Feasibility, safety, and impact of the RTS,S/AS01E malaria vaccine when implemented through national immunisation programmes: evaluation of cluster-randomised introduction of the vaccine in Ghana, Kenya, and Malawi.

BACKGROUND: The RTS,S/AS01E malaria vaccine (RTS,S) was introduced by national immunisation programmes in Ghana, Kenya, and Malawi in 2019 in large-scale pilot schemes. We aimed to address questions about feasibility and impact, and to assess safety signals that had been observed in the phase 3 trial that included an excess of meningitis and cerebral malaria cases in RTS,S recipients, and the possibility of an excess of deaths among girls who received RTS,S than in controls, to inform decisions about wider use. METHODS: In this prospective evaluation, 158 geographical clusters (66 districts in Ghana; 46 sub-counties in Kenya; and 46 groups of immunisation clinic catchment areas in Malawi) were randomly assigned to early or delayed introduction of RTS,S, with three doses to be administered between the ages of 5 months and 9 months and a fourth dose at the age of approximately 2 years. Primary outcomes of the evaluation, planned over 4 years, were mortality from all causes except injury (impact), hospital admission with severe malaria (impact), hospital admission with meningitis or cerebral malaria (safety), deaths in girls compared with boys (safety), and vaccination coverage (feasibility). Mortality was monitored in children aged 1-59 months throughout the pilot areas. Surveillance for meningitis and severe malaria was established in eight sentinel hospitals in Ghana, six in Kenya, and four in Malawi. Vaccine uptake was measured in surveys of children aged 12-23 months about 18 months after vaccine introduction. We estimated that sufficient data would have accrued after 24 months to evaluate each of the safety signals and the impact on severe malaria in a pooled analysis of the data from the three countries. We estimated incidence rate ratios (IRRs) by comparing the ratio of the number of events in children age-eligible to have received at least one dose of the vaccine (for safety outcomes), or age-eligible to have received three doses (for impact outcomes), to that in non-eligible age groups in implementation areas with the equivalent ratio in comparison areas. To establish whether there was evidence of a difference between girls and boys in the vaccine's impact on mortality, the female-to-male mortality ratio in age groups eligible to receive the vaccine (relative to the ratio in non-eligible children) was compared between implementation and comparison areas. Preliminary findings contributed to WHO's recommendation in 2021 for widespread use of RTS,S in areas of moderate-to-high malaria transmission. FINDINGS: By April 30, 2021, 652 673 children had received at least one dose of RTS,S and 494 745 children had received three doses. Coverage of the first dose was 76% in Ghana, 79% in Kenya, and 73% in Malawi, and coverage of the third dose was 66% in Ghana, 62% in Kenya, and 62% in Malawi. 26 285 children aged 1-59 months were admitted to sentinel hospitals and 13 198 deaths were reported through mortality surveillance. Among children eligible to have received at least one dose of RTS,S, there was no evidence of an excess of meningitis or cerebral malaria cases in implementation areas compared with comparison areas (hospital admission with meningitis: IRR 0·63 [95% CI 0·22-1·79]; hospital admission with cerebral malaria: IRR 1·03 [95% CI 0·61-1·74]). The impact of RTS,S introduction on mortality was similar for girls and boys (relative mortality ratio 1·03 [95% CI 0·88-1·21]). Among children eligible for three vaccine doses, RTS,S introduction was associated with a 32% reduction (95% CI 5-51%) in hospital admission with severe malaria, and a 9% reduction (95% CI 0-18%) in all-cause mortality (excluding injury). INTERPRETATION: In the first 2 years of implementation of RTS,S, the three primary doses were effectively deployed through national immunisation programmes. There was no evidence of the safety signals that had been observed in the phase 3 trial, and introduction of the vaccine was associated with substantial reductions in hospital admission with severe malaria. Evaluation continues to assess the impact of four doses of RTS,S. FUNDING: Gavi, the Vaccine Alliance; the Global Fund to Fight AIDS, Tuberculosis and Malaria; and Unitaid.

Could Less Be More? Accounting for Fractional-Dose Regimens and Different Number of Vaccine Doses When Measuring the Impact of the RTS,S/AS01E Malaria Vaccine.

BACKGROUND: The RTS,S/AS01E (RTS,S) malaria vaccine is recommended for children in malaria endemic areas. This phase 2b trial evaluates RTS,S fractional- and full-dose regimens in Ghana and Kenya. METHODS: In total, 1500 children aged 5-17 months were randomized (1:1:1:1:1) to receive RTS,S or rabies control vaccine. RTS,S groups received 2 full RTS,S doses at months 0 and 1 and either full (groups R012-20, R012-14-26) or fractional doses (one-fifth; groups Fx012-14-26, Fx017-20-32). RESULTS: At month 32 post-dose 1, vaccine efficacy against clinical malaria (all episodes) ranged from 38% (R012-20; 95% confidence interval [CI]: 24%-49%) to 53% (R012-14-26; 95% CI: 42%-62%). Vaccine impact (cumulative number of cases averted/1000 children vaccinated) was 1344 (R012-20), 2450 (R012-14-26), 2273 (Fx012-14-26), and 2112 (Fx017-20-32). To account for differences in vaccine volume (fractional vs full dose; post hoc analysis), we estimated cases averted/1000 RTS,S full-dose equivalents: 336 (R012-20), 490 (R012-14-26), 874 (Fx012-14-26), and 880 (Fx017-20-32). CONCLUSIONS: Vaccine efficacy was similar across RTS,S groups. Vaccine impact accounting for full-dose equivalence suggests that using fractional-dose regimens could be a viable dose-sparing strategy. If maintained through trial end, these observations underscore the means to reduce cost per regimen thus maximizing impact and optimizing supply. CLINICAL TRIALS REGISTRATION: NCT03276962 (ClinicalTrials.gov).

