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 .

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.

Bio-efficacy of Olyset® Plus, PermaNet® 3.0 and Interceptor® G2 on pyrethroid-resistant populations of Anopheles gambiae s.l. prior to the June 2023 net distribution campaign in Benin, West Africa.

BACKGROUND: This study investigates the effectiveness of new-generation mosquito nets, like Olyset® Plus and PermaNet® 3.0, and dual-action nets such as Interceptor® G2, against pyrethroid-resistant Anopheles gambiae mosquitoes following the 2023 mass distribution of long-lasting insecticidal nets in Benin. METHODS: We tested wild mosquito populations from six communes in Benin against various pyrethroid (permethrin 0.75%, alphacypermethrin 0.05%, and deltamethrin 0.05%) using WHO tube tests. Additionally, we exposed mosquitoes to chlorfenapyr 100 µg/ml using the CDC bottle bioassay method. A subset of mosquitoes underwent biochemical and PCR tests to check the overexpression of metabolic enzymes and the Kdr L1014F mutation. We evaluated the effectiveness of Olyset® Plus, PermaNet® 3.0, and Interceptor® G2 nets using cone and tunnel tests on both laboratory and field populations of An. gambiae. RESULTS: Overall, the highest mortality rate was 60% with pyrethroid and 98 to100% with chlorfenapyr. In cone tests, all three types of nets induced mortality rates above 80% in the susceptible laboratory strain of An. gambiae. Notably, Olyset® Plus showed the highest mortality rates for pyrethroid-resistant mosquitoes in cone tests, ranging from 81.03% (95% CI: 68.59-90.13) in Djougou to 96.08% (95% CI: 86.54-99.52) in Akpro-Missérété. PermaNet® 3.0 had variable rates, from 42.5% (95% CI: 27.04-59.11) in Djougou to 58.54% (95% CI: 42.11-73.68) in Porto-Novo. However, revealed good results for Interceptor® G2, with 94% (95% CI: 87.40-97.77) mortality and 89.09% blood sampling inhibition in local populations of An. gambiae. In comparison, Interceptor® had lower rates of 17% (95% CI: 10.23-25.82) and 60%, respectively. CONCLUSION: These results suggest that tunnel tests are effective for evaluating dual-active ingredient nets. Additionally, Interceptor® G2 and PBO nets like Olyset® Plus could be considered as alternatives against pyrethroid-resistant mosquitoes.

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.

The impact of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr long-lasting insecticidal nets on density of primary malaria vectors Anopheles gambiae s.s. and Anopheles coluzzii in Benin: a secondary analysis of a cluster randomised controlled trial.

BACKGROUND: Long-lasting insecticidal nets (LLINs) may have different impacts on distinct mosquito vector species. We assessed the efficacy of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr LLINs on the density of Anopheles gambiae s.s. and An. coluzzii compared to pyrethroid-only nets in a three-arm cluster randomised control trial in Benin. METHODS: Indoor and outdoor collections of adult mosquitoes took place in 60 clusters using human landing catches at baseline and every 3 months for 2 years. After morphological identification, around 15% of randomly selected samples of An. gambiae s.l. were dissected to determine parity, species (using PCR). RESULTS: Overall, a total of 46,613 mosquito specimens were collected at baseline and 259,250 in the eight quarterly collections post-net distribution. Post-net distribution, approximately 70% of the specimens of An. gambiae s.l. speciated were An. coluzzii, while the rest were mostly composed of An. gambiae s.s. with a small proportion (< 1%) of hybrids (An. gambiae/coluzzii). There was no evidence of a significant reduction in vector density indoors in either primary vector species [An. coluzzii: DR (density ratio) = 0.62 (95% CI 0.21-1.77), p = 0.3683 for the pyrethroid-pyriproxyfen LLIN and DR = 0.56 (95% CI 0.19-1.62), p = 0.2866 for the pyrethroid-chlorfenapyr LLIN, An. gambiae s.s.: DR = 0.52 (95% CI 0.18-1.46), p = 0.2192 for the pyrethroid-pyriproxyfen LLIN and DR = 0.53 (95% CI 0.19-1.46), p = 0.2222 for the pyrethroid-chlorfenapyr]. The same trend was observed outdoors. Parity rates of An. gambiae s.l. were also similar across study arms. CONCLUSIONS: Compared with pyrethroid-only LLINs, pyrethroid-chlorfenapyr LLINs and pyrethroid-pyriproxyfen LLINs performed similarly against the two primary mosquito species An. gambiae s.s. and An. coluzzii in Benin.

