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) - 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.

Characterizing pyrethroid resistance and mechanisms in Anopheles gambiae (s.s.) and Anopheles arabiensis from 11 districts in Uganda.

Insecticide resistance threatens recent progress on malaria control in Africa. To characterize pyrethroid resistance in Uganda, Anopheles gambiae (s.s.) and Anopheles arabiensis were analyzed from 11 sites with varied vector control strategies. Mosquito larvae were collected between May 2018 and December 2020. Sites were categorized as receiving no indoor-residual spraying ('no IRS', n â€‹= â€‹3); where IRS was delivered from 2009 to 2014 and in 2017 and then discontinued ('IRS stopped', n â€‹= â€‹4); and where IRS had been sustained since 2014 ('IRS active', n â€‹= â€‹4). IRS included bendiocarb, pirimiphos methyl and clothianidin. All sites received long-lasting insecticidal nets (LLINs) in 2017. Adult mosquitoes were exposed to pyrethroids; with or without piperonyl butoxide (PBO). Anopheles gambiae (s.s.) and An. arabiensis were identified using PCR. Anopheles gambiae (s.s.) were genotyped for Vgsc-995S/F, Cyp6aa1, Cyp6p4-I236M, ZZB-TE, Cyp4j5-L43F and Coeae1d, while An. arabiensis were examined for Vgsc-1014S/F. Overall, 2753 An. gambiae (s.l.), including 1105 An. gambiae (s.s.) and 1648 An. arabiensis were evaluated. Species composition varied by site; only nine An. gambiae (s.s.) were collected from 'IRS active' sites, precluding species-specific comparisons. Overall, mortality following exposure to permethrin and deltamethrin was 18.8% (148/788) in An. gambiae (s.s.) and 74.6% (912/1222) in An. arabiensis. Mortality was significantly lower in An. gambiae (s.s.) than in An. arabiensis in 'no IRS' sites (permethrin: 16.1 vs 67.7%, P â€‹< â€‹0.001; deltamethrin: 24.6 vs 83.7%, P â€‹< â€‹0.001) and in 'IRS stopped' sites (permethrin: 11.3 vs 63.6%, P â€‹< â€‹0.001; deltamethrin: 25.6 vs 88.9%, P â€‹< â€‹0.001). When PBO was added, mortality increased for An. gambiae (s.s.) and An. arabiensis. Most An. gambiae (s.s.) had the Vgsc-995S/F mutation (95% frequency) and the Cyp6p4-I236M resistance allele (87%), while the frequency of Cyp4j5 and Coeae1d were lower (52% and 55%, respectively). Resistance to pyrethroids was widespread and higher in An. gambiae (s.s.). Where IRS was active, An. arabiensis dominated. Addition of PBO to pyrethroids increased mortality, supporting deployment of PBO LLINs. Further surveillance of insecticide resistance and assessment of associations between genotypic markers and phenotypic outcomes are needed to better understand mechanisms of pyrethroid resistance and to guide vector control.

