ELISA and qRT-PCR-Based Diagnosis of Dengue and Chikungunya Virus Co- Infections during the 2023-24 Outbreak in Punjab, Pakistan

Abstract Dengue virus (DENV) and Chikungunya virus (CHIKV) pose significant public health threats in tropical and subtropical regions, including Pakistan. The coexistence of Aedes aegypti and Aedes albopictus vectors in these areas facilitate viral co-circulation and human co-infection. This study investigated the prevalence, clinical manifestations, and diagnostic challenges of DENV and CHIKV infections during the 2023–2024 outbreaks in Punjab, Pakistan.We screened 1,152 suspected cases using ELISA for DENV NS1 antigen and CHIKV IgM antibodies. Confirmatory testing and DENV serotyping were performed using quantitative reverse transcription PCR (qRT-PCR).Results indicate DENV infection in 45% of cases, CHIKV in 25%, and co-infections in 11%. A shift in predominant DENV serotypes was observed, with DENV-1 and DENV-2 replacing the previously dominant DENV-3. Co-infected patients exhibited more severe clinical symptoms, including prolonged fever, intense arthralgia, and hematological abnormalities, compared to those with single infections. qRT-PCR demonstrated higher sensitivity than ELISA for early DENV detection.These findings highlight the risk of misdiagnosis, particularly for CHIKV in dengue-negative patients, and underscore the importance of dual diagnostic testing to improve case identification and patient management. The results provide crucial insights for strengthening diagnostic protocols and public health responses to arboviral outbreaks in endemic regions.