Tribal gaps in Malaria in relation to KAP in East Godavari District, Andhra Pradesh.

Objectives: To assess the level of GAPS in knowledge, attitudes and practices among tribal people regarding malaria disease. To assess the interassociation among knowledge, attitude and practices of scores. Materials and Methods: A cross-sectional study was performed in tribal areas of East Godavari District for a period of one year during 2013. Sample size was calculated as 1136. Multistage Random sampling method was used to identify the 4 PHC's in 4 directions of tribal areas and sample was equally distributed. In that study subjects were selected by using a simple Random method. Data were collected by using a pre-tested, semi-structured questionnaire and results were displayed. Setting: Tribal area of east Godavari district, Andhra Pradesh State, India. Participants: Study subjects were selected by simple random method. Results: Knowledge-practice gap for source of mosquito breeding 5.99%, for the prevention of mosquito breeding sources and for the implementation of minor engineering measures it is 10.51%. For bush cutting, the knowledge practice gap is -20.07%. For potted plants, the knowledge practice gap is -8.45%. For broken pots, it is 68.93%. IRS is performed outside the house not inside the house. The KAP-GAP for screening is nil. Covering oneself with a blanket to prevent man-mosquito contact is 6.96%. Conclusions: In our study, we found Know-do gap or KAP-GAP in all aspects of prevention of mosquito breeding places and personal protective measures. Regarding the interassociation among knowledge, attitudes and practices, they were associated with each other with significant difference.

EaveTubes for control of vector-borne diseases in Côte d'Ivoire: study protocol for a cluster randomized controlled trial.

BACKGROUND: Vector control tools, long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS), have significantly contributed to malaria prevention efforts in sub-Saharan Africa. However, insecticide resistance has seriously hampered their efficacy in recent years and new tools are essential to further progress. In2Care® EaveTubes (ETs) are an inexpensive, new resistance-breaking vector control product under World Health Organization (WHO) evaluation informed by mosquito ecology to efficiently target malaria vectors. By installing ETs in the walls of the house at the eave level that funnel the natural airflow, mosquitoes are drawn in by the same heat and odor cues that typically attract them through open eaves. Once inside an ET, mosquitoes are exposed to insecticide-treated netting placed inside the ET. The aim of this study is to test whether ETs as stand-alone tool have an effect on the epidemiology of malaria in villages where houses have been modified with the ET intervention. METHODS: A two-armed, cluster randomized controlled trial will be conducted to evaluate the effect of ETs on clinical malaria incidence in children living in Côte d'Ivoire. Thirty-four villages will be selected based on population size and the proportion of houses suitable for modification with ETs (17 treatment arms (ETs + LLINs, 17 control arms (LLINs only)). Based on the population census, 55 households per cluster with eligible children (i.e., between the ages of 6 months to 8 years old at the start of the study) will be randomly selected for recruitment into the active detection cohorts. In the treatment arm, we will enroll eligible children who reside in ET-treated houses. The intervention and control cohorts will be followed for 4 months for baseline covariate measurements and 24 months with intervention. During case detection visits, blood samples will be taken from all febrile children and tested for malaria infection with rapid diagnostic tests (RDTs). All positive clinical malaria infections will be treated. To estimate the impact of the ET on malaria vector densities, entomological measurements (indoor sampling with CDC traps) will be conducted monthly in 20 clusters (10 ET, 10 Control) in 10 randomly selected households per cluster. To estimate the infectiousness of malaria vectors, sporozoite rates will be measured in subsets of the collected mosquito samples. DISCUSSION: Findings will serve as an efficacy trial of ETs and will be submitted to the WHO Vector Control Advisory Group (VCAG) for assessment of public health value. Entomological outcomes will also be measured as proxies of malaria transmission to help develop guidelines for the evaluation of future In2Care® ETs products. TRIAL REGISTRATION: ClinicalTrials.gov NCT05736679. Registered on 10 February 2023.