Effect of long-lasting insecticidal nets with and without piperonyl butoxide on malaria indicators in Uganda (LLINEUP): final results of a cluster-randomised trial embedded in a national distribution campaign.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the foundation of malaria control but resistance of mosquito vectors to pyrethroids threatens their effectiveness. We embedded a cluster-randomised trial into Uganda's 2017-18 campaign to distribute LLINs. LLINs with piperonyl butoxide (PBO) reduced parasite prevalence more effectively than conventional LLINs (without PBO) for 18 months. Here, we report the final 25-month survey results. METHODS: LLINEUP was a cluster-randomised trial conducted in 48 districts in eastern and western Uganda. 104 health subdistricts (clusters) without ongoing or planned indoor residual spraying with pirimiphos-methyl (Actellic, Basel, Switzerland) were eligible for inclusion in the trial. Clusters were randomly assigned to PBO LLINs (PermaNet 3.0 or Olyset Plus) and conventional LLINs (PermaNet 2.0 or Olyset Net) with proportionate randomisation using STATA version 14.2. LLINs were delivered from March 25, 2017, to March 18, 2018. Between April 23, 2019, and Sept 13, 2019, community surveys were conducted in 50 randomly selected households per cluster; ten households per cluster were randomly selected for entomology surveys. Mosquitoes were collected in the morning from indoor surfaces of households using Prokopack aspirators. Due to COVID-19 restrictions, only 90 of the 104 clusters were surveyed at 25 months. The primary outcome was parasite prevalence by microscopy in children aged 2-10 years, assessed in the as-treated population, determined using the results from the 6-month household survey on the type of LLINs received in each cluster. This trial is registered with ISRCTN, ISRCTN17516395, and is now completed. FINDINGS: In the as-treated analysis, two clusters were excluded (no predominant LLIN received) and four were reassigned; 40 PBO LLIN clusters (30 PermaNet 3.0, ten Olyset Plus) and 48 non-PBO LLIN (36 PermaNet 2.0, 12 Olyset Net) were included. Parasite prevalence was 17·1% (506 of 2958 participants) in the PBO group and 19·8% (701 of 3534) in the non-PBO group (prevalence ratio adjusted for baseline 0·80 [95% CI 0·69-0·93], p=0·0048). Comparing within-treatment group parasite prevalence to baseline, parasite prevalence ratios were lower in the PBO groups at all timepoints, but the difference was greatest at 6 months (PBO LLINs parasite prevalence at baseline 28·8% [1001 of 3472, 95% CI 27·3-30·4] vs at 6 months 12·0% [361 of 3009, 10·9-13·2], prevalence ratio [PR] 0·43 [95% CI 0·36-0·52], p<0·0001; non-PBO LLINs parasite prevalence at baseline 25·4% [1015 of 4004, 24·0-26·7] vs 6 months 14·8% [526 of 3551, 13·7-16·0], PR 0·60 [0·54-0·68], p<0·0001) and 25 months (PBO LLINs parasite prevalence at 25 months 17·1% [506 of 2958, 15·8-18·5], PR 0·63 [95% CI 0·57-0·71], p<0·0001; non-PBO LLINs parasite prevalence at 25 months 19·8% [701 of 3534, 18·5-21·2], PR 0·79 [0·73-0·86], p<0·0001). INTERPRETATION: In Uganda, PBO LLINs outperformed pyrethroid-only LLINs for 25 months. WHO concluded that PBO LLINs are more effective against malaria than non-PBO LLINs when resistance to pyrethroids is high and issued a conditional recommendation suggesting PBO LLINs should be deployed in areas of pyrethroid resistance. FUNDING: The Against Malaria Foundation, UK Department for International Development, Innovative Vector Control Consortium, and Bill and Melinda Gates Foundation.

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.

LLIN evaluation in Uganda project (LLINEUP): The fabric integrity, chemical content and bioefficacy of long-lasting insecticidal nets treated with and without piperonyl butoxide across two years of operational use in Uganda.

Long-lasting insecticidal nets (LLINs) supplemented with the synergist piperonyl butoxide have been developed in response to growing pyrethroid resistance; however, their durability in the field remains poorly described. A pragmatic cluster-randomised trial was embedded into Uganda's 2017-2018 LLIN distribution to compare the durability of LLINs with and without PBO. A total of 104 clusters (health sub-districts) were included with each receiving one of four LLIN products, two with pyrethroid + PBO (Olyset Plus and PermaNet 3.0) and two pyrethroid-only (Olyset Net and PermaNet 2.0). Nets were sampled at baseline, 12 and 25 months post-distribution to assess physical condition, chemical content, and bioefficacy. Physical condition was quantified using proportionate Hole Index and chemical content measured using high-performance liquid chromatography. Bioefficacy was assessed with three-minute World Health Organisation (WHO) Cone and Wireball assays using pyrethroid-resistant Anopheles gambiae, with 1-h knockdown and 24-h mortality recorded. There was no difference in physical durability between LLIN products assessed (P = 0.644). The pyrethroid content of all products remained relatively stable across time-points but PBO content declined by 55% (P < 0.001) and 58% (P < 0.001) for Olyset Plus and PermaNet 3.0 respectively. Both PBO LLINs were highly effective against pyrethroid-resistant mosquitoes when new, knocking down all mosquitoes. However, bioefficacy declined over time with Olyset Plus knocking down 45.72% (95% CI: 22.84-68.62%, P = 0.021) and Permanent 3.0 knocking down 78.57% (95% CI: 63.57-93.58%, P < 0.001) after 25 months. Here we demonstrate that both Olyset Plus and PermaNet 3.0 are as durable as their pyrethroid-only equivalents and had superior bioefficacy against pyrethroid-resistant An. gambiae. However, the superiority of PBO-LLINs decreased with operational use, correlating with a reduction in total PBO content. This decline in bioefficacy after just two years is concerning and there is an urgent need to assess the durability of PBO LLINs in other settings.

Incremental cost and cost-effectiveness of the addition of indoor residual spraying with pirimiphos-methyl in sub-Saharan Africa versus standard malaria control: results of data collection and analysis in the Next Generation Indoor Residual Sprays (NgenIRS) project, an economic-evaluation.