Malaria Chemoprevention in the Postdischarge Management of Severe Anemia.

BACKGROUND: Children who have been hospitalized with severe anemia in areas of Africa in which malaria is endemic have a high risk of readmission and death within 6 months after discharge. No prevention strategy specifically addresses this period. METHODS: We conducted a multicenter, two-group, randomized, placebo-controlled trial in nine hospitals in Kenya and Uganda to determine whether 3 months of malaria chemoprevention could reduce morbidity and mortality after hospital discharge in children younger than 5 years of age who had been admitted with severe anemia. All children received standard in-hospital care for severe anemia and a 3-day course of artemether-lumefantrine at discharge. Two weeks after discharge, children were randomly assigned to receive dihydroartemisinin-piperaquine (chemoprevention group) or placebo, administered as 3-day courses at 2, 6, and 10 weeks after discharge. Children were followed for 26 weeks after discharge. The primary outcome was one or more hospital readmissions for any reason or death from the time of randomization to 6 months after discharge. Conditional risk-set modeling for recurrent events was used to calculate hazard ratios with the use of the Prentice-Williams-Peterson total-time approach. RESULTS: From May 2016 through May 2018, a total of 1049 children underwent randomization; 524 were assigned to the chemoprevention group and 525 to the placebo group. From week 3 through week 26, a total of 184 events of readmission or death occurred in the chemoprevention group and 316 occurred in the placebo group (hazard ratio, 0.65; 95% confidence interval [CI], 0.54 to 0.78; P<0.001). The lower incidence of readmission or death in the chemoprevention group than in the placebo group was restricted to the intervention period (week 3 through week 14) (hazard ratio, 0.30; 95% CI, 0.22 to 0.42) and was not sustained after that time (week 15 through week 26) (hazard ratio, 1.13; 95% CI, 0.87 to 1.47). No serious adverse events were attributed to dihydroartemisinin-piperaquine. CONCLUSIONS: In areas with intense malaria transmission, 3 months of postdischarge malaria chemoprevention with monthly dihydroartemisinin-piperaquine in children who had recently received treatment for severe anemia prevented more deaths or readmissions for any reason after discharge than placebo. (Funded by the Research Council of Norway and the Centers for Disease Control and Prevention; ClinicalTrials.gov number, NCT02671175.).

Evaluation of community-based vector surveillance system for routine entomological monitoring under low malaria vector densities and high bed net coverage in western Kenya.

BACKGROUND: Entomological surveillance is traditionally conducted by supervised teams of trained technicians. However, it is expensive and limiting in the number of sites visited. Surveillance through community-based collectors (CBC) may be more cost-effective and sustainable for longitudinal entomological monitoring. This study evaluated the efficiency of CBCs in monitoring mosquito densities compared to quality-assured sampling conducted by experienced entomology technicians. METHODS: Entomological surveillance employing CBCs was conducted in eighteen clusters of villages in western Kenya using indoor and outdoor CDC light traps and indoor Prokopack aspiration. Sixty houses in each cluster were enrolled and sampled once every month. Collected mosquitoes were initially identified to the genus level by CBCs, preserved in 70% ethanol and transferred to the laboratory every 2 weeks. Parallel, collections by experienced entomology field technicians were conducted monthly by indoor and outdoor CDC light traps and indoor Prokopack aspiration and served as a quality assurance of the CBCs. RESULTS: Per collection, the CBCs collected 80% fewer Anopheles gambiae sensu lato (s.l.) [RR = 0.2; (95% CI 0.14-0.27)] and Anopheles coustani [RR = 0.2; (95% CI 0.06-0.53)] and 90% fewer Anopheles funestus [RR = 0.1; (95% CI 0.08-0.19)] by CDC light traps compared to the quality assured (QA) entomology teams. Significant positive correlations were however observed between the monthly collections by CBCs and QA teams for both An. gambiae and An. funestus. In paired identifications of pooled mosquitoes, the CBCs identified 4.3 times more Anopheles compared to experienced technicians. The cost per person-night was lower in the community-based sampling at $9.1 compared to $89.3 by QA per collection effort. CONCLUSION: Unsupervised community-based mosquito surveillance collected substantially fewer mosquitoes per trap-night compared to quality-assured collection by experienced field teams, while consistently overestimating the number of Anopheles mosquitoes during identification. However, the numbers collected were significantly correlated between the CBCs and the QA teams suggesting that trends observed by CBCs and QA teams were similar. Further studies are needed to evaluate whether adopting low-cost, devolved supervision with spot checks, coupled with remedial training of the CBCs, can improve community-based collections to be considered a cost-effective alternative to surveillance conducted by experienced entomological technicians.

