Randomized, open-label, phase 2a study to evaluate the contribution of artefenomel to the clinical and parasiticidal activity of artefenomel plus ferroquine in African patients with uncomplicated Plasmodium falciparum malaria.

BACKGROUND: The contribution of artefenomel to the clinical and parasiticidal activity of ferroquine and artefenomel in combination in uncomplicated Plasmodium falciparum malaria was investigated. METHODS: This Phase 2a, randomized, open-label, parallel-group study was conducted from 11th September 2018 to 6th November 2019 across seven centres in Benin, Burkina Faso, Gabon, Kenya, and Uganda. Patients aged ≥ 14-69 years with microscopically confirmed infection (≥ 3000 to ≤ 50,000 parasites/µL blood) were randomized 1:1:1:1 to 400 mg ferroquine, or 400 mg ferroquine plus artefenomel 300, 600, or 1000 mg, administered as a single oral dose. The primary efficacy analysis was a logistic regression evaluating the contribution of artefenomel exposure to Day 28 PCR-adjusted adequate clinical and parasitological response (ACPR). Safety was also evaluated. RESULTS: The randomized population included 140 patients. For the primary analysis in the pharmacokinetic/pharmacodynamic efficacy population (N = 121), the contribution of artefenomel AUC0-∞ to Day 28 PCR-adjusted ACPR was not demonstrated when accounting for ferroquine AUC0-d28, baseline parasitaemia, and other model covariates: odds ratio 1.1 (95% CI 0.98, 1.2; P = 0.245). In the per-protocol population, Day 28 PCR-adjusted ACPR was 80.8% (21/26; 95% CI 60.6, 93.4) with ferroquine alone and 90.3% (28/31; 95% CI 74.2, 98.0), 90.9% (30/33; 95% CI 75.7, 98.1) and 87.1% (27/31; 95% CI 70.2, 96.4) with 300, 600, and 1000 mg artefenomel, respectively. Median time to parasite clearance (Kaplan-Meier) was 56.1 h with ferroquine, more rapid with artefenomel, but similar for all doses (30.0 h). There were no deaths. Adverse events (AEs) of any cause occurred in 51.4% (18/35) of patients with ferroquine 400 mg alone, and 58.3% (21/36), 66.7% (24/36), and 72.7% (24/33) with 300, 600, and 1000 mg artefenomel, respectively. All AEs were of mild-to-moderate severity, and consistent with the known profiles of the compounds. Vomiting was the most reported AE. There were no cases of QTcF prolongation ≥ 500 ms or > 60 ms from baseline. CONCLUSION: The contribution of artefenomel exposure to the clinical and parasitological activity of ferroquine/artefenomel could not be demonstrated in this study. Parasite clearance was faster with ferroquine/artefenomel versus ferroquine alone. All treatments were well tolerated. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03660839 (7 September, 2018).

Impact of population based indoor residual spraying with and without mass drug administration with dihydroartemisinin-piperaquine on malaria prevalence in a high transmission setting: a quasi-experimental controlled before-and-after trial in northeastern Uganda.

BACKGROUND: Declines in malaria burden in Uganda have slowed. Modelling predicts that indoor residual spraying (IRS) and mass drug administration (MDA), when co-timed, have synergistic impact. This study investigated additional protective impact of population-based MDA on malaria prevalence, if any, when added to IRS, as compared with IRS alone and with standard of care (SOC). METHODS: The 32-month quasi-experimental controlled before-and-after trial enrolled an open cohort of residents (46,765 individuals, 1st enumeration and 52,133, 4th enumeration) of Katakwi District in northeastern Uganda. Consented participants were assigned to three arms based on residential subcounty at study start: MDA+IRS, IRS, SOC. IRS with pirimiphos methyl and MDA with dihydroartemisinin- piperaquine were delivered in 4 co-timed campaign-style rounds 8 months apart. The primary endpoint was population prevalence of malaria, estimated by 6 cross-sectional surveys, starting at baseline and preceding each subsequent round. RESULTS: Comparing malaria prevalence in MDA+IRS and IRS only arms over all 6 surveys (intention-to-treat analysis), roughly every 6 months post-interventions, a geostatistical model found a significant additional 15.5% (95% confidence interval (CI): [13.7%, 17.5%], Z = 9.6, p = 5e-20) decrease in the adjusted odds ratio (aOR) due to MDA for all ages, a 13.3% reduction in under 5's (95% CI: [10.5%, 16.8%], Z = 4.02, p = 5e-5), and a 10.1% reduction in children 5-15 (95% CI: [8.5%, 11.8%], Z = 4.7, p = 2e-5). All ages residents of the MDA + IRS arm enjoyed an overall 80.1% reduction (95% CI: [80.0%, 83.0%], p = 0.0001) in odds of qPCR confirmed malaria compared with SOC residents. Secondary difference-in-difference analyses comparing surveys at different timepoints to baseline showed aOR (MDA + IRS vs IRS) of qPCR positivity between 0.28 and 0.66 (p < 0.001). Of three serious adverse events, one (nonfatal) was considered related to study medications. Limitations include the initial non-random assignment of study arms, the single large cluster per arm, and the lack of an MDA-only arm, considered to violate equipoise. CONCLUSIONS: Despite being assessed at long time points 5-7 months post-round, MDA plus IRS provided significant additional protection from malaria infection over IRS alone. Randomized trials of MDA in large areas undergoing IRS recommended as well as cohort studies of impact on incidence. TRIAL REGISTRATION: This trial was retrospectively registered 11/07/2018 with the Pan African Clinical Trials Registry (PACTR201807166695568).

