Plasmodium falciparum and Plasmodium vivax malaria were approximately similar.The northwestern border of Thailand is an area of low seasonal malaria transmission. Until recent successful malaria elimination activities, malaria was a major cause of disease and death. Historically the incidences of symptomatic All malaria cases managed in the Shoklo Malaria Research Unit along the Thailand-Myanmar border between 2000 and 2016 were reviewed.P. vivax and 94 467 for symptomatic P. falciparum malaria. Overall, 4844 (5.1%) patients with P. falciparum malaria were admitted to field hospitals, of whom 66 died, compared with 278 (0.34%) with P. vivax malaria, of whom 4 died . Applying the 2015 World Health Organization severe malaria criteria, 68 of 80 841 P. vivax admissions (0.08%) and 1482 of 94 467 P. falciparum admissions (1.6%) were classified as severe. Overall, patients with P. falciparum malaria were 15  times more likely than those with P. vivax malaria to require hospital admission, 19 (14.6–23.8) times more likely to develop severe malaria, and ≥14 (5.1–38.7) times more likely to die.There were 80 841 consultations for symptomatic P. falciparum and P. vivax infections were important causes of hospitalization, but life-threatening P. vivax illness was rare.In this area, both  Plasmodium falciparum and 80 841 with Plasmodium vivax malaria, patients with P. falciparum were 15 times more likely to require hospital admission, 19 times more likely to develop severe malaria, and 14 times more likely to die.Among 94 467 patients with  Plasmodium vivax was known as “benign tertian malaria” to differentiate it from the commonly fatal “malignant tertian” malaria caused by Plasmodium falciparum ) than with P. falciparum malaria (650 of 2097 [31%])  (P. vivax (58 of 102 [57%]), and significantly less (123 of 650 [19%]) for P. falciparum. Many of the post-partum P. vivax patients admitted had reasons for admission that were unrelated to malaria. In hospitalized P. vivax malaria cases, the presenting mean hematocrit was 4.2% lower in pregnant women  and 7.3% lower in post-partum women  than for nonpregnant or non post-partum patients. Chronic disease  was much more common in patients hospitalized with P. vivax than in those hospitalized with P. falciparum   than those with P. falciparum (4 [4–6] days); P < .001) (In adults (aged >15 years) hospitalized for suspected severe malaria, more women were admitted with  < .001) . Pregnan < .001) . Patient < .001) .P. falciparum and 4 with P. vivax infection. One P. vivax malaria death occurred in a 54-year old man with Gram-negative meningitis; Escherichia coli grew in an immediate post-mortem cerebrospinal fluid culture. The parasite density was 1 in 500 white blood cells (16/µL). This suggests that the parasitemia was incidental to the fatal bacterial meningitis. Two other P. vivax deaths occurred in patients with low parasite densities (<3300/µL), leukocytosis, and a clinical diagnosis of sepsis, although blood cultures were negative. Both patients were anemic and required blood transfusions. The fourth P. vivax malaria death occurred in a malnourished woman (HIV and tuberculosis negative) in the third trimester of pregnancy ) met the WHO criteria for severe malaria  of those with P. falciparum malaria  (P. vivax cases. Pulmonary edema was a more common manifestation of severity in P. vivax (27 of 68 [40%]) than in P. falciparum malaria (167 of 1482 [11%]) . Severe anemia was also more common among the patients with severe vivax malaria (18 of 68 [26%]) than among those with severe falciparum malaria (167 of 1482 [11%])   . ImpaireP = .01) . The promalaria. .P. vivax (63 vs 67 of 278) and P. falciparum cases (1463 vs 1482 of 4844) classified as severe malaria were largely unchanged (P. vivax cases (68 of 80 841) compared with 15.7/1000 cases (14.9–16.5) P. falciparum cases (1482 of 94 467). Thus, for outpatients presenting with malaria, the risk of severe malaria was 19  times greater in P. falciparum than in P. vivax infections (P < .001).Applying the stricter research criteria for severe malaria , the pronchanged . Based oP. vivax malaria was very common along the Thailand-Myanmar border, severe vivax malaria was rare, as it is elsewhere in the Greater Mekong subregion. Nearly all malaria deaths in this region result from P. falciparum. The relatively low mortality rate of severe falciparum malaria in this series (4.4%) is explained by the high proportion of cases with prostration or otherwise uncomplicated hyperparasitemia, both of which carry a relatively good prognosis, and prompt treatment with artesunate [P. vivax malaria [P. vivax and P. falciparum is generally low and unstable, as it is in the Greater Mekong subregion [Although tesunate , 29. Our malaria . In Indiubregion , 22.P. vivax malaria has been reported extensively [P. vivax infections had severe malaria [P. falciparum malaria in our study and considerably exceeds the proportion observed for P. vivax. The case-specific mortality rate of acute vivax malaria on the Thailand-Myanmar border is >100 times lower than reported from Bikaner in Northwest India, 6 times lower than reported from Manaus