Malaria in pregnancy (MiP) studies assessing the clinical performance of highly sensitive rapid diagnostic tests (HS-RDT) for Plasmodium falciparum detection.

BACKGROUND: Rapid diagnostic tests (RDTs) are effective tools to diagnose and inform the treatment of malaria in adults and children. The recent development of a highly sensitive rapid diagnostic test (HS-RDT) for Plasmodium falciparum has prompted questions over whether it could improve the diagnosis of malaria in pregnancy and pregnancy outcomes in malaria endemic areas. METHODS: This landscape review collates studies addressing the clinical performance of the HS-RDT. Thirteen studies were identified comparing the HS-RDT and conventional RDT (co-RDT) to molecular methods to detect malaria in pregnancy. Using data from five completed studies, the association of epidemiological and pregnancy-related factors on the sensitivity of HS-RDT, and comparisons with co-RDT were investigated. The studies were conducted in 4 countries over a range of transmission intensities in largely asymptomatic women. RESULTS: Sensitivity of both RDTs varied widely (HS-RDT range 19.6 to 85.7%, co-RDT range 22.8 to 82.8% compared to molecular testing) yet HS-RDT detected individuals with similar parasite densities across all the studies including different geographies and transmission areas [geometric mean parasitaemia around 100 parasites per µL (p/µL)]. HS-RDTs were capable of detecting low-density parasitaemias and in one study detected around 30% of infections with parasite densities of 0-2 p/µL compared to the co-RDT in the same study which detected around 15%. CONCLUSION: The HS-RDT has a slightly higher analytical sensitivity to detect malaria infections in pregnancy than co-RDT but this mostly translates to only fractional and not statistically significant improvement in clinical performance by gravidity, trimester, geography or transmission intensity. The analysis presented here highlights the need for larger and more studies to evaluate incremental improvements in RDTs. The HS-RDT could be used in any situation where co-RDT are currently used for P. falciparum diagnosis, if storage conditions can be adhered to.

Diagnostic Performance of Loop-Mediated Isothermal Amplification and Ultrasensitive Rapid Diagnostic Tests for Malaria Screening Among Pregnant Women in Kenya.

BACKGROUND: Screen-and-treat strategies with sensitive diagnostic tests may reduce malaria-associated adverse pregnancy outcomes. We conducted a diagnostic accuracy study to evaluate new point-of-care tests to screen pregnant women for malaria at their first antenatal visit in western Kenya. METHODS: Consecutively women were tested for Plasmodium infection by expert microscopy, conventional rapid diagnostic test (cRDT), ultra sensitive RDT (usRDT), and loop-mediated isothermal amplification (LAMP). Photoinduced electron-transfer polymerase chain reaction (PET-PCR) served as the reference standard. Diagnostic performance was calculated and modelled at low parasite densities. RESULTS: Between May and September 2018, 172 of 482 screened participants (35.7%) were PET-PCR positive. Relative to PET-PCR, expert microscopy was least sensitive (40.1%; 95% confidence interval [CI], 32.7%-47.9%), followed by cRDT (49.4%; 95% CI, 41.7%-57.1), usRDT (54.7%; 95% CI, 46.9%-62.2%), and LAMP (68.6%; 95% CI, 61.1%-75.5%). Test sensitivities were comparable in febrile women (n = 90). Among afebrile women (n = 392), the geometric-mean parasite density was 29 parasites/µL and LAMP (sensitivity = 61.9%) and usRDT (43.2%) detected 1.74 (95% CI, 1.31-2.30) and 1.21 (95% CI, 88-2.21) more infections than cRDT (35.6%). Per our model, tests performed similarly at densities >200 parasites/µL. At 50 parasites/µL, the sensitivities were 45%, 56%, 62%, and 74% with expert microscopy, cRDT, usRDT, and LAMP, respectively. CONCLUSIONS: This first-generation usRDT provided moderate improvement in detecting low-density infections in afebrile pregnant women compared to cRDTs.

Efficacy of Single-Dose Primaquine With Artemisinin Combination Therapy on Plasmodium falciparum Gametocytes and Transmission: An Individual Patient Meta-Analysis.