Efficacy of Cipargamin (KAE609) in a Randomized, Phase II Dose-Escalation Study in Adults in Sub-Saharan Africa With Uncomplicated Plasmodium falciparum Malaria.

BACKGROUND: Cipargamin (KAE609) is a potent antimalarial in a phase II trial. Here we report efficacy, pharmacokinetics, and resistance marker analysis across a range of cipargamin doses. These were secondary endpoints from a study primarily conducted to assess the hepatic safety of cipargamin (hepatic safety data are reported elsewhere). METHODS: This phase II, multicenter, randomized, open-label, dose-escalation trial was conducted in sub-Saharan Africa in adults with uncomplicated Plasmodium falciparum malaria. Cipargamin monotherapy was given as single doses up to 150 mg or up to 50 mg once daily for 3 days, with artemether-lumefantrine as control. Key efficacy endpoints were parasite clearance time (PCT), and polymerase chain reaction (PCR)-corrected and uncorrected adequate clinical and parasitological response (ACPR) at 14 and 28 days. Pharmacokinetics and molecular markers of drug resistance were also assessed. RESULTS: All single or multiple cipargamin doses ≥50 mg were associated with rapid parasite clearance, with median PCT of 8 hours versus 24 hours for artemether-lumefantrine. PCR-corrected ACPR at 14 and 28 days was >75% and 65%, respectively, for each cipargamin dose. A treatment-emerging mutation in the Pfatp4 gene, G358S, was detected in 65% of treatment failures. Pharmacokinetic parameters were consistent with previous data, and approximately dose proportional. CONCLUSIONS: Cipargamin, at single doses of 50 to 150 mg, was associated with very rapid parasite clearance, PCR-corrected ACPR at 28 days of >65% in adults with uncomplicated P. falciparum malaria, and recrudescent parasites frequently harbored a treatment-emerging mutation. Cipargamin will be further developed with a suitable combination partner. CLINICAL TRIALS REGISTRATION: ClinicalTrials.gov (NCT03334747).

Factors influencing consistent use of bed nets for the control of malaria among children under 5 years in Soroti District, North Eastern Uganda.

BACKGROUND: The use of insecticide-treated bed nets has been proven to be effective in reducing malaria transmission in highly endemic areas. Use of long-lasting insecticidal nets (LLINs) has been embraced by many malaria endemic countries. LLINs are up to 95% effective in inhibiting blood feeding, when used consistently even after 7 years. The challenge, however, is enhancing their consistent use, especially by the most vulnerable groups (children under 5 years and pregnant women). The study established factors associated with consistent use of bed nets for malaria control among children under 5 years in Soroti district. METHODS: The study employed a cross-sectional design, with multi-stage sampling of households. A total of 400 households (HH) were sampled and the HH head in each household interviewed. Key informant interviews (KIIs) were conducted with 7 key informants who were knowledgeable on the subject matter. Data analysis was done using SPSS 17.0 at Univariate, Bivariate and Multivariable levels; after entry and cleaning. Key informants' data were summarized manually; verbatim quotes and text used to reinforce quantitative data in line with objectives. RESULTS: Only 56.8% of the 690 children under 5 years used bed nets consistently. The factors affecting consistent bed net use were age of the child, their use of bed nets the previous night, occupation of caretaker, respondents' perceived susceptibility, perceived risk of getting malaria, size and shape of the bed nets. Rectangular nets were difficult to hang daily in huts according to most key informants. CONCLUSION: Consistent bed net use among under fives is still below the RBM target of 85% by 2015 and can be enhanced by providing conical bed nets and setting aside a health education programme to emphasize the effectiveness of even one mosquito in spreading malaria at night to the entire household and ability of bed nets to stop transmission better than other methods.

Changing Prevalence of Potential Mediators of Aminoquinoline, Antifolate, and Artemisinin Resistance Across Uganda.