Long-Lasting Insecticide-Treated Nets Combined or Not with Indoor Residual Spraying May Not Be Sufficient to Eliminate Malaria: A Case-Control Study, Benin, West Africa.

In sub-Saharan Africa, despite the implementation of multiple control interventions, the prevalence of malaria infection and clinical cases remains high. The primary tool for vector control against malaria in this region is the use of long-lasting insecticide-treated nets (LLINs) combined or not with indoor residual spraying (IRS) to achieve a synergistic effect in protection. The objective of this study was to assess the effectiveness of LLINs, with or without IRS, protected against Plasmodium falciparum infection and uncomplicated clinical cases (UCC) of malaria in Benin. A case-control study was conducted, encompassing all age groups, in the urban area of Djougou and the rural area of Cobly. A cross-sectional survey was conducted that included 2080 individuals in the urban area and 2770 individuals in the rural area. In the urban area, sleeping under LLINs did not confer significant protection against malaria infection and UCC when compared to no intervention. However, certain neighbourhoods benefited from a notable reduction in infection rates ranging from 65% to 85%. In the rural area, the use of LLINs alone, IRS alone, or their combination did not provide additional protection compared to no intervention. IRS alone and LLINs combined with IRS provided 61% and 65% protection against malaria infection, respectively, compared to LLINs alone. The effectiveness of IRS alone and LLINs combined with IRS against UCC was 52% and 54%, respectively, when compared to LLINs alone. In both urban and rural areas, the use of LLINs alone, IRS alone, and their combination did not demonstrate significant individual protection against malaria infection and clinical cases when compared to no intervention. In the conditions of this study, LLINs combined or not with IRS are not effective enough to eliminate malaria. In addition to the interventions, this study identified factors associated with malaria in Benin as housing design, neglected social groups like gender-marginalised individuals and adolescents, and socio-economic conditions acting as barriers to effective malaria prevention. Addressing these factors is crucial in order to facilitate malaria elimination efforts in sub-Saharan Africa.

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

Background: 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 the synergist piperonyl butoxide (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, cross-sectional entomological surveys were carried out at baseline and then every 6 months for two years. In each survey, ten households per HSD were randomly selected for indoor household entomological collections. Results: Overall, 5395 female Anopheles mosquitoes were collected from 5046 households. The proportion of mosquitoes infected 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. Conclusions: Distribution of LLINs in Uganda was associated with reductions in parasite prevalence and vector density, but the proportion of infective mosquitoes remained stable, 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.

Can insecticide resistance increase malaria transmission? A genetics-epidemiology mathematical modeling approach.

The great successes recorded in the fight against malaria over the last two decades, resulting from the wide scale implementation of insecticide-based interventions in malaria-endemic areas, has prompted a renewed global effort to eradicate malaria. The widespread emergence of insecticide resistance in the population of adult female malaria mosquitoes is considered to pose a potential challenge to such effort. In this study, we address one of the key questions in malaria ecology, namely whether or not insecticide resistance increase malaria transmission. We developed a genetics-epidemiology modeling framework that incorporates a detailed genotype structure of the gene that confers insecticide resistance in mosquitoes, malaria epidemiology in mosquitoes and humans (stratified based on whether or not they are protected by Long-lasting insecticide-treated nets (LLINs) indoors), genotype-specific mosquito repellance property of LLINs and mosquito biting behavior (indoor and outdoor bites). Conditions for the existence and local asymptotic stability of the various disease-free equilibria (by genotype) of the resulting genetic-epidemiology model are derived. This study identifies four parameters of the model that play a crucial role on quantifying the impact of insecticide resistance on malaria transmission, namely the parameters related to the level of the dominance of the resistant allele in heterozygous mosquitoes, the coverage of long-lasting insecticidal nets in the community, the probability of endophilic mosquitoes to successfully take a bloodmeal indoors and the proportion of new adult mosquitoes that are endophilic. We showed that, depending on the values of these four identified parameters, insecticide resistance can increase, decrease, or have no effect on malaria transmission. Our simulations show that malaria eradication can indeed be achieved using the currently-available chemical insecticides, even in the wake of the prevailing widespread insecticide resistance in malaria-endemic areas, if the insecticide-based interventions implemented can result in the attainment of the optimal values of the four identified parameters in malaria-endemic areas.