BACKGROUND: Since the World Health Organization recommended single low-dose (0.25 mg/kg) primaquine (PQ) in combination with artemisinin-based combination therapies (ACTs) in areas of low transmission or artemisinin-resistant Plasmodium falciparum, several single-site studies have been conducted to assess efficacy. METHODS: An individual patient meta-analysis to assess gametocytocidal and transmission-blocking efficacy of PQ in combination with different ACTs was conducted. Random effects logistic regression was used to quantify PQ effect on (1) gametocyte carriage in the first 2 weeks post treatment; and (2) the probability of infecting at least 1 mosquito or of a mosquito becoming infected. RESULTS: In 2574 participants from 14 studies, PQ reduced PCR-determined gametocyte carriage on days 7 and 14, most apparently in patients presenting with gametocytemia on day 0 (odds ratio [OR], 0.22; 95% confidence interval [CI], .17-.28 and OR, 0.12; 95% CI, .08-.16, respectively). Rate of decline in gametocyte carriage was faster when PQ was combined with artemether-lumefantrine (AL) compared to dihydroartemisinin-piperaquine (DP) (P = .010 for day 7). Addition of 0.25 mg/kg PQ was associated with near complete prevention of transmission to mosquitoes. CONCLUSIONS: Transmission blocking is achieved with 0.25 mg/kg PQ. Gametocyte persistence and infectivity are lower when PQ is combined with AL compared to DP.

Plasmodium falciparum pfhrp2 and pfhrp3 Gene Deletions from Persons with Symptomatic Malaria Infection in Ethiopia, Kenya, Madagascar, and Rwanda.

Histidine-rich protein 2 (HRP2)-based rapid diagnostic tests detect Plasmodium falciparum malaria and are used throughout sub-Saharan Africa. However, deletions in the pfhrp2 and related pfhrp3 (pfhrp2/3) genes threaten use of these tests. Therapeutic efficacy studies (TESs) enroll persons with symptomatic P. falciparum infection. We screened TES samples collected during 2016-2018 in Ethiopia, Kenya, Rwanda, and Madagascar for HRP2/3, pan-Plasmodium lactate dehydrogenase, and pan-Plasmodium aldolase antigen levels and selected samples with low levels of HRP2/3 for pfhrp2/3 genotyping. We observed deletion of pfhrp3 in samples from all countries except Kenya. Single-gene deletions in pfhrp2 were observed in 1.4% (95% CI 0.2%-4.8%) of Ethiopia samples and in 0.6% (95% CI 0.2%-1.6%) of Madagascar samples, and dual pfhrp2/3 deletions were noted in 2.0% (95% CI 0.4%-5.9%) of Ethiopia samples. Although this study was not powered for precise prevalence estimates, evaluating TES samples revealed a low prevalence of pfhrp2/3 deletions in most sites.

Efficacy of Artemether-Lumefantrine and Dihydroartemisinin-Piperaquine for the Treatment of Uncomplicated Plasmodium falciparum Malaria among Children in Western Kenya, 2016 to 2017.

Antimalarial resistance threatens global malaria control efforts. The World Health Organization (WHO) recommends routine antimalarial efficacy monitoring through a standardized therapeutic efficacy study (TES) protocol. From June 2016 to March 2017, children with uncomplicated P. falciparum mono-infection in Siaya County, Kenya were enrolled into a standardized TES and randomized (1:1 ratio) to a 3-day course of artemether-lumefantrine (AL) or dihydroartemisinin-piperaquine (DP). Efficacy outcomes were measured at 28 and 42 days. A total of 340 children were enrolled. All but one child cleared parasites by day 3. PCR-corrected adequate clinical and parasitological response (ACPR) was 88.5% (95% CI: 80.9 to 93.3%) at day 28 for AL and 93.0% (95% CI: 86.9 to 96.4%) at day 42 for DP. There were 9.6 times (95% CI: 3.4 to 27.2) more reinfections in the AL arm compared to the DP arm at day 28, and 3.1 times (95% CI: 1.9 to 4.9) more reinfections at day 42. Both AL and DP were efficacious (per WHO 90% cutoff in the confidence interval) and well tolerated for the treatment of uncomplicated malaria in western Kenya, but AL efficacy appears to be waning. Further efficacy monitoring for AL, including pharmacokinetic studies, is recommended.

Safety of single-dose primaquine as a Plasmodium falciparum gametocytocide: a systematic review and meta-analysis of individual patient data.

BACKGROUND: In 2012, the World Health Organization (WHO) recommended single low-dose (SLD, 0.25 mg/kg) primaquine to be added as a Plasmodium (P.) falciparum gametocytocide to artemisinin-based combination therapy (ACT) without glucose-6-phosphate dehydrogenase (G6PD) testing, to accelerate malaria elimination efforts and avoid the spread of artemisinin resistance. Uptake of this recommendation has been relatively slow primarily due to safety concerns. METHODS: A systematic review and individual patient data (IPD) meta-analysis of single-dose (SD) primaquine studies for P. falciparum malaria were performed. Absolute and fractional changes in haemoglobin concentration within a week and adverse effects within 28 days of treatment initiation were characterised and compared between primaquine and no primaquine arms using random intercept models. RESULTS: Data comprised 20 studies that enrolled 6406 participants, of whom 5129 (80.1%) had received a single target dose of primaquine ranging between 0.0625 and 0.75 mg/kg. There was no effect of primaquine in G6PD-normal participants on haemoglobin concentrations. However, among 194 G6PD-deficient African participants, a 0.25 mg/kg primaquine target dose resulted in an additional 0.53 g/dL (95% CI 0.17-0.89) reduction in haemoglobin concentration by day 7, with a 0.27 (95% CI 0.19-0.34) g/dL haemoglobin drop estimated for every 0.1 mg/kg increase in primaquine dose. Baseline haemoglobin, young age, and hyperparasitaemia were the main determinants of becoming anaemic (Hb < 10 g/dL), with the nadir observed on ACT day 2 or 3, regardless of G6PD status and exposure to primaquine. Time to recovery from anaemia took longer in young children and those with baseline anaemia or hyperparasitaemia. Serious adverse haematological events after primaquine were few (9/3, 113, 0.3%) and transitory. One blood transfusion was reported in the primaquine arms, and there were no primaquine-related deaths. In controlled studies, the proportions with either haematological or any serious adverse event were similar between primaquine and no primaquine arms. CONCLUSIONS: Our results support the WHO recommendation to use 0.25 mg/kg of primaquine as a P. falciparum gametocytocide, including in G6PD-deficient individuals. Although primaquine is associated with a transient reduction in haemoglobin levels in G6PD-deficient individuals, haemoglobin levels at clinical presentation are the major determinants of anaemia in these patients. TRIAL REGISTRATION: PROSPERO, CRD42019128185.

