Anopheles gambiae (s.l.) exhibit high intensity pyrethroid resistance throughout Southern and Central Mali (2016-2018): PBO or next generation LLINs may provide greater control

Abstract

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 chlorfenapyr 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.

Keywords: Anopheles gambiae; CDC bottle bioassay; Indoor residual spraying; Long-lasting insecticidal net; Mali; Piperonyl butoxide; Resistance intensity; Susceptibility test; Vector control; WHO tube test.

Fig. 1

Map showing the insecticide monitoring sites in Mali

Fig. 2

Percent mortality of An. gambiae (s.l.) after 30 min exposure to 10× the diagnostic concentration of permethrin (215 µg ai/bottle) in CDC bottle bioassays in 2016 and 2017. Abbreviation: NA, no data

Fig. 3

Percent mortality of An. gambiae (s.l.) after 30 min exposure to 10× the diagnostic concentration of deltamethrin (125 µg ai/bottle) in bottle bioassays in 2016 and 2017. Abbreviation: NA, no data

Fig. 4

Percent mortality of An. gambiae (s.l.) tested in WHO tube tests using 1× (0.05%), 5× (0.25%) and 10× (0.50%) the diagnostic concentration of alpha-cypermethrin in 2018

Fig. 5

Percent mortality of An. gambiae (s.l.) tested in WHO tube tests using 1× (0.75%), 5× (3.75%) and 10× (7.5%) the diagnostic concentration of permethrin in 2018

Fig. 6

Percent mortality of An. gambiae (s.l.) tested in WHO tubes using 1× (0.05%), 5× (0.25%) and 10× (0.5%) the diagnostic concentration of deltamethrin in 2018

Fig. 7

Percent mortality (24 h) of An. gambiae (s.l.) tested with permethrin (0.75%) alone and after pre-exposure to PBO (4%) synergist using WHO tube tests (bars for the same site sharing the same superscript letter (a or b) do not differ significantly, P > 0.05)

Fig. 8

Percent mortality (24 h) of An. gambiae (s.l.) tested with deltamethrin (0.05%) alone and after pre-exposure to PBO (4%) synergist using WHO tube tests (bars for the same site sharing the same superscript letter (a or b) do not differ significantly, P > 0.05). Abbreviation: NA, no data

Fig. 9

Results of An. gambiae (s.l.) (field-collected as larvae) and An. coluzzii Ngousso (susceptible insectary strain) susceptibility tests against chlorfenapyr (100 µg ai/bottle) in 2017

Fig. 10

Mortality of An. gambiae (s.l.) (collected as larvae) from four IRS sites tested against clothianidin 2% in WHO tube tests in 2018

Fig. 11

Percent mortality of An. gambiae (s.l.) in WHO tube test (24 h mortality) and CDC bottle bioassay (30 min mortality) to permethrin and deltamethrin in Koulikoro and Niono. Statistical comparison was made comparing results of WHO tube tests and CDC bottle bioassay by site and dose tested (bars for the same site sharing the same superscript letter (a or b) do not differ significantly, P > 0.05). Abbreviation: NA, no data (2× dose not tested for WHO tube test)

Fig. 12

An. gambiae (s.l.) sibling species composition and vgsc-1014F and vgsc-1014S frequency in the six surveyed regions in 2017 (top) and 2018 (bottom)