An. gambiae s.l. intensity of resistance to permethrin and deltamethrin in CDC bottle bioassays in 2016 and 2017
Figure 2 shows mortality rates of An. gambiae s.l. following 30 minutes exposure to permethrin 10× (215µg ai/bottle) and figure 3 for deltamethrin 10× (125µg (ai)/bottle). More data are provided showing results of 1×, 2× and 5× as a supplementary file (Table S1). In all sites An. gambiae s.l. showed high intensity resistance to permethrin and deltamethrin in 2016 and 2017. Overall, mortality rates to permethrin 10× ranged from 28% to 93% in 2016 and from 35% to 85% in 2017 (Figure 2). With deltamethrin 10×, mortality rates varied from 53% to 91% in 2016 and from 72% to 97% in 2017 (Figure 3). Mean trends showed there may have been a slight increase in permethrin resistance intensity in 2017 compared to 2016, but for deltamethrin trends were similar in 2016 and 2017.
An. gambiae s.l. intensity of resistance to alpha-cypermethrin, permethrin and deltamethrin in WHO tube tests in 2018.
Figures 4, 5 and 6 show resistance intensity results for An. gambiae s.l. against alpha-cypermethrin, permethrin, and deltamethrin at 1×, 5× and 10× the WHO diagnostic concentration. High intensity resistance was recorded in all sites to alpha-cypermethrin (mortality <98% at 10× dose) (Figure 4). The mean % mortality across all sites for alpha-cypermethrin was 24.8% at 1×, 56.9% at 5× and 79.2% at 10× the diagnostic concentration.
Resistance intensity was also high at all sites to permethrin and deltamethrin (Figures 5 and 6). The mean % mortality across all sites for permethrin was 33.6% at 1×, 76.9% at 5× and 90.9% at 10×. For deltamethrin the mean % mortality was 49.2% at 1×, 80.9% at 5× and 93.5% at 10×. Crude mortality was <98% in all sites, indicating high intensity resistance..
Synergist assays using piperonyl-butoxide (PBO) and pyrethroids
Results in Figures 7a and 7b show that overexpression of mixed function oxidases (MFOs) is an important resistance mechanism in Mali, as shown by significantly greater mortality rates after PBO pre-exposure. Figure 7a shows that pre-exposure to PBO resulted in significantly greater mortality than for permethrin alone, in 13 of 14 sites (P<0.05), with Koulikoro the only site where there was no apparent response to PBO. Across all sites mean mortality was 33.7% with permethrin compared with 71.8% when pre-exposed to PBO, representing a 2.13 fold increase in mortality (P<0.0001).
Figure 7b shows that for all 14 sites there was a significant increase in mortality caused by deltamethrin following PBO pre-exposure. Although significantly increased mortality rates were obtained in nearly all sites for both insecticides after pre-exposure of An. gambiae s.l. to PBO, susceptibility was not fully restored in any sites. Mortality levels did increase to >90% in two sites with PBO + permethrin (Bamako and Bla) and four sites (Niono, Bougouni, Bankass and Bamako) with PBO + deltamethrin. Across all sites mean mortality was 48.2% with deltamethrin compared with 81.3% when pre-exposed to PBO, representing a 1.69 fold increase in mortality (P<0.0001).
Susceptibility of An. gambiae s.l. to pirimiphos-methyl (0.25%) in WHO susceptibility tube tests in 2016, 2017and 2018
In all years, susceptibility (mortality rate ≥98%) to pirimiphos-methyl (0.25%) was observed in all sites where pirimiphos-methyl CS has previously been sprayed for malaria control, including Koulikoro, Baroueli, Djenné, Bandiagara and Bankass. Susceptibility was also recorded in all other sites, except for Selingue, where possible resistance was noted in 2018 (96.7% mortality) (Table 2: supplementary file).
Susceptibility of An. gambiae s.l. to bendiocarb (0.1%) in WHO susceptibility tube tests in 2016, 2017and 2018
In 2016, susceptibility (mortality rate ≥98%) was obtained with bendiocarb (0.1%) in all sites except for Niono (95%) and Bougouni (92%), where possible resistance was observed. In 2017, susceptibility was observed in 5 sites (Fana, Koulikoro, Bla, Djenné and Bankass), with possible resistance (90-97% mortality) in 4 sites (Kita, Kati, Bamako and Bandiagara) and resistance (<90%) in 3 sites (Barouéli, Sélingué and Bougouni). In 2018, susceptibility was noted in all 6 sites where testing was conducted (Bla, Selingue, Bougouni, Djenné, Bandiagara and Bankass). Mortality rates to bendiocarb in the surveyed sites are summarized in Table 3: supplementary file.
