A Cost-Effective Method for Laboratory Preparation of Insecticide Test Papers to Monitor Resistance in Culex Mosquito Populations

Monitoring resistance to insecticides in mosquito populations is a critical component of global efforts to control vector-borne diseases. The World Health Organization (WHO) tube bioassay serves as the international standard for this purpose. However, a significant practical barrier exists for many laboratories, particularly those operating in regions with limited resources. The assay’s dependence on commercially pre-treated filter papers, which can be costly and subject to lengthy procurement delays, often hinders the timely surveillance of resistance. This delay can result in the continued application of ineffective insecticides, undermining control programs. To overcome this challenge, we developed and validated a standardized, in-house protocol for preparing insecticide-impregnated papers. The method utilizes precise dilutions of technical-grade insecticides specifically lambda-cyhalothrin and dichlorvos in acetone. These solutions are then uniformly applied to standard Whatman filter papers using a simple, reproducible pin-and-foam support system. Subsequent bioassays were conducted following established WHO tube procedures on field-collected Culex mosquitoes. Results from these tests proved to be both consistent and reliable, effectively differentiating between susceptible and resistant mosquito populations. Exposure to high, diagnostic concentrations (0.7% lambda-cyhalothrin and 7.0% dichlorvos) resulted in mortality rates at or above 98%, confirming susceptibility. In contrast, exposure to lower concentrations (0.4% lambda-cyhalothrin and 3.0% dichlorvos) yielded significantly reduced mortality rates of 52.63% and 66.66%, respectively, clearly indicating resistance. Control mortality remained below 5%, validating the assay conditions. This protocol offers a viable, affordable, and accessible alternative to proprietary test kits. It empowers local laboratories to conduct independent, routine resistance monitoring, thereby facilitating prompt, evidence-based decisions for vector control, especially in settings where standard commercial kits are unavailable or impractical.