Safety and efficacy of malaria vaccine candidate R21/Matrix-M in African children: a multicentre, double-blind, randomised, phase 3 trial.

BACKGROUND: Recently, we found that a new malaria vaccine, R21/Matrix-M, had over 75% efficacy against clinical malaria with seasonal administration in a phase 2b trial in Burkina Faso. Here, we report on safety and efficacy of the vaccine in a phase 3 trial enrolling over 4800 children across four countries followed for up to 18 months at seasonal sites and 12 months at standard sites. METHODS: We did a double-blind, randomised, phase 3 trial of the R21/Matrix-M malaria vaccine across five sites in four African countries with differing malaria transmission intensities and seasonality. Children (aged 5-36 months) were enrolled and randomly assigned (2:1) to receive 5 µg R21 plus 50 µg Matrix-M or a control vaccine (licensed rabies vaccine [Abhayrab]). Participants, their families, investigators, laboratory teams, and the local study team were masked to treatment. Vaccines were administered as three doses, 4 weeks apart, with a booster administered 12 months after the third dose. Half of the children were recruited at two sites with seasonal malaria transmission and the remainder at standard sites with perennial malaria transmission using age-based immunisation. The primary objective was protective efficacy of R21/Matrix-M from 14 days after third vaccination to 12 months after completion of the primary series at seasonal and standard sites separately as co-primary endpoints. Vaccine efficacy against multiple malaria episodes and severe malaria, as well as safety and immunogenicity, were also assessed. This trial is registered on ClinicalTrials.gov, NCT04704830, and is ongoing. FINDINGS: From April 26, 2021, to Jan 12, 2022, 5477 children consented to be screened, of whom 1705 were randomly assigned to control vaccine and 3434 to R21/Matrix-M; 4878 participants received the first dose of vaccine. 3103 participants in the R21/Matrix-M group and 1541 participants in the control group were included in the modified per-protocol analysis (2412 [51·9%] male and 2232 [48·1%] female). R21/Matrix-M vaccine was well tolerated, with injection site pain (301 [18·6%] of 1615 participants) and fever (754 [46·7%] of 1615 participants) as the most frequent adverse events. Number of adverse events of special interest and serious adverse events did not significantly differ between the vaccine groups. There were no treatment-related deaths. 12-month vaccine efficacy was 75% (95% CI 71-79; p<0·0001) at the seasonal sites and 68% (61-74; p<0·0001) at the standard sites for time to first clinical malaria episode. Similarly, vaccine efficacy against multiple clinical malaria episodes was 75% (71-78; p<0·0001) at the seasonal sites and 67% (59-73; p<0·0001) at standard sites. A modest reduction in vaccine efficacy was observed over the first 12 months of follow-up, of similar size at seasonal and standard sites. A rate reduction of 868 (95% CI 762-974) cases per 1000 children-years at seasonal sites and 296 (231-362) at standard sites occurred over 12 months. Vaccine-induced antibodies against the conserved central Asn-Ala-Asn-Pro (NANP) repeat sequence of circumsporozoite protein correlated with vaccine efficacy. Higher NANP-specific antibody titres were observed in the 5-17 month age group compared with 18-36 month age group, and the younger age group had the highest 12-month vaccine efficacy on time to first clinical malaria episode at seasonal (79% [95% CI 73-84]; p<0·001) and standard (75% [65-83]; p<0·001) sites. INTERPRETATION: R21/Matrix-M was well tolerated and offered high efficacy against clinical malaria in African children. This low-cost, high-efficacy vaccine is already licensed by several African countries, and recently received a WHO policy recommendation and prequalification, offering large-scale supply to help reduce the great burden of malaria in sub-Saharan Africa. FUNDING: The Serum Institute of India, the Wellcome Trust, the UK National Institute for Health Research Oxford Biomedical Research Centre, and Open Philanthropy.

Seasonal Malaria Vaccination with or without Seasonal Malaria Chemoprevention.