BACKGROUND: Malaria is a major cause of morbidity and mortality globally, especially in sub-Saharan Africa. Widespread resistance to pyrethroids threatens the gains achieved by vector control. To counter resistance to pyrethroids, third-generation indoor residual spraying (3GIRS) products have been developed. This study details the results of a multi-country cost and cost-effectiveness analysis of indoor residual spraying (IRS) programmes using Actellic®300CS, a 3GIRS product with pirimiphos-methyl, in sub-Saharan Africa in 2017 added to standard malaria control interventions including insecticide-treated bed nets versus standard malaria control interventions alone. METHODS: An economic evaluation of 3GIRS using Actellic®300CS in a broad range of sub-Saharan African settings was conducted using a variety of primary data collection and evidence synthesis methods. Four IRS programmes in Ghana, Mali, Uganda, and Zambia were included in the effectiveness analysis. Cost data come from six IRS programmes: one in each of the four countries where effect was measured plus Mozambique and a separate programme conducted by AngloGold Ashanti Malaria Control in Ghana. Financial and economic costs were quantified and valued. The main indicator for the cost was cost per person targeted. Country-specific case incidence rate ratios (IRRs), estimated by comparing IRS study districts to adjacent non-IRS study districts or facilities, were used to calculate cases averted in each study area. A deterministic analysis and sensitivity analysis were conducted in each of the four countries for which effectiveness evaluations were available. Probabilistic sensitivity analysis was used to generate plausibility bounds around the incremental cost-effectiveness ratio estimates for adding IRS to other standard interventions in each study setting as well as jointly utilizing data on effect and cost across all settings. RESULTS: Overall, IRRs from each country indicated that adding IRS with Actellic®300CS to the local standard intervention package was protective compared to the standard intervention package alone (IRR 0.67, [95% CI 0.50-0.91]). Results indicate that Actellic®300CS is expected to be a cost-effective (> 60% probability of being cost-effective in all settings) or highly cost-effective intervention across a range of transmission settings in sub-Saharan Africa. DISCUSSION: Variations in the incremental costs and cost-effectiveness likely result from several sources including: variation in the sprayed wall surfaces and house size relative to household population, the underlying malaria burden in the communities sprayed, the effectiveness of 3GIRS in different settings, and insecticide price. Programmes should be aware that current recommendations to rotate can mean variation and uncertainty in budgets; programmes should consider this in their insecticide-resistance management strategies. CONCLUSIONS: The optimal combination of 3GIRS delivery with other malaria control interventions will be highly context specific. 3GIRS using Actellic®300CS is expected to deliver acceptable value for money in a broad range of sub-Saharan African malaria transmission settings.

Resurgence of malaria in Uganda despite sustained indoor residual spraying and repeated long lasting insecticidal net distributions.

Five years of sustained indoor residual spraying (IRS) of insecticide from 2014 to 2019, first using a carbamate followed by an organophosphate, was associated with a marked reduction in the incidence of malaria in five districts of Uganda. We assessed changes in malaria incidence over an additional 21 months, corresponding to a change in IRS formulations using clothianidin with and without deltamethrin. Using enhanced health facility surveillance data, our objectives were to 1) estimate the impact of IRS on monthly malaria case counts at five surveillance sites over a 6.75 year period, and 2) compare monthly case counts at five facilities receiving IRS to ten facilities in neighboring districts not receiving IRS. For both objectives, we specified mixed effects negative binomial regression models with random intercepts for surveillance site adjusting for rainfall, season, care-seeking, and malaria diagnostic. Following the implementation of IRS, cases were 84% lower in years 4-5 (adjusted incidence rate ratio [aIRR] = 0.16, 95% CI 0.12-0.22), 43% lower in year 6 (aIRR = 0.57, 95% CI 0.44-0.74), and 39% higher in the first 9 months of year 7 (aIRR = 1.39, 95% CI 0.97-1.97) compared to pre-IRS levels. Cases were 67% lower in IRS sites than non-IRS sites in year 6 (aIRR = 0.33, 95% CI 0.17-0.63) but 38% higher in the first 9 months of year 7 (aIRR = 1.38, 95% CI 0.90-2.11). We observed a resurgence in malaria to pre-IRS levels despite sustained IRS. The timing of this resurgence corresponded to a change of active ingredient. Further research is needed to determine causality.

Effectiveness of in-service training plus the collaborative improvement strategy on the quality of routine malaria surveillance data: results of a pilot study in Kayunga District, Uganda.

