LLIN Evaluation in Uganda Project (LLINEUP): modelling the impact of COVID-19-related disruptions on delivery of long-lasting insecticidal nets on malaria indicators in Uganda.

BACKGROUND: Disruptions in malaria control due to COVID-19 mitigation measures were predicted to increase malaria morbidity and mortality in Africa substantially. In Uganda, long-lasting insecticidal nets (LLINs) are distributed nationwide every 3-4 years, but the 2020-2021 campaign was altered because of COVID-19 restrictions so that the timing of delivery of new nets was different from the original plans made by the National Malaria Control Programme. METHODS: A transmission dynamics modelling exercise was conducted to explore how the altered delivery of LLINs in 2020-2021 impacted malaria burden in Uganda. Data were available on the planned LLIN distribution schedule for 2020-2021, and the actual delivery. The transmission model was used to simulate 100 health sub-districts, and parameterized to match understanding of local mosquito bionomics, net use estimates, and seasonal patterns based on data collected in 2017-2019 during a cluster-randomized trial (LLINEUP). Two scenarios were compared; simulated LLIN distributions matching the actual delivery schedule, and a comparable scenario simulating LLIN distributions as originally planned. Model parameters were otherwise matched between simulations. RESULTS: Approximately 70% of the study population received LLINs later than scheduled in 2020-2021, although some areas received LLINs earlier than planned. The model indicates that malaria incidence in 2020 was substantially higher in areas that received LLINs late. In some areas, early distribution of LLINs appeared less effective than the original distribution schedule, possibly due to attrition of LLINs prior to transmission peaks, and waning LLIN efficacy after distribution. On average, the model simulations predicted broadly similar overall mean malaria incidence in 2021 and 2022. After accounting for differences in cluster population size and LLIN distribution dates, no substantial increase in malaria burden was detected. CONCLUSIONS: The model results suggest that the disruptions in the 2020-2021 LLIN distribution campaign in Uganda did not substantially increase malaria burden in the study areas.

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 .

House modifications as a malaria control tool: how does local context shape participants' experience and interpretation in Uganda?

BACKGROUND: Evidence that house design can provide protection from malaria is growing. Housing modifications such as screening windows, doors, and ceilings, and attaching insecticide-impregnated materials to the eaves (the gap between the top of the wall and bottom of the roof), can protect against malaria. To be effective at scale, however, these modifications must be adopted by household residents. There is evidence that housing modifications can be acceptable, but in-depth knowledge on the experiences and interpretation of modifications is lacking. This qualitative study was carried out to provide a holistic account of the relationship between experiences and interpretations of four types of piloted housing modifications and the local context in Jinja, Uganda. METHODS: Qualitative research was conducted between January to June 2021, before and during the installation of four types of housing modifications. The methods included nine weeks of participant observations in two study villages, nine focus group discussions with primary caregivers and heads of households (11-12 participants each), and nine key informant interviews with stakeholders and study team members. RESULTS: Most residents supported the modifications. Experiences and interpretation of the housing modifications were shaped by the different types of housing in the area and the processes through which residents finished their houses, local forms of land and property ownership, and cultural and spiritual beliefs about houses. CONCLUSIONS: To maximize the uptake and benefit of housing modifications against malaria, programme development needs to take local context into account. Forms of local land and house ownership, preferences, the social significance of housing types, and religious and spiritual ideas shape the responses to housing modifications in Jinja. These factors may be important in other setting. Trial registration Trial registration number is NCT04622241. The first draft was posted on November 9th 2020.

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.

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 (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.

Antibiotic use among children under five years with diarrhea in rural communities of Gulu, northern Uganda: a cross-sectional study.