BACKGROUND: Malaria control remains a challenge in many parts of the Sahel and sub-Sahel regions of Africa. METHODS: We conducted an individually randomized, controlled trial to assess whether seasonal vaccination with RTS,S/AS01E was noninferior to chemoprevention in preventing uncomplicated malaria and whether the two interventions combined were superior to either one alone in preventing uncomplicated malaria and severe malaria-related outcomes. RESULTS: We randomly assigned 6861 children 5 to 17 months of age to receive sulfadoxine-pyrimethamine and amodiaquine (2287 children [chemoprevention-alone group]), RTS,S/AS01E (2288 children [vaccine-alone group]), or chemoprevention and RTS,S/AS01E (2286 children [combination group]). Of these, 1965, 1988, and 1967 children in the three groups, respectively, received the first dose of the assigned intervention and were followed for 3 years. Febrile seizure developed in 5 children the day after receipt of the vaccine, but the children recovered and had no sequelae. There were 305 events of uncomplicated clinical malaria per 1000 person-years at risk in the chemoprevention-alone group, 278 events per 1000 person-years in the vaccine-alone group, and 113 events per 1000 person-years in the combination group. The hazard ratio for the protective efficacy of RTS,S/AS01E as compared with chemoprevention was 0.92 (95% confidence interval [CI], 0.84 to 1.01), which excluded the prespecified noninferiority margin of 1.20. The protective efficacy of the combination as compared with chemoprevention alone was 62.8% (95% CI, 58.4 to 66.8) against clinical malaria, 70.5% (95% CI, 41.9 to 85.0) against hospital admission with severe malaria according to the World Health Organization definition, and 72.9% (95% CI, 2.9 to 92.4) against death from malaria. The protective efficacy of the combination as compared with the vaccine alone against these outcomes was 59.6% (95% CI, 54.7 to 64.0), 70.6% (95% CI, 42.3 to 85.0), and 75.3% (95% CI, 12.5 to 93.0), respectively. CONCLUSIONS: Administration of RTS,S/AS01E was noninferior to chemoprevention in preventing uncomplicated malaria. The combination of these interventions resulted in a substantially lower incidence of uncomplicated malaria, severe malaria, and death from malaria than either intervention alone. (Funded by the Joint Global Health Trials and PATH; ClinicalTrials.gov number, NCT03143218.).

Plasma biomarkers of hemoglobin loss in Plasmodium falciparum-infected children identified by quantitative proteomics.

Anemia is common among young children infected with Plasmodium falciparum and severe malarial anemia (SMA) is a major cause of their mortality. Two major mechanisms cause malarial anemia: hemolysis of uninfected as well as infected erythrocytes and insufficient erythropoiesis. In a longitudinal birth cohort in Mali, we commonly observed marked hemoglobin reductions during P falciparum infections with a small proportion that progressed to SMA. We sought biomarkers of these processes using quantitative proteomic analysis on plasma samples from 9 P falciparum-infected children, comparing those with reduced hemoglobin (with or without SMA) vs those with stable hemoglobin. We identified higher plasma levels of circulating 20S proteasome and lower insulin-like growth factor-1 (IGF-1) levels in children with reduced hemoglobin. We confirmed these findings in independent enzyme-linked immunosorbent assay-based validation studies of subsets of children from the same cohort (20S proteasome, N = 71; IGF-1, N = 78). We speculate that circulating 20S proteasome plays a role in digesting erythrocyte membrane proteins modified by oxidative stress, resulting in hemolysis, whereas decreased IGF-1, a critical factor for erythroid maturation, might contribute to insufficient erythropoiesis. Quantitative plasma proteomics identified soluble mediators that may contribute to the major mechanisms underlying malarial anemia. This study was registered at www.clinicaltrials.gov as #NCT01168271.

Population pharmacokinetics of primaquine and its metabolites in African males.

BACKGROUND: Primaquine (PQ) is the prototype 8-aminoquinoline drug, a class which targets gametocytes and hypnozoites. The World Health Organization (WHO) recommends adding a single low dose of primaquine to the standard artemisinin-based combination therapy (ACT) in order to block malaria transmission in regions with low malaria transmission. However, the haemolytic toxicity is a major adverse outcome of primaquine in glucose-6-phosphate dehydrogenase (G6PD)-deficient subjects. This study aimed to characterize the pharmacokinetic properties of primaquine and its major metabolites in G6PD-deficient subjects. METHODS: A single low-dose of primaquine (0.4-0.5 mg/kg) was administered in twenty-eight African males. Venous and capillary plasma were sampled up to 24 h after the drug administration. Haemoglobin levels were observed up to 28 days after drug administration. Only PQ, carboxy-primaquine (CPQ), and primaquine carbamoyl-glucuronide (PQCG) were present in plasma samples and measured using liquid chromatography mass spectrometry. Drug and metabolites' pharmacokinetic properties were investigated using nonlinear mixed-effects modelling. RESULTS: Population pharmacokinetic properties of PQ, CPQ, and PQCG can be described by one-compartment disposition kinetics with a transit-absorption model. Body weight was implemented as an allometric function on the clearance and volume parameters for all compounds. None of the covariates significantly affected the pharmacokinetic parameters. No significant correlations were detected between the exposures of the measured compounds and the change in haemoglobin or methaemoglobin levels. There was no significant haemoglobin drop in the G6PD-deficient patients after administration of a single low dose of PQ. CONCLUSIONS: A single low-dose of PQ was haematologically safe in this population of G6PD-normal and G6PD-deficient African males without malaria. Trial registration NCT02535767.