Residual malaria transmission: Magnitude and drivers of persistent Plasmodium infections despite high coverage of control interventions in Burkina Faso, West Africa.

This study collected baseline data on malaria vectors to characterize the drivers and the factors of persistent malaria transmission in two villages in the western part of Burkina Faso. Mosquitoes were collected in each village using the Human landing catch and pyrethrum spray catch and identified using the morphological keys. Molecular analyses were performed for the identification of An. gambiae complex species, the detection of Plasmodium infection and kdr-995F mutation. Anopheles mosquito larvae were also collected in the same villages, reared to adult's stage for the WHO tube and cone tests performing. The physical integrity of the LLINs already used by people in each village was assessed using the proportional hole index (pHI). An. gambiae s.l. was the main malaria vector accounting for 79.82% (5560/6965) of all collected mosquitoes. The biting pattern of An. gambiae s.l. was almost constant during the survey with an early aggressiveness before 8 p.m. and later biting activity after 6 a.m. The EIR varied from 0.13 to 2.55 infected bites per human per night (average: 1.03 infected bites per human per night). An. gambiae s.l. populations were full susceptible to Chlorpyrifos-methyl (0.4%) and Malathion (5%) with high kdr-995F mutation frequencies (>0.8). The physical integrity assessment showed high proportion of good nets in Santidougou compared to those collected in Kimidougou. This study highlighted a persistence of malaria transmission despite the intense use of vector control tools as LLINs and IRS by correlating mosquito biting time and human behavior. It provided a baseline guide for the monitoring of the residual malaria transmission in sub-Saharan Africa and encouraging the development of new alternative strategies to support the current malaria control tools.

Durable wall lining for malaria control in Liberia: results of a cluster randomized trial.

BACKGROUND: Malaria control in Liberia depends upon universal coverage with pyrethroid-impregnated long-lasting insecticidal nets (LLINs). Despite regular mass distribution, LLIN coverage and usage is patchy. Pyrethroid resistance in malaria vectors may further reduce LLIN efficacy. Durable Wall Lining (DWL), a novel material treated with two non-pyrethroid class insecticides, was designed to be installed onto the surface of inner walls, and cover openings and ceiling surfaces of rural houses. OBJECTIVES: AIM: To determine the malaria control efficacy of DWL. PRIMARY OBJECTIVE: To determine if DWL has an additional protective effect in an area of pyrethroid resistance. SECONDARY OBJECTIVES: To compare surface bio-availability of insecticides and entomological effectiveness over the study duration. DESIGN: A cluster randomized trial. PARTICIPANTS: Children aged 2-59 months. CONTROL ARM: 50 houses per 20 clusters, all of which received LLIN within the previous 12 months. ACTIVE ARM: 50 houses per 20 experimental clusters, all of which received LLINs with the previous 12 months, and had internal walls and ceilings lined with DWL. RANDOMISATION: Cluster villages were randomly allocated to control or active arms, and paired on 4 covariates. MAIN OUTCOME MEASURES: PRIMARY MEASURE: Prevalence of infection with P. falciparum in children aged 2 to 59 months. SECONDARY MEASURE: Surface bioavailability and entomological effectiveness of DWL active ingredients. RESULTS: Plasmodium falciparum prevalence in active clusters after 12 months was 34.6% compared to 40.1% in control clusters (p = 0.052). The effect varied with elevation and was significant (RR = 1.3, p = 0.022) in 14 pairs of upland villages. It was not significant (RR = 1.3, p = 0.344) in 6 pairs of coastal villages. Pooled risk ratio (RR) was calculated in SAS (Cary, NC, USA) using the Cochran-Mantel-Haenszel (CMH) test for upland and coastal cluster pairs. DWL efficacy was sustained at almost 100% for 12 months. CONCLUSIONS: Findings indicate that DWL is a scalable and effective malaria control intervention in stable transmission areas with pyrethroid-resistant vectors, where LLIN usage is difficult to achieve, and where local housing designs include large gable and eve openings. Trial registration ClinicalTrials.gov identifier: NCT02448745 (19 May 2015): https://clinicaltrials.gov/ct2/show/NCT02448745.

