Complex non-falciparum transmission and widespread insecticide resistance in Penka-Michel, a hyperendemic malaria focus of the Cameroon highlands

A 12-month longitudinal entomological study (September 2023-August 2024) in the Western Highlands of Cameroon (Penka-Michel) investigated malaria transmission dynamics and insecticide resistance markers, including knockdown resistance mutations (L1014F, N1575Y) and metabolic resistance genes (G119S- Ace1 , E205D -CYP6P3 , L119F -GSTe2 , CYP6P9a/b ). Malaria transmission has depended on the coexistence of three species: Anopheles funestus (43.7%), Anopheles gambiae (42.9%), and Anopheles ziemanni (13.5%). The site was hyperendemic with annual EIR of 839.5 infective-bites/human/night. Plasmodium falciparum predominated (70.2%), yet a significant non- falciparum burden was observed ( P. malariae , 27.5%; P. ovale , 22.1%). An. funestus was the principal driver (IR = 7.8%, EIR = 0.9 infective bites/human/night), with P. falciparum and P. ovale curtisi peaking in the later rainy season (November), while An. gambiae (IR = 5.4%; EIR = 0.6 ib/h/n) vectored P. malariae (14.5%) primarily, followed by a sustained peak in the dry season (January). Widespread insecticide resistance was observed, with resistant homozygotes detected in 70% of infected mosquito samples. We observed associations of L119F-GSTe2 mutations with P. ovale curtisi transmission (IRR = 1.15, p  = 0.03), and L1014F-kdr with P. malariae (IRR = 2.2, p  = 0.04). These dynamics highlight a complex multi-species system in which seasonal vector efficiency and insecticide resistance sustain hyperendemicity of the often-overlooked non- falciparum malaria species.