT-PCR is essential.