Temporal trends in molecular markers of drug resistance in Plasmodium falciparum in human blood and profiles of corresponding resistant markers in mosquito oocysts in Asembo, western Kenya.

BACKGROUND: Over the last two decades, the scale-up of vector control and changes in the first-line anti-malarial, from chloroquine (CQ) to sulfadoxine-pyrimethamine (SP) and then to artemether-lumefantrine (AL), have resulted in significant decreases in malaria burden in western Kenya. This study evaluated the long-term effects of control interventions on molecular markers of Plasmodium falciparum drug resistance using parasites obtained from humans and mosquitoes at discrete time points. METHODS: Dried blood spot samples collected in 2012 and 2017 community surveys in Asembo, Kenya were genotyped by Sanger sequencing for markers associated with resistance to SP (Pfdhfr, Pfdhps), CQ, AQ, lumefantrine (Pfcrt, Pfmdr1) and artemisinin (Pfk13). Temporal trends in the prevalence of these markers, including data from 2012 to 2017 as well as published data from 1996, 2001, 2007 from same area, were analysed. The same markers from mosquito oocysts collected in 2012 were compared with results from human blood samples. RESULTS: The prevalence of SP dhfr/dhps quintuple mutant haplotype C50I51R59N108I164/S436G437E540A581A613 increased from 19.7% in 1996 to 86.0% in 2012, while an increase in the sextuple mutant haplotype C50I51R59N108I164/H436G437E540A581A613 containing Pfdhps-436H was found from 10.5% in 2012 to 34.6% in 2017. Resistant Pfcrt-76 T declined from 94.6% in 2007 to 18.3% in 2012 and 0.9% in 2017. Mutant Pfmdr1-86Y decreased across years from 74.8% in 1996 to zero in 2017, mutant Pfmdr1-184F and wild Pfmdr1-D1246 increased from 17.9% to 58.9% in 2007 to 55.9% and 90.1% in 2017, respectively. Pfmdr1 haplotype N86F184S1034N1042D1246 increased from 11.0% in 2007 to 49.6% in 2017. No resistant mutations in Pfk13 were found. Prevalence of Pfdhps-436H was lower while prevalence of Pfcrt-76 T was higher in mosquitoes than in human blood samples. CONCLUSION: This study showed an increased prevalence of dhfr/dhps resistant markers over 20 years with the emergence of Pfdhps-436H mutant a decade ago in Asembo. The reversal of Pfcrt from CQ-resistant to CQ-sensitive genotype occurred following 19 years of CQ withdrawal. No Pfk13 markers associated with artemisinin resistance were detected, but the increased haplotype of Pfmdr1 N86F184S1034N1042D1246 was observed. The differences in prevalence of Pfdhps-436H and Pfcrt-76 T SNPs between two hosts and the role of mosquitoes in the transmission of drug resistant parasites require further investigation.

Novel application of one-step pooled molecular testing and maximum likelihood approaches to estimate the prevalence of malaria parasitaemia among rapid diagnostic test negative samples in western Kenya.