Natural History of Malaria Infections During Early Childhood in Twins.

BACKGROUND: The frequency and clinical presentation of malaria infections show marked heterogeneity in epidemiological studies. However, deeper understanding of this variability is hampered by the difficulty in quantifying all relevant factors. Here, we report the history of malaria infections in twins, who are exposed to the same in utero milieu, share genetic factors, and are similarly exposed to vectors. METHODS: Data were obtained from a Malian longitudinal birth cohort. Samples from 25 twin pairs were examined for malaria infection and antibody responses. Bayesian models were developed for the number of infections during follow-up. RESULTS: In 16 of 25 pairs, both children were infected and often developed symptoms. In 8 of 25 pairs, only 1 twin was infected, but usually only once or twice. Statistical models suggest that this pattern is not inconsistent with twin siblings having the same underlying infection rate. In a pair with discordant hemoglobin genotype, parasite densities were consistently lower in the child with hemoglobin AS, but antibody levels were similar. CONCLUSIONS: By using a novel design, we describe residual variation in malaria phenotypes in naturally matched children and confirm the important role of environmental factors, as suggested by the between-twin pair heterogeneity in malaria history.

IFNγ, TNFα polymorphisms and IFNγ serum levels are associated with the clearance of drug-resistant P. falciparum in Malian children.

Host immunity has been suggested to clear drug-resistant parasites in malaria-endemic settings. However, the immunogenetic mechanisms involved in parasite clearance are poorly understood. Characterizing the host's immunity and genes involved in controlling the parasitic infection can inform the development of blood-stage malaria vaccines. This study investigates host regulatory cytokines and immunogenomic factors associated with the clearance of Plasmodium falciparum carrying a chloroquine resistance genotype. Biological samples from participants of previous drug efficacy trials conducted in two Malian localities were retrieved. The P. falciparum chloroquine resistance transporter (Pfcrt) gene was genotyped using parasite DNA. Children carrying parasites with the mutant allele (Pfcrt-76T) were classified based on their ability to clear their parasites. The levels of the different cytokines were measured in serum. The polymorphisms of specific human genes involved in malaria susceptibility were genotyped using human DNA. The prevalence of the Pfcrt-76T was significantly higher in Kolle than in Bandiagara (81.6 % vs 38.6 %, p < 10-6). The prevalence of children who cleared their mutant parasites was significantly higher in Bandiagara than in Kolle (82.2 % vs 67.4 %, p < 0.05). The genotyping of host genes revealed that IFN-γ -874 T and TNF-α -308A alleles were positively associated with parasite clearance. Cytokine profiling revealed that IFN-γ level was positively associated with parasite clearance (p = 0.04). This study highlights the role of host's immunity and immunogenetic factors to clear resistant parasites, suggesting further characterization of these polymorphisms may help to develop novel approaches to antiparasitic treatment strategies.

Improving social justice in observational studies: protocol for the development of a global and Indigenous STROBE-equity reporting guideline.

