Background: Millions of pyrethroid LLINs have been distributed in Mali during the past 20 years which, along with agricultural use, has increased the selection pressure on malaria vector populations. This study investigated pyrethroid resistance intensity and susceptible status of malaria vectors to alternative insecticides to guide choice of insecticides for LLINs and IRS for effective control of malaria vectors. Methods: For 3 years between 2016 and 2018, susceptibility testing was conducted annually in 14–16 sites covering southern and central Mali. Anopheles gambiae (s.l.) were collected from larval sites and adult mosquitoes exposed in WHO tube tests to diagnostic doses of bendiocarb (0.1%) and pirimiphos-methyl (0.25%). Resistance intensity tests were conducted using CDC bottle bioassays (2016–2017) and WHO tube tests (2018) at 1×, 2×, 5×, and 10× the diagnostic concentration of permethrin, deltamethrin and alpha-cypermethrin. WHO tube tests were conducted with pre-exposure to the synergist PBO followed by permethrin or deltamethrin. Chlorfenapyr was tested in CDC bottle bioassays at 100 µg active ingredient per bottle and clothianidin at 2% in WHO tube tests. PCR was performed to identify species within the An. gambiae complex. Results: In all sites An. gambiae (s.l.) showed high intensity resistance to permethrin and deltamethrin in CDC bottle bioassay tests in 2016 and 2017. In 2018, the WHO intensity tests resulted in survivors at all sites for permethrin, deltamethrin and alpha-cypermethrin when tested at 10× the diagnostic dose. Across all sites mean mortality was 33.7% with permethrin (0.75%) compared with 71.8% when pre-exposed to PBO (4%), representing a 2.13-fold increase in mortality. A similar trend was recorded for deltamethrin. There was susceptibility to pirimiphos-methyl, chlorfenapyr and clothianidin in all surveyed sites, including current IRS sites in Mopti Region. An. coluzzii was the primary species in 4 of 6 regions. Conclusions: Widespread high intensity pyrethroid resistance was recorded during 2016–2018 and is likely to compromise the effectiveness of pyrethroid LLINs in Mali. PBO or chlrofenapyr LLINs should provide improved control of An. gambiae (s.l.). Clothianidin and pirimiphos-methyl insecticides are currently being used for IRS as part of a rotation strategy based on susceptibility being confirmed in this study.

BackgroundFollowing agricultural use and large-scale distribution of insecticide treated nets (ITNs), malaria vector resistance to pyrethroids is widespread in sub-Saharan Africa. Interceptor® G2 is a new dual active ingredient (AI) ITN treated with alpha-cypermethrin and chlorfenapyr for the control of pyrethroid-resistant malaria vectors. In anticipation of these new nets being more widely distributed, testing was conducted to develop a chlorfenapyr susceptibility bioassay protocol and gather susceptibility information. MethodsBottle bioassay tests were conducted using five concentrations of chlorfenapyr at 12.5, 25, 50, 100 and 200µg AI/bottle in ten countries in sub-Saharan Africa using 13,639 wild collected An. gambiae s.l. (56 vector populations per dose) and 4,494 pyrethroid susceptible insectary mosquitoes from 8 colonized strains. In parallel, susceptibility tests were conducted using a provisional discriminating concentration of 100µg AI/bottle in 16 countries using 23,422 wild collected pyrethroid resistant An. gambiae s.l. (259 vector populations). Exposure time was 60 minutes, with mortality recorded at 24, 48 and 72 hours after exposure. ResultsMedian mortality rates (up to 72h after exposure) of insectary colony mosquitoes was 100% at all five concentrations tested, but the lowest dose to kill all mosquitoes tested was 50µg AI/bottle. The median 72h mortality of wild An. gambiae s.l. in 10 countries was 71.5%, 90.5%, 96.5%, 100% and 100% at concentrations of 12.5, 25, 50, 100 and 200µg AI/bottle, respectively. Log-probit analysis of the five concentrations tested determined that the LC95 of wild An. gambiae s.l. was 67.9µg AI/bottle (95% CI: 48.8-119.5). The discriminating concentration of 203.8µg AI/bottle (95% CI: 146-359) was calculated by multiplying the LC95 by three. However, the difference in mortality between 100 and 200µg AI/bottle was minimal and large-scale testing using 100µg AI/bottle with wild An. gambiae s.l. in 16 countries showed that this concentration was generally suitable, with a median mortality rate of 100% at 72h.ConclusionsThis study determined that 200µg AI/bottle chlorfenapyr in bottle bioassays is the most suitable discriminating concentration for monitoring susceptibility of wild An. gambiae s.l., using mortality recorded up to 72h. Testing in 16 countries in sub-Saharan Africa demonstrated vector susceptibility to chlorfenapyr, including mosquitoes with multiple resistance mechanisms to pyrethroids.