Understanding the current state-of-the-art of long-lasting insecticide nets and potential for sustainable alternatives.

Long-lasting insecticide-treated nets (LLINs) are widely distributed to communities where malaria is a major cause of mortality, especially to those under the age of 5 years-old. To protect people from this illness, LLINs provide physical and chemical barriers by containing insecticides within the matrix of the polymer fibers or on the surface. Synthetic polymers including polyethylene and polyester are common material choices for these nets, and pyrethroids, along with other additives, are the insecticides of choice for this application. Many studies have shown the effectiveness of these nets on the impact of malaria is highly significant, but there is a demand for more durable nets that last longer than only a few years as the available products are rated for 2-3 years of use. Improvements in this area would increase cost effectiveness, because better durability would reduce the frequency of manufacturing and worldwide shipping. Additionally, due to the plastic fibers, the waste can build quickly, damaging the environment. To deal with the sustainability and durability issues, biodegradable and renewable materials should be chosen as an alternative.

LLIN Evaluation in Uganda Project (LLINEUP2)-Factors associated with coverage and use of long­lasting insecticidal nets following the 2020-21 national mass distribution campaign: a cross-sectional survey of 12 districts.

BACKGROUND: In 2020-2021, long-lasting insecticidal nets (LLINs) were distributed nationwide in Uganda during the COVID-19 pandemic. A cross-sectional survey was conducted in 12 districts to evaluate the impact of the campaign 1-5 months after LLIN distribution. METHODS: During April-May 2021, households were randomly selected from target areas (1-7 villages) surrounding 12 government-run health facilities established as Malaria Reference Centres; at least 50 households were enrolled per cluster. Outcomes included household ownership of LLINs distributed through the universal coverage campaign (UCC) (at least one UCC LLIN), adequate coverage of UCC LLINs (at least one UCC LLIN per 2 residents), and use of LLINs (resident slept under a LLIN the previous night). Multivariate logistic regression models were used to identify household- and individual-level factors associated with outcomes, controlling for clustering around health facilities. RESULTS: In total, 634 households, with 3342 residents and 1631 bed-nets, were included. Most households (93.4%) owned at least 1 UCC LLIN, but only 56.8% were adequately covered by UCC LLINs. In an adjusted analysis, the factor most strongly associated with adequate coverage by UCC LLINs was fewer household residents (1-4 vs 7-14; adjusted odds ratio [aOR] 12.96, 95% CI 4.76-35.26, p < 0.001; 5-6 vs 7-14 residents; aOR 2.99, 95% CI 1.21-7.42, p = 0.018). Of the 3166 residents of households that owned at least one UCC LLIN, only 1684 (53.2%) lived in adequately covered households; 89.9% of these used an LLIN the previous night, compared to 1034 (69.8%) of 1482 residents living in inadequately covered households. In an adjusted analysis, restricted to residents of inadequately covered households, LLIN use was higher in children under-five than those aged 5-15 years (aOR 3.04, 95% CI 2.08-4.46, p < 0.001), and higher in household heads than distantly-related residents (aOR 3.94, 95% CI 2.38-6.51, p < 0.001). CONCLUSIONS: Uganda's 2021-21 campaign was successful, despite the COVID-19 pandemic. In future campaigns, strategies should be adopted to ensure high LLIN coverage, particularly for larger households. A better understanding of the drivers of LLIN use within households is needed to guide future interventions, educational messages, and behaviour change communication strategies; school-aged children and distantly-related residents appear vulnerable and could be targeted.