BACKGROUND: Addressing persistent and pervasive health inequities is a global moral imperative, which has been highlighted and magnified by the societal and health impacts of the COVID-19 pandemic. Observational studies can aid our understanding of the impact of health and structural oppression based on the intersection of gender, race, ethnicity, age and other factors, as they frequently collect this data. However, the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guideline, does not provide guidance related to reporting of health equity. The goal of this project is to develop a STROBE-Equity reporting guideline extension. METHODS: We assembled a diverse team across multiple domains, including gender, age, ethnicity, Indigenous background, disciplines, geographies, lived experience of health inequity and decision-making organizations. Using an inclusive, integrated knowledge translation approach, we will implement a five-phase plan which will include: (1) assessing the reporting of health equity in published observational studies, (2) seeking wide international feedback on items to improve reporting of health equity, (3) establishing consensus amongst knowledge users and researchers, (4) evaluating in partnership with Indigenous contributors the relevance to Indigenous peoples who have globally experienced the oppressive legacy of colonization, and (5) widely disseminating and seeking endorsement from relevant knowledge users. We will seek input from external collaborators using social media, mailing lists and other communication channels. DISCUSSION: Achieving global imperatives such as the Sustainable Development Goals (e.g., SDG 10 Reduced inequalities, SDG 3 Good health and wellbeing) requires advancing health equity in research. The implementation of the STROBE-Equity guidelines will enable a better awareness and understanding of health inequities through better reporting. We will broadly disseminate the reporting guideline with tools to enable adoption and use by journal editors, authors, and funding agencies, using diverse strategies tailored to specific audiences.

Plasmodium falciparum in Aotus nancymaae: A New Model for Placental Malaria.

Plasmodium falciparum-infected erythrocytes that display the variant surface antigen VAR2CSA bind chondroitin sulfate A (CSA) to sequester in placental intervillous spaces, causing severe sequelae for mother and offspring. Here, we establish a placental malaria (PM) monkey model. Pregnant Aotus infected with CSA-binding P. falciparum CS2 parasites during the third trimester developed pronounced sequestration of late-stage parasites in placental intervillous spaces that express VAR2CSA and bind specifically to CSA. Similar to immune multigravid women, a monkey infected with P. falciparum CS2 parasites over successive pregnancies acquired antibodies against VAR2CSA, with potent functional activity that was boosted upon subsequent pregnancy infections. Aotus also developed functional antibodies after multiple acute PM episodes and subsequent VAR2CSA immunization. In summary, P. falciparum infections in pregnant Aotus monkeys recapitulate all the prominent features of human PM infection and immunity, and this model can be useful for basic mechanistic studies and preclinical studies to qualify candidate PM vaccines. Clinical Trials Registration: NCT02471378.

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.

Rapidly Increasing Severe Acute Respiratory Syndrome Coronavirus 2 Seroprevalence and Limited Clinical Disease in 3 Malian Communities: A Prospective Cohort Study.

BACKGROUND: The extent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure and transmission in Mali and the surrounding region is not well understood. We aimed to estimate the cumulative incidence of SARS-CoV-2 in 3 communities and understand factors associated with infection. METHODS: Between July 2020 and January 2021, we collected blood samples and demographic, social, medical, and self-reported symptoms information from residents aged 6 months and older over 2 study visits. SARS-CoV-2 antibodies were measured using a highly specific 2-antigen enzyme-linked immunosorbent assay optimized for use in Mali. We calculated cumulative adjusted seroprevalence for each community and evaluated factors associated with serostatus at each visit by univariate and multivariate analysis. RESULTS: Overall, 94.8% (2533/2672) of participants completed both study visits. A total of 31.3% (837/2672) were aged <10 years, 27.6% (737/2672) were aged 10-17 years, and 41.1% (1098/2572) were aged ≥18 years. The cumulative SARS-CoV-2 exposure rate was 58.5% (95% confidence interval, 47.5-69.4). This varied between sites and was 73.4% in the urban community of Sotuba, 53.2% in the rural town of Bancoumana, and 37.1% in the rural village of Donéguébougou. Study site and increased age were associated with serostatus at both study visits. There was minimal difference in reported symptoms based on serostatus. CONCLUSIONS: The true extent of SARS-CoV-2 exposure in Mali is greater than previously reported and may now approach hypothetical "herd immunity" in urban areas. The epidemiology of the pandemic in the region may be primarily subclinical and within background illness rates.

The Anti-Circumsporozoite Antibody Response of Children to Seasonal Vaccination With the RTS,S/AS01E Malaria Vaccine.

