Reduced bio-efficacy of aged PermaNet^® 3.0 Nets against local pyrethroid-resistant Anopheles arabiensis in Western Kenya

Background: Long-lasting insecticidal nets (LLINs) are central to malaria control and designed to protect for up to three years; however, increasing pyrethroid resistance undermines their effectiveness. Piperonyl butoxide (PBO) LLINs, designed to enhance pyrethroid efficacy, are recommended in high-resistance areas, yet their long-term operational bio-efficacy remains unclear. This study evaluated the killing efficacy of PBO LLINs over three years of field use compared to standard LLINs and monitored insecticide resistance in local Anopheles gambiae s.l. populations. Methods: During a trial in Muhoroni, western Kenya (2021–2024), standard and PBO LLINs were collected at 6, 18, and 36 months of use. Residual bio-efficacy was assessed using WHO cone bioassays against a susceptible Anopheles gambiae s.s. Kisumu strain and field An. gambiae s.l. populations. WHO tube and bottle assays determined insecticide resistance, while synergist assay assessed metabolic resistance. Quantitative polymerase chain reaction detected target-site mutations (kdr-1014 F/S and Ace-1 G119S). Results: Molecular identification confirmed all An. gambiae s.l. were Anopheles arabiensis, showing increasing pyrethroid resistance, with deltamethrin mortality declining from 96.3% (2021) to 22.7% (2024) while remaining susceptible to pirimiphos-methyl and clothianidin. PBO pre-exposure restored deltamethrin mortality from 22.7% to 98.9%. The frequency of 1014F increased from 0.09 to 0.17 and 1014S from 0.04 to 0.06, with no Ace-1 mutations detected. Standard LLINs retained >80% efficacy against the susceptible strain for 18 months but were below threshold against field mosquitoes even when new. PBO LLINs were effective at baseline against field populations but declined sharply, with mortality dropping to 24% by 6 months. Overall, both net types exhibited a marked decline in killing efficacy over time against field mosquitoes, with mortality falling < 20% within six months. Conclusion: Anopheles arabiensis showed increasing pyrethroid resistance, driven largely by metabolic mechanisms. Standard LLINs showed suboptimal killing efficacy against field populations, while PBO LLINs achieved high baseline efficacy but declined significantly by six months. These findings indicate that PBO LLINs can improve protection against resistant vectors but may be insufficient in high-resistance areas, underscoring the need for alternative non-pyrethroid interventions, strategic deployment, and revised LLIN re-distribution cycles aligned with their functional lifespan.