BACKGROUND: Surveillance data are essential for malaria control, but quality is often poor. The aim of the study was to evaluate the effectiveness of the novel combination of training plus an innovative quality improvement method-collaborative improvement (CI)-on the quality of malaria surveillance data in Uganda. METHODS: The intervention (training plus CI, or TCI), including brief in-service training and CI, was delivered in 5 health facilities (HFs) in Kayunga District from November 2015 to August 2016. HF teams monitored data quality, conducted plan-do-study-act cycles to test changes, attended periodic learning sessions, and received CI coaching. An independent evaluation was conducted to assess data completeness, accuracy, and timeliness. Using an interrupted time series design without a separate control group, data were abstracted from 156,707 outpatient department (OPD) records, laboratory registers, and aggregated monthly reports (MR) for 4 time periods: baseline-12 months, TCI scale-up-5 months; CI implementation-9 months; post-intervention-4 months. Monthly OPD register completeness was measured as the proportion of patient records with a malaria diagnosis with: (1) all data fields completed, and (2) all clinically-relevant fields completed. Accuracy was the relative difference between: (1) number of monthly malaria patients reported in OPD register versus MR, and (2) proportion of positive malaria tests reported in the laboratory register versus MR. Data were analysed with segmented linear regression modelling. RESULTS: Data completeness increased substantially following TCI. Compared to baseline, all-field completeness increased by 60.1%-points (95% confidence interval [CI]: 46.9-73.2%) at mid-point, and clinically-relevant completeness increased by 61.6%-points (95% CI: 56.6-66.7%). A relative - 57.4%-point (95% confidence interval: - 105.5, - 9.3%) change, indicating an improvement in accuracy of malaria test positivity reporting, but no effect on data accuracy for monthly malaria patients, were observed. Cost per additional malaria patient, for whom complete clinically-relevant data were recorded in the OPD register, was $3.53 (95% confidence interval: $3.03, $4.15). CONCLUSIONS: TCI improved malaria surveillance completeness considerably, with limited impact on accuracy. Although these results are promising, the intervention's effectiveness should be evaluated in more HFs, with longer follow-up, ideally in a randomized trial, before recommending CI for wide-scale use.

The impact of stopping and starting indoor residual spraying on malaria burden in Uganda.

The scale-up of malaria control efforts has led to marked reductions in malaria burden over the past twenty years, but progress has slowed. Implementation of indoor residual spraying (IRS) of insecticide, a proven vector control intervention, has been limited and difficult to sustain partly because questions remain on its added impact over widely accepted interventions such as bed nets. Using data from 14 enhanced surveillance health facilities in Uganda, a country with high bed net coverage yet high malaria burden, we estimate the impact of starting and stopping IRS on changes in malaria incidence. We show that stopping IRS was associated with a 5-fold increase in malaria incidence within 10 months, but reinstating IRS was associated with an over 5-fold decrease within 8 months. In areas where IRS was initiated and sustained, malaria incidence dropped by 85% after year 4. IRS could play a critical role in achieving global malaria targets, particularly in areas where progress has stalled.

Plasmodium falciparum parasite prevalence in East Africa: Updating data for malaria stratification.

The High Burden High Impact (HBHI) strategy for malaria encourages countries to use multiple sources of available data to define the sub-national vulnerabilities to malaria risk, including parasite prevalence. Here, a modelled estimate of Plasmodium falciparum from an updated assembly of community parasite survey data in Kenya, mainland Tanzania, and Uganda is presented and used to provide a more contemporary understanding of the sub-national malaria prevalence stratification across the sub-region for 2019. Malaria prevalence data from surveys undertaken between January 2010 and June 2020 were assembled form each of the three countries. Bayesian spatiotemporal model-based approaches were used to interpolate space-time data at fine spatial resolution adjusting for population, environmental and ecological covariates across the three countries. A total of 18,940 time-space age-standardised and microscopy-converted surveys were assembled of which 14,170 (74.8%) were identified after 2017. The estimated national population-adjusted posterior mean parasite prevalence was 4.7% (95% Bayesian Credible Interval 2.6-36.9) in Kenya, 10.6% (3.4-39.2) in mainland Tanzania, and 9.5% (4.0-48.3) in Uganda. In 2019, more than 12.7 million people resided in communities where parasite prevalence was predicted ≥ 30%, including 6.4%, 12.1% and 6.3% of Kenya, mainland Tanzania and Uganda populations, respectively. Conversely, areas that supported very low parasite prevalence (<1%) were inhabited by approximately 46.2 million people across the sub-region, or 52.2%, 26.7% and 10.4% of Kenya, mainland Tanzania and Uganda populations, respectively. In conclusion, parasite prevalence represents one of several data metrics for disease stratification at national and sub-national levels. To increase the use of this metric for decision making, there is a need to integrate other data layers on mortality related to malaria, malaria vector composition, insecticide resistance and bionomic, malaria care-seeking behaviour and current levels of unmet need of malaria interventions.

Preventive malaria treatment among school-aged children in sub-Saharan Africa: a systematic review and meta-analyses.