BACKGROUND: A trial in African children showed that combining seasonal vaccination with the RTS,S/AS01E vaccine with seasonal malaria chemoprevention reduced the incidence of uncomplicated and severe malaria compared with either intervention given alone. Here, we report on the anti-circumsporozoite antibody response to seasonal RTS,S/AS01E vaccination in children in this trial. METHODS: Sera from a randomly selected subset of children collected before and 1 month after 3 priming doses of RTS,S/AS01E and before and 1 month after 2 seasonal booster doses were tested for anti-circumsporozoite antibodies using enzyme-linked immunosorbent assay. The association between post-vaccination antibody titer and incidence of malaria was explored. RESULTS: A strong anti-circumsporozoite antibody response to 3 priming doses of RTS,S/AS01E was seen (geometric mean titer, 368.9 enzyme-linked immunosorbent assay units/mL), but titers fell prior to the first booster dose. A strong antibody response to an annual, pre-malaria transmission season booster dose was observed, but this was lower than after the primary vaccination series and lower after the second than after the first booster dose (ratio of geometric mean rise, 0.66; 95% confidence interval [CI], .57-.77). Children whose antibody response was in the upper tercile post-vaccination had a lower incidence of malaria during the following year than children in the lowest tercile (hazard ratio, 0.43; 95% CI, .28-.66). CONCLUSIONS: Seasonal vaccination with RTS,S/AS01E induced a strong booster antibody response that was lower after the second than after the first booster dose. The diminished antibody response to the second booster dose was not associated with diminished efficacy. CLINICAL TRIALS REGISTRATION: NCT03143218.

Effect of Adding Azithromycin to Seasonal Malaria Chemoprevention.

BACKGROUND: Mass administration of azithromycin for trachoma control led to a sustained reduction in all-cause mortality among Ethiopian children. Whether the addition of azithromycin to the monthly sulfadoxine-pyrimethamine plus amodiaquine used for seasonal malaria chemoprevention could reduce mortality and morbidity among African children was unclear. METHODS: We randomly assigned children 3 to 59 months of age, according to household, to receive either azithromycin or placebo, together with sulfadoxine-pyrimethamine plus amodiaquine, during the annual malaria-transmission season in Burkina Faso and Mali. The drug combinations were administered in four 3-day cycles, at monthly intervals, for three successive seasons. The primary end point was death or hospital admission for at least 24 hours that was not due to trauma or elective surgery. Data were recorded by means of active and passive surveillance. RESULTS: In July 2014, a total of 19,578 children were randomly assigned to receive seasonal malaria chemoprevention plus either azithromycin (9735 children) or placebo (9843 children); each year, children who reached 5 years of age exited the trial and new children were enrolled. In the intention-to-treat analysis, the overall number of deaths and hospital admissions during three malaria-transmission seasons was 250 in the azithromycin group and 238 in the placebo group (events per 1000 child-years at risk, 24.8 vs. 23.5; incidence rate ratio, 1.1; 95% confidence interval [CI], 0.88 to 1.3). Results were similar in the per-protocol analysis. The following events occurred less frequently with azithromycin than with placebo: gastrointestinal infections (1647 vs. 1985 episodes; incidence rate ratio, 0.85; 95% CI, 0.79 to 0.91), upper respiratory tract infections (4893 vs. 5763 episodes; incidence rate ratio, 0.85; 95% CI, 0.81 to 0.90), and nonmalarial febrile illnesses (1122 vs. 1424 episodes; incidence rate ratio, 0.79; 95% CI, 0.73 to 0.87). The prevalence of malaria parasitemia and incidence of adverse events were similar in the two groups. CONCLUSIONS: Among children in Burkina Faso and Mali, the addition of azithromycin to the antimalarial agents used for seasonal malaria chemoprevention did not result in a lower incidence of death or hospital admission that was not due to trauma or surgery than antimalarial agents plus placebo, although a lower disease burden was noted with azithromycin than with placebo. (Funded by the Joint Global Health Trials scheme; ClinicalTrials.gov number, NCT02211729.).

The duration of protection against clinical malaria provided by the combination of seasonal RTS,S/AS01E vaccination and seasonal malaria chemoprevention versus either intervention given alone.