Persistently high proportions of plasmodium-infected Anopheles funestus mosquitoes in two villages in the Kilombero valley, South-Eastern Tanzania.

Background: In south-eastern Tanzania where insecticide-treated nets have been widely used for >20 years, malaria transmission has greatly reduced but remains highly heterogenous over small distances. This study investigated the seasonal prevalence of Plasmodium sporozoite infections in the two main malaria vector species, Anopheles funestus and Anopheles arabiensis for 34 months, starting January 2018 to November 2020. Methods: Adult mosquitoes were collected using CDC-light traps and Prokopack aspirators inside local houses in Igumbiro and Sululu villages, where earlier surveys had found very high densities of An. funestus. Collected females were sorted by taxa, and the samples examined using ELISA assays for detecting Plasmodium circumsporozoite protein in their salivary glands. Results: Of 7859 An. funestus tested, 4.6% (n = 365) were positive for Pf sporozoites in the salivary glands. On the contrary, only 0.4% (n = 9) of the 2382 An. arabiensis tested were positive. The sporozoite prevalence did not vary significantly between the villages or seasons. Similarly, the proportions of parous females of either species were not significantly different between the two villages (p > 0.05) but was slightly higher in An. funestus (0.50) than in An. arabiensis (0.42). Analysis of the 2020 data determined that An. funestus contributed 97.7% of all malaria transmitted in households in these two villages. Conclusions: In contexts where individual vector species mediate most of the pathogen transmission, it may be most appropriate to pursue a species-focused approach to better understand the ecology of the dominant vectors and target them with effective interventions to suppress transmission. Despite the ongoing efforts on tackling malaria in the two study villages, there is still persistently high Plasmodium infection prevalence in local populations of An. funestus, which now carry ~97% of all malaria infections and mediates intense year-round transmission. Further reduction in malaria burden in these or other similar settings requires effective targeting of An. funestus.

Resistance intensity status of Anopheles gambiae s.l. species at KOLOKOPE, eastern plateau Togo: A potential site to assess new vector control tools.

According to WHO recommendations, the deployment of the next generation of Long-Lasting Insecticidal Nets (LLINs) for malaria vector control requires appropriate investigations on the insecticide resistance profile of the vector. Most of the next generation of LLINs are impregnated with a combination of pyrethroid insecticides and piperonyl butoxide (PBO), a synergist with an additional impact on the increase in the mortality rate of Anopheles gambiae s.l. (Diptera: Culicidae). Kolokopé is a cotton-growing area in the central region of Togo characterized by an intensive use of agricultural pesticides and insecticides where there is a phase II experimental hut station. For the characterization of the site, WHO susceptibility tests using diagnostic doses of ten insecticides, PBO synergist assays and intensity assays of three pyrethroids (5x and 10x) were conducted on adult female mosquitoes obtained from larvae collected around the site. Anopheles gambiae s.l. from Kolokopé showed high resistance to pyrethroids and DDT, but to a lesser extent to carbamates and organophosphates. Likewise, high intensity of resistance to pyrethroid was observed with less than 40% mortality at 10x deltamethrin, 52 and 29% mortality at 10x permethrin and 10x alphacypermethrin, respectively. Also, PBO treatment resulted in increased mortality which was higher than the mortality rate at 10x doses of pyrethroids. The high pyrethroid intensity resistance recorded at Kolokopé could be mainly due to the selection pressure on An. gambiae s.l. caused by the excessive use of insecticide in agriculture. These results can be used to assess the next generation of LLINs either in experimental hut or at a community trial.

Evaluation of the residual efficacy and physical durability of five long-lasting insecticidal nets (LLINs) in Senegal.