BACKGROUND: The burden of malaria infection in sub-Saharan Africa among school-aged children aged 5-15 years is underappreciated and represents an important source of human-to-mosquito transmission of Plasmodium falciparum. Additional interventions are needed to control and eliminate malaria. We aimed to assess whether preventive treatment of malaria might be an effective means of reducing P falciparum infection and anaemia in school-aged children and lowering parasite transmission. METHODS: In this systematic review and two meta-analyses, we searched the online databases PubMed, Embase, Cochrane CENTRAL, and Clinicaltrials.gov for intervention studies published between Jan 1, 1990, and Dec 14, 2018. We included randomised studies that assessed the effect of antimalarial treatment among asymptomatic school-aged children aged 5-15 years in sub-Saharan Africa on prevalence of P falciparum infection and anaemia, clinical malaria, and cognitive function. We first extracted data for a study-level meta-analysis, then contacted research groups to request data for an individual participant data meta-analysis. Outcomes of interest included prevalence of P falciparum infection detected by microscopy, anaemia (study defined values or haemoglobin less than age-adjusted and sex-adjusted values), clinical malaria (infection and symptoms on the basis of study-specific definitions) during follow-up, and code transmission test scores. We assessed effects by treatment type and duration of time protected, and explored effect modification by transmission setting. For study-level meta-analysis, we calculated risk ratios for binary outcomes and standardised mean differences for continuous outcomes and pooled outcomes using fixed-effect and random-effects models. We used a hierarchical generalised linear model for meta-analysis of individual participant data. This study is registered with PROSPERO, CRD42016030197. FINDINGS: Of 628 studies identified, 13 were eligible for the study-level meta-analysis (n=16 309). Researchers from 11 studies contributed data on at least one outcome (n=15 658) for an individual participant data meta-analysis. Interventions and study designs were highly heterogeneous; overall risk of bias was low. In the study-level meta-analysis, treatment was associated with reductions in P falciparum prevalence (risk ratio [RR] 0·27, 95% CI 0·17-0·44), anaemia (0·77, 0·65-0·91), and clinical malaria (0·40, 0·28-0·56); results for cognitive outcomes are not presented because data were only available for three trials. In our individual participant data meta-analysis, we found treatment significantly decreased P falciparum prevalence (adjusted RR [ARR] 0·46, 95% CI 0·40-0·53; p<0·0001; 15 648 individuals; 11 studies), anaemia (ARR 0·85, 0·77-0·92; p<0·0001; 15 026 individuals; 11 studies), and subsequent clinical malaria (ARR 0·50, 0·39-0·60; p<0·0001; 1815 individuals; four studies) across transmission settings. We detected a marginal effect on cognitive function in children older than 10 years (adjusted mean difference in standardised test scores 0·36, 0·01-0·71; p=0·044; 3962 individuals; five studies) although we found no significant effect when combined across all ages. INTERPRETATION: Preventive treatment of malaria among school-aged children significantly decreases P falciparum prevalence, anaemia, and risk of subsequent clinical malaria across transmission settings. Policy makers and programme managers should consider preventive treatment of malaria to protect this age group and advance the goal of malaria elimination, while weighing these benefits against potential risks of chemoprevention. FUNDING: US National Institutes of Health and Burroughs Wellcome Fund/ASTMH Fellowship.

Effect of long-lasting insecticidal nets with and without piperonyl butoxide on malaria indicators in Uganda (LLINEUP): a pragmatic, cluster-randomised trial embedded in a national LLIN distribution campaign.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the primary malaria prevention tool, but their effectiveness is threatened by pyrethroid resistance. We embedded a pragmatic cluster-randomised trial into Uganda's national LLIN campaign to compare conventional LLINs with those containing piperonyl butoxide (PBO), a synergist that can partially restore pyrethroid susceptibility in mosquito vectors. METHODS: 104 health sub-districts, from 48 districts in Uganda, were randomly assigned to LLINs with PBO (PermaNet 3.0 and Olyset Plus) and conventional LLINs (PermaNet 2.0 and Olyset Net) by proportionate randomisation using an iterative process. At baseline 6, 12, and 18 months after LLIN distribution, cross-sectional surveys were done in 50 randomly selected households per cluster (5200 per survey); a subset of ten households per cluster (1040 per survey) were randomly selected for entomological surveys. The primary outcome was parasite prevalence by microscopy in children aged 2-10 years, assessed in the as-treated population at 6, 12, and 18 months. This trial is registered with ISRCTN, ISRCTN17516395. FINDINGS: LLINs were delivered to households from March 25, 2017, to March 18, 2018, 32 clusters were randomly assigned to PermaNet 3.0, 20 to Olyset Plus, 37 to PermaNet 2.0, and 15 to Olyset Net. In the as-treated analysis, three clusters were excluded because no dominant LLIN was received, and four clusters were reassigned, resulting in 49 PBO LLIN clusters (31 received PermaNet 3.0 and 18 received Olyset Plus) and 52 non-PBO LLIN clusters (39 received PermaNet 2.0 and 13 received Olyset Net). At 6 months, parasite prevalence was 11% (386/3614) in the PBO group compared with 15% (556/3844) in the non-PBO group (prevalence ratio [PR] adjusted for baseline values 0·74, 95% CI 0·62-0·87; p=0·0003). Parasite prevalence was similar at month 12 (11% vs 13%; PR 0·73, 95% CI 0·63-0·85; p=0·0001) and month 18 (12% vs 14%; PR 0·84, 95% CI 0·72-0·98; p=0·029). INTERPRETATION: In Uganda, where pyrethroid resistance is high, PBO LLINs reduced parasite prevalence more effectively than did conventional LLINs for up to 18 months. This study provides evidence needed to support WHO's final recommendation on use of PBO LLINs. FUNDING: The Against Malaria Foundation, UK Department for International Development, Innovative Vector Control Consortium, and Bill and Melinda Gates Foundation.