BACKGROUND: Diarrhea is the second leading cause of mortality in children under 5 years of age globally, and the risk of death increases with practices such as restriction of fluid intake and inappropriate use of antibiotics. We determined the prevalence of antibiotic use in managing diarrhea in children under 5 years of age in rural communities of Gulu district, northern Uganda. METHOD: A cross-sectional study among children under 5 years with diarrhea, from households selected using multi-stage sampling. A researcher administered questionnaire was used to obtain data from caregivers of these children. RESULTS: Of the 856 children recruited, 318 (37.1%, 318/856) had experienced diarrhea, where 263 (82.7%, 263/318) had diarrhea with acute respiratory infections (ARIs), and 55 (17.3%, 55/318) had diarrhea without ARIs. The majority (89.6%, 285/318) of the children had non-bloody diarrhea. A high proportion (82.8%) of the children with non-bloody diarrhea also had ARIs. Bloody diarrhea was reported for 33 (10.4%) children including those with ARIs, and only 6 of these (18.2%) children had bloody diarrhea without ARIs. Of the 318 children with diarrhea, over half (52%, CI: 46-57) were administered antibiotics. Of the 55 children who had diarrhea without ARIs, over a third (38%, CI: 26-51) were administered antibiotics. Similarly, of the 263 children with diarrhea and ARIs, 54% (CI: 48-60) were treated with antibiotics. The determinants of antibiotic use included; children living in peri-urban settings (AOR: 3.41, CI: 1.65-7.08, P = 0.001), getting treatment from health facility (AOR: 1.76, CI: 1.06-2.93, P = 0.029), and having diarrhea with ARIs (AOR: 3.09, CI: 1.49-6.42, P = 0.003). CONCLUSION: Antibiotic use is common among children under 5 years with diarrhea in rural communities of northern Uganda.

Alcohol consumption, hypertension and obesity: Relationship patterns along different age groups in Uganda.

The prevalence of non-communicable diseases including hypertension and obesity is rising and alcohol consumption is a predisposing factor. This study explored the effect of alcohol consumption patterns on the hypertension-age group and obesity-age group relationships. The data were extracted from the 2014 National NCD Survey of adults aged 18-69 years. Hypertension was defined as a condition of having systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg while obesity was defined as having a body mass index ≥30 kg/m2. Frequent alcohol consumption was measured as alcohol use ≥3 times a week. Multivariable log binomial regression analysis was used to assess independent relationship between the outcomes and alcohol consumption. The prevalences of hypertension, frequent alcohol consumption and obesity increased across age groups but were divergent towards last age group. Hypertension prevalence ratios were higher with higher age groups among moderate and nondrinkers but not among frequent drinkers. Alcohol drinking pattern modified the age hypertension relationship in a model with ungrouped age. The drinking pattern did not modify obesity-age relationship. Alcohol consumption pattern appeared to modify the hypertension-age group relationship. However, more research is needed to explain why prevalence ratios are higher with higher age groups among moderate drinkers and abstainers while they stagnate among the frequent drinkers. There was no evidence to show the effect of alcohol consumption on obesity-age group relationship.

Use of antibacterials in the management of symptoms of acute respiratory tract infections among children under five years in Gulu, northern Uganda: Prevalence and determinants.

Inappropriate use of antibacterials is a major public health challenge as it can promote emergence of resistance, wastage of financial resources, morbidity and mortality. In this study, we determined the prevalence and factors associated with antibacterial use in managing symptoms of acute respiratory tract infections (ARIs) in households in rural communities of Gulu district, northern Uganda. A cross-sectional study was conducted among households selected using multi-stage sampling. Data were collected through interviews with care-givers of children under five years, using a structured interviewer administered questionnaire. Out of the 856 children who had symptoms of ARIs, 515 (60.2%; CI: 54.5%-65.6%) were treated with antibacterials. The most commonly used antibacterials were amoxicillin (55.2%, n = 358), cotrimoxazole (15.4%, n = 100) and metronidazole (11.4%, n = 74). The determinants of antibacterial use included; getting treatment from a health facility (AOR: 1.85, CI: 1.34-2.56, P < 0.001), households located in peri-urban area (AOR: 2.54, CI: 1.34-4.84, P = 0.005), and a child having cough (AOR: 7.02, CI: 4.36-11.31, P < 0.001). The prevalence of antibacterial use among children under five years with symptoms of ARIs is high in communities of Gulu district, northern Uganda. Getting treatment from a health facility, if a household was located in a peri-urban area and having a cough are positive predictors of antibacterial use. There is need for targeted education on appropriate antibacterial use in rural communities and hospital settings where over prescription is most likely especially in treating symptoms of ARIs among children under five years.

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.

Associations between red blood cell variants and malaria among children and adults from three areas of Uganda: a prospective cohort study.