BACKGROUND: Detection of malaria parasitaemia in samples that are negative by rapid diagnostic tests (RDTs) requires resource-intensive molecular tools. While pooled testing using a two-step strategy provides a cost-saving alternative to the gold standard of individual sample testing, statistical adjustments are needed to improve accuracy of prevalence estimates for a single step pooled testing strategy. METHODS: A random sample of 4670 malaria RDT negative dried blood spot samples were selected from a mass testing and treatment trial in Asembo, Gem, and Karemo, western Kenya. Samples were tested for malaria individually and in pools of five, 934 pools, by one-step quantitative polymerase chain reaction (qPCR). Maximum likelihood approaches were used to estimate subpatent parasitaemia (RDT-negative, qPCR-positive) prevalence by pooling, assuming poolwise sensitivity and specificity was either 100% (strategy A) or imperfect (strategy B). To improve and illustrate the practicality of this estimation approach, a validation study was constructed from pools allocated at random into main (734 pools) and validation (200 pools) subsets. Prevalence was estimated using strategies A and B and an inverse-variance weighted estimator and estimates were weighted to account for differential sampling rates by area. RESULTS: The prevalence of subpatent parasitaemia was 14.5% (95% CI 13.6-15.3%) by individual qPCR, 9.5% (95% CI (8.5-10.5%) by strategy A, and 13.9% (95% CI 12.6-15.2%) by strategy B. In the validation study, the prevalence by individual qPCR was 13.5% (95% CI 12.4-14.7%) in the main subset, 8.9% (95% CI 7.9-9.9%) by strategy A, 11.4% (95% CI 9.9-12.9%) by strategy B, and 12.8% (95% CI 11.2-14.3%) using inverse-variance weighted estimator from poolwise validation. Pooling, including a 20% validation subset, reduced costs by 52% compared to individual testing. CONCLUSIONS: Compared to individual testing, a one-step pooled testing strategy with an internal validation subset can provide accurate prevalence estimates of PCR-positivity among RDT-negatives at a lower cost.

Impact of Community-Based Mass Testing and Treatment on Malaria Infection Prevalence in a High-Transmission Area of Western Kenya: A Cluster Randomized Controlled Trial.

BACKGROUND: Global gains toward malaria elimination have been heterogeneous and have recently stalled. Interventions targeting afebrile malaria infections may be needed to address residual transmission. We studied the efficacy of repeated rounds of community-based mass testing and treatment (MTaT) on malaria infection prevalence in western Kenya. METHODS: Twenty clusters were randomly assigned to 3 rounds of MTaT per year for 2 years or control (standard of care for testing and treatment at public health facilities along with government-sponsored mass long-lasting insecticidal net [LLIN] distributions). During rounds, community health volunteers visited all households in intervention clusters and tested all consenting individuals with a rapid diagnostic test. Those positive were treated with dihydroartemisinin-piperaquine. Cross-sectional community infection prevalence surveys were performed in both study arms at baseline and each year after 3 rounds of MTaT. The primary outcome was the effect size of MTaT on parasite prevalence by microscopy between arms by year, adjusted for age, reported LLIN use, enhanced vegetative index, and socioeconomic status. RESULTS: Demographic and behavioral characteristics, including LLIN usage, were similar between arms at each survey. MTaT coverage across the 3 annual rounds ranged between 75.0% and 77.5% in year 1, and between 81.9% and 94.3% in year 2. The adjusted effect size of MTaT on the prevalence of parasitemia between arms was 0.93 (95% confidence interval [CI], .79-1.08) and 0.92 (95% CI, .76-1.10) after year 1 and year 2, respectively. CONCLUSIONS: MTaT performed 3 times per year over 2 years did not reduce malaria parasite prevalence in this high-transmission area. CLINICAL TRIALS REGISTRATION: NCT02987270.

Plasmodium falciparum kelch 13 Mutations, 9 Countries in Africa, 2014-2018.

The spread of drug resistance to antimalarial treatments poses a serious public health risk globally. To combat this risk, molecular surveillance of drug resistance is imperative. We report the prevalence of mutations in the Plasmodium falciparum kelch 13 propeller domain associated with partial artemisinin resistance, which we determined by using Sanger sequencing samples from patients enrolled in therapeutic efficacy studies from 9 sub-Saharan countries during 2014-2018. Of the 2,865 samples successfully sequenced before treatment (day of enrollment) and on the day of treatment failure, 29 (1.0%) samples contained 11 unique nonsynonymous mutations and 83 (2.9%) samples contained 27 unique synonymous mutations. Two samples from Kenya contained the S522C mutation, which has been associated with delayed parasite clearance; however, no samples contained validated or candidate artemisinin-resistance mutations.

Safety, Tolerability, and Immunogenicity of Plasmodium falciparum Sporozoite Vaccine Administered by Direct Venous Inoculation to Infants and Young Children: Findings From an Age De-escalation, Dose-Escalation, Double-blind, Randomized Controlled Study in Western Kenya.

BACKGROUND: The whole Plasmodium falciparum sporozoite (PfSPZ) vaccine is being evaluated for malaria prevention. The vaccine is administered intravenously for maximal efficacy. Direct venous inoculation (DVI) with PfSPZ vaccine has been safe, tolerable, and feasible in adults, but safety data for children and infants are limited. METHODS: We conducted an age de-escalation, dose-escalation randomized controlled trial in Siaya County, western Kenya. Children and infants (aged 5-9 years, 13-59 months, and 5-12 months) were enrolled into 13 age-dose cohorts of 12 participants and randomized 2:1 to vaccine or normal saline placebo in escalating doses: 1.35 × 105, 2.7 × 105, 4.5 × 105, 9.0 × 105, and 1.8 × 106 PfSPZ, with the 2 highest doses given twice, 8 weeks apart. Solicited adverse events (AEs) were monitored for 8 days after vaccination, unsolicited AEs for 29 days, and serious AEs throughout the study. Blood taken prevaccination and 1 week postvaccination was tested for immunoglobulin G antibodies to P. falciparum circumsporozoite protein (PfCSP) using enzyme-linked immunosorbent assay. RESULTS: Rates of AEs were similar in vaccinees and controls for solicited (35.7% vs 41.5%) and unsolicited (83.9% vs 92.5%) AEs, respectively. No related grade 3 AEs, serious AEs, or grade 3 laboratory abnormalities occurred. Most (79.0%) vaccinations were administered by a single DVI. Among those in the 9.0 × 105 and 1.8 × 106 PfSPZ groups, 36 of 45 (80.0%) vaccinees and 4 of 21 (19.0%) placebo controls developed antibodies to PfCSP (P < .001). CONCLUSIONS: PfSPZ vaccine in doses as high as 1.8 × 106 can be administered to infants and children by DVI, and was safe, well tolerated, and immunogenic. CLINICAL TRIALS REGISTRATION: NCT02687373.