BACKGROUND: In Uganda, artemether-lumefantrine is recommended for malaria treatment and sulfadoxine-pyrimethamine for chemoprevention during pregnancy, but drug resistance may limit efficacies. METHODS: Genetic polymorphisms associated with sensitivities to key drugs were characterized in samples collected from 16 sites across Uganda in 2018 and 2019 by ligase detection reaction fluorescent microsphere, molecular inversion probe, dideoxy sequencing, and quantitative polymerase chain reaction assays. RESULTS: Considering transporter polymorphisms associated with resistance to aminoquinolines, the prevalence of Plasmodium falciparum chloroquine resistance transporter (PfCRT) 76T decreased, but varied markedly between sites (0-46% in 2018; 0-23% in 2019); additional PfCRT polymorphisms and plasmepsin-2/3 amplifications associated elsewhere with resistance to piperaquine were not seen. For P. falciparum multidrug resistance protein 1, in 2019 the 86Y mutation was absent at all sites, the 1246Y mutation had prevalence ≤20% at 14 of 16 sites, and gene amplification was not seen. Considering mutations associated with high-level sulfadoxine-pyrimethamine resistance, prevalences of P. falciparum dihydrofolate reductase 164L (up to 80%) and dihydropteroate synthase 581G (up to 67%) were high at multiple sites. Considering P. falciparum kelch protein propeller domain mutations associated with artemisinin delayed clearance, prevalence of the 469Y and 675V mutations has increased at multiple sites in northern Uganda (up to 23% and 41%, respectively). CONCLUSIONS: We demonstrate concerning spread of mutations that may limit efficacies of key antimalarial drugs.

Genetic diversity and genetic relatedness in Plasmodium falciparum parasite population in individuals with uncomplicated malaria based on microsatellite typing in Eastern and Western regions of Uganda, 2019-2020.

BACKGROUND: Genetic diversity and parasite relatedness are essential parameters for assessing impact of interventions and understanding transmission dynamics of malaria parasites, however data on its status in Plasmodium falciparum populations in Uganda is limited. Microsatellite markers and DNA sequencing were used to determine diversity and molecular characterization of P. falciparum parasite populations in Uganda. METHODS: A total of 147 P. falciparum genomic DNA samples collected from cross-sectional surveys in symptomatic individuals of 2-10 years were characterized by genotyping of seven highly polymorphic neutral microsatellite markers (n = 85) and genetic sequencing of the Histidine Rich Protein 2 (pfhrp2) gene (n = 62). ArcGIS was used to map the geographical distribution of isolates while statistical testing was done using Student's t-test or Wilcoxon's rank-sum test and Fisher's exact test as appropriate at P ≤ 0.05. RESULTS: Overall, 75.5% (95% CI 61.1-85.8) and 24.5% (95% CI14.2-38.9) of parasites examined were of multiclonal (mixed genotype) and single clone infections, respectively. Multiclonal infections occurred more frequently in the Eastern region 73.7% (95% CI 48.8-89.1), P < 0.05. Overall, multiplicity of infection (MOI) was 1.9 (95% CI 1.7-2.1), P = 0.01 that was similar between age groups (1.8 vs 1.9), P = 0.60 and regions (1.9 vs 1.8), P = 0.43 for the < 5 and ≥ 5 years and Eastern and Western regions, respectively. Genomic sequencing of the pfhrp2 exon2 revealed a high level of genetic diversity reflected in 96.8% (60/62) unique sequence types. Repeat type AHHAAAHHATD and HRP2 sequence Type C were more frequent in RDT-/PCR + samples (1.9% vs 1.5%) and (13% vs 8%), P < 0.05 respectively. Genetic relatedness analysis revealed small clusters of gene deleted parasites in Uganda, but no clustering with Eritrean parasites. CONCLUSION: High level of genetic diversity of P. falciparum parasites reflected in the frequency of multiclonal infections, multiplicity of infection and variability of the pfhrp2 gene observed in this study is consistent with the high malaria transmission intensity in these settings. Parasite genetic analysis suggested spontaneous emergence and clonal expansion of pfhrp2 deleted parasites that require close monitoring to inform national malaria diagnosis and case management policies.

Hepatic safety and tolerability of cipargamin (KAE609), in adult patients with Plasmodium falciparum malaria: a randomized, phase II, controlled, dose-escalation trial in sub-Saharan Africa.

