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Summary 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.

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 distribution to compare the durability of LLINs with and without PBOA total of 104 clusters 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 WHO Cone and Wireball assays using pyrethroid-resistant An. gambiae, with 1hr knockdown and 24hr 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 timepoints but PBO content declined by 55% (pHere 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.

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.

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.

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 ℃, 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 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.

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

IntroductionUganda is experiencing a significant increase in the prevalence of non-communicable diseases including hypertension and obesity. Frequent alcohol use is also highly prevalent in Uganda and is a key risk factor for both hypertension and obesity. This study determines the trends of frequent alcohol consumption, hypertension and obesity across different age groups, and the extent to which alcohol consumption affects the two.MethodsThe data were extracted from the 2014 National Non-communicable Diseases Risk Factor Survey (N=3,987) conducted among adults aged 18 to 69 years. Hypertension was defined as systolic blood pressure ≥140mmHG or diastolic blood pressure ≥90. Obesity was defined as body mass index >30 kg/m2. Frequent alcohol consumption was defined as alcohol use 3 or more times a week. Multivariable log binomial regression analysis was carried out for each of the two outcome variables against age group and controlled for frequency of alcohol consumption and few other independent factors. Non-parametric tests were used to compare trends of prevalence ratios across age groups. Modified Poisson regression was use in few instances when the model failed to converge.ResultsThe results showed increasing trend in the prevalence of hypertension and frequent alcohol consumption but a declining trend for obesity along different age groups (pConclusionThe results call for more research to understand the effect of alcohol on the hypertension-age relationship, and the obesity-age relationship. Why prevalence ratios for hypertension decline among those who take alcohol most frequently is another issue that needs further research.

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.

Contains fulltext : 172114.pdf (Publisher’s version ) (Open Access) 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.

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., In this prospective observational study, Grant Dorsey and colleagues measure changes in malaria burden after long-lasting insecticidal net distribution and indoor residual spraying at three sites of in Uganda., Author Summary Why Was This Study Done? Long-lasting insecticidal nets (LLINs), which prevent mosquitoes from biting people while they sleep, and indoor residual spraying of insecticides (IRS) in houses, which prevents mosquitoes from resting in houses, are the main tools used to prevent malaria in Africa. Although LLINs and IRS have been shown to be effective, changes in the behavior of mosquitoes and people as well as the emergence of mosquitoes resistant to insecticides could compromise the benefits of these interventions. Recently the government of Uganda distributed free LLINs throughout the country and began IRS in selected areas. In this “real world” setting, it is important to monitor for changes in the burden of malaria following the scale-up of LLIN distribution and IRS. What Did the Researchers Do and Find? The researchers conducted comprehensive malaria surveillance between October 1, 2011, and March 31, 2016, at three sites in Uganda that differed in the intensity of malaria transmission. Between 2013 and 2014, LLINs were distributed to the entire population at all three sites, and in December 2014, IRS with an insecticide different from that used in the LLINs was started in the site with the highest level of malaria transmission. The researchers found that following LLIN distribution, there were only modest declines in some measures of malaria burden at the three sites, and no changes in other measures. In contrast, following the addition of IRS at the highest transmission site, all measures of malaria burden declined dramatically. The research also documented a high level of resistance to the type of insecticide used in LLINS at all three sites. What Do These Findings Mean? These findings suggest that in countries like Uganda, which has a heavy burden of malaria, LLINs alone may not be adequate to substantially drive down the burden of malaria, and the addition of IRS using a different insecticide may be needed to have a major impact. However, it should be noted that IRS is more expensive and harder to implement than distributing LLINs, and generally needs to be repeated every 6–12 months to have a sustained effect.

The intensification of control interventions has led to marked reductions in malaria burden in some settings, but not others. To provide a comprehensive description of malaria epidemiology in Uganda, we conducted surveillance studies over 24 months in 100 houses randomly selected from each of three subcounties: Walukuba (peri-urban), Kihihi (rural), and Nagongera (rural). Annual entomological inoculation rate (aEIR) was estimated from monthly Centers for Disease Control and Prevention (CDC) light trap mosquito collections. Children aged 0.5-10 years were provided long-lasting insecticidal nets (LLINs) and followed for measures of parasite prevalence, anemia and malaria incidence. Estimates of aEIR were 2.8, 32.0, and 310 infectious bites per year, and estimates of parasite prevalence 7.4%, 9.3%, and 28.7% for Walukuba, Kihihi, and Nagongera, respectively. Over the 2-year study, malaria incidence per person-years decreased in Walukuba (0.51 versus 0.31, P = 0.001) and increased in Kihihi (0.97 versus 1.93, P < 0.001) and Nagongera (2.33 versus 3.30, P < 0.001). Of 2,582 episodes of malaria, only 8 (0.3%) met criteria for severe disease. The prevalence of anemia was low and not associated with transmission intensity. In our cohorts, where LLINs and prompt effective treatment were provided, the risk of complicated malaria and anemia was extremely low. However, malaria incidence was high and increased over time at the two rural sites, suggesting improved community-wide coverage of LLIN and additional malaria control interventions are needed in Uganda.