BACKGROUND: A recent trial of 5920 children in Burkina Faso and Mali showed that the combination of seasonal vaccination with the RTS,S/AS01E malaria vaccine (primary series and two seasonal boosters) and seasonal malaria chemoprevention (four monthly cycles per year) was markedly more effective than either intervention given alone in preventing clinical malaria, severe malaria, and deaths from malaria. METHODS: In order to help optimise the timing of these two interventions, trial data were reanalysed to estimate the duration of protection against clinical malaria provided by RTS,S/AS01E when deployed seasonally, by comparing the group who received the combination of SMC and RTS,S/AS01E with the group who received SMC alone. The duration of protection from SMC was also estimated comparing the combined intervention group with the group who received RTS,S/AS01E alone. Three methods were used: Piecewise Cox regression, Flexible parametric survival models and Smoothed Schoenfeld residuals from Cox models, stratifying on the study area and using robust standard errors to control for within-child clustering of multiple episodes. RESULTS: The overall protective efficacy from RTS,S/AS01E over 6 months was at least 60% following the primary series and the two seasonal booster doses and remained at a high level over the full malaria transmission season. Beyond 6 months, protective efficacy appeared to wane more rapidly, but the uncertainty around the estimates increases due to the lower number of cases during this period (coinciding with the onset of the dry season). Protection from SMC exceeded 90% in the first 2-3 weeks post-administration after several cycles, but was not 100%, even immediately post-administration. Efficacy begins to decline from approximately day 21 and then declines more sharply after day 28, indicating the importance of preserving the delivery interval for SMC cycles at a maximum of four weeks. CONCLUSIONS: The efficacy of both interventions was highest immediately post-administration. Understanding differences between these interventions in their peak efficacy and how rapidly efficacy declines over time will help to optimise the scheduling of SMC, malaria vaccination and the combination in areas of seasonal transmission with differing epidemiology, and using different vaccine delivery systems. TRIAL REGISTRATION: The RTS,S-SMC trial in which these data were collected was registered at clinicaltrials.gov: NCT03143218.

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.

CXCR4 and MIF are required for neutrophil extracellular trap release triggered by Plasmodium-infected erythrocytes.

Neutrophil extracellular traps (NETs) evolved as a unique effector mechanism contributing to resistance against infection that can also promote tissue damage in inflammatory conditions. Malaria infection can trigger NET release, but the mechanisms and consequences of NET formation in this context remain poorly characterized. Here we show that patients suffering from severe malaria had increased amounts of circulating DNA and increased neutrophil elastase (NE) levels in plasma. We used cultured erythrocytes and isolated human neutrophils to show that Plasmodium-infected red blood cells release macrophage migration inhibitory factor (MIF), which in turn caused NET formation by neutrophils in a mechanism dependent on the C-X-C chemokine receptor type 4 (CXCR4). NET production was dependent on histone citrullination by peptidyl arginine deiminase-4 (PAD4) and independent of reactive oxygen species (ROS), myeloperoxidase (MPO) or NE. In vitro, NETs functioned to restrain parasite dissemination in a mechanism dependent on MPO and NE activities. Finally, C57/B6 mice infected with P. berghei ANKA, a well-established model of cerebral malaria, presented high amounts of circulating DNA, while treatment with DNAse increased parasitemia and accelerated mortality, indicating a role for NETs in resistance against Plasmodium infection.

Resurgent and delayed malaria.

The populations of moderate or highly malaria endemic areas gradually acquire some immunity to malaria as a result of repeated exposure to the infection. When this exposure is reduced as a result of effective malaria control measures, subjects who benefitted from the intervention may consequently be at increased risk of malaria if the intervention is withdrawn, especially if this is done abruptly, and an effective malaria vector remains. There have been many examples of this occurring in the past, a phenomenon often termed 'rebound malaria', with the incidence of malaria rebounding to the level present before the intervention was introduced. Because the main clinical burden of malaria in areas with a high level of malaria transmission is in young children, malaria control efforts have, in recent decades, focussed on this group, with substantial success being obtained with interventions such as insecticide treated mosquito nets, chemoprevention and, most recently, malaria vaccines. These are interventions whose administration may not be sustained. This has led to concerns that in these circumstances, the overall burden of malaria in children may not be reduced but just delayed, with the main period of risk being in the period shortly after the intervention is no longer given. Although dependent on the same underlying process as classical 'resurgent' malaria, it may be helpful to differentiate the two conditions, describing the later as 'delayed malaria'. In this paper, some of the evidence that delayed malaria occurs is discussed and potential measures for reducing its impact are suggested.

Effect of three years' seasonal malaria chemoprevention on molecular markers of resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine and amodiaquine in Ouelessebougou, Mali.