BACKGROUND: The novel anti-malarial cipargamin (KAE609) has potent, rapid activity against Plasmodium falciparum. Transient asymptomatic liver function test elevations were previously observed in cipargamin-treated subjects in two trials: one in malaria patients in Asia and one in volunteers with experimentally induced malaria. In this study, the hepatic safety of cipargamin given as single doses of 10 to 150 mg and 10 to 50 mg once daily for 3 days was assessed. Efficacy results, frequency of treatment-emerging mutations in the atp4 gene and pharmacokinetics have been published elsewhere. Further, the R561H mutation in the k13 gene, which confers artemisinin-resistance, was associated with delayed parasite clearance following treatment with artemether-lumefantrine in Rwanda in this study. This was also the first study with cipargamin to be conducted in patients in sub-Saharan Africa. METHODS: This was a Phase II, multicentre, randomized, open-label, dose-escalation trial in adults with uncomplicated falciparum malaria in five sub-Saharan countries, using artemether-lumefantrine as control. The primary endpoint was ≥ 2 Common Terminology Criteria for Adverse Events (CTCAE) Grade increase from baseline in alanine aminotransferase (ALT) or aspartate transaminase (AST) during the 4-week trial. RESULTS: Overall, 2/135 patients treated with cipargamin had ≥ 2 CTCAE Grade increases from baseline in ALT or AST compared to 2/51 artemether-lumefantrine patients, with no significant difference between any cipargamin treatment group and the control group. Cipargamin exposure was comparable to or higher than those in previous studies. Hepatic adverse events and general safety and tolerability were similar for all cipargamin doses and artemether-lumefantrine. Cipargamin was well tolerated with no safety concerns. CONCLUSIONS: This active-controlled, dose escalation study was a detailed assessment of the hepatic safety of cipargamin, across a wide range of doses, in patients with uncomplicated falciparum malaria. Comparison with previous cipargamin trials requires caution as no clear conclusion can be drawn as to whether hepatic safety and potential immunity to malaria would differ with ethnicity, patient age and or geography. Previous concerns regarding hepatic safety may have been confounded by factors including malaria itself, whether natural or experimental infection, and should not limit the further development of cipargamin. Trial registration ClinicalTrials.gov number: NCT03334747 (7 Nov 2017), other study ID CKAE609A2202.

Efficacy and safety of artemether-lumefantrine and dihydroartemisinin-piperaquine for the treatment of uncomplicated Plasmodium falciparum malaria and prevalence of molecular markers associated with artemisinin and partner drug resistance in Uganda.

BACKGROUND: In Uganda, artemether-lumefantrine (AL) is first-line therapy and dihydroartemisinin-piperaquine (DP) second-line therapy for the treatment of uncomplicated malaria. This study evaluated the efficacy and safety of AL and DP in the management of uncomplicated falciparum malaria and measured the prevalence of molecular markers of resistance in three sentinel sites in Uganda from 2018 to 2019. METHODS: This was a randomized, open-label, phase IV clinical trial. Children aged 6 months to 10 years with uncomplicated falciparum malaria were randomly assigned to treatment with AL or DP and followed for 28 and 42 days, respectively. Genotyping was used to distinguish recrudescence from new infection, and a Bayesian algorithm was used to assign each treatment failure a posterior probability of recrudescence. For monitoring resistance, Pfk13 and Pfmdr1 genes were Sanger sequenced and plasmepsin-2 copy number was assessed by qPCR. RESULTS: There were no early treatment failures. The uncorrected 28-day cumulative efficacy of AL ranged from 41.2 to 71.2% and the PCR-corrected cumulative 28-day efficacy of AL ranged from 87.2 to 94.4%. The uncorrected 28-day cumulative efficacy of DP ranged from 95.8 to 97.9% and the PCR-corrected cumulative 28-day efficacy of DP ranged from 98.9 to 100%. The uncorrected 42-day efficacy of DP ranged from 73.5 to 87.4% and the PCR-corrected 42-day efficacy of DP ranged from 92.1 to 97.5%. There were no reported serious adverse events associated with any of the regimens. No resistance-associated mutations in the Pfk13 gene were found in the successfully sequenced samples. In the AL arm, the NFD haplotype (N86Y, Y184F, D1246Y) was the predominant Pfmdr1 haplotype, present in 78 of 127 (61%) and 76 of 110 (69%) of the day 0 and day of failure samples, respectively. All the day 0 samples in the DP arm had one copy of the plasmepsin-2 gene. CONCLUSIONS: DP remains highly effective and safe for the treatment of uncomplicated malaria in Uganda. Recurrent infections with AL were common. In Busia and Arua, the 95% confidence interval for PCR-corrected AL efficacy fell below 90%. Further efficacy monitoring for AL, including pharmacokinetic studies, is recommended. Trial registration The trail was also registered with the ISRCTN registry with study Trial No. PACTR201811640750761.

Relationships Between Measures of Malaria at Delivery and Adverse Birth Outcomes in a High-Transmission Area of Uganda.

BACKGROUND: Clinical trials of interventions for preventing malaria in pregnancy often use measures of malaria at delivery as their primary outcome. Although the objective of these interventions is to improve birth outcomes, data on associations between different measures of malaria at delivery and adverse birth outcomes are limited. METHODS: Data came from 637 Ugandan women enrolled in a randomized controlled trial of intermittent preventive treatment of malaria in pregnancy. Malaria at delivery was detected using peripheral and placental blood microscopy, placental blood loop-mediated isothermal amplification (LAMP), and placental histopathology. Multivariate analyses were used to estimate associations between measures of malaria at delivery and risks of low birth weight (LBW), small for gestational age (SGA), and preterm birth (PTB). RESULTS: Detection of malaria parasites by microscopy or LAMP was not associated with adverse birth outcomes. Presence of malaria pigment detected by histopathology in ≥30% of high-powered fields was strongly associated with LBW (adjusted risk ratio [aRR] = 3.42, P = .02) and SGA (aRR = 4.24, P < .001) but not PTB (aRR = 0.88, P = .87). CONCLUSIONS: A semiquantitative classification system based on histopathologically detected malaria pigment provided the best surrogate measure of adverse birth outcomes in a high-transmission setting and should be considered for use in malaria in pregnancy intervention studies.