Naturally acquired immunity to malaria emerges slowly in malaria-endemic populations. In areas of high transmission, adults experience relatively few clinical episodes, compared with children, despite similar exposure to parasites, indicating that the adult immune system is able to control parasite burden and/or immunopathology associated with disease. Murine models have shown that cytokine-producing CD4+ T cells are critical for protection from malaria [1, 2]. In naturally exposed humans, interferon γ (IFN-γ) production by CD4+ T cells in response to merozoite [3, 4] and sporozoite [5–7] antigens has been associated with protection from disease. The importance of Plasmodium falciparum–specific CD4+ T-cell responses was further demonstrated by a study of naive adults vaccinated with an ultra-low dose of blood-stage parasites, which found that the CD4+ T-cell response was protective from malaria, even in the absence of antibody [8]. Advances in multiparametric flow cytometry have made it clear that assessment of pathogen-specific T cells by a single parameter is often inadequate for identifying correlates of protection. Antigen-specific T cells are functionally heterogeneous and can exhibit a complex variety of effector mechanisms. Mounting evidence from many infectious models suggests that the quality, rather than the quantity, of response is critical for immunity [9]. For instance, multifunctional CD4+ T cells coproducing IFN-γ, tumor necrosis factor α (TNF-α), and interleukin 2 (IL-2) correlate with vaccine-mediated protection from Leishmania major [10] and are implicated in protection induced by vaccinia virus immunization [11]. Multifunctional CD4+ T cells that coproduce TNF-α and/or IL-2 in conjunction with IFN-γ are thought to have increased function [12] and are associated with nonprogression of human immunodeficiency virus (HIV) infection [13]. In P. falciparum malaria, sterilizing immunity induced by experimental vaccination with whole sporozoites is associated with the induction of CD4+ T cells that coproduce IFN-γ, IL-2, and/or TNF-α [14–16]. CD4+ T-cell responses of a similar phenotype have been implicated in protection from malaria conferred by the vaccine RTS,S [17–19]. Among naturally exposed children, several effector phenotypes of P. falciparum–specific CD4+ T cells have been described, including distinct subsets producing inflammatory (TNF-α and IFN-γ) and regulatory (interleukin 10 [IL-10]) cytokines [20–22]. Recent studies suggest that the effector phenotype may be dependent on parasite exposure intensity, as recent symptomatic malaria is associated with increased frequencies of IFN-γ/IL-10–coproducing cells and a reduced frequency of TNF-α–producing cells [20, 21]. Murine models of malaria and other parasitic infections indicate that IFN-γ/IL-10–coproducing cells play an essential role in protecting the host from immunopathology, although this may come at the cost of reduced or delayed parasite clearance [23–25]. The identification of CD4+ T-cell responses in children susceptible to symptomatic malaria that differ from those in immune adults may provide important information regarding the mechanisms mediating protection. In this study, we performed a detailed assessment of P. falciparum–specific CD4+ T-cell responses among children and their adult caregivers in Uganda, in areas of high or low parasite transmission. We hypothesized that the frequency and functional characteristics of P. falciparum–specific CD4+ T-cell responses among clinically immune adults may differ from those of children who remain vulnerable to symptomatic malaria.

