Anopheles gambiae s.s. was the dominant malaria vector in Sierra Leone followed by An. coluzzii. Consistent with previous studies, they were the only species sampled in larval habitats [13]. The main malaria vector in Sierra Leone, An. gambiae s.l., was highly resistant to the three pyrethroid insecticides tested with partial restoration of susceptibility following pre-exposure to PBO. The presence of survivors following exposure to 10x the discriminating doses is indicative of the high intensity of resistance to pyrethroids. The observed range of enhancement of mortality due to pre-exposure to PBO was similar across the three pyrethroids tested.
This assessment also demonstrated the kdr L1014F resistance mutation to be present at high frequency, which is not surprising, considering the high level of phenotypic resistance to pyrethroids. Though not frequently identified in previous samples, this report is a first of kdr L1014S and N1575Y mutations in Sierra Leone. The N1575Y mutation, located in the Vgsc and believed to have a synergistic effect on pyrethroid and DDT resistance when combined with the L1014F mutation was previously reported from Burkina Faso, Benin, Cameroon and Côte d’Ivoire [31, 33–35].
The detection of the N1575Y mutation in Sierra Leone requires more investigation to better characterize its expected synergistic relationship with 1014F kdr. The presence of this additional mechanism that could further reduce insecticide efficacy in the already pyrethroid-resistant mosquitoes in Sierra Leone is concerning. Thus, monitoring of N1575Y should continue in order to understand its contribution to insecticide resistance among local vectors. This resistance mechanism could spread very rapidly [31] and threaten the malaria vector control efforts in Sierra Leone which had relied primarily on the distribution of pyrethroid-treated ITNs. This finding also provides evidence for justifying the need to move to the distribution of non-pyrethroid nets.
As the effect of PBO exposure was similar across the three pyrethroids tested, nets treated with any of the pyrethroids plus PBO might have similar effects and can be used as alternative tools in the national Sierra Leone Insecticide Resistance Management Plan. Indeed, NMCP distributed PermaNet 3.0 (deltamethrin + PBO) and Olyset Plus (permethrin + PBO) in the 2020 mass net campaign and these contributed to malaria reduction in Sierra Leone [36]. These data not only yielded the evidence that PBO nets could provide better protection than pyrethroid nets without PBO in Sierra Leone, but also demonstrated that in the absence of non-pyrethroid ITNs available at the time of this assessment, Sierra Leone had the flexibility to choose PBO ITNs treated with any of the three pyrethroids. Sierra Leone was the world’s first country to provide population-level coverage nationally with next-generation PBO ITNs during a mass distribution campaign and evaluations to measure their durability and performance are underway. The NMCP has now incorporated these data and subsequent data on insecticide resistance to procure IG2 nets for 2023 mass ITN campaign.
Anopheles gambiae s.l. was susceptible to pirimiphos-methyl (organophosphate), chlorfenapyr (pyrrole) and clothianidin (neonicotinoid). However, there was an indication of possible resistance to bendiocarb (carbamate). As the NMCP prepared to implement IRS in Bo and Bombali districts in May/June 2021 at the start of the rainy season, the findings from this assessment provided critical data that guided the selection of an appropriate insecticide for the IRS program. Thus, clothianidin was selected for IRS because vectors were fully susceptible to it. There was no phenotypic resistance to pirimiphos-methyl and low-level resistance to bendiocarb was reported. However, carbamates and organophosphates are known to share the Ace-1 pathway as a resistance mechanism [37]. With the reported phenotypic resistance to bendiocarb and the reported prevalence of the G119S mutation at 13.6% in the vector population, this mutation needs close monitoring to guide future IRS in Sierra Leone. Moreover, the combination of IRS using pirimiphos-methyl and PBO nets have been suggested to be antagonistic [38]. Thus, with the mass distribution of PBO nets that occurred in Sierra Leone in 2020 and continued PBO ITN distribution through some routine channels, and considering this potential antagonism between vector control interventions, caution should be used in the selection of pirimiphos-methyl for IRS. The susceptibility of the vector to chlorfenapyr also suggests that its use for IRS or as part of ITN treatment is a possibility when it becomes available.
The presence of three or more Ace-1 alleles in An. gambiae s.s. mosquitoes was previously documented in several countries in West Africa [39–41]. However, prior to the present assessment, little information was available for Sierra Leone. Although the G119S mutation is indicative of the phenotypic resistance to bendiocarb in this study, the high number of wild types that survived exposure to bendiocarb indicates involvement of other resistance mechanisms in Sierra Leone. The relatively low frequency of the G119S mutation (0.14; N = 26) was consistent with the low phenotypic resistance to bendiocarb and full susceptibility to pirimiphos-methyl observed in this assessment.
The absence of the G119S mutation in An. coluzzii cannot be explained by geographic variation, as both species were sympatric and collected from the same breeding sites. This finding is different from previous reports from West Africa, which observed this mutation in higher frequencies in both species: 0.32 in An. gambiae s.s. and 0.04 in An. coluzzii from Burkina Faso [40], 0.31 in An. gambiae s.s. and 0.35 in An. coluzzii from Côte d’Ivoire [42, 43], and 0.24 in An. gambiae s.s. and 0.04 in An. coluzzii from Ghana [39]. The G119S mutation has been associated with a high fitness cost in Culex pipiens populations [37, 44]. Indications of a high fitness cost were also similar in An. gambiae s.s. as the frequency of the Ace-1 mutation in mosquito populations was observed to decline rapidly after a few generations without selection pressure from organophosphates or carbamates [40, 45]. In Burkina Faso, an excess of heterozygous genotypes in S form populations likely indicated that a fitness cost is associated with the mutation when present in a homozygous form. However, the findings from this assessment were different, such that all the Ace-1 G119S mutations were detected in the homozygous form with complete deficiency of heterozygotes and in significant deviation from the Hardy-Weinberg equilibrium. Nevertheless, the phenomenon of heterozygote deficiency in the G119S mutation is not new, as other studies have reported such a deficit in some sites in Cameroon [41] but does suggest a need for further exploration.
In the study by Elanga-Ndille et al. [41] in Cameroon, each sequenced individual specimen possessed at least two distinct Ace-1 resistant alleles and one susceptible allele. This was a possible explanation for why most mosquitoes that remained alive after carbamate exposure were genotyped as homozygote resistant with a lack of heterozygotes: mosquitoes with two copies of the gene seemed to have three resistant alleles vs. only one susceptible allele. It is hypothesized that this duplication decreases the fitness cost associated with the resistant genotype [45, 46] thereby hindering carbamate-based vector control strategies [35]. Since no mosquitoes survived the pirimiphos-methyl bioassays and the few that survived the bendiocarb exposure were not tested separately, we were unable to investigate the link between the G119S mutation and phenotypic resistance to carbamates and organophosphates. However, six of the 13 mosquitoes from the bioassay with bendiocarb were carrying the Ace-1 G119S mutation homozygous allele.
The Ace-1 G119S mutation (associated with a low level of resistance to carbamate insecticides) was also present in An. gambiae s.s. but not in An. coluzzii. The G119S Ace-1 mutation was not associated with pirimiphos-methyl resistance. However, further bioassays and screening for the Ace-1 resistance allele on a wider scale would be required to understand the implications of the current status of the Ace-1 mutation for the efficacy of organophosphate insecticide use in vector control in Sierra Leone.