The duration of chemoprophylaxis against malaria after treatment with artesunate-amodiaquine and artemether-lumefantrine and the effects of pfmdr1 86Y and pfcrt 76T: a meta-analysis of individual patient data.

BACKGROUND: The majority of Plasmodium falciparum malaria cases in Africa are treated with the artemisinin combination therapies artemether-lumefantrine (AL) and artesunate-amodiaquine (AS-AQ), with amodiaquine being also widely used as part of seasonal malaria chemoprevention programs combined with sulfadoxine-pyrimethamine. While artemisinin derivatives have a short half-life, lumefantrine and amodiaquine may give rise to differing durations of post-treatment prophylaxis, an important additional benefit to patients in higher transmission areas. METHODS: We analyzed individual patient data from 8 clinical trials of AL versus AS-AQ in 12 sites in Africa (n = 4214 individuals). The time to PCR-confirmed reinfection after treatment was used to estimate the duration of post-treatment protection, accounting for variation in transmission intensity between settings using hidden semi-Markov models. Accelerated failure-time models were used to identify potential effects of covariates on the time to reinfection. The estimated duration of chemoprophylaxis was then used in a mathematical model of malaria transmission to determine the potential public health impact of each drug when used for first-line treatment. RESULTS: We estimated a mean duration of post-treatment protection of 13.0 days (95% CI 10.7-15.7) for AL and 15.2 days (95% CI 12.8-18.4) for AS-AQ overall. However, the duration varied significantly between trial sites, from 8.7-18.6 days for AL and 10.2-18.7 days for AS-AQ. Significant predictors of time to reinfection in multivariable models were transmission intensity, age, drug, and parasite genotype. Where wild type pfmdr1 and pfcrt parasite genotypes predominated (<=20% 86Y and 76T mutants, respectively), AS-AQ provided ~ 2-fold longer protection than AL. Conversely, at a higher prevalence of 86Y and 76T mutant parasites (> 80%), AL provided up to 1.5-fold longer protection than AS-AQ. Our simulations found that these differences in the duration of protection could alter population-level clinical incidence of malaria by up to 14% in under-5-year-old children when the drugs were used as first-line treatments in areas with high, seasonal transmission. CONCLUSION: Choosing a first-line treatment which provides optimal post-treatment prophylaxis given the local prevalence of resistance-associated markers could make a significant contribution to reducing malaria morbidity.

Molecular surveillance reveals the presence of pfhrp2 and pfhrp3 gene deletions in Plasmodium falciparum parasite populations in Uganda, 2017-2019.

BACKGROUND: Histidine-rich protein-2 (HRP2)-based rapid diagnostic tests (RDTs) are the only RDTs recommended for malaria diagnosis in Uganda. However, the emergence of Plasmodium falciparum histidine rich protein 2 and 3 (pfhrp2 and pfhrp3) gene deletions threatens their usefulness as malaria diagnostic and surveillance tools. The pfhrp2 and pfhrp3 gene deletions surveillance was conducted in P. falciparum parasite populations in Uganda. METHODS: Three-hundred (n = 300) P. falciparum isolates collected from cross-sectional malaria surveys in symptomatic individuals in 48 districts of eastern and western Uganda were analysed for the presence of pfhrp2 and pfhrp3 genes. Presence of parasite DNA was confirmed by PCR amplification of the 18s rRNA gene, msp1 and msp2 single copy genes. Presence or absence of deletions was confirmed by amplification of exon1 and exon2 of pfhrp2 and pfhrp3 using gene specific PCR. RESULTS: Overall, pfhrp2 and pfhrp3 gene deletions were detected in 29/300 (9.7%, 95% CI 6.6-13.6%) parasite isolates. The pfhrp2 gene was deleted in 10/300 (3.3%, 95% CI 1.6-6.0%) isolates, pfhrp3 in 9/300 (3.0%, 95% CI 1.4-5.6%) while both pfhrp2 and pfhrp3 were deleted in 10/300 (3.3%, 95% CI 1.6-6.0%) parasite isolates. Proportion of pfhrp2/3 deletions was higher in the eastern 14.7% (95% CI 9.7-20.0%) compared to the western region 3.1% (95% CI 0.8-7.7%), p = 0.001. Geographical location was associated with gene deletions aOR 6.25 (2.02-23.55), p = 0.003. CONCLUSIONS: This is the first large-scale survey reporting the presence of pfhrp2/3 gene deletions in P. falciparum isolates in Uganda. Roll out of RDTs for malaria diagnosis should take into consideration the existence of pfhrp2/3 gene deletions particularly in areas where they were detected. Periodic pfhrp2/3 surveys are recommended to inform future decisions for deployment of alternative RDTs.

Rapid shifts in the age-specific burden of malaria following successful control interventions in four regions of Uganda.