BACKGROUND: Multiple red blood cell (RBC) variants appear to offer protection against the most severe forms of Plasmodium falciparum malaria. Associations between these variants and uncomplicated malaria are less clear. METHODS: Data from a longitudinal cohort study conducted in 3 sub-counties in Uganda was used to quantify associations between three red blood cell variants Hb [AA, AS, S (rs334)], alpha thalassaemia 3.7 kb deletion, and glucose-6-phosphate dehydrogenase deficiency A-(G6PD 202A genotype) and malaria incidence, parasite prevalence, parasite density (a measure of anti-parasite immunity) and body temperature adjusted for parasite density (a measure of anti-disease immunity). All analyses were adjusted for age, average household entomological inoculation rate, and study site. Results for all variants were compared to those for wild type genotypes. RESULTS: In children, HbAS was associated, compared to wild type, with a lower incidence of malaria (IRR = 0.78, 95% CI 0.66-0.92, p = 0.003), lower parasite density upon infection (PR = 0.66, 95% CI 0.51-0.85, p = 0.001), and lower body temperature for any given parasite density (- 0.13 â„ƒ, 95% CI - 0.21, - 0.05, p = 0.002). In children, HbSS was associated with a lower incidence of malaria (IRR = 0.17, 95% CI 0.04-0.71, p = 0.02) and lower parasite density upon infection (PR = 0.31, 95% CI 0.18-0.54, p < 0.001). α-/αα thalassaemia, was associated with higher parasite prevalence in both children and adults (RR = 1.23, 95% CI 1.06-1.43, p = 0.008 and RR = 1.52, 95% CI 1.04-2.23, p = 0.03, respectively). G6PD deficiency was associated with lower body temperature for any given parasite density only among male hemizygote children (- 0.19 â„ƒ, 95% CI - 0.31, - 0.06, p = 0.003). CONCLUSION: RBC variants were associated with non-severe malaria outcomes. Elucidation of the mechanisms by which they confer protection will improve understanding of genetic protection against malaria.

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.

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.

Association Between Recent Overnight Travel and Risk of Malaria: A Prospective Cohort Study at 3 Sites in Uganda.

Background: Human movement can undermine malaria control efforts. However, understanding of the association between travel and malaria infection in Africa is limited. We evaluated the association between recent overnight travel and malaria incidence in Uganda. Methods: All children aged 0.5-10 years and 1 adult living in 266 randomly selected households within 3 different regions of Uganda were followed up prospectively. Information on overnight travel was collected in 2015-2016. Malaria, defined as fever with parasites detected by microscopy, was measured using passive surveillance. Results: At least 1 overnight trip was reported by 64 of 275 (23.3%) participants in Walukuba, 37 of 317 (11.7%) in Nagongera, and 19 of 314 (6.1%) in Kihihi. Among individuals who traveled, the incidence of malaria was higher in the first 60 days after traveling, compared with periods without recent travel at all 3 sites (overall, 1.15 vs 0.33 episodes per person-year; incidence rate ratio, 3.53; 95% confidence interval, 1.85-6.73; P < .001). Risk factors for malaria within 60 days after overnight travel included young age (19.5% in children vs 4.9% in adults; odds ratio, 5.29; 95% confidence interval, 1.34-21.0; P = .02) and not using an insecticide-treated net during travel (18.0% for no use vs 4.1% for any use; 5.10; 1.07-24.5; P = .04). Conclusions: Recent overnight travel was associated with a higher incidence of malaria. Individuals who travel may represent a high-risk group that could be targeted for malaria control interventions, particularly use of insecticide-treated nets.

LLIN Evaluation in Uganda Project (LLINEUP): factors associated with ownership and use of long-lasting insecticidal nets in Uganda: a cross-sectional survey of 48 districts.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are a key malaria control intervention. To investigate factors associated with ownership and use of LLINs in Uganda, a cross-sectional community survey was conducted in March-June 2017, approximately 3 years after a national Universal Coverage Campaign (UCC). METHODS: Households from 104 clusters (health sub-districts) in 48 districts were randomly selected using two-staged cluster sampling; 50 households were enrolled per cluster. Outcomes were household ownership of LLINs (at least one LLIN), adequate LLIN coverage (at least one LLIN per 2 residents), and use of LLINs (resident slept under a LLIN the previous night). Associations between variables of interest and outcomes were made using multivariate logistic regression. RESULTS: In total, 5196 households, with 29,627 residents and 6980 bed-nets, were included in the analysis. Overall, 65.0% of households owned at least one LLIN (down from 94% in 2014). In the adjusted analysis, factors most strongly associated with LLIN ownership were living in a wealthier household (highest tercile vs lowest; adjusted odds ratio [aOR] 1.94, 95% CI 1.66-2.28, p < 0.001) and time since the last UCC (29-37 vs 42-53 months; aOR 1.91, 95% CI 1.60-2.28, p < 0.001). Only 17.9% of households had adequate LLIN coverage (down from 65% in 2014). Factors most strongly associated with adequate coverage were fewer residents (2-4 vs ≥ 7; aOR 6.52, 95% CI 5.13-8.29, p < 0.001), living in a wealthier household (highest tercile vs lowest; aOR: 2,32, 95% CI 1.88-2.85, p < 0.001) and time since the last UCC (29-37 vs 42-53 months; aOR 2.13, 95% CI 1.61-2.81, p < 0.001). Only 39.5% of residents used a LLIN the previous night. Age was strongly associated with LLIN use, as were household wealth and time since the last UCC. Children < 5 years (44.7%) and residents > 15 years (44.1%) were more likely to use nets than children aged 5-15 years (30.7%; < 5 years: aOR 1.71, 95% CI 1.62-1.81, p < 0.001; > 15 years: aOR 1.37, 95% CI 1.29-1.45, p < 0.001). CONCLUSIONS: Long-lasting insecticidal net ownership and coverage have reduced markedly in Uganda since the last net distribution campaign in 2013/14. Houses with many residents, poorer households, and school-aged children should be targeted to improve LLIN coverage and use. Trial registration This study is registered with ISRCTN (17516395).