Intermittent preventive treatment of malaria delivered to primary schoolchildren provided effective individual protection in Jinja, Uganda: secondary outcomes of a cluster-randomized trial (START-IPT).

BACKGROUND: Intermittent preventive treatment (IPT) of malaria is recommended as policy for certain high-risk populations, but not currently for schoolchildren. A cluster-randomized trial was conducted to evaluate the effect of IPT with dihydroartemisinin-piperaquine (DP) on primary schoolchildren in Jinja, Uganda. Results of the impact of IPT of schoolchildren on community-level transmission have been reported previously. Here, secondary outcomes from a school-based survey are presented. METHODS: Eighty-four clusters (one primary school plus 100 households) were randomized to intervention and control (1:1 ratio). Participants from intervention schools received monthly IPT with DP for up to 6 rounds (June-December 2014). At endline (November-December 2014), randomly selected children from all 84 schools were surveyed (13 per school) and thick blood smears were done. Those with fever or history of fever were tested with rapid diagnostic tests (RDTs) for malaria. Haemoglobin was measured in every fifth participant. Outcome measures included prevalence of asexual parasites and gametocytes (by microscopy), and prevalence of anaemia. Prevalence outcomes were analysed using generalized linear Poisson models with log link function, incorporating a cluster-level random intercept and quantified using prevalence risk ratios. RESULTS: Among 23,280 students listed on the 42 intervention school registers, 10,079 (43.3%) aged 5-20 years were enrolled into the IPT intervention and received at least one dose of DP; of these, 9286 (92.1%) received at least one full (3-day) course. In total, 1092 children were enrolled into the final school survey (546 per arm) and had a thick blood smear done; of these, 255 had haemoglobin measured (129 intervention, 126 control). Children in the intervention arm were less likely to have asexual parasites (9.2% intervention vs 44.1% control, adjusted risk ratio [aRR] 0.22 [95% CI 0.16-0.30] p < 0.001), gametocytes (3.1% intervention vs 9.5% control, aRR 0.34 [95% CI 0.20-0.56] p < 0.001), fever (20.2% intervention vs 56.2% control, aRR 0.35 [95% CI 0.25-0.50] p < 0.001), or symptomatic malaria (5.1% intervention vs 35.7% control, aRR 0.14 [95% CI 0.08-0.26] p < 0.001). Prevalence of anaemia and mean haemoglobin were similar in both study arms. CONCLUSIONS: School-aged children are a major reservoir of malaria parasites. Delivering IPT to schoolchildren would benefit individual children and may reduce transmission. School-based IPT could help to intensify malaria control toward elimination, and should be considered for policies and programmes. Trial registration Clinicaltrials.gov (NCT02009215), Registered 11 December 2013. https://clinicaltrials.gov/ct2/show/NCT02009215.

LLIN Evaluation in Uganda Project (LLINEUP): factors associated with childhood parasitaemia and anaemia 3 years after a national long-lasting insecticidal net distribution campaign: a cross-sectional survey.