BACKGROUND: The preventive and curative strategies of malaria are based on promoting the use of long-lasting insecticidal nets (LLINs) and treating confirmed cases with artemisinin-based combination therapy. These strategies have led to a sharp decline in the burden of malaria, which remains a significant public health problem in sub-Saharan countries. The objective of this study was to determine and compare the residual efficacy of LLINs recommended by the World Health Organization. METHODS: The study was conducted in six villages in two sites in Senegal located in the Sahelo-Sudanian area of the Thiès region, 70 km from Dakar and in Mbagame, a semi-urban zone in the Senegal River Valley. A census was conducted of all sleeping places in each household to be covered by LLINs. Five brands of LLIN were distributed, and every six months, retention rates, net use, maintenance, physical integrity, insecticide chemical content, and biological efficacy were examined for each type of LLIN. RESULTS: A total of 3012 LLINs were distributed in 1249 households in both sites, with an average coverage rate of 94% (95% CI 92.68-95.3). After 36 months, the average retention rate was 12.5% and this rate was respectively 20.5%, 15.1%, 10%, 7%, and 3% for Olyset Net®, Dawa Plus® 2.0, PermaNet® 2.0, NetProtect® and Life Net®, respectively. The proportion of LLINs with holes and the average number of holes per mosquito net increased significantly during each follow-up, with a large predominance of size 1 (small) holes for all types of LLINs distributed. During the three-year follow-up, bioassay mortality rates of a susceptible strain of insectary reared Anopheles coluzzii decreased in the following net types: in Dawa Plus® 2.0 (100% to 51.7%), PermaNet® 2.0 (96.6% to 83%), and Olyset Net® (96.6% to 33.3%). Mortality rates remained at 100% in Life Net® over the same time period. After 36 months, the average insecticide content per brand of LLIN decreased by 40.9% for Dawa Plus® 2.0, 31% for PermaNet® 2.0, 39.6% for NetProtect® and 51.9% for Olyset Net® and 40.1% for Life Net. CONCLUSIONS: Although some net types retained sufficient insecticidal activity, based on all durability parameters measured, none of the net types survived longer than 2 years.

Qualitative study on the use and maintenance of long-lasting insecticidal nets (LLINs) in Bouaké (Côte d'Ivoire), 17 months after the last mass distribution campaign.

BACKGROUND: The use of long-lasting insecticide-treated nets (LLINs) is one of the main malaria prevention method promoted by the World Health Organization (WHO) in Côte d'Ivoire. LLIN-coverage has reached 95% since 2015 and nearly 16 million LLINs were distributed in 2017. Despite these efforts, malaria incidence at the national level remains high (120‰ in 2012 to 164‰ in 2017) although this could be partly explained by increased screening efforts. This study aimed at determining what preventative measures were used against mosquito bites, as well as LLIN maintenance practices used by the inhabitants of the city of Bouaké, capital city of the Gbêkê region with a malaria incidence of 257‰ in 2017. METHODS: A descriptive qualitative investigation took place in Bouaké, in four neighbourhoods that were selected through purposive sampling based on their social composition. Data were collected using an interview guide based on convenience sampling. RESULTS: The results of the study reveal that LLINs are the most reported used malaria prevention measure (66.4%). Environmental health (28.8%) came second in their declarations, smoke coils (23.5%) third and aerosol cans (18.8%) last. The percentage of respondents who answered that they had slept under an LLIN the previous night was 53%. 57.7% reported that they wash their LLINs, 12.1% that they do not wash them, and 4% that they replace dirty LLINs with new ones. The LLINs washing methods described by the respondents did not comply with the WHO recommendations and there was no mention of LLINs repairs. CONCLUSION: Despite mass distributions of LLINs in Côte d'Ivoire, this key malaria control tool remains under-used by the population. Regarding LLIN maintenance, more than half of the population reports that they wash their nets while not complying with recommended practices or repairing them.

