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Peer-reviewed journal article

Significant variations in tolerance to clothianidin and pirimiphos-methyl in Anopheles gambiae and Anopheles funestus populations during a dramatic malaria resurgence despite sustained indoor residual spraying in Uganda

Ambrose OruniiD, Emmanuel ArinaitweiD, James Adiga, Geoffrey Otto, Patrick Kyagamba, Joseph Okoth, Daniel Ayo, Jackson Asiimwe Rwatooro, Maato ZediiD, John C. RekiD, Kyle J. WalkeriD, Ashlee Braithwaite, Jonathan KayondoiD, Melissa D. ConradiD, Teun BousemaiD, Mark J. I. PaineiD, Hanafy M. IsmailiD, Paul J. KrezanoskiiD, Charles Sinclair WondjiiD, Moses Robert KamyaiD, Grant DorseyiD, Martin J. DonnellyiD

Abstract

A dramatic malaria resurgence occurred in areas of Uganda between 2020 and 2022 coincident with the switch to clothianidin-based formulations for indoor residual spraying. During the resurgence, Anopheles funestus numbers increased but when an alternative insecticide, pirimiphos methyl, was reintroduced in 2023, both malaria cases and An. funestus mosquito density fell. In this study, we investigated possible causes of the resurgence by assessing; 1) whether sufficient quantities of insecticide were sprayed; 2) the residual insecticide bioefficacy against wild mosquitoes and; 3) the insecticide susceptibility of vector populations using standard test tube assays and wall cone assays. In 2023, after adjusting for extraction efficiency, 70-80% of the houses had optimal residual concentrations of insecticides (clothianidin >0.3g/m2; pirimiphos methyl >0.5g/m2) with significant variations between sampling rounds and wall types. Mud walls had the lowest residual concentration of insecticides, and the lowest observed mortality in wall cone assays, compared to fired bricks with plaster/cement/paint. In the studies of residual bio efficacy, by World Health Organization (WHO) definitions, An. funestus showed resistance to clothianidin (90% mortality) when exposed to wall surfaces up to 7 months post-spray. In WHO tube tests, variations were observed in susceptibility to clothianidin in An. funestus populations using dose- and time-response assays (80-98% mortality). In 2022, An. gambiae was largely susceptible to the clothianidin-based formulation Sumishield (85-90% mortality) although the levels dropped slightly in 2023 (60-85% mortality) mainly in mud and pole houses. In contrast, An. gambiae was mildly susceptible to the pirimiphos methyl-based formulation Actellic ([~]80% mortality) and time response assays showed An. gambiae populations had very low knockdown and mortality at lower exposure time compared to An. funestus. Regression models showed a positive association between residual insecticide concentration (RIC) and mortality in houses sprayed with Sumishield but not Actellic houses. Despite the possible variations observed in spray operations, the study revealed that An. funestus exhibited a higher tolerance to clothianidin-based formulations compared to An. gambiae, and this might have driven the malaria resurgence observed in Uganda. However, there are signals of An. gambiae resistance to pirimiphos-methyl which will require further investigation and monitoring.

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Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (17 Feb 2025) · CC BY 4.0

In Tororo, Uganda, An. funestus tolerated clothianidin-sprayed walls better than An. gambiae yet was killed on pirimiphos-methyl walls up to 7 months post-spray, implicating it in the malaria resurgence.

In Tororo, malaria incidence rose more than 8-fold (0.36 vs 2.97 episodes per person-year) from 2021 to 2022 after Uganda switched indoor residual spraying (IRS) to clothianidin, although a 16-country survey had found Anopheles largely susceptible to it, and fell after pirimiphos-methyl returned in 2023. The work tests whether under-dosed spraying or vector tolerance explains this, pairing high-performance liquid chromatography of wall residues with wall cone and tube bioassays in both main vectors.

Key results

  1. ShownWild An. funestus had lower knockdown and mortality than An. gambiae on clothianidin (Sumishield) walls in 2022 and lower mortality in 2023. Its mortality fell below the <80% WHO resistance threshold the authors cite, while An. gambiae was largely susceptible.

    2022, 6-7 months post-spray in the same 9 houses: An. funestus vs An. gambiae knockdown 7.3% ± 0.4 vs 50.9% ± 2.5 (P = 0.00013) and 48-h mortality 76.1% ± 3.8 vs 98.2% ± 4.9 (P = 0.00043). 2023: An. funestus mortality below 75% at all time points, lower than Kisumu (ANOVA F = 8.82, df = 15, P = 0.011); An. gambiae above 80% to 12 months except 59.1% ± 5.75 at 5-6 months. · Trends in mosquito exposure to IRS chemicals on…; Abstract

  2. SuggestedOn pirimiphos-methyl (Actellic) walls in 2023 the species pattern reversed. An. funestus was killed for about 7 months before mortality declined, whereas wild An. gambiae showed reduced mortality from the first month, which the authors read as mild tolerance.

    An. funestus (three exposure rounds only): 92.37% ± 3.56 mortality at 6-7 months, then 60.29% ± 4.49 at 8-9 months and 51.62% ± 5.1 at 10-11 months. Wild An. gambiae: knockdown under 40% and mortality under 80% from month 1 to month 12, differing significantly from the Kisumu strain (ANOVA F = 6.85, df = 9, P = 0.031). · Trends in mosquito exposure to IRS chemicals on…; Abstract

  3. ShownBoth active ingredients were detected in every sprayed house. After correcting for glue-dot extraction efficiency in 2023, about three-quarters of Actellic surfaces met the target dose, while Sumishield results shifted sharply between sampling rounds and residues depended on wall type.