in Brazil [P. vivax cases in India, suggesting greater P. vivax virulence there, or unusual susceptibility, or whether there is an ascertainment bias in the diagnoses. In contrast, the island of New Guinea has markedly higher P. vivax transmission than in other malaria-endemic areas of the world. Young children are affected particularly, and frequent P. falciparum and multiple relapsing P. vivax infections result in severe anemia and an increased risk of death [Symptomatic malaria occurs at all ages, and severe ensively . A 2021  malaria . This isn Brazil , and haln Brazil   did not have severe malaria, and their prognosis was very good. Pregnancy and the postpartum period were particular risk factors for P. vivax–associated severe anemia hospitalizations. This reflects both the cumulative impact of recurrent P. vivax malaria and the higher risk of anemia in this population generally [P. falciparum malaria) both have a relatively good prognosis, provided that there is ready access to diagnosis and treatment (with artesunate) and that blood transfusions can be given [P. vivax malaria , all but 1 patient survived [Although some patients with acute enerally . Severe be given , 29, 36.survived , 29, 37.P. vivax malaria” reporting differences between malaria regions. These relate to the criteria used, definitions applied, and use of proxy indicators to determine severity. For example, severe thrombocytopenia is often used as a severity criterion and accounts for a large proportion of reported “severe vivax” cases [P. vivax case patients have severe anemia , which iagnosed . Other fP. vivax recurrence rates, especially when anti-relapse treatment is not given, and it thereby increases the prevalence and severity of anemia. Chloroquine resistance may be a contributor to reported severe P. vivax malaria in India, although the levels of resistance reported to date in South Asia and the Greater Mekong subregion are low [Antimalarial resistance increases  are low .P. vivax and other common infections.This study had several limitations. This was a retrospective evaluation, so procedures varied in time and place, and missing inpatient records were not included and outcomes were sometimes not known for patients referred to tertiary hospitals. Full blood cell counts were routinely available only in the latter half of the study period. Results of biochemical investigations were not available. Pulmonary edema could not be confirmed with chest radiography, and pulse oximetry was not routinely available. Investigations to exclude alternative diagnoses limited our ability to differentiate causation between severe P. vivax malaria is common, but severe infections and death resulting from P. vivax are rare. Further investigation is needed to determine why there are differences in the apparent proportions of severe P. vivax malaria across different geographic regions, transmission settings, and relapse intervals.In conclusion, in northwest Thailand along the Myanmar border, Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.ciad262_Supplementary_DataClick here for additional data file.


The efficacy and effectiveness of antimalarial drugs are threatened by increasing levels of resistance and therefore require continuous monitoring. Chemoprevention is increasingly deployed as a malaria control measure, but there are no generally accepted methods of assessment. We propose a simple method of grading the parasitological response to chemoprevention  that is based on pharmacometric assessment. Seasonal malaria chemoprevention (SMC) is the administration of treatment doses of antimalarial drugs to children at monthly intervals to suppress malaria infections in areas of highly seasonal transmission. It is therefore a form of antimalarial chemoprophylaxis. For the most part, SMC (formerly known as intermittent preventive treatment in children [IPTc]) has used a combination of three drugs, sulphadoxine–pyrimethamine with amodiaquine (SPAQ), in children 3–59 months of age. SMC has been deployed across the Sahel region of Africa, a belt of intense seasonal malaria transmission. SPAQ is given monthly for 3–5 months . The excMeanwhile, SMC deployment has been scaled up and now has spread across Africa. In 2022 the WHO broadened substantially their original recommendations for SMC (which were confined to the Sahel) to other geographic regions in Africa (where drug resistance is worse) and removed restrictions on the number of monthly cycles or age , 5. SimiFunding to support malaria control is not unlimited. Clearly it is important for national control programs, agencies and donors to know that the preventive interventions they are providing do work and that they are not supporting interventions that are ineffective. Continued effective suppression of malaria by SMC justifies the considerable expense and effort. But if the current chemoprevention interventions start to lose their effectiveness, this should be recognized quickly and alternative approaches should be deployed. Historically, the use of failing or useless antimalarial drugs has continued for many years and has resulted in substantial human and financial costs . This