Reduced performance of community bednets against pyrethroid-resistant Anopheles funestus and Anopheles gambiae, major malaria vectors in Cameroon.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are a vital tool in the fight against malaria vectors. However, their efficacy in the field can be impacted by several factors, including patterns of usage, net age, mosquito resistance and the delayed mortality effect, all of which could influence malaria transmission. We have investigated the effectiveness of the various brands of LLINs available in markets and households in Cameroon on pyrethroid-resistant mosquitoes and assessed their post-exposure effect. METHODS: Following quality control assessment on a susceptible laboratory mosquito strain, we evaluated the immediate and delayed mortality effects of exposure to LLINs (both newly bough LLINst and used ones collected from households in Elende village, Cameroon, in 2019) using standard WHO cone tests on Anopheles gambiae and Anopheles funestus populations collected from the Centre region of Cameroon. Alive female mosquitoes were genotyped for various resistance markers at different time points post-exposure to evaluate the impact of insecticide resistance on the efficacy of bednets. RESULTS: The laboratory-susceptible strain experienced high mortality rates when exposed to all pyrethroid-only brands of purchased nets (Olyset® Net, Super Net, PermaNet® 2.0, Yorkool®, Royal Sentry®) (Mean±SEM: 68.66 ± 8.35% to 93.33 ± 2.90%). However, low mortality was observed among wild An. funestus mosquitoes exposed to the bednets (0 ± 0 to 28 ± 6.7%), indicating a reduced performance of these nets against field mosquitoes. Bednets collected from households also showed reduced efficacy on the laboratory strain (mortality: 19-66%), as well as displaying a significant loss of efficacy against the local wild strains (mortality: 0 ± 0% to 4 ± 2.6% for An. gambiae sensu lato and 0 ± 0% to 8 ± 3.2% for An. funestus). However, compared to the unexposed group, mosquitoes exposed to bednets showed a significantly reduced longevity, indicating that the efficacy of these nets was not completely lost. Mosquitoes with the CYP6P9a-RR and L119F-GSTe2 mutations conferring pyrethroid resistance showed greater longevity after exposure to the Olyset net than their susceptible counterparts, indicating the impact of resistance on bednet efficacy and delayed mortality. CONCLUSION: These findings show that although standard bednets drastically lose their efficacy against pyrethroid-resistant field mosquitoes, they still are able to induce delayed mortality in exposed populations. The results of this study also provide evidence of the actual impact of resistance on the quality and efficacy of LLINs in use in the community, with mosquitoes carrying the CYP6P9a-RR and L119F-GSTe2 mutations conferring pyrethroid resistance living longer than their susceptible counterparts. These results highlight the need to use new-generation nets that do not rely solely on pyrethroids.

Pyrethroid Resistance Intensity in Anopheles gambiae s.l. from Different Agricultural Production Zones in Benin, West Africa.

Agricultural production activities usually occur in Benin with the use of a huge amount of insecticides including pyrethroids for pest control. It is therefore important to regularly monitor pyrethroid resistance intensity in Anopheles gambiae s.l., the main malaria vector. This study was conducted in cereal, cotton, rice growing, and urban market gardening areas throughout the country in 2018 and 2019. Females An. gambiae s.l. field-collected as larvae were exposed to deltamethrin 1 × (0.05%), 2 × (0.1%), 5 × (0.25%), and 10 × (0.5%) and permethrin 1 × (0.75%), 2 × (1.5%), 5 × (3.75%), and 10 × (7.5%). Synergist assays were also performed using World Health Organization articles combining piperonyl butoxide (PBO) (4%) + deltamethrin 1 × and, PBO (4%) + Permethrin 1 × . Molecular species and L1014F kdr mutation were identified using PCR. Expression of metabolic enzymes was also assessed through biochemical tests. After exposure to permethrin and deltamethrin 10 × , An. gambiae s.l. displayed mortality rates <98%. Synergist assays induced significantly higher mortality rates than pyrethroids alone (p < 0.05). An. gambiae s.l. complex was composed of An. gambiae s.s., Anopheles coluzzii, and Anopheles arabiensis, with mean frequency of the L1014F kdr mutation >75%. Overexpression of nonspecific α and ß esterases was observed in the cereal, cotton, and urban market gardening areas, while an overexpression of mixed function oxidases was observed in the cotton and rice growing areas. Overall, An. gambiae s.l. showed high resistance intensity to both deltamethrin and permethrin. The synergist and biochemical tests performed suggest that PBO long-lasting insecticidal nets may provide a greater control of pyrethroid-resistant mosquitoes.