Rapid improvements to rural Ugandan housing and their association with malaria from intense to reduced transmission: a cohort study.

BACKGROUND: Rapid population growth in Africa requires an urgent expansion and improvement of housing options. Improving housing presents a promising opportunity for malaria control by reducing indoor exposure to mosquitoes. We measured recent changes in house design in rural Uganda and evaluated their association with malaria in relation to a mass scale-up of control efforts. METHODS: This analysis was part of a cohort study designed to compare temporal changes in malaria incidence from a cohort of children and adults with temporal changes in malaria test positivity rate from health facility surveillance. All children aged 6 months to 10 years (n=384) living in 107 households in Nagongera sub-country, Tororo, Uganda, were given long-lasting insecticide-treated nets and followed between Aug 19, 2011, and June 30, 2017. Repeat rounds of indoor residual spraying of insecticide were initiated on Dec 5, 2014. Socioeconomic data were collected at two timepoints (Sept 25-Oct 9, 2013 and June 21-July 11, 2016) and houses were classified as modern (cement, wood, or metal walls, tiled or metal roof, and closed eaves) or traditional (all other homes). Associations between house design and three outcomes were evaluated before and after the introduction of indoor residual spraying: human biting rate estimated monthly in each household using US Centers for Disease Control and Prevention light traps; parasite prevalence measured routinely by microscopy every 3 months before indoor residual spraying and monthly after indoor residual spraying; and malaria incidence measured by passive surveillance. FINDINGS: The implementation of indoor residual spraying was associated with significant declines in human biting rate (33·5 vs 2·7 Anopheles per house per night after indoor residual spraying, p<0·0001), parasite prevalence (32·0% vs 14·0%, p<0·0001), and malaria incidence (3·0 vs 0·5 episodes per person-year at risk, p<0·0001). The prevalence of modern housing increased from 23·4% in 2013 to 45·4% in 2016 (p=0·001). Compared with traditional houses, modern houses were associated with a 48% reduction in human biting rate before indoor residual spraying (adjusted incidence rate ratio [aIRR] 0·52, 95% CI 0·36-0·73, p=0·0002), and a 73% reduction after indoor residual spraying (aIRR 0·27, 0·17-0·42, p<0·0001). Before indoor residual spraying, there was no association between house type and parasite prevalence, but after indoor residual spraying there was a 57% reduction in the odds of parasitaemia in modern houses compared with traditional houses, controlling for age, sex, and socioeconomic position (adjusted odds ratio 0·43, 95% CI 0·24-0·77, p=0·004). House type was not associated with malaria incidence before or after indoor residual spraying. INTERPRETATION: House design improved rapidly in rural Uganda and was associated with additional reductions in mosquito density and parasite prevalence following the introduction of indoor residual spraying. Changes to house design in endemic Africa, including closing eaves and the replacement of traditional building materials, might help further the gains achieved with more widely accepted malaria control interventions. FUNDING: US National Institutes of Health, Bill & Melinda Gates Foundation, and Medical Research Council UK.

Both inflammatory and regulatory cytokine responses to malaria are blunted with increasing age in highly exposed children.