Background Verbal autopsy (VA) procedures can be used to estimate cause of death in settings with inadequate vital registries. However, the sensitivity of VA for determining malaria-specific mortality may be low, and may vary with transmission intensity. We assessed the diagnostic accuracy of VA procedures as compared to hospital medical records for determining cause of death in children under five in three different malaria transmission settings in Uganda, including Tororo (high), Kampala (medium), and Kisoro (low). Methods and Findings Caretakers of children who died in participating hospitals were interviewed using a standardized World Health Organization questionnaire. Medical records from the child's hospitalization were also reviewed. Causes of death based on the VA questionnaires and the medical records were assigned independently by physician reviewers and then compared. A total of 719 cases were included in the final analysis, 67 in Tororo, 600 in Kampala, and 52 in Kisoro. Malaria was classified as the underlying or contributory cause of death by review of medical records in 33 deaths in Tororo, 60 in Kampala, and 0 in Kisoro. The sensitivity of VA procedures for determining malaria deaths in Tororo was 61% (95% CI 44–78%) and 50% in Kampala (95% CI 37–63%). Specificity for determining malaria deaths in Tororo and Kampala was high (>88%), but positive predictive value varied widely, from 83% in Tororo to 34% in Kampala (difference 49%, 95% CI 31–67, p

FoxP3+ regulatory CD4 T cells (Tregs) help to maintain the delicate balance between pathogen-specific immunity and immune-mediated pathology. Prior studies suggest that Tregs are induced by P. falciparum both in vivo and in vitro; however, the factors influencing Treg homeostasis during acute and chronic infections, and their role in malaria immunopathogenesis, remain unclear. We assessed the frequency and phenotype of Tregs in well-characterized cohorts of children residing in a region of high malaria endemicity in Uganda. We found that both the frequency and absolute numbers of FoxP3+ Tregs in peripheral blood declined markedly with increasing prior malaria incidence. Longitudinal measurements confirmed that this decline occurred only among highly malaria-exposed children. The decline of Tregs from peripheral blood was accompanied by reduced in vitro induction of Tregs by parasite antigen and decreased expression of TNFR2 on Tregs among children who had intense prior exposure to malaria. While Treg frequencies were not associated with protection from malaria, there was a trend toward reduced risk of symptomatic malaria once infected with P. falciparum among children with lower Treg frequencies. These data demonstrate that chronic malaria exposure results in altered Treg homeostasis, which may impact the development of antimalarial immunity in naturally exposed populations., Author Summary In malaria endemic regions, immunity is slow to develop and does not provide substantial protection against reinfection. Rather, following repeated exposure, older children and adults eventually develop protection from most symptomatic manifestations of the infection. This may be due in part to the induction of immunoregulatory mechanisms by the P. falciparum parasite, such as FoxP3+ regulatory T cells (Tregs). Prior human studies have shown that Tregs are induced by malaria parasites both in vivo and in vitro, but the role of these cells in immunity in children who are chronically exposed to malaria remains unclear. In this study, we assessed the frequency and features of Tregs among children from areas of high malaria transmission in Uganda. We found that this regulatory T cell population declined markedly with increasing malaria episodes. This loss was associated with decreased expression of TNFR2, which is a protein implicated in stability of Tregs. Additionally, T cells from highly malaria exposed children demonstrated a reduced propensity to differentiate into Tregs following parasite stimulation. Together our data suggest that repeated episodes of malaria alter Treg homeostasis, which may influence the development of immunity to malaria in children.

To assess different methods for determining cause of death from verbal autopsy (VA) questionnaire data, the intra-rater reliability of Physician-Certified Verbal Autopsy (PCVA) and the accuracy of PCVA, expert-derived (non-hierarchical) and data-driven (hierarchal) algorithms were assessed for determining common causes of death in Ugandan children. A verbal autopsy validation study was conducted from 2008-2009 in three different sites in Uganda. The dataset included 104 neonatal deaths (0-27 days) and 615 childhood deaths (1-59 months) with the cause(s) of death classified by PCVA and physician review of hospital medical records (the ‘reference standard’). Of the original 719 questionnaires, 141 (20%) were selected for a second review by the same physicians; the repeat cause(s) of death were compared to the original,and agreement assessed using the Kappa statistic.Physician reviewers’ refined non-hierarchical algorithms for common causes of death from existing expert algorithms, from which, hierarchal algorithms were developed. The accuracy of PCVA, non-hierarchical, and hierarchical algorithms for determining cause(s) of death from all 719 VA questionnaires was determined using the reference standard. Overall, intra-rater repeatability was high (83% agreement, Kappa 0.79 [95% CI 0.76-0.82]). PCVA performed well, with high specificity for determining cause of neonatal (>67%), and childhood (>83%) deaths, resulting in fairly accurate cause-specific mortality fraction (CSMF) estimates. For most causes of death in children, non-hierarchical algorithms had higher sensitivity, but correspondingly lower specificity, than PCVA and hierarchical algorithms, resulting in inaccurate CSMF estimates. Hierarchical algorithms were specific for most causes of death, and CSMF estimates were comparable to the reference standard and PCVA. Inter-rater reliability of PCVA was high, and overall PCVA performed well. Hierarchical algorithms performed better than non-hierarchical algorithms due to higher specificity and more accurate CSMF estimates. Use of PCVA to determine cause of death from VA questionnaire data is reasonable while automated data-driven algorithms are improved.