Assessment of molecular markers of anti-malarial drug resistance among children participating in a therapeutic efficacy study in western Kenya.

BACKGROUND: Anti-malarial drug resistance remains a major threat to global malaria control efforts. In Africa, Plasmodium falciparum remains susceptible to artemisinin-based combination therapy (ACT), but the emergence of resistant parasites in multiple countries in Southeast Asia and concerns over emergence and/or spread of resistant parasites in Africa warrants continuous monitoring. The World Health Organization recommends that surveillance for molecular markers of resistance be included within therapeutic efficacy studies (TES). The current study assessed molecular markers associated with resistance to Artemether-lumefantrine (AL) and Dihydroartemisinin-piperaquine (DP) from samples collected from children aged 6-59 months enrolled in a TES conducted in Siaya County, western Kenya from 2016 to 2017. METHODS: Three hundred and twenty-three samples collected pre-treatment (day-0) and 110 samples collected at the day of recurrent parasitaemia (up to day 42) were tested for the presence of drug resistance markers in the Pfk13 propeller domain, and the Pfmdr1 and Pfcrt genes by Sanger sequencing. Additionally, the Pfpm2 gene copy number was assessed by real-time polymerase chain reaction. RESULTS: No mutations previously associated with artemisinin resistance were detected in the Pfk13 propeller region. However, other non-synonymous mutations in the Pfk13 propeller region were detected. The most common mutation found on day-0 and at day of recurrence in the Pfmdr1 multidrug resistance marker was at codon 184F. Very few mutations were found in the Pfcrt marker (< 5%). Within the DP arm, all recrudescent cases (8 sample pairs) that were tested for Pfpm2 gene copy number had a single gene copy. None of the associations between observed mutations and treatment outcomes were statistically significant. CONCLUSION: The results indicate absence of Pfk13 mutations associated with parasite resistance to artemisinin in this area and a very high proportion of wild-type parasites for Pfcrt. Although the frequency of Pfmdr1 184F mutations was high in these samples, the association with treatment failure did not reach statistical significance. As the spread of artemisinin-resistant parasites remains a possibility, continued monitoring for molecular markers of ACT resistance is needed to complement clinical data to inform treatment policy in Kenya and other malaria-endemic regions.

Human Direct Skin Feeding Versus Membrane Feeding to Assess the Mosquitocidal Efficacy of High-Dose Ivermectin (IVERMAL Trial).

BACKGROUND: Ivermectin is being considered for mass drug administration for malaria, due to its ability to kill mosquitoes feeding on recently treated individuals. In a recent trial, 3-day courses of 300 and 600 mcg/kg/day were shown to kill Anopheles mosquitoes for at least 28 days post-treatment when fed patients' venous blood using membrane feeding assays. Direct skin feeding on humans may lead to higher mosquito mortality, as ivermectin capillary concentrations are higher. We compared mosquito mortality following direct skin and membrane feeding. METHODS: We conducted a mosquito feeding study, nested within a randomized, double-blind, placebo-controlled trial of 141 adults with uncomplicated malaria in Kenya, comparing 3 days of ivermectin 300 mcg/kg/day, ivermectin 600 mcg/kg/day, or placebo, all co-administered with 3 days of dihydroartemisinin-piperaquine. On post-treatment day 7, direct skin and membrane feeding assays were conducted using laboratory-reared Anopheles gambiae sensu stricto. Mosquito survival was assessed daily for 28 days post-feeding. RESULTS: Between July 20, 2015, and May 7, 2016, 69 of 141 patients participated in both direct skin and membrane feeding (placebo, n = 23; 300 mcg/kg/day, n = 24; 600 mcg/kg/day, n = 22). The 14-day post-feeding mortality for mosquitoes fed 7 days post-treatment on blood from pooled patients in both ivermectin arms was similar with direct skin feeding (mosquitoes observed, n = 2941) versus membrane feeding (mosquitoes observed, n = 7380): cumulative mortality (risk ratio 0.99, 95% confidence interval [CI] 0.95-1.03, P = .69) and survival time (hazard ratio 0.96, 95% CI 0.91-1.02, P = .19). Results were consistent by sex, by body mass index, and across the range of ivermectin capillary concentrations studied (0.72-73.9 ng/mL). CONCLUSIONS: Direct skin feeding and membrane feeding on day 7 resulted in similar mosquitocidal effects of ivermectin across a wide range of drug concentrations, suggesting that the mosquitocidal effects seen with membrane feeding accurately reflect those of natural biting. Membrane feeding, which is more patient friendly and ethically acceptable, can likely reliably be used to assess ivermectin's mosquitocidal efficacy. CLINICAL TRIALS REGISTRATION: NCT02511353.