BACKGROUND: Malaria control using long-lasting insecticidal nets (LLINs) and indoor residual spraying of insecticide (IRS) has been associated with reduced transmission throughout Africa. However, the impact of transmission reduction on the age distribution of malaria cases remains unclear. METHODS: Over a 10-year period (January 2009 to July 2018), outpatient surveillance data from four health facilities in Uganda were used to estimate the impact of control interventions on temporal changes in the age distribution of malaria cases using multinomial regression. Interventions included mass distribution of LLINs at all sites and IRS at two sites. RESULTS: Overall, 896,550 patient visits were included in the study; 211,632 aged < 5 years, 171,166 aged 5-15 years and 513,752 > 15 years. Over time, the age distribution of patients not suspected of malaria and those malaria negative either declined or remained the same across all sites. In contrast, the age distribution of suspected and confirmed malaria cases increased across all four sites. In the two LLINs-only sites, the proportion of malaria cases in < 5 years decreased from 31 to 16% and 35 to 25%, respectively. In the two sites receiving LLINs plus IRS, these proportions decreased from 58 to 30% and 64 to 47%, respectively. Similarly, in the LLINs-only sites, the proportion of malaria cases > 15 years increased from 40 to 61% and 29 to 39%, respectively. In the sites receiving LLINs plus IRS, these proportions increased from 19 to 44% and 18 to 31%, respectively. CONCLUSIONS: These findings demonstrate a shift in the burden of malaria from younger to older individuals following implementation of successful control interventions, which has important implications for malaria prevention, surveillance, case management and control strategies.

Spatial-temporal patterns of malaria incidence in Uganda using HMIS data from 2015 to 2019.

BACKGROUND: As global progress to reduce malaria transmission continues, it is increasingly important to track changes in malaria incidence rather than prevalence. Risk estimates for Africa have largely underutilized available health management information systems (HMIS) data to monitor trends. This study uses national HMIS data, together with environmental and geographical data, to assess spatial-temporal patterns of malaria incidence at facility catchment level in Uganda, over a recent 5-year period. METHODS: Data reported by 3446 health facilities in Uganda, between July 2015 and September 2019, was analysed. To assess the geographic accessibility of the health facilities network, AccessMod was employed to determine a three-hour cost-distance catchment around each facility. Using confirmed malaria cases and total catchment population by facility, an ecological Bayesian conditional autoregressive spatial-temporal Poisson model was fitted to generate monthly posterior incidence rate estimates, adjusted for caregiver education, rainfall, land surface temperature, night-time light (an indicator of urbanicity), and vegetation index. RESULTS: An estimated 38.8 million (95% Credible Interval [CI]: 37.9-40.9) confirmed cases of malaria occurred over the period, with a national mean monthly incidence rate of 20.4 (95% CI: 19.9-21.5) cases per 1000, ranging from 8.9 (95% CI: 8.7-9.4) to 36.6 (95% CI: 35.7-38.5) across the study period. Strong seasonality was observed, with June-July experiencing highest peaks and February-March the lowest peaks. There was also considerable geographic heterogeneity in incidence, with health facility catchment relative risk during peak transmission months ranging from 0 to 50.5 (95% CI: 49.0-50.8) times higher than national average. Both districts and health facility catchments showed significant positive spatial autocorrelation; health facility catchments had global Moran's I = 0.3 (p < 0.001) and districts Moran's I = 0.4 (p < 0.001). Notably, significant clusters of high-risk health facility catchments were concentrated in Acholi, West Nile, Karamoja, and East Central - Busoga regions. CONCLUSION: Findings showed clear countrywide spatial-temporal patterns with clustering of malaria risk across districts and health facility catchments within high risk regions, which can facilitate targeting of interventions to those areas at highest risk. Moreover, despite high and perennial transmission, seasonality for malaria incidence highlights the potential for optimal and timely implementation of targeted interventions.

Comparative Efficacy of Artemether-Lumefantrine and Dihydroartemisinin-Piperaquine for the Treatment of Uncomplicated Malaria in Ugandan Children.

BACKGROUND: In Uganda, artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DHA-PQ) showed excellent treatment efficacy for uncomplicated malaria in prior trials. Because the frequency of resistance to artemisinins and piperaquine is increasing in Southeast Asia and the prevalence of Plasmodium falciparum polymorphisms associated with resistance has changed, we reassessed treatment efficacies at 3 sites in Uganda. METHODS: For this randomized, single-blinded clinical trial, children aged 6-59 months with uncomplicated falciparum malaria were assigned treatment with AL or DHA-PQ and followed for 42 days. Primary end points were risks of recurrent parasitemia, either unadjusted or adjusted to distinguish recrudescence from new infection. We assessed selection by study regimens of relevant P. falciparum genetic polymorphisms associated with drug resistance. RESULTS: Of 599 patients enrolled, 578 completed follow-up. There were no early treatment failures. The risk of recurrent parasitemia was lower with DHA-PQ as compared to AL at all 3 sites at 42 days (26.0% vs 47.0%; P < .001). Recrudescent infections were uncommon in both the DHA-PQ and AL arms (1.1% and 2.2%, respectively; P = .25). Neither regimen selected for pfcrt or pfmdr1 polymorphisms associated with drug resistance. CONCLUSIONS: AL and DHA-PQ remain effective for the treatment of malaria in Uganda. Neither regimen selected for genetic polymorphisms associated with drug resistance. CLINICAL TRIALS REGISTRATION: ISRCTN15793046.

