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Chlorfenapyr bednets effectively overcome pyrethroid resistance escalation in highly resistant Anopheles malaria vectors in Uganda

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Abstract

Escalating insecticide resistance threatens the efficacy of LLINs, undermining malaria control in Africa. We conducted the first experimental hut trials in Uganda using highly resistant free-flying wild Anopheles mosquitoes and F2 hybrids of FANG and Uganda An. funestus to evaluate the performance of bednets. The interceptor G2 (chlorfenapyr) bednet demonstrated superior efficacy compared to Interceptor (pyrethroid-only) net [mortality odds ratio (OR): 18.7 (8.05–48.6) P < 0.0001], achieving an overall mortality rate of 70.6% and 63.2% against An. funestus and An. gambiae respectively. In contrast, PermaNet 3.0 and Olyset Plus (piperonyl butoxide (PBO)) and Royal Guard (pyriproxyfen (PPF)-treated) bednets exhibited significantly lower mortality against An. funestus [Olyset Plus: 36.1%, PermaNet 3.0: 31.0% and Royal Guard (37.6%], though performance against An. gambiae was moderate [PermaNet 3.0: 61.4%, Olyset Plus: 50.0%, Royal Guard: 51.6%]. Interceptor net produced the lowest mortality (~ 25%) against both species. Regarding blood-feeding inhibition (BFI), PBO nets, particularly Olyset Plus, outperformed Interceptor G2 and Royal Guard, while Interceptor produced minimal BFI (< 36%). Further evaluation of Royal Guard’s PPF effect on oviposition revealed no significant reduction in oviposition rates compared to controls with An. funestus (63.9% vs. 63.3%, P > 0.05). Genetic analysis using the hybrid crosses revealed that pyrethroid resistance markers (4.3 Kb-SV and G454A-Cyp9K1) were significantly associated with mosquito survival and blood-feeding success against PermaNet 2.0 (pyrethroid-only) and PermaNet 3.0 but showed no significant association with Interceptor G2 net. These findings support Interceptor G2 as a promising intervention for regions dominated by both highly resistant An. funestus s.l. and An. gambiae s.l. Piperonyl butoxide and PPF nets emerge as a good alternative for areas mostly dominated by resistant An. gambiae s.l. populations. Critically, the demonstrated variable impact of insecticide resistance on bednet efficacy underscores the imperative need for a comprehensive vector distribution mapping, continuous field efficacy assessments, and systematic resistance monitoring. This evidence-based triad should guide strategic LLIN distribution and rotations to sustain malaria control efficacy in resistance-prone settings.

Data availability

All datasets generated or analysed during this study are included in this published article and its supplementary files.

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Acknowledgements

We extend our heartfelt gratitude to the Village Health Teams (VHTs) and assistants in Mayuge district for their invaluable support in recruiting volunteers. We deeply appreciate the volunteers who participated in the hut trial and assisted with mosquito collection. We also express our sincere thanks to the technicians and administration at the Centre for Research in Infectious Diseases (CRID) in Cameroon for their contributions to mosquito rearing and laboratory work, and to the Uganda Virus Research Institute (UVRI) in Entebbe for their efforts in organizing field activities and preparing samples for shipping.

Funding

This study was funded by BMGF (INV-006003) and Wellcome Trust (217188/Z/19/Z).

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Authors and Affiliations

  1. Entomology Department, Uganda Virus Research Institute, P.O. BOX 49, Entebbe, Uganda

    Ambrose Oruni & Jonathan Kayondo

  2. Centre for Research in Infectious Diseases, LSTM-Research Unit, P.O BOX 3591, Yaoundé, Cameroon

    Ambrose Oruni, Benjamin D. Menze, Yvan G. Fotso-Toguem, Vanessa B. Ngannang-Fezeu, Riccado F. Thiomela, Magellan Tchouakui & Charles S. Wondji

  3. Liverpool School of Tropical Medicine, Vector Biology Department, Liverpool, L3 5QA, UK

    Ambrose Oruni, Jack Hearn & Charles S. Wondji

  4. Centre of Epidemiology and Planetary Health, School of Veterinary Medicine, Scotland’s Rural College, Inverness, UK

    Jack Hearn

  5. International Institute of Tropical Agriculture (IITA), P.O. Box 2008, Yaoundé, Cameroon

    Charles S. Wondji

Authors

  1. Ambrose Oruni
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  2. Benjamin D. Menze
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  6. Magellan Tchouakui
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  7. Jack Hearn
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  8. Jonathan Kayondo
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  9. Charles S. Wondji
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Contributions

C.S.W. conceived and designed the research with inputs from B.D.M, M.T and J.K. A.O carried out the resarch, field work, sample processing, laboratory analysis, data entry, data analysis and writing the first draft of the manuscript. A.O was assisted by B.D.M and R.F.T in the field and V.B.N-F in the laboratory. M.T., J.H, J.K and C.S.W supervised the study and revision of the first draft of the manuscript. All authors contributed to the writing of the final draft of the manuscript. All authors read, revised and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to
Ambrose Oruni or Charles S. Wondji.

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Competing interests

The authors declare no competing interests.

Ethics approval

The protocol to conduct this study was approved by The Uganda Virus Research Institute Research Ethics Committee (UVRI REC) (Ref: GC/127/833) and Uganda National Council for Science and Technology (UNCST) (HS2063ES). Prior to trials, written, informed and signed consents were obtained from the volunteers (sleepers). All the volunteers involved in the study were supervised, followed up and treated when showing signs and symptoms of malaria. All methods in this trial were performed in accordance with the relevant guidelines and regulations.

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Oruni, A., Menze, B.D., Fotso-Toguem, Y.G. et al. Chlorfenapyr bednets effectively overcome pyrethroid resistance escalation in highly resistant Anopheles malaria vectors in Uganda.
Sci Rep (2026). https://doi.org/10.1038/s41598-025-34493-3

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  • DOI: https://doi.org/10.1038/s41598-025-34493-3

Keywords

  • Experimental huts
  • New generation-LLINs
  • Interceptor G2
  • Malaria vectors

  • Anopheles funestus

  • Anopheles gambiae
  • Resistance escalation
  • Uganda

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