How important is the angle of tilt in the WHO cone bioassay?

The World Health Organization (WHO) cone bioassay plays an integral role in the evaluation of the efficacy of long-lasting insecticidal nets as well as insecticides used in indoor residual spraying. The test is used on a variety of treated substrates, such as pieces of bed nets, mud, cement and wood. The cone setup assumes a wide variety of angles under different settings in which it is applied. However, the guidelines provided for the performance of the assay do not specify the angle at which the test must be performed.; Laboratory colonies of Anopheles gambiae Kisumu-1 and Anopheles stephensi STI were tested in the WHO cone bioassay at four different angles (0°, 45°, 60° and 90°) following the WHO guidelines against net pieces of Olyset Plus and Netprotect. The tests were repeated after 20 washes of the nets. Individual mosquitoes were also exposed at 0° and 60° and the amount of time each spent in contact with the net was recorded.; Mosquitoes spent more time on the net at 60° as compared to 0° (coefficient = 45.8, 95 % CI 34.6-55.6, p < 0.001) and were more likely to die when the test was done at 45° (OR 3.3, 95 % CI 1.7-6.3, p = 0.001), 60° (OR 3.1, 95 % CI 1.7-5.9, p < 0.001) and 90° (OR 6.0, 95 % CI 1.9-18.5, p = 0.002) as compared to 0°.; The angle at which the test is performed significantly affects the amount of time mosquitoes spend resting on the nets, and subsequently mortality. Angle must thus be considered as an important component in the performance of the assay and duly incorporated into the guidelines.