Implementing population-based mass drug administration for malaria: experience from a high transmission setting in North Eastern Uganda.

BACKGROUND: Mass drug administration (MDA) is a suggested mean to accelerate efforts towards elimination and attainment of malaria-free status. There is limited evidence of suitable methods of implementing MDA programme to achieve a high coverage and compliance in low-income countries. The objective of this paper is to assess the impact of this MDA delivery strategy while using coverage measured as effective population in the community and population available. METHODS: Population-based MDA was implemented as a part of a larger program in a high transmission setting in Uganda. Four rounds of interventions were implemented over a period of 2 years at an interval of 6 to 8 months. A housing and population census was conducted to establish the eligible population. A team of 19 personnel conducted MDA at established village meeting points as distribution sites at every village. The first dose of dihydroartemisinin-piperaquine (DHA-PQ) was administered via a fixed site distribution strategy by directly observed treatment on site, the remaining doses were taken at home and a door-to-door follow up strategy was implemented by community health workers to monitor adherence to the second and third doses. RESULTS: Based on number of individuals who turned up at the distribution site, for each round of MDA, effective coverage was 80.1%, 81.2%, 80.0% and 80% for the 1st, 2nd, 3rd and 4th rounds respectively. However, coverage based on available population at the time of implementing MDA was 80.1%, 83.2%, 82.4% and 82.9% for rounds 1, 2, 3 and 4, respectively. Intense community mobilization using community structures and mass media facilitated community participation and adherence to MDA. CONCLUSION: A hybrid of fixed site distribution and door-to-door follow up strategy of MDA delivery achieved a high coverage and compliance and seemed feasible. This model can be considered in resource-limited settings.

Effects and factors associated with indoor residual spraying with Actellic 300 CS on malaria morbidity in Lira District, Northern Uganda.

BACKGROUND: Indoor residual spraying (IRS) with Actellic 300 CS was conducted in Lira District between July and August 2016. No formal assessment has been conducted to estimate the effect of spraying with Actellic 300 CS on malaria morbidity in the Ugandan settings. This study assessed malaria morbidity trends before and after IRS with Actellic 300 CS in Lira District in Northern Uganda. METHODS: The study employed a mixed methods design. Malaria morbidity records from four health facilities were reviewed, focusing on 6 months before and after the IRS intervention. The outcome of interest was malaria morbidity defined as; proportion of outpatient attendance due to total malaria, proportion of outpatient attendance due to confirmed malaria and proportion of malaria case numbers confirmed by microscopy or rapid diagnostic test. Since malaria morbidity was based on count data, an ordinary Poisson regression model was used to obtain percentage point change (pp) in monthly malaria cases before and after IRS. A household survey was also conducted in 159 households to determine IRS coverage and factors associated with spraying. A modified Poisson regression model was fitted to determine factors associated with household spray status. RESULTS: The proportion of outpatient attendance due to malaria dropped from 18.7% before spraying to 15.1% after IRS. The proportion of outpatient attendance due to confirmed malaria also dropped from 5.1% before spraying to 4.0% after the IRS intervention. There was a decreasing trend in malaria test positivity rate (TPR) for every unit increase in month after spraying. The decreasing trend in TPR was more prominent 5-6 months after the IRS intervention (Adj. pp = - 0.60, P-value = 0.015; Adj. pp = - 1.19, P-value < 0.001). The IRS coverage was estimated at 89.3%. Households of respondents who were formally employed or owned any form of business were more likely to be unsprayed; (APR = 5.81, CI 2.72-12.68); (APR = 3.84, CI 1.20-12.31), respectively. CONCLUSION: Coverage of IRS with Actellic 300 CS was high and was associated with a significant decline in malaria related morbidity 6 months after spraying.

Systematic review of the status of pfhrp2 and pfhrp3 gene deletion, approaches and methods used for its estimation and reporting in Plasmodium falciparum populations in Africa: review of published studies 2010-2019.