    Glue-dot extraction efficiency 61.27% (clothianidin) and 10.65% (pirimiphos-methyl). Actellic houses (n=49): ~25% of surfaces under 0.5 g/m^2 at both 0 and 3 months. Sumishield houses (n=9): ~70% under 0.3 g/m^2 at 0 months but ~10% at 3 months, with more residue at 3 months (P < 0.001). Burnt-brick-with-mud walls held more than mud-and-pole walls (P = 0.0078 at 0 months, P = 0.0072 at 3 months). · Concentration of IRS chemicals sprayed on walls…; Discussion; Abstract

  4. SuggestedIn bottle assays, An. funestus susceptibility to clothianidin varied through 2023: weak in May and high by August and November. An. gambiae had significantly higher knockdown at all three time points but significantly higher mortality only in May, and the dose-response assays showed no species difference.

    May 2023: An. funestus 60-min knockdown 16.7% ± 0.5, 5-day mortality 83.3%; An. gambiae knockdown 35-55% (t = 5.7, P = 0.0006), mortality 78-98% (t = 2.9, P = 0.0132). August and November: An. funestus mortality 95-100% and 90-98%, no significant difference (P = 0.2623; P = 0.0577). Knockdown stayed significantly lower in An. funestus in August (t = 6.01, P = 0.000477) and November (t = 2.80288, P = 0.015523). · Temporal monitoring of insecticide resistance to…; Abstract

  5. SuggestedWall insecticide levels were positively associated with mosquito mortality in Sumishield (clothianidin) houses in 2022, but not in Actellic (pirimiphos-methyl) houses in 2023, where the relation was unexpectedly negative.

    Binomial logistic regression with household as a random effect gave significant positive residue-mortality associations in 2022 (Sumishield) but an unexpected negative relation in 2023 Actellic houses. Correlations between residues at different months ranged from none to strong, with limited predictive value (Table 2; coefficients are not given in the text). · Correlation analysis between insecticide wall…; Data analysis; Abstract

Methods, data and reproducibility

Over 2022-2023, wall residues of clothianidin and pirimiphos-methyl in 60 Tororo households under indoor residual spraying were quantified by HPLC (glue dots, corrected in 2023 against sprayed filter papers), and bioefficacy was measured with WHO wall cone assays (15 unsprayed Busia houses as controls) plus time- and dose-response tube and bottle assays on An. gambiae and An. funestus.

  • Tororo district households (PRISM Border Cohort) sprayed with Sumishield in 2022 (51 houses) and with Actellic (49) or Sumishield (9) in 2023 · n = 60 households
  • Busia district households that never received IRS, used as wall cone assay controls · n = 15 households
  • 2022 wall residue samples (glue discs, middle of bedroom wall) at 4, 5 and 7 months post-spray · n = 180 samples (Sumishield houses n=51, unsprayed n=9)
  • 2023 wall residue samples (filter papers during spraying, glue dots after) from living room and bedroom at 0 and 3 months post-spray · n = Actellic n=49, Sumishield n=9, unsprayed n=2 houses
  • Field An. gambiae: F0 adults from larvae near sprayed Tororo houses, plus 2023 F1 adults from indoor resting collections in Mayuge · An. arabiensis (55.4%) and An. gambiae s.s. (35.8%) in typed sample
  • Laboratory strains: BusiaUg colony (unselected since 2019) and Kisumu reference strain · Anopheles gambiae s.s.
  • An. funestus F1 progeny from indoor resting collections in Busia (2022) and Mayuge (2023) · Anopheles funestus
Methods & tools
WHO wall cone bioassay, WHO tube assay, CDC bottle assay, HPLC (Dionex UltiMate 3000), Hypersil Gold C18 column, Chromeleon 7.3, R / RStudio 1.4.1106, Prokopack aspirator, Bostik glue dots, Whatman filter paper, Ultrawave U Series Sonicator
Reagents & environment
Analysis in R/RStudio version 1.4.1106; chromatograms processed in Chromeleon 7.3.
Reproducibility gaps
No analysis code is shared. Data are said to be in the article and supplementary files, with no repository, URL or accession.
Limitations (6)
  • The 2022 residues could not be sampled at the time of spraying, so they were not corrected for extraction efficiency; uncorrected, no 2022 surface reached 0.3 g/m^2. · stated by the authors
  • In 2023 only 9 houses, all mud and pole, received Sumishield, and their residues were unexpectedly higher at 3 than at 0 months, possibly from uneven spraying or field sampling error. · stated by the authors
  • Rearing problems limited An. funestus wall exposures to one time point in 2022 and three in 2023, and poor egg laying in Busia collections forced a switch to Mayuge mosquitoes in 2023; burnt-brick-with-mud houses numbered only 3. · stated by the authors
  • Results are location specific and should not be generalized, since clothianidin IRS has worked elsewhere (Benin, Cameroon); the mechanism behind An. funestus tolerance to clothianidin is unknown. · stated by the authors
  • Test mosquitoes did not all come from the sprayed Tororo houses: An. funestus came from Busia and Mayuge, and the 'An. gambiae' group mixed F0, F1 and colony insects, mostly An. arabiensis. This complicates comparisons between years and between species. · noted in this summary
  • The Kisumu 'susceptible' reference kept at Nagongera was resistant to deltamethrin, permethrin and DDT, probably from contamination, which weakens it as a baseline for the wall cone comparisons. · noted in this summary

Who should read:Read this if you work on insecticide resistance surveillance or IRS policy and want field data on how An. funestus and An. gambiae respond to clothianidin versus pirimiphos-methyl on real house walls.

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