Nationwide assessment of pyrethroid resistance escalation and investigation of its molecular basis in Anopheles funestus from Cameroon

Background : Malaria prevention still relies greatly on vector control interventions. However, increasing levels of resistance to pyrethroids across Africa have significantly reduced the efficacy of pyrethroid-based interventions leading to a stagnation and/or an increase of malaria burden. Consequently, it is imperative to nation-widely characterize the extent and molecular basis of this resistance. Methods : This study was conducted from 2020 to 2021 in a South-North transect across Cameroon to assess patterns of resistance to insecticides in Anopheles funestus, establish its impact on efficacy of insecticide-treated nets and investigate underlying molecular mechanisms. Taqman assay and nested PCR were used to determine Plasmodium sporozoite prevalence. Genotyping of known genetic markers as well as species identification was done using PCR technique while quantitative PCR was used to determine gene expression. Results: High pyrethroid resistance intensity was noticed in all sites with mortalities ranging from 80-93.9%, 84.9-96.7% and 82% for permethrin, deltamethrin and alphacypermethrin at 10x concentration respectively. This high level of resistance led to dramatic inefficacy of pyrethroid-only nets with 0-17% mortality recorded 24-hour post exposure while PBO-based nets displayed optimal efficacy. The L119F- GSTe2 allele was higher in the South (56-68%) compared to the North (20-37%) meanwhile the P450-linked 4.3kb structural variant was fixed contrasting with the absence of the CYP6P9a/b-R , 6.5kb insertion and N485I- Ace1 alleles. Furthermore, the L119F- GSTe2 allele confers significant ability to mosquito to survive permethrin. In addition, the CYP325A, CYP6P5, CYP6P9a/b, and the Carb2514 were the most overexpressed genes in pyrethroid resistant mosquitoes. However, no further association was noticed between these alleles/genes and increasing doses of pyrethroids. Conclusion : This study confirms the escalation of pyrethroid resistance across Cameroon and the inefficacy of pyrethroid-only nets and highlights genes potentially implicated in the aggravation of insecticide resistance.