BACKGROUND: In 2012, seasonal malaria chemoprevention (SMC) was recommended as policy for malaria control by the World Health Organization (WHO) in areas of highly seasonal malaria transmission across the Sahel sub-region in Africa along with monitoring of drug resistance. We assessed the long-term impact of SMC on Plasmodium falciparum resistance to sulfadoxine-pyrimethamine (SP) and amodiaquine (AQ) over a 3-year period of SMC implementation in the health district of Ouelessebougou, Mali. METHODS: In 8 randomly selected sub-districts of Ouelessebougou, Mali, children aged 0-5 years were randomly selected during cross-sectional surveys at baseline (August 2014) and 1, 2 and 3 years post-SMC, at the beginning and end of the malaria transmission season. Blood smears and blood spots on filter paper were obtained and frequencies of mutation in P. falciparum genes related to resistance to SP and AQ (Pfdhfr, Pfdhps, Pfmdr1, and Pfcrt) were assessed by PCR amplification on individual samples and PCR amplification followed by deep sequencing on pooled (by site and year) samples. RESULTS: At each survey, approximately 50-100 individual samples were analysed by PCR amplification and a total of 1,164 samples were analysed by deep sequencing with an average read depth of 18,018-36,918 after pooling by site and year. Most molecular markers of resistance did not increase in frequency over the period of study (2014-2016). After 3 years of SMC, the frequencies of Pfdhps 540E, Pfdhps 437G and Pfcrt K76T remained similar compared to baseline (4.0 vs 1.4%, p = 0.41; 74.5 vs 64.6%, p = 0.22; 71.3 vs 67.4%, p = 0.69). Nearly all samples tested carried Pfdhfr 59R, and this proportion remained similar 3 years after SMC implementation (98.8 vs 100%, p = 1). The frequency of Pfmdr1 N86Y increased significantly over time from 5.6% at baseline to 18.6% after 3 years of SMC (p = 0.016). Results of pooled analysis using deep sequencing were consistent with those by individual analysis with standard PCR, but also indicated for the first time the presence of mutations at the Pfdhps A581G allele at a frequency of 11.7% after 2 years of SMC, as well as the Pfdhps I431V allele at frequencies of 1.6-9.3% following 1 and 2 years of SMC, respectively. CONCLUSION: Two and 3 years of SMC implementation were associated with increased frequency of the Pfmdr1 N86Y mutation but not Pfdhps 540E, Pfdhps 437G and Pfcrt K76T. The first-time detection of the Pfdhps haplotype bearing the I431V and A581G mutations in Mali, even at low frequency, warrants further long-term surveillance.

Persistence of Plasmodium falciparum HRP-2 antigenaemia after artemisinin combination therapy is not associated with gametocytes.

BACKGROUND: In some settings, sensitive field diagnostic tools may be needed to achieve elimination of falciparum malaria. To this end, rapid diagnostic tests (RDTs) based on the detection of the Plasmodium falciparum protein HRP-2 are being developed with increasingly lower limits of detection. However, it is currently unclear how parasite stages that are unaffected by standard drug treatments may contribute to HRP-2 detectability and potentially confound RDT results even after clearance of blood stage infection. This study assessed the detectability of HRP-2 in periods of post-treatment residual gametocytaemia. METHODS: A cohort of 100 P. falciparum infected, gametocyte positive individuals were treated with or without the gametocytocidal drug primaquine (PQ), alongside standard artemisinin-based combination therapy (ACT), in the context of a randomised clinical trial in Ouelessebougou, Mali. A quantitative ELISA was used to measure levels of HRP-2, and compared time to test negativity using a standard and ultra-sensitive RDT (uRDT) between residual gametocyte positive and negative groups. RESULTS: Time to test negativity was longest by uRDT, followed by ELISA and then standard RDT. No significant difference in time to negativity was found between the treatment groups with and without residual gametocytes: uRDT (HR 0.79 [95% CI 0.52-1.21], p = 0.28), RDT (HR 0.77 [95% CI 0.51-1.15], p = 0.20) or ELISA (HR 0.88 [95% CI 0.59-1.32], p = 0.53). Similarly, no difference was observed when adjusting for baseline asexual parasite density. Quantified levels of HRP-2 over time were similar between groups, with differences attributable to asexual parasite densities. Furthermore, no difference in levels of HRP-2 was found between individuals who were or were not infectious to mosquitoes (OR 1.19 [95% CI 0.98-1.46], p = 0.077). CONCLUSIONS: Surviving sexual stage parasites after standard ACT treatment do not contribute to the persistence of HRP-2 antigenaemia, and appear to have little impact on RDT results.

