Study sites
As part of the PMI VectorLink project, insecticide resistance monitoring is conducted annually in partner countries in sub-Saharan Africa with commonly used insecticides for malaria vector control. In anticipation of SumiShield™ 50WG (November, 2017) and Fludora Fusion™ WP-SB (December, 2018) receiving WHO PQ listing, PMI VectorLink entomologists and partners collected baseline data to determine vector susceptibility status to clothianidin. Each of the 16 countries conducted testing in at least one location using field collected Anopheles mosquitoes along with an insectary reared susceptible strain. Figure 1 shows the location of sites in West and Central Africa and Fig. 2 shows sites in East and Southern Africa where clothianidin susceptibility bioassays were conducted.
Preparation of insecticide-treated papers
Insecticide susceptibility tests are normally conducted using pre-treated filter papers that are prepared by a WHO collaborating institution (Universiti Sains, Malaysia) and distributed to field sites [26]. Diagnostic doses are determined by WHO through testing of multiple mosquito species in different countries. However, as of 2019 there was no published guidance from WHO regarding clothianidin susceptibility test procedures or diagnostic concentrations. Therefore, a protocol was developed and optimized by Sumitomo Chemical Company (SCC). SCC conducted WHO cylinder tests to determine a suitable solvent and diagnostic dose for clothianidin susceptibility tests. A dose range of 0.125%, 0.25%, 0.5%, 1%, 2% and 4% w/v (weight per volume) clothianidin active ingredient were tested in WHO cylinder tests against 6 insectary strains of An. gambiae and Anopheles arabiensis. Acetone, hexane and water were tested as solvents. Results showed that both hexane and water were suitable solvents (poor results were obtained using acetone) for evaluating clothianidin in WHO cylinder tests and that 1% w/v clothianidin provided 100% mortality against all 6 strains (including pyrethroid resistant strains). The same result was observed using either technical grade clothianidin or formulated SumiShield™ 50WG. The diagnostic dose was set at 2% w/v clothianidin (i.e. twice the minimum dose that killed 100%), with water or hexane as suitable solvents (Ohashi, pers. commun.).
Therefore, in each site, filter papers were treated in situ with 2% w/v clothianidin (equivalent of 734 mg/m2), by treating each filter paper with 26.4 mg of SumiShield™ 50WG, diluted in distilled water. SumiShield™ 50WG was used instead of technical grade material, due to availability of formulated product. However, a limitation of using formulated material is greater batch-to-batch variation than technical grade active ingredient. Whatman filter papers (grade 1) were cut to 12 cm by 15 cm to be the same size as WHO filter papers used in tube tests. An insecticide solution was prepared by adding 264 mg of SumiShield™ 50WG granules to 20 ml distilled water in a Falcon™ tube and shaking until fully dissolved. The filter paper was supported on a bed of nails (which were hammered into a piece of wood at equal heights) during insecticide application, to allow for even absorption into the filter paper. A pipette was used to transfer 2 ml of insecticide solution evenly onto the filter paper by carefully dispensing rows of droplets until there were no dry sections of the filter paper at a target dosage of 13.2 mg/active ingredient per paper (see video [27]). Treated filter papers were left in a dark cupboard to dry overnight. As the stability of SumiShield™ 50WG on filter papers has not been established, all tests were conducted within 24 h of treating the papers.
Insecticide susceptibility tests
Insecticide susceptibility tests were conducted according to established WHO protocols, with modifications made to the holding period [26]. In general, a total of 100 (depending on availability) An. gambiae s.l. or Anopheles funestus s.l. were exposed for 60 min in 4 replicates of 25 mosquitoes, with an additional 1 or 2 replicates of 25 mosquitoes used for the negative control (paper treated with distilled water). Results for the negative control bioassays are presented in supplementary file 1. After exposure, mosquitoes were transferred to clean holding tubes and provided with sugar solution. Mortality was recorded every 24 h after exposure for a maximum of 7 days, or until 100% mortality was reached. Tests were conducted in the morning and holding conditions were intended to be within WHO guidelines of 27 °C ± 2 °C and relative humidity of 75% ± 20%. Temperature and humidity were monitored, however, in most cases could not be accurately controlled, as tests with wild collected mosquitoes were generally conducted under field conditions. A summary of max/min temperature and humidity is available as Additional file 1: Table S1.
Mosquito species tested
Tests were conducted using insectary-reared susceptible Anopheles as well as wild collected malaria vector species. Insectary-reared mosquitoes included susceptible An. gambiae sensu stricto (s.s.) Kisumu (Burkina Faso, Burundi, Ghana, Nigeria, Tanzania, Uganda, Zambia), Anopheles coluzzii Yaoundé (DR Congo, Mali, Senegal) and An. arabiensis KGB (Ethiopia and Zimbabwe), based on previous molecular analysis as part of routine insectary colony checks. Insectary colonies were not tested in Kenya, Liberia, Madagascar, and Mozambique.
Larval collections were made at various times between September 2016 and December 2017 for each study site using larval dippers. Larvae were subsequently transported to a field insectary for rearing, usually using field collected water, with larvae fed using Tetramin® fish food. Emerging adult mosquitoes were provided with sugar solution until they were used in insecticide susceptibility tests when aged 2 to 5 days. Wild Anopheles were identified morphologically as An. gambiae s.l. in 14 of 16 countries, and wild caught F0 An. funestus s.l. adults of unknown age were tested in Mozambique and Zambia. Molecular species analysis of wild mosquitoes tested was not conducted.
Data analysis
Insecticide susceptibility results were presented as percentage mortality every 24 h after bioassay exposure for a maximum of 7 days. If negative control mortality was greater than 20%, the data was discarded and tests repeated. Control mortality results are presented for all tests in Additional file 1: Table S2. WHO criteria were used for interpretation, with 98–100% mortality indicating susceptibility; 90–97% indicating possible resistance, with resistance genes to be confirmed; and mortality < 90% indicating resistance [28]. PoloPlus (LeOra Software) was used to conduct probit analysis on the logarithmic scale to calculate the time taken for a fixed concentration of clothianidin to kill a defined proportion of insects, known as lethal time (LT). For example, the LT50 is the lethal time predicted for 50% mortality to be reached.