Pyrethroid resistance and gene expression profile of a new resistant An. gambiae colony from Uganda reveals multiple resistance mechanisms and overexpression of Glutathione-S-Transferases linked to survival of PBO-pyrethroid combination.

Background: The effectiveness of long-lasting insecticidal nets (LLINs) are being threatened by growing resistance to pyrethroids. To restore their efficacy, a synergist, piperonyl butoxide (PBO) which inhibits cytochrome P450s has been incorporated into pyrethroid treated nets. A trial of PBO-LLINs was conducted in Uganda from 2017 and we attempted to characterize mechanisms of resistance that could impact intervention efficacy. Methods: We established an Anopheles gambiae s.s colony in 2018 using female mosquitoes collected from Busia district in eastern Uganda. We first assessed the phenotypic resistance profile of this colony using WHO tube and net assays using a deltamethrin dose-response approach. The Busia colony was screened for known resistance markers and RT-qPCR targeting 15 genes previously associated with insecticide resistance was performed. Results: The Busia colony had very high resistance to deltamethrin, permethrin and DDT. In addition, the colony had moderate resistance to alpha-cypermethrin and lambda-cyhalothrin but were fully susceptible to bendiocarb and fenitrothion. Exposure to PBO in combination with permethrin and deltamethrin resulted in higher mortality rates in both net and tube assays, with a higher mortality observed in net assays than tube assays. The kdr marker, Vgsc-995S was at very high frequency (91.7-98.9%) whilst the metabolic markers Coeae1d and Cyp4j5-L43F were at very low (1.3% - 11.5%) and moderate (39.5% - 44.7%) frequencies respectively. Our analysis showed that gene expression pattern in mosquitoes exposed to deltamethrin, permethrin or DDT only were similar in comparison to the susceptible strain and there was significant overexpression of cytochrome P450s, glutathione-s-transferases (GSTs) and carboxyl esterases (COEs). However, mosquitoes exposed to both PBO and pyrethroid strikingly and significantly only overexpressed closely related GSTs compared to unexposed mosquitoes while major cytochrome P450s were underexpressed. Conclusions: The high levels of pyrethroid resistance observed in Busia appears associated with a wide range of metabolic gene families.

Susceptibility of Anopheles gambiae to Natural Plasmodium falciparum Infection: A Comparison between the Well-Established Anopheles gambiae s.s Line and a Newly Established Ugandan Anopheles gambiae s.s. Line.

Much of our understanding of malaria transmission comes from mosquito feeding assays using Anopheles mosquitoes from colonies that are well adapted to membrane feeding. This raises the question whether results from colony mosquitoes lead to overestimates of outcomes in wild Anopheles mosquitoes. We successfully established an Anopheles colony using progeny of wild Anopheles gambiae s.s. mosquitoes (Busia mosquitoes) and directly compared their susceptibility to infection with Plasmodium falciparum with the widely used An. gambiae s.s. mosquitoes (Kisumu mosquitoes) using gametocyte-infected Ugandan donor blood. The proportion of infectious feeds did not differ between Busia (71.8%, 23/32) and Kisumu (68.8%, 22/32, P = 1.00) mosquitoes. When correcting for random effects of donor blood, we observed a 23% higher proportion of infected Busia mosquitoes than infected Kisumu mosquitoes (RR, 1.23; 95% CI, 1.10-1.38, P < 0.001). This study suggests that feeding assays with Kisumu mosquitoes do not overestimate outcomes in wild An. gambiae s.s. mosquitoes, the mosquito species most relevant to malaria transmission in Uganda.

Dramatic resurgence of malaria after 7 years of intensive vector control interventions in Eastern Uganda.