BACKGROUND: Young children are at greatest risk for malaria-associated morbidity and mortality. The immune response of young children differs in fundamental ways from that of adults, and these differences likely contribute to the increased susceptibility of children to severe malaria and to their delayed development of immunity. Elevated levels of pro-inflammatory cytokines and chemokines in the peripheral blood during acute infection contribute to the control of parasitaemia, but are also responsible for much of the immunopathology seen during symptomatic disease. Clinical immunity to malaria may depend upon the ability to regulate these pro-inflammatory responses, possibly through mechanisms of immunologic tolerance. In order to explore the effect of age on the immune response to malaria and the development of clinical immunity, cytokines and chemokines were measured in the plasma of children at day 0 of an acute malaria episode and during convalescence. RESULTS: Younger children presenting with acute malaria exhibited much higher levels of TNF, IL2, and IL6, as well as increased Th1 associated chemokines IP10, MIG, and MCP1, compared to older children with acute malaria. Additionally, the regulatory cytokines IL10 and TNFRI were dramatically elevated in younger children compared to older children during acute infection, indicating that regulatory as well as pro-inflammatory cytokine responses are dampened in later childhood. CONCLUSIONS: Together these data suggest that there is a profound blunting of the cytokine and chemokine response to malaria among older children residing in endemic settings, which may be due to repeated malaria exposure, intrinsic age-based differences in the immune response, or both.

Vδ2+ T cell response to malaria correlates with protection from infection but is attenuated with repeated exposure.

Vδ2+ γδ T cells are semi-innate T cells that expand markedly following P. falciparum (Pf) infection in naïve adults, but are lost and become dysfunctional among children repeatedly exposed to malaria. The role of these cells in mediating clinical immunity (i.e. protection against symptoms) to malaria remains unclear. We measured Vδ2+ T cell absolute counts at acute and convalescent malaria timepoints (n = 43), and Vδ2+ counts, cellular phenotype, and cytokine production following in vitro stimulation at asymptomatic visits (n = 377), among children aged 6 months to 10 years living in Uganda. Increasing age was associated with diminished in vivo expansion following malaria, and lower Vδ2 absolute counts overall, among children living in a high transmission setting. Microscopic parasitemia and expression of the immunoregulatory markers Tim-3 and CD57 were associated with diminished Vδ2+ T cell pro-inflammatory cytokine production. Higher Vδ2 pro-inflammatory cytokine production was associated with protection from subsequent Pf infection, but also with an increased odds of symptoms once infected. Vδ2+ T cells may play a role in preventing malaria infection in children living in endemic settings; progressive loss and dysfunction of these cells may represent a disease tolerance mechanism that contributes to the development of clinical immunity to malaria.

Measures of Malaria Burden after Long-Lasting Insecticidal Net Distribution and Indoor Residual Spraying at Three Sites in Uganda: A Prospective Observational Study.