Background: Good house construction may reduce the risk of malaria by limiting the entry of mosquito vectors. We assessed how house design may affect mosquito house entry and malaria risk in Uganda. Methods: 100 households were enrolled in each of three sub-counties: Walukuba, Jinja district; Kihihi, Kanungu district; and Nagongera, Tororo district. CDC light trap collections of mosquitoes were done monthly in all homes. All children aged six months to ten years (n = 878) were followed prospectively for a total of 24 months to measure parasite prevalence every three months and malaria incidence. Homes were classified as modern (cement, wood or metal walls; and tiled or metal roof; and closed eaves) or traditional (all other homes). Results: A total of 113,618 female Anopheles were collected over 6,765 nights. 6,816 routine blood smears were taken of which 1,061 (15.6%) were malaria parasite positive. 2,582 episodes of uncomplicated malaria were diagnosed after 1,569 person years of follow-up, giving an overall incidence of 1.6 episodes per person year at risk. The human biting rate was lower in modern homes than in traditional homes (adjusted incidence rate ratio (IRR) 0.48, 95% confidence interval (CI) 0.37–0.64, p

Background: sub-Saharan Africa is expected to show the greatest rates of urbanization over the next 50 years. Urbanization has shown a substantial impact in reducing malaria transmission due to multiple factors, including unfavourable habitats for Anopheles mosquitoes, generally healthier human populations, better access to healthcare, and higher housing standards. Statistical relationships have been explored at global and local scales, but generally only examining the effects of urbanization on single malaria metrics. In this study, associations between multiple measures of urbanization and a variety of malaria metrics were estimated at local scales.Methods: cohorts of children and adults from 100 households across each of three contrasting sub-counties of Uganda (Walukuba, Nagongera and Kihihi) were followed for 24 months. Measures of urbanicity included density of surrounding households, vegetation index, satellite-derived night-time lights, land cover, and a composite urbanicity score. Malaria metrics included the household density of mosquitoes (number of female Anopheles mosquitoes captured), parasite prevalence and malaria incidence. Associations between measures of urbanicity and malaria metrics were made using negative binomial and logistic regression models.Results: one site (Walukuba) had significantly higher urbanicity measures compared to the two rural sites. In Walukuba, all individual measures of higher urbanicity were significantly associated with a lower household density of mosquitoes. The higher composite urbanicity score in Walukuba was also associated with a lower household density of mosquitoes (incidence rate ratio = 0.28, 95 % CI 0.17–0.48, p < 0.001) and a lower parasite prevalence (odds ratio, OR = 0.44, CI 0.20–0.97, p = 0.04). In one rural site (Kihihi), only a higher density of surrounding households was associated with a lower parasite prevalence (OR = 0.15, CI 0.07–0.34, p < 0.001). And, in only one rural site (Nagongera) was living where NDVI ?0.45 associated with higher incidence of malaria (IRR = 1.35, CI 1.35–1.70, p = 0.01).Conclusions: urbanicity has been shown previously to lead to a reduction in malaria transmission at large spatial scales. At finer scales, individual household measures of higher urbanicity were associated with lower mosquito densities and parasite prevalence only in the site that was generally characterized as being urban. The approaches outlined here can help better characterize urbanicity at the household level and improve targeting of control interventions

Additional file 1: Table S1. Prevalence of red blood cell variants stratified by study site and age group.

Additional file 5: Table S1. Resistance and polytene chromosome allele frequencies in Anopheles gambiae (s.s.) analysed as a function of sub-region using a generalized linear model.

Additional file 5: Table S1. Resistance and polytene chromosome allele frequencies in Anopheles gambiae (s.s.) analysed as a function of sub-region using a generalized linear model.

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 15 years (44.1%) were more likely to use nets than children aged 5–15 years (30.7%; 15 years: aOR 1.37, 95% CI 1.29–1.45, p 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)

Additional file 1: Table S1. Prevalence of red blood cell variants stratified by study site and age group.