Background: Tororo District, Uganda experienced a dramatic decrease in malaria burden from 2015-19 following 5 years of indoor residual spraying (IRS) with carbamate (Bendiocarb) and then organophosphate (Actellic) insecticides. However, a marked resurgence occurred in 2020, which coincided with a change to a clothianidin-based IRS formulations (Fludora Fusion/SumiShield). To quantify the magnitude of the resurgence, investigate causes, and evaluate the impact of a shift back to IRS with Actellic in 2023, we assessed changes in malaria metrics in regions within and near Tororo District. Methods: Malaria surveillance data from Nagongera Health Center, Tororo District was included from 2011-2023. In addition, a cohort of 667 residents from 84 houses was followed from August 2020 through September 2023 from an area bordering Tororo and neighboring Busia District, where IRS has never been implemented. Cohort participants underwent passive surveillance for clinical malaria and active surveillance for parasitemia every 28 days. Mosquitoes were collected in cohort households every 2 weeks using CDC light traps. Female Anopheles were speciated and tested for sporozoites and phenotypic insecticide resistance. Temporal comparisons of malaria metrics were stratified by geographic regions. Findings: At Nagongera Health Center average monthly malaria cases varied from 419 prior to implementation of IRS; to 56 after 5 years of IRS with Bendiocarb and Actellic; to 1591 after the change in IRS to Fludora Fusion/SumiShield; to 155 after a change back to Actellic. Among cohort participants living away from the border in Tororo, malaria incidence increased over 8-fold (0.36 vs. 2.97 episodes per person year, p<0.0001) and parasite prevalence increased over 4-fold (17% vs. 70%, p<0.0001) from 2021 to 2022 when Fludora Fusion/SumiShield was used. Incidence decreased almost 5-fold (2.97 vs. 0.70, p<0.0001) and prevalence decreased by 39% (70% vs. 43%, p<0.0001) after shifting back to Actellic. There was a similar pattern among those living near the border in Tororo, with increased incidence between 2021 and 2022 (0.93 vs. 2.40, p<0.0001) followed by a decrease after the change to Actellic (2.40 vs. 1.33, p<0.001). Among residents of Busia, malaria incidence did not change significantly over the 3 years of observation. Malaria resurgence in Tororo was temporally correlated with the replacement of An. gambiae s.s. by An. funestus as the primary vector, with a marked decrease in the density of An. funestus following the shift back to IRS with Actellic. In Busia, An. gambiae s.s. remained the primary vector throughout the observation period. Sporozoite rates were approximately 50% higher among An. funestus compared to the other common malaria vectors. Insecticide resistance phenotyping of An. funestus revealed high tolerance to clothianidin, but full susceptibility to Actellic. Conclusions: A dramatic resurgence of malaria in Tororo was temporally associated with a change to clothianidin-based IRS formulations and emergence of An. funestus as the predominant vector. Malaria decreased after a shift back to IRS with Actellic. This study highlights the ability of malaria vectors to rapidly circumvent control efforts and the importance of high-quality surveillance systems to assess the impact of malaria control interventions and generate timely, actionable data.

LLIN Evaluation in Uganda Project (LLINEUP)-effects of a vector control trial on Plasmodium infection prevalence and genotypic markers of insecticide resistance in Anopheles vectors from 48 districts of Uganda.

Pyrethroid bednets treated with the synergist piperonyl butoxide (PBO) offer the possibility of improved vector control in mosquito populations with metabolic resistance. In 2017-2019, we conducted a large-scale, cluster-randomised trial (LLINEUP) to evaluate long-lasting insecticidal nets (LLINs) treated with a pyrethroid insecticide plus PBO (PBO LLINs), as compared to conventional, pyrethroid-only LLINs across 104 health sub-districts (HSDs) in Uganda. In LLINEUP, and similar trials in Tanzania, PBO LLINs were found to provide greater protection against malaria than conventional LLINs, reducing parasitaemia and vector density. In the LLINEUP trial, we conducted cross-sectional household entomological surveys at baseline and then every 6 months for two years, which we use here to investigate longitudinal changes in mosquito infection rate and genetic markers of resistance. Overall, 5395 female Anopheles mosquitoes were collected from 5046 households. The proportion of mosquitoes infected (PCR-positive) with Plasmodium falciparum did not change significantly over time, while infection with non-falciparum malaria decreased in An. gambiae s.s., but not An. funestus. The frequency of genetic markers associated with pyrethroid resistance increased significantly over time, but the rate of change was not different between the two LLIN types. The knock-down resistance (kdr) mutation Vgsc-995S declined over time as Vgsc-995F, the alternative resistance mutation at this codon, increased. Vgsc-995F appears to be spreading into Uganda. Distribution of LLINs in Uganda was previously found to be associated with reductions in parasite prevalence and vector density, but here we show that the proportion of infective mosquitoes remained stable across both PBO and non-PBO LLINs, suggesting that the potential for transmission persisted. The increased frequency of markers of pyrethroid resistance indicates that LLIN distribution favoured the evolution of resistance within local vectors and highlights the potential benefits of resistance management strategies.Trial registration: This study is registered with ISRCTN, ISRCTN17516395. Registered 14 February 2017, http://www.isrctn.com/ISRCTN17516395 .