Impact of seasonal RTS,S/AS01E vaccination plus seasonal malaria chemoprevention on the nutritional status of children in Burkina Faso and Mali.

BACKGROUND: A recent trial in Burkina Faso and Mali showed that combining seasonal RTS,S/AS01E malaria vaccination with seasonal malaria chemoprevention (SMC) substantially reduced the incidence of uncomplicated and severe malaria in young children compared to either intervention alone. Given the possible negative effect of malaria on nutrition, the study investigated whether these children also experienced lower prevalence of acute and chronic malnutrition. METHODS: In Burkina Faso and Mali 5920 children were randomized to receive either SMC alone, RTS,S/AS01E alone, or SMC combined with RTS,S/AS01E for three malaria transmission seasons (2017-2019). After each transmission season, anthropometric measurements were collected from all study children at a cross-sectional survey and used to derive nutritional status indicators, including the binary variables wasted and stunted (weight-for-height and height-for-age z-scores below - 2, respectively). Binary and continuous outcomes between treatment groups were compared by Poisson and linear regression. RESULTS: In 2017, compared to SMC alone, the combined intervention reduced the prevalence of wasting by approximately 12% [prevalence ratio (PR) = 0.88 (95% CI 0.75, 1.03)], and approximately 21% in 2018 [PR = 0.79 (95% CI 0.62, 1.01)]. Point estimates were similar for comparisons with RTS,S/AS01E, but there was stronger evidence of a difference. There was at least a 30% reduction in the point estimates for the prevalence of severe wasting in the combined group compared to the other two groups in 2017 and 2018. There was no difference in the prevalence of moderate or severe wasting between the groups in 2019. The prevalence of stunting, low-MUAC-for-age or being underweight did not differ between groups for any of the three years. The prevalence of severe stunting was higher in the combined group compared to both other groups in 2018, and compared to RTS,S/AS01E alone in 2017; this observation does not have an obvious explanation and may be a chance finding. Overall, malnutrition was very common in this cohort, but declined over the study as the children became older. CONCLUSIONS: Despite a high burden of malnutrition and malaria in the study populations, and a major reduction in the incidence of malaria in children receiving both interventions, this had only a modest impact on nutritional status. Therefore, other interventions are needed to reduce the high burden of malnutrition in these areas. TRIAL REGISTRATION: https://www.clinicaltrials.gov/ct2/show/NCT03143218 , registered 8th May 2017.

Severe Acute Respiratory Syndrome Coronavirus 2 Seroassay Performance and Optimization in a Population With High Background Reactivity in Mali.

BACKGROUND: False positivity may hinder the utility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological tests in sub-Saharan Africa. METHODS: From 312 Malian samples collected before 2020, we measured antibodies to the commonly tested SARS-CoV-2 antigens and 4 other betacoronaviruses by enzyme-linked immunosorbent assay (ELISA). In a subset of samples, we assessed antibodies to a panel of Plasmodium falciparum antigens by suspension bead array and functional antiviral activity by SARS-CoV-2 pseudovirus neutralization assay. We then evaluated the performance of an ELISA using SARS-CoV-2 spike protein and receptor-binding domain developed in the United States using Malian positive and negative control samples. To optimize test performance, we compared single- and 2-antigen approaches using existing assay cutoffs and population-specific cutoffs. RESULTS: Background reactivity to SARS-CoV-2 antigens was common in prepandemic Malian samples. The SARS-CoV-2 reactivity varied between communities, increased with age, and correlated negligibly/weakly with other betacoronavirus and P falciparum antibodies. No prepandemic samples demonstrated functional activity. Regardless of the cutoffs applied, test specificity improved using a 2-antigen approach. Test performance was optimal using a 2-antigen assay with population-specific cutoffs (sensitivity, 73.9% [95% confidence interval {CI}, 51.6-89.8]; specificity, 99.4% [95% CI, 97.7-99.9]). CONCLUSIONS: We have addressed the problem of SARS-CoV-2 seroassay performance in Africa by using a 2-antigen assay with cutoffs defined by performance in the target population.