BACKGROUND: Recent reductions in malaria burden have been attributed largely to long-lasting insecticidal nets (LLINs). In March-June 2017, approximately 3 years after a national LLIN distribution campaign, a cross-sectional community survey was conducted to investigate factors associated with malaria parasitaemia and anaemia, in advance of Uganda's 2017-2018 LLIN campaign. METHODS: Households from 104 clusters in 48 districts were randomly selected using two-staged cluster sampling; 50 households were enrolled per cluster. Eligible children aged 2-10 years had blood obtained for a thick blood smear and those aged 2-4 years had haemoglobin measured. Associations between outcomes and variables of interest were assessed using log-binomial regression with generalized estimating equations to adjust for household clustering. RESULTS: In total, 5196 households, 8834 children with blood smear results, and 3753 with haemoglobin results were included. Only 16% of children lived in households with adequate LLIN coverage. Overall, parasite prevalence was 26.0%, ranging from 8.0% in the South West to 53.1% in East Central. Limiting data to children 2-4 years of age, parasite prevalence was 21.4%, up from 16.9% in 2014-2015 following the national LLIN campaign. In a multivariate analysis, factors associated with parasitaemia included region (East-Central vs South-Western; adjusted prevalence ratio [aPR] 6.45, 95% CI 5.55-7.50; p < 0.001), older age (8-10 vs 2-3 years; aPR 1.57, 95% CI 1.43-1.72; p < 0.001), living in a poorer household (poorest vs least poor tercile; aPR 2.32, 95% CI 2.05-2.63; p < 0.001), one constructed of traditional materials (aPR 1.13, 95% CI 1.03-1.24; p = 0.008), or without adequate LLIN coverage (aPR 1.30, 95% CI 1.14-1.48; p < 0.001). Overall, the prevalence of anaemia (haemoglobin < 10 g/dL) was 15.1% and varied geographically. In a multivariate analysis, factors associated with anaemia included region, younger age, living in a traditional house, and parasitaemia, which was the strongest predictor (aPR 2.50, 95% CI 2.12-2.95; p < 0.001). CONCLUSIONS: Three years after a national LLIN campaign, LLIN coverage was low and parasite prevalence had increased. Parasite prevalence varied widely across Uganda; older children, those living in poorer households, and those with inadequate LLIN coverage, were at highest risk of parasitaemia. LLINs may need to be distributed more frequently through mass campaigns or continuously through sustainable mechanisms. Targeting interventions to geographic areas and populations at highest risk should also be considered.

pfhrp2 and pfhrp3 Gene Deletions That Affect Malaria Rapid Diagnostic Tests for Plasmodium falciparum: Analysis of Archived Blood Samples From 3 African Countries.

BACKGROUND: Malaria rapid diagnostic tests (mRDTs) that target histidine-rich protein 2 (HRP2) are important tools for Plasmodium falciparum diagnosis. Parasites with pfhrp2/3 gene deletions threaten the use of these mRDTs and have been reported in Africa, Asia, and South America. We studied blood samples from 3 African countries to determine if these gene deletions were present. METHODS: We analyzed 911 dried blood spots from Ghana (n = 165), Tanzania (n = 176), and Uganda (n = 570). Plasmodium falciparum infection was confirmed by 18S rDNA polymerase chain reaction (PCR), and pfhrp2/3 genes were genotyped. True pfhrp2/3 gene deletions were confirmed if samples were (1) microscopy positive; (2) 18S rDNA PCR positive; (3) positive for merozoite surface protein genes by PCR or positive by loop-mediated isothermal amplification; or (4) quantitative PCR positive with >5 parasites/µL. RESULTS: No pfhrp2/3 deletions were detected in samples from Ghana, but deletions were identified in Tanzania (3 pfhrp2; 2 pfhrp3) and Uganda (7 pfhrp2; 2 pfhrp3). Of the 10 samples with pfhrp2 deletions, 9 tested negative by HRP2-based mRDT. CONCLUSIONS: The presence of pfhrp2/3 deletions in Tanzania and Uganda, along with reports of pfhrp2/3-deleted parasites in neighboring countries, reinforces the need for systematic surveillance to monitor the reliability of mRDTs in malaria-endemic countries.

LLIN Evaluation in Uganda Project (LLINEUP) - Impact of long-lasting insecticidal nets with, and without, piperonyl butoxide on malaria indicators in Uganda: study protocol for a cluster-randomised trial.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are a key malaria control intervention, but their effectiveness is threatened by resistance to pyrethroid insecticides. Some new LLINs combine pyrethroids with piperonyl butoxide (PBO), a synergist that can overcome P450-based metabolic resistance to pyrethroids in mosquitoes. In 2017-2018, the Ugandan Ministry of Health distributed LLINs with and without PBO through a national mass-distribution campaign, providing a unique opportunity to rigorously evaluate PBO LLINs across different epidemiological settings. METHODS/DESIGN: Together with the Ministry of Health, we embedded a cluster-randomised trial to evaluate the impact of LLINs delivered in the 2017-2018 national campaign. A total of 104 clusters (health sub-districts) in Eastern and Western Uganda were involved, covering 48 of 121 (40%) districts. Using adaptive randomisation driven by the number of LLINs available, clusters were assigned to receive one of four types of LLINs, including two brands with PBO: 1) PermaNet 3.0 (n = 32) and 2) Olyset Plus (n = 20); and two without PBO: 3) PermaNet 2.0 (n = 37) and 4) Olyset Net (n = 15). We are conducting cross-sectional community surveys in 50 randomly selected households per cluster (5200 households per survey) and entomological surveillance for insecticide resistance in up to 10 randomly selected households enrolled in the community surveys per cluster (1040 households per survey) at baseline and 6, 12, and 18 months after LLIN distribution. Net durability and bio-efficacy will be assessed in 400 nets withdrawn from households with replacement at 12 months. The primary trial outcome is parasite prevalence as measured by microscopy in children aged 2-10 years in the follow-up surveys. DISCUSSION: PBO LLINs are a promising new tool to reduce the impact of pyrethroid resistance on malaria control. The World Health Organization has issued a preliminary endorsement of PBO LLINs, but additional epidemiological evidence of the effect of PBO LLINs is urgently needed. The results of this innovative, large-scale trial embedded within a routine national distribution campaign will make an important contribution to the malaria control policy in Uganda and throughout Africa, where pyrethroid resistance in malaria vectors has increased dramatically. This model of evaluation could be a paradigm for future assessment of malaria control interventions. TRIAL REGISTRATION: ISRCTN, ISRCTN17516395 . Registered on 14 February 2017. WORLD HEALTH ORGANIZATION TRIAL REGISTRATION DATA SET: See Additional file 1.