shSMC differs fundamentally from the treatment of clinical malaria. In SMC, treatment doses of antimalarials are given to healthy children. Many of these children are infected with malaria parasites, but at densities in the blood that do not make them ill. In most cases they have already controlled their malaria infections. As a result, when they take an antimalarial drug, the therapeutic response is substantially better than in a symptomatic infection, which, by definition, has not been controlled by the host and is usually associated with parasite numbers that are orders of magnitude higher , 8. NevePharmacometric antimalarial resistance monitoring (PARM) describes a simple method of assessing chemopreventive antimalarial drug efficacy . It reliWe propose a simple grading system to interpret the results of PARM and thus assess SMC effectiveness Figure . This syThe proposed grading depends primarily on the prevalence of filter paper–based qPCR-estimated parasitaemia 28 d after starting SMC. This does not discriminate between asexual and sexual parasitaemia. This value is interpreted in relation to simultaneously measured drug levels. Finding recurrent parasitaemia in the presence of expected drug concentrations suggests resistance. Detection of gametocytaemia by microscopy informs the estimation of drug resistance selection pressures and transmissibility, but it is not used in the proposed grading.Even with fully sensitive malaria parasites and highly effective antimalarial medicines there are always some subjects who have low drug exposures  . An effeAs chemoprevention begins to fail (either because of drug resistance or low drug exposures), the proportion of day 28 detectable infections increases to 5–10%. Gametocytaemia is present in a few subjects but gametocyte densities are below the level of microscopy detection and unlikely to transmit. Suppression of further asexual-stage multiplication by the next chemoprevention dose prevents generation of transmissible gametocyte densities.The proportion of day 28 detectable infections is 10–20% and some subjects do have microscopy-detectable gametocytaemia at day 28, which is presumably transmissible. This is evidence of the selective pressure on drug resistance. Nearly all recurrent infections are new infections.The proportion of day 28 detectable infections is 20–30% and many of these breakthrough infections now have microscopy-detectable gametocytaemia. Some of the breakthrough infections may be associated with illness. The majority of recurrent infections are still new infections.The proportion of day 28 detectable infections now exceeds 30% and many of these infections have microscopy-detectable gametocytaemia. Some recurrences are recrudescences, but most recurrent infections are new infections and many are symptomatic.Chemoprevention is completely ineffective. The proportion of day 28 subjects who are parasitaemic is similar to that at baseline and there is little or no reduction in parasite prevalence or densities during the 28 d.This proposed approach to the assessment of malaria chemoprevention, based on basic pharmacometric principles , is desiSMC has been an excellent malaria control intervention across the Sahel and has provided substantial benefit in reducing childhood malaria . SPAQ isSMC and PMC should only be deployed with drugs that are effective in areas where there is high transmission and a high burden of illness. In this context, a substantial proportion of the target population is parasitaemic (by qPCR) at the beginning of the malaria transmission season. Obviously, if only a minority of subjects are parasitaemic before SMC is deployed and transmission is relatively low or the study is done at the wrong time in relation to the transmission season, then there will be few breakthrough infections at day 28. It could thus be concluded incorrectly that SMC was effective. Measuring the baseline prevalence addresses this question, although in some areas with intensely seasonal transmission the first round of SMC might be given just before the rapid increase in malaria prevalence.In an attempt to justify continued use of the failing antimalarial drug chloroquine, it was once argued that control of parasitaemia was sufficient to justify antimalarial drug deployment and it was suggested that clinical benefits could be dissociated from parasitological responses in malaria . This is


