A closer look at the WHO cone bioassay: video analysis of the hidden effects of a human host on mosquito behaviour and insecticide contact

Abstract

Background The WHO cone test is one of three tests currently used to evaluate the efficacy of insecticide-treated bed nets (ITNs). It generates two test outputs, knockdown and 24-h mortality, both indicative of immediate toxicity but that reveal little about the nature of mosquito and ITN interaction or how results translate to real-world settings. Methods A human arm held 5 mm behind the net surface acted as a host attractant during cone tests and a smartphone was used to capture mosquito behaviour in the cone. Post-exposure blood feeding and survival for nine days were recorded; ingested blood meal size was determined by measuring excreted haematin. Four strains of Anopheles gambiae (insecticide susceptible: Kisumu and N’gousso; insecticide resistant: Banfora and VK7) were tested with and without the host attractant using untreated, Permanet 2.0 and Olyset nets. Video recordings were scan sampled every five seconds to record mosquito positions on either the net, in flight or in contact with the cone. Generalized estimating equations were used to analyse all data except survival within nine days which was analysed using Weighted Cox Regression. Results Net contact was the most frequently recorded behaviour in all Anopheles spp. strains on all nets. Adding the human host as attractant triggered excitatory behaviours: in all strains, the magnitude of net contact was significantly decreased compared to tests without a host. ITN exposure altered the observed behaviour of the two susceptible strains, which exhibited a decreased response to the host during ITN tests. The resistant strains did not alter their behaviour during ITN tests. Significantly less net contact was observed during Olyset Net tests compared to Permanet 2.0. The host presence affected survival after exposure: Banfora and VK7 mosquitoes exposed to Permanet 2.0 with a host lived longer compared to tests performed without a host. However, mosquitoes that blood-fed and survived long enough to digest the blood meal did not exhibit significantly reduced longevity regardless of the presence of the host attractant. Conclusions Simple modifications to the WHO cone test and extension of post-test monitoring beyond the current 24 h enable detailed behavioural characterizations of individual ITNs to be compiled. The effects observed from testing with a host and including blood feeding suggest that more representative estimates of true of ITN efficacy are gained with these modifications than when using the current testing protocol.

Impacts of dual active-ingredient bed nets on the behavioural responses of pyrethroid resistant Anopheles gambiae determined by room-scale infrared video tracking

“…All video analysis was carried out using bespoke software written in Matlab (Mathworks) developed by collaborators at Warwick University [ 25 ]. Video segmentation, then compression to.mp4 files was performed before all videos were manually reviewed and cleaned to remove false tracks and human movement using ‘Sequential File Processing’ software [ 26 ]. Data extracted includes trajectory duration, distance travelled the number, duration and location of contacts with the bed net, time to first contact and track velocity, all of which have been previously described by Parker et al .…”

“…Additional track joining and the deletion of false tracks created by volunteer and camera noise was performed in ‘Post Processing’. Activity was categorized into behavioural modes (Table 2 ) using existing quantification algorithms [ 26 ] and recorded as occurring in one of ten non-overlapping regions of the bed net. Since many mosquitoes were released into the room in all tests, tracking individual mosquitoes was not possible, hence analysis was performed on flight tracks with each track from entry into and exit out of the field of view analysed separately.…”

“…The recording field of view was divided into 16 regions as previously described [ 26 ]. Ten of these regions were on the net surface; six on top of the bed net, two on the front of the net and one at either side.…”

A bioassay method validation framework for laboratory and semi-field tests used to evaluate vector control tools

“…1 ) is also described. Case study 2 (Additional file 4 ) contains the study design for the external validation of the VCT PLUS , an adaptation of the WHO cone test that is used to quantify mosquito behavioural responses to ITNs [ 79 ]. The process contains replication and comparison sub-studies and data analyses based on precision and to the 24 h mortality agreement between the VCT PLUS and WHO cone tests using the Bland–Altman plot.…”

A bioassay method validation framework for laboratory and semi-field tests used to evaluate vector control tools

“…Case Study 1 (Additional le 3) contains a description and worked example of the commercial validation of the Insecticide Quanti cation Kit, a test used to quantify residues applied to wall surfaces during IRS[57, 58]. Case study 2 (Additional le 4) contains the study design for the external validation of the ViCTA Plus, an adaptation of the WHO cone test that is used to quantify mosquito behavioural responses to ITNs[59].…”