Community-based intermittent mass testing and treatment for malaria in an area of high transmission intensity, western Kenya: development of study site infrastructure and lessons learned.

BACKGROUND: Malaria transmission is high in western Kenya and the asymptomatic infected population plays a significant role in driving the transmission. Mathematical modelling and simulation programs suggest that interventions targeting asymptomatic infections through mass testing and treatment (MTaT) or mass drug administration (MDA) have the potential to reduce malaria transmission when combined with existing interventions. OBJECTIVE: This paper describes the study site, capacity development efforts required, and lessons learned for implementing a multi-year community-based cluster-randomized controlled trial to evaluate the impact of MTaT for malaria transmission reduction in an area of high transmission in western Kenya. METHODS: The study partnered with Kenya's Ministry of Health (MOH) and other organizations on community sensitization and engagement to mobilize, train and deploy community health volunteers (CHVs) to deliver MTaT in the community. Within the health facilities, the study availed staff, medical and laboratory supplies and strengthened health information management system to monitor progress and evaluate impact of intervention. RESULTS: More than 80 Kenya MOH CHVs, 13 clinical officers, field workers, data and logistical staff were trained to carry out MTaT three times a year for 2 years in a population of approximately 90,000 individuals. A supply chain management was adapted to meet daily demands for large volumes of commodities despite the limitation of few MOH facilities having ideal storage conditions. Modern technology was adapted more to meet the needs of the high daily volume of collected data. CONCLUSIONS: In resource-constrained settings, large interventions require capacity building and logistical planning. This study found that investing in relationships with the communities, local governments, and other partners, and identifying and equipping the appropriate staff with the skills and technology to perform tasks are important factors for success in delivering an intervention like MTaT.

Modelling the relationship between malaria prevalence as a measure of transmission and mortality across age groups.

BACKGROUND: Parasite prevalence has been used widely as a measure of malaria transmission, especially in malaria endemic areas. However, its contribution and relationship to malaria mortality across different age groups has not been well investigated. Previous studies in a health and demographic surveillance systems (HDSS) platform in western Kenya quantified the contribution of incidence and entomological inoculation rates (EIR) to mortality. The study assessed the relationship between outcomes of malaria parasitaemia surveys and mortality across age groups. METHODS: Parasitological data from annual cross-sectional surveys from the Kisumu HDSS between 2007 and 2015 were used to determine malaria parasite prevalence (PP) and clinical malaria (parasites plus reported fever within 24 h or temperature above 37.5 °C). Household surveys and verbal autopsy (VA) were used to obtain data on all-cause and malaria-specific mortality. Bayesian negative binomial geo-statistical regression models were used to investigate the association of PP/clinical malaria with mortality across different age groups. Estimates based on yearly data were compared with those from aggregated data over 4 to 5-year periods, which is the typical period that mortality data are available from national demographic and health surveys. RESULTS: Using 5-year aggregated data, associations were established between parasite prevalence and malaria-specific mortality in the whole population (RRmalaria = 1.66; 95% Bayesian Credible Intervals: 1.07-2.54) and children 1-4 years (RRmalaria = 2.29; 1.17-4.29). While clinical malaria was associated with both all-cause and malaria-specific mortality in combined ages (RRall-cause = 1.32; 1.01-1.74); (RRmalaria = 2.50; 1.27-4.81), children 1-4 years (RRall-cause = 1.89; 1.00-3.51); (RRmalaria = 3.37; 1.23-8.93) and in older children 5-14 years (RRall-cause = 3.94; 1.34-11.10); (RRmalaria = 7.56; 1.20-39.54), no association was found among neonates, adults (15-59 years) and the elderly (60+ years). Distance to health facilities, socioeconomic status, elevation and survey year were important factors for all-cause and malaria-specific mortality. CONCLUSION: Malaria parasitaemia from cross-sectional surveys was associated with mortality across age groups over 4 to 5 year periods with clinical malaria more strongly associated with mortality than parasite prevalence. This effect was stronger in children 5-14 years compared to other age-groups. Further analyses of data from other HDSS sites or similar platforms would be useful in investigating the relationship between malaria and mortality across different endemicity levels.

Capacity Development through the US President's Malaria Initiative-Supported Antimalarial Resistance Monitoring in Africa Network.

Antimalarial drug resistance is an evolving global health security threat to malaria control. Early detection of Plasmodium falciparum resistance through therapeutic efficacy studies and associated genetic analyses may facilitate timely implementation of intervention strategies. The US President's Malaria Initiative-supported Antimalarial Resistance Monitoring in Africa Network has assisted numerous laboratories in partner countries in acquiring the knowledge and capability to independently monitor for molecular markers of antimalarial drug resistance.