Multi-district molecular surveillance of dengue virus in Pakistan reveals serotype cycling and recurrent DENV-2 dominance, 2021–2025

Dengue virus (DENV) remains a major public health concern in Pakistan, with recurrent seasonal outbreaks and shifting serotype patterns. Understanding temporal and geographic trends in circulating serotypes is essential for improving surveillance and outbreak preparedness. This study aimed to characterize the spatio-temporal distribution and serotype dynamics of DENV detected through molecular surveillance in Pakistan between 2021 and 2025. NS1-positive dengue cases were tested at the National Institute of Health (NIH), Islamabad, using real-time reverse transcription polymerase chain reaction (qRT-PCR) for serotyping. Demographic, temporal, and geographic data were analyzed to assess the distribution of DENV infections and circulating serotypes over the study period. Among 1,644 NS1-positive dengue cases tested, 1,162 were confirmed by qRT-PCR. DENV-2 was the predominant serotype overall, accounting for 78.4% of confirmed infections, followed by DENV-1 (21.4%) and DENV-3 (0.09%). Temporal analysis showed that DENV-2 predominated during 2021 and 2022, was temporarily replaced by DENV-1 in 2023, and re-emerged as the dominant serotype in 2024 and 2025. Dengue transmission demonstrated strong seasonal patterns, with most cases occurring during the post-monsoon months of September and October. Gender-Age distribution showed majority of cases in males (66%), particularly those aged 21-40 years. Geographically, most confirmed cases were reported from Rawalpindi and Islamabad, reflecting the primary referral catchment of the surveillance system, while additional cases were detected from several other districts, including Peshawar, Nowshera, Mansehra, and Karachi. These findings highlight dynamic shifts in DENV serotype circulation in Pakistan and emphasize the need for sustained molecular surveillance to guide public health responses.