Longitudinal entomological study using molecular xenomonitoring of Wuchereria bancrofti in Culex quinquefasciatus in three Nepalese districts: Implications for lymphatic filariasis transmission assessment and elimination

Background: In Nepal, lymphatic filariasis (LF), caused by Wuchereria bancrofti, remains a priority neglected tropical disease targeted for elimination by 2030. Despite large-scale mass drug administration (MDA) and post-MDA surveillance, focal transmission may persist. Molecular xenomonitoring (MX) of mosquito vectors is a complementary method for detecting ongoing transmission. This study assessed mosquito abundance and species composition and compared gravid traps and CDC light traps for capturing Culex quinquefasciatus, the primary LF vector. It examined spatial and seasonal vector patterns, W. bancrofti infection rates, and climatic associations across three LF endemic districts. Methodology: A longitudinal entomological survey was conducted from September 2022 to September 2023 in selected sites of Dhading, Jhapa, and Kapilvastu districts using gravid traps and CDC light traps. Mosquitoes were morphologically identified to species level, and female Cx. quinquefasciatus were pooled (up to 25 mosquitoes) for detection of W. bancrofti DNA using real-time PCR. Mosquito abundance, species composition, trap efficiency, spatial variation (within and between locations), temporal trends (daily, weekly, monthly, seasonal), infection rate (pool positivity, and maximum likelihood estimate (MLE) of infection using PoolTestR package in R software), and climatic associations (precipitation, temperature, humidity) were analysed. Results: A total of 60,922 mosquitoes belonging to eight genera (Culex, Anopheles, Aedes, Armigeres, Malaya, Mansonia, Tripteroides, and Toxorhynchites) were identified. Of these, 16,044 were female Cx. quinquefasciatus. Gravid traps showed higher relative trapping efficiency of female Cx. quinquefasciatus than light traps, with the highest efficiency observed in Jhapa (3.60:1). Temporal trends varied across locations; however, overall Cx. quinquefasciatus abundance peaked during the spring and summer months (March-May), coinciding with the monsoon period, and was positively correlated with climatic factors. W. bancrofti infection rates varied by location and trap type, with gravid traps yielding higher infection rates, particularly in Jhapa. A total of 785 pools of Cx. quinquefasciatus were screened. Pool positivity and infection prevalence (MLE) ranged from 10.3% to 17.6% and 0.5% to 0.9% in Dhading, 12.5% to 36.8% and 0.9% to 2.0% in Jhapa, and 5.3% to 20.0% and 0.3% to 1.5% in Kapilvastu, respectively. Conclusions: The study highlights ongoing transmission in three LF-endemic areas of Nepal, emphasising the need for continued surveillance in high-risk areas and periods. Gravid trap-based xenomonitoring could complement community-based W. bancrofti surveillance by identifying hotspots and optimising the timing and targeting of LF elimination interventions.