Susceptibility of An. gambiae s.l. to chlorfenapyr (100 µg ai/bottle) in CDC bottle bioassays
Figure 8 displays mortality rates obtained 24, 48 and 72 hours after exposing An. gambiae s.l. from Djenné, Mopti, Bandiagara and Bankass to chlorfenapyr at 100µg ai/bottle in 2017. After 48 hours, susceptibility (mortality rate ≥ 98 percent) with both field and insectary An. coluzzii Ngousso (susceptible insectary strain) was determined at all sites, except Bandiagara. A. gambiae s.l. mortality was <98% in Bandiagara after 48h but did reach 98% at 72 hours (the diagnostic time). Therefore, susceptibility to chlorfenapyr was recorded in all sites within 72h of exposure.
Susceptibility of An. gambiae s.l. to clothianidin (2%) in WHO susceptibility tube tests in 2018
Figure 9 shows mortality rates following exposure to clothianidin 2% of An. gambiae s.l. (collected as larvae) from four IRS sites (Djenné, Mopti, Bandiagara and Bankass). Parallel tests were done with the same papers using the susceptible insectary strain of An. coluzzii Ngousso. Twenty-four hours after exposure, mortality rates were 90% for the insectary strain and between 44-90% for wild An. gambiae s.l. For the insectary strain, 99% mortality was observed three days after exposure, with mortality rates slightly lower for wild An. gambiae s.l. One hundred percent mortality was recorded for insectary and wild An. gambiae s.l., five days after exposure, indicating susceptibility to clothianidin in all four IRS sites. Mortality rates in negative controls were low and varied from 0% to 10% after five days.
Comparison of CDC bottle bioassays and WHO susceptibility tube tests for determining pyrethroid resistance intensity
Figure 10 shows percentage mortality of An. gambiae s.l. to permethrin (a) and deltamethrin (b) at doses of 1×, 2×, 5× and 10× the diagnostic concentration, using both bottle bioassays (30 minutes mortality) and WHO susceptibility tube tests (24h mortality) in Koulikoro and Niono in 2018. Both methods indicate high intensity pyrethroid resistance in Koulikoro and Niono (mortality <98% at 10×). Testing conducted in Niono consistently produced higher mortality rates for both permethrin and deltamethrin with WHO tube tests as compared to CDC bottle bioassays at all doses (p<0.05). In Koulikoro the 5× and 10× doses of permethrin produced higher mortality for permethrin in WHO tube tests than CDC bottle bioassay. However, there was only a difference at the 1× dose with deltamethrin in Koulikoro.
Molecular species identification of the An. gambiae species complex
In 2017 and 2018, adult An. gambiae s.l. specimens (collected as larvae) used for susceptibility tests from sentinel sites (approx. 50 mosquitoes/site) located in 6 regions (Figure 1), were tested by PCR for species identification. Figure 11 summarizes An. gambiae s.l. sibling species composition by region and L1014F/S frequency. An. coluzzii was the primary vector in 4 regions (Koulikoro, Ségou, Mopti and Bamako) in 2017 and 2018. In the southern region of Sikasso, slightly more than half were An. gambiae, with just over 40% being An. coluzzii in both years. Some hybrid samples (gambiae/coluzzii) were recorded at low frequency (≤2% by region).
In Kayes Region, the composition changed from predominantly An. gambiae in 2017 to An. arabiensis in 2018. However, the sites in the region changed during this period with Kita maintained in both years and the western Kayes site (Figure 1) only surveyed during 2018 (which accounted for most An. arabiensis). An. arabiensis was present at relatively low frequency in all other regions (2-10%). Between 3-24% of samples did not amplify by PCR using primers of the An. gambiae s.l. complex. Samples may not have amplified due to degraded DNA or due to morphological mis-identification (non An. gambiae s.l.).
The frequency of vgsc-1014F and 1014S alleles are summarized as tables in Figure 11 for the main vector species of each region. In 2017, the vgsc-1014S allele was absent in most regions, only being detected at a frequency of 0.01 in Koulikoro. The vgsc 1014F allele was present at moderate to high frequency in all regions for An. gambiae (0.47-0.95) than An. coluzzii (0.58-0.77).