LLIN Evaluation in Uganda Project (LLINEUP): a cross-sectional survey of species diversity and insecticide resistance in 48 districts of Uganda.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the principal tool for malaria control in Africa and are presently treated with a single class of insecticide; however, increasing levels of insecticide resistance threaten their success. In response to this threat nets have been developed that incorporate the synergist, piperonyl butoxide (PBO), which inhibits the activity of cytochrome P450s which is one main mechanisms of insecticide resistance, allowing resistance to pyrethroids to be reversed. However, data on the value and cost effectiveness of these nets is lacking. A large-scale cluster randomised trial of conventional LLINs and PBO-LLINs was conducted in Uganda in 104 health sub-districts (HSDs) in 2017-2019. Prior to the mass distribution of LLINs, a baseline entomological survey was carried out, the results of which are reported herein. Ten households from each HSD were randomly selected for entomological surveillance at baseline which included household mosquito collections. RESULTS: Prior to LLIN distribution entomological collections were carried out in 1029 houses across the 104 HSDs. Anopheles gambiae (s.l.) was the principal vector in all but 9 of the 71 HSDs that yielded vector species. Molecular analysis found An. gambiae (s.s.) to be the predominant vector collected. Plasmodium falciparum was detected in 5.5% of An. gambiae (s.s.) and in 4.0% of An. funestus (s.s.) examined. Infection rates of other plasmodium species (P. vivax, P. ovale and P. malariae) were lower with infection rates of 1.2% and 1.7% for An. gambiae (s.s.) and An. funestus (s.s.), respectively. The knockdown resistance (kdr) mutation Vgsc-L1014S was found at very high frequency in An. gambiae (s.s.) with the Vgsc-L1014F mutation at low frequency and the wild-type allele virtually absent. In An. arabiensis the wild-type allele was predominant. The resistance-associated alleles, Cyp4j5-L43F and Coeae1d were found at moderate frequencies which varied across the study site. Vgsc-N1575Y mutation was not found in any samples examined. CONCLUSIONS: No significant differences between planned intervention arms was observed in vector densities, sporozoite infection rate or insecticide resistance marker frequency across the study site prior to the distribution of LLINs. Very high levels of kdr resistance were observed in all areas; however, the resistance-associated markers Cyp4j5-L43F and Coeae1d were found at varying frequencies across the study site which may have implications for the effectiveness of standard LLINs. Trial registration This study is registered with ISRCTN, ISRCTN17516395. Registered 14 February 2017, http://www.isrctn.com/ISRCTN17516395.

Cost for sickle cell disease screening using isoelectric focusing with dried blood spot samples and estimation of price thresholds for a point-of-care test in Uganda.

BACKGROUND: Early identification through newborn screening is the first step in active management of sickle cell disease (SCD). Uganda currently screens newborns and infants under 2 years for SCD in high HIV-burden districts using isoelectric focusing with dried blood spot samples. Our analysis sought to estimate the costs per child screened for SCD using this method in Uganda and then to use those data to estimate the price threshold for screening with a point-of-care (POC) test. METHODS: We estimated the financial and economic costs per child screened for SCD using data from health facilities and the Central Public Health Laboratory. These costs included sample collection, transportation, and laboratory processing. Price thresholds for a POC test were estimated using two scenarios. RESULTS: The price threshold of an SCD POC test used for diagnosis would be $3.77 when taking into account only financial costs and $5.14 when taking into account economic costs. Thresholds for a POC test used for screening would be $3.07-$3.51 and $4.38-$5.09, respectively, depending on test specificity. CONCLUSION: The price threshold of a POC test for SCD will depend on the assumptions on how it will be used - either as a screening or diagnostic test. If used for screening, test specificity will have significant impact. Results from this type of costing study can allow developers to incorporate quantitatively estimated price thresholds for innovative products into target product profiles early in the product development cycle.