Background: Millions of pyrethroid LLINs have been distributed in Mali during the past 20 years which, along with agricultural use, has increased the selection pressure on malaria vector populations. This study investigated pyrethroid resistance intensity and susceptible status of malaria vectors to alternative insecticides to guide choice of insecticides for LLINs and IRS for effective control of malaria vectors.Methods: For 3 years between 2016 and 2018, susceptibility testing was conducted annually in 14-16 sites covering southern and central Mali. Anopheles gambiae s.l. were collected from larval sites and adult mosquitoes exposed in WHO tube tests to diagnostic doses of bendiocarb (0.1%) and pirimiphos-methyl (0.25%). Resistance intensity tests were conducted using CDC bottle bioassays (2016-17) and WHO tube tests (2018) at 1×, 2×, 5×, and 10× the diagnostic concentration of permethrin, deltamethrin and alpha-cypermethrin. WHO tube tests were conducted with pre-exposure to the synergist PBO followed by permethrin or deltamethrin. Chlorfenapyr was tested in CDC bottle bioassays at 100µg active ingredient per bottle and clothianidin at 2% in WHO tube tests. PCR was performed to identify species within the An. gambiae complex.Results: In all sites An. gambiae s.l. showed high intensity resistance to permethrin and deltamethrin in CDC bottle bioassay tests in 2016 and 2017. In 2018, WHO intensity tests resulted in survivors at all sites for permethrin, deltamethrin and alpha-cypermethrin when tested at 10× the diagnostic dose. Across all sites mean mortality was 33.7% with permethrin (0.75%) compared with 71.8% when pre-exposed to PBO (4%), representing a 2.13 fold increase in mortality. A similar trend was recorded for deltamethrin. There was susceptibility to pirimiphos-methyl, chlorfenapyr and clothianidin in all surveyed sites, including current IRS sites in Mopti Region. An. coluzzii was the primary species in 4 of 6 regions.Conclusions: Widespread high intensity pyrethroid resistance was recorded during 2016-18 and is likely to compromise the effectiveness of pyrethroid LLINs in Mali. PBO or chlrofenapyr LLINs should provide improved control of An. gambiae s.l. Clothianidin and pirimiphos-methyl insecticides are currently being used for IRS as part of a rotation strategy based on susceptibility being confirmed in this study.

Background The effectiveness of long-lasting insecticidal nets (LLINs) mainly relies on their physical integrity, an important indicator of the LLINs durability in households. The present study aims to assess this physical integrity for three widely used LLIN products: DawaPlus®2.0, PermaNet®2.0 (polyester) and DuraNet® (polyethylene), distributed in Benin during the mass campaign of 2014. Methods The study was conducted from October 2014 to June 2017 in three districts of Benin: Tori-Bossito, Toffo, and Ouesse. Nine hundred LLINs (a cohort of 300 LLINs per product) in use and found hanging on sleeping materials were selected and tagged. Every six months, the LLIN attrition and their physical condition was monitored. The holes were counted by category allowed to calculate the proportionate hole index (pHI) and classify the LLINs into "good ", "serviceable" or "torn" categories. Factors associated with loss of integrity have also been identified following World Health Organization Guidelines. Results After 30 months of use, 55.9% (n= 503; 95% CI: 52.6-59.2) of the LLINs were lost. Attritions due to physical damage were similar between the LLIN products and were respectively 28.3% for PermaNet® 2.0, 29.7% for DawaPlus® 2.0, and 31% for DuraNet® (p˃0.05). The mean pHI was significantly higher for DuraNet® LLINs (pHI= 1431) than DawaPlus® 2.0 (pHI =366) and PermaNet® 2.0 (pHI =321) (<0.001). A significant difference was also observed between the proportion of LLINs in “torn” condition and included 8.7% of PermaNet® 2.0, 14.3% of DawaPlus® 2.0, and 34.1% of DuraNet® (p <0.001). The use of LLINs every night, the frequency of washing and other factors were significantly associated with the physical damage of the LLINs (p <0.001). Conclusion These results suggest that the physical barrier conferred by the LLIN can be significantly affected during the normal course of its use depending on the type of product distributed. National malaria programs must, therefore, consider the physical integrity performance in local conditions in the choice of LLIN product to be distributed. Keywords: LLINs, physical integrity, pHI, malaria, Benin.