BACKGROUND: Long-lasting insecticidal nets (LLINs) and indoor residual spraying of insecticide (IRS) are the primary vector control interventions used to prevent malaria in Africa. Although both interventions are effective in some settings, high-quality evidence is rarely available to evaluate their effectiveness following deployment by a national malaria control program. In Uganda, we measured changes in key malaria indicators following universal LLIN distribution in three sites, with the addition of IRS at one of these sites. METHODS AND FINDINGS: Comprehensive malaria surveillance was conducted from October 1, 2011, to March 31, 2016, in three sub-counties with relatively low (Walukuba), moderate (Kihihi), and high transmission (Nagongera). Between 2013 and 2014, universal LLIN distribution campaigns were conducted in all sites, and in December 2014, IRS with the carbamate bendiocarb was initiated in Nagongera. High-quality surveillance evaluated malaria metrics and mosquito exposure before and after interventions through (a) enhanced health-facility-based surveillance to estimate malaria test positivity rate (TPR), expressed as the number testing positive for malaria/number tested for malaria (number of children tested for malaria: Walukuba = 42,833, Kihihi = 28,790, and Nagongera = 38,690); (b) cohort studies to estimate the incidence of malaria, expressed as the number of episodes per person-year [PPY] at risk (number of children observed: Walukuba = 340, Kihihi = 380, and Nagongera = 361); and (c) entomology surveys to estimate household-level human biting rate (HBR), expressed as the number of female Anopheles mosquitoes collected per house-night of collection (number of households observed: Walukuba = 117, Kihihi = 107, and Nagongera = 107). The LLIN distribution campaign substantially increased LLIN coverage levels at the three sites to between 65.0% and 95.5% of households with at least one LLIN. In Walukuba, over the 28-mo post-intervention period, universal LLIN distribution was associated with no change in the incidence of malaria (0.39 episodes PPY pre-intervention versus 0.20 post-intervention; adjusted rate ratio [aRR] = 1.02, 95% CI 0.36-2.91, p = 0.97) and non-significant reductions in the TPR (26.5% pre-intervention versus 26.2% post-intervention; aRR = 0.70, 95% CI 0.46-1.06, p = 0.09) and HBR (1.07 mosquitoes per house-night pre-intervention versus 0.71 post-intervention; aRR = 0.41, 95% CI 0.14-1.18, p = 0.10). In Kihihi, over the 21-mo post-intervention period, universal LLIN distribution was associated with a reduction in the incidence of malaria (1.77 pre-intervention versus 1.89 post-intervention; aRR = 0.65, 95% CI 0.43-0.98, p = 0.04) but no significant change in the TPR (49.3% pre-intervention versus 45.9% post-intervention; aRR = 0.83, 95% 0.58-1.18, p = 0.30) or HBR (4.06 pre-intervention versus 2.44 post-intervention; aRR = 0.71, 95% CI 0.30-1.64, p = 0.40). In Nagongera, over the 12-mo post-intervention period, universal LLIN distribution was associated with a reduction in the TPR (45.3% pre-intervention versus 36.5% post-intervention; aRR = 0.82, 95% CI 0.76-0.88, p < 0.001) but no significant change in the incidence of malaria (2.82 pre-intervention versus 3.28 post-intervention; aRR = 1.10, 95% 0.76-1.59, p = 0.60) or HBR (41.04 pre-intervention versus 20.15 post-intervention; aRR = 0.87, 95% CI 0.31-2.47, p = 0.80). The addition of three rounds of IRS at ~6-mo intervals in Nagongera was followed by clear decreases in all outcomes: incidence of malaria (3.25 pre-intervention versus 0.63 post-intervention; aRR = 0.13, 95% CI 0.07-0.27, p < 0.001), TPR (37.8% pre-intervention versus 15.0% post-intervention; aRR = 0.54, 95% CI 0.49-0.60, p < 0.001), and HBR (18.71 pre-intervention versus 3.23 post-intervention; aRR = 0.29, 95% CI 0.17-0.50, p < 0.001). High levels of pyrethroid resistance were documented at all three study sites. Limitations of the study included the observational study design, the lack of contemporaneous control groups, and that the interventions were implemented under programmatic conditions. CONCLUSIONS: Universal distribution of LLINs at three sites with varying transmission intensity was associated with modest declines in the burden of malaria for some indicators, but the addition of IRS at the highest transmission site was associated with a marked decline in the burden of malaria for all indicators. In highly endemic areas of Africa with widespread pyrethroid resistance, IRS using alternative insecticide formulations may be needed to achieve substantial gains in malaria control.

Characterizing microscopic and submicroscopic malaria parasitaemia at three sites with varied transmission intensity in Uganda.

BACKGROUND: Parasite prevalence is a key metric used to quantify the burden of malaria and assess the impact of control strategies. Most published estimates of parasite prevalence are based on microscopy and likely underestimate true prevalence. METHODS: Thick smear microscopy was performed in cohorts of children (aged 6 month to 10 years) and adults every 90 days over 2 years, at three sites of varying transmission intensity in Uganda. Microscopy-negative samples were tested for sub-microscopic parasitaemia using loop-mediated isothermal amplification (LAMP). Generalized estimating equation models were used to evaluate associations between age and parasitaemia, factors associated with sub-microscopic infection and associations between parasitaemia and haemoglobin. RESULTS: A total of 9260 samples were collected from 1245 participants. Parasite prevalence among children across the three sites was 7.4, 9.4 and 28.8 % by microscopy and 21.3, 31.8 and 69.0 % by microscopy plus LAMP. Parasite prevalence among adults across the three sites was 3.1, 3.0 and 5.2 % by microscopy and 18.8, 24.2 and 53.5 % by microscopy plus LAMP. Among those with parasitaemia, adults and persons recently treated with anti-malarial therapy had the highest prevalence of sub-microscopic infection. Children with sub-microscopic or microscopic parasitaemia had lower mean haemoglobin levels compared to children with no detectable parasites. CONCLUSIONS: Across a range of transmission intensities in Uganda, microscopy vastly underestimated parasite prevalence, especially among adults.