BACKGROUND: Malaria rapid diagnostic tests based on histidine-rich protein-2 have played a vital role in improving malaria case management and surveillance particularly in Africa, where Plasmodium falciparum is predominant. However, their usefulness has been threatened by the emergence of gene deletion on P. falciparum histidine rich protein 2 (pfhrp2) and P. falciparum histidine rich protein 3 (pfhrp3). Use of standard and recommended methods is key for accurate investigation, confirmation and reporting of pfhrp2 and pfhrp3 gene deletion. METHODS: A systematic review was conducted to assess the status, methods and approaches that have been used for investigation, confirmation and reporting of pfhrp2 and pfhrp3 gene deletion in Africa. An online search was done using PubMed and MEDLINE Google Scholar for all articles published in English on pfhrp2/3 gene deletion in Africa. Relevant articles that met the inclusion criteria were summarized and assessed based on the protocol recommended by the World Health Organization for confirmation and reporting of pfhrp2/3 gene deletion. RESULTS: The search identified a total of 18 articles out of which 14 (77.7%) fulfilled the criteria for inclusion and were retained for review. The articles were distributed across 12 countries where the pfhrp2 and pfhrp3 gene deletion studies were conducted and reported. The level of pfhrp2/3 gene deletion across selected studies in Africa ranged from the highest 62% to the lowest 0.4%. There was wide variation in methods and approaches including study designs, size and sampling and whether both pfhrp2 and pfhrp3 double deletions or pfhrp2 single deletion were investigated, with a wide variation in laboratory methods. CONCLUSION: Based on the review, there is evidence of the presence of pfhrp2/3 gene-deleted P. falciparum parasites in Africa. The approaches and methods used for investigation, confirmation and reporting of pfhrp2/3 deleted parasites have varied between studies and across countries. Countries that are considering plans to investigate, confirm and report pfhrp2/3 deletion should use recommended standard and harmonized methods to prevent unnecessary recommendations for costly switch of RDTs in Africa.

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.

THE REAL McCOIL: A method for the concurrent estimation of the complexity of infection and SNP allele frequency for malaria parasites.

As many malaria-endemic countries move towards elimination of Plasmodium falciparum, the most virulent human malaria parasite, effective tools for monitoring malaria epidemiology are urgent priorities. P. falciparum population genetic approaches offer promising tools for understanding transmission and spread of the disease, but a high prevalence of multi-clone or polygenomic infections can render estimation of even the most basic parameters, such as allele frequencies, challenging. A previous method, COIL, was developed to estimate complexity of infection (COI) from single nucleotide polymorphism (SNP) data, but relies on monogenomic infections to estimate allele frequencies or requires external allele frequency data which may not available. Estimates limited to monogenomic infections may not be representative, however, and when the average COI is high, they can be difficult or impossible to obtain. Therefore, we developed THE REAL McCOIL, Turning HEterozygous SNP data into Robust Estimates of ALelle frequency, via Markov chain Monte Carlo, and Complexity Of Infection using Likelihood, to incorporate polygenomic samples and simultaneously estimate allele frequency and COI. This approach was tested via simulations then applied to SNP data from cross-sectional surveys performed in three Ugandan sites with varying malaria transmission. We show that THE REAL McCOIL consistently outperforms COIL on simulated data, particularly when most infections are polygenomic. Using field data we show that, unlike with COIL, we can distinguish epidemiologically relevant differences in COI between and within these sites. Surprisingly, for example, we estimated high average COI in a peri-urban subregion with lower transmission intensity, suggesting that many of these cases were imported from surrounding regions with higher transmission intensity. THE REAL McCOIL therefore provides a robust tool for understanding the molecular epidemiology of malaria across transmission settings.

Community facilitators and barriers to a successful implementation of mass drug administration and indoor residual spraying for malaria prevention in Uganda: a qualitative study.

BACKGROUND: There is growing interest to add mass drug administration (MDA) to the already existing malaria prevention strategies, such as indoor residual spraying (IRS). However, successful MDA and IRS requires high population-wide coverage, emphasizing the importance of community acceptance. This study's objectives were to identify community-level facilitators and barriers during the implementation of both MDA and IRS in communities with high malaria transmission intensity. METHODS: This was a qualitative study conducted in two sub-counties in Katakwi district. Kapujan sub-county residents received two rounds of IRS and MDA while Toroma sub-county residents received two rounds of IRS only. Key informant interviews and focus group discussions were conducted with key influential district and sub-county personnel and community members. Data were analysed using thematic analysis. Transcripts and interview notes from the in-depth interviews were analysed using a coding scheme developed from pre-defined topics together with themes emerging from the data. The Nvivo software program was used to aggregate the data by codes and to present study findings. RESULTS: Overall, 14 key informants were interviewed: 4 from Katakwi district and 5 each from Kapujan and Toroma sub-counties. Five focus group discussions were conducted: 4 with community members (men and women), 2 in each sub-county and one with medical staff of Toroma health centre IV. Important themes for consideration raised by the respondents include community sensitization, conducting implementation during the low activity dry season, involvement of government and local leadership, use of the competent locally composed team, community knowledge of malaria effects and consequences, combining interventions and evidence of malaria reduction from interventions. Potential barriers such as spreading of misinformation regarding interventions, the strong unpleasant smell from Actellic and inadequate duration of engagement with the community should be taken into consideration. CONCLUSION: This study documents important community engagement strategies that need to be considered when implementing malaria MDA in combination with IRS, for malaria prevention in such settings. This information is useful for malaria programmes, especially during the design and implementation of such community level interventions.