Plasmodium falciparum. Candidates were validated in plasma samples from a cohort of pediatric patients with malaria from Mozambique, resulting in the identification of several markers with capacity to distinguish uncomplicated from severe malaria, the most potent being the metallopeptidase ADAMTS18. Two other biomarkers, Angiopoietin-like-4 and Inhibin-βE were able to differentiate children with cerebral malaria within the severe malaria group, showing increased sensitivity after combination in a biomarker signature. The validation of the predicted candidate biomarkers in plasma of children with severe and cerebral malaria underscores the power of this transcriptomic approach and indicates that a specific endothelial response to P. falciparum–infected erythrocytes is linked to the pathophysiology of severe malaria. Malaria can quickly progress from an uncomplicated infection into a life-threatening severe disease. However, the unspecificity of early symptoms often makes it difficult to identify patients at high risk of developing severe disease. Additionally, one of the most feared malaria complications — cerebral malaria — is challenging to diagnose, often resulting in treatment delays that can lead to adverse outcomes. To identify candidate biomarkers for the prognosis and/or diagnosis of severe and cerebral malaria, we have analyzed the transcriptomic response of human brain microvascular endothelial cells to erythrocytes infected with   Plasmodium falciparum, uncomplicated malaria (UM) may rapidly progress into severe malaria (SM), which is associated with different complications, including cerebral malaria (CM). This is one of its most feared complications, with a high risk of death (18%–30%), and is also associated with long-term cognitive and neurological deficits in up to 30%–50% of survivors , the protein commonly used in RDTs, have evidenced high percentages of false-negative P. falciparum diagnoses in some endemic areas  , resulted in the identification of 3 potentially novel candidate biomarkers for pediatric SM: ADAM metallopeptidase with thrombospondin type 1 motif 18 (ADAMTS18), Angiopoietin-like-4 (ANGPTL4), and Inhibin-βE (INHBE). Furthermore, 2 of them, ANGPTL4 and INHBE, are also able to significantly differentiate between pediatric CM and noncerebral SM.Here we have used this transcriptomic (RNA-Seq) analysis of human brain microvascular endothelial cells (HBMEC)  incubates (iRBC) , a systeP. falciparum, but not by inflammatory stimuli, that encode putative secretory proteins. After confirmation that the candidate proteins are secreted in response to P. falciparum in vitro, they were validated in a pediatric cohort of malaria patients with different degrees of severity , compared with control RBCL  (https://doi.org/10.1172/jci.insight.172845DS1).We have analyzed the transcriptome of HBMEC incubated with rol RBCL  to identrol RBCL , 25 and  < 0.05)   by iRBCL compared with TNF, a potent inflammatory stimulus that is highly induced during SM and was also included in the transcriptomic analysis  Supplem. Finally2 fold change = 0.48) (2 fold change = –0.49). We therefore investigated the secretory profile of Angiopoietin-2 in HBMEC to determine whether this marker is secreted in response to inflammatory stimuli, as previously described  , but it  = 0.48) , since iescribed , and/or um–iRBCL , and thiP. falciparum–iRBCL. For this purpose, we determined the levels of each candidate protein in the culture media between 1 and 24 hours after incubation of HBMEC with P. falciparum–iRBCL, using uninfected RBCL as a negative control. Out of the 14 candidates, 7 proteins were confirmed to be secreted by HBMEC in response to P. falciparum–iRBCL at the protein level (We next tested whether the proteins encoded by the selected candidate biomarker genes  are indein level . Seven cin level .n = 136) or diagnosed with UM and treated as outpatients (n = 128). Patients with SM include children with different complications (frequently more than 1 complication in a single patient): CM (n = 23), prostration (n = 96), multiple seizures (n = 60), severe anemia (n = 35), hypoglycemia (n = 10), or acidosis (n = 53). When children with CM presented more than 1 complication, they were classified in the CM group  of age admitted to Manhiça District Hospital in southern Mozambique with SM  for ADAMTS18; 0.68 (P < 0.0001) for ANGPTL4; and 0.63 (P = 0.0004) for INHBE. Moreover, we observed a significant correlation between ADAMTS18 and ANGPTL4 levels across the cohort . To a lesser extent, INHBE levels were also correlated with these biomarkers . As expected because of the correlation between them, the combination of these candidates into a single biomarker signature did not improve AUC.The levels of these candidate biomarkers were then tested in the entire cohort to compare the groups with UM versus SM. We observed significant differences in the levels of ADAMTS18, ANGPTL4, and INHBE between UM and SM , with anP. falciparum biomass, measured as levels of HRP-2, since malaria severity is strongly associated with this parameter , while ADAMTS18 had a weaker correlation , while INHBE was not significantly correlated. No correlation was found between any of the candidate biomarkers and circulating parasitemia determined by quantitative PCR (qPCR).We also determined the relation of the candidate biomarkers to arameter . We obseum–iRBCL , correlaWe also analyzed the relation of the candidate biomarkers with other malaria complications, finding a significant association of the 3 candidate biomarkers with neurological complications such as CM and prostration. ADAMTS18 and ANGPTL4 were also correlated with acidosis and severe anemia. Jaundice was only associated with ANGPTL4, and multiple seizures was associated with ADAMTS18 and INHBE .P < 0.005) and 0.70 (P < 0.002), respectively (P = 0.0574). Analysis of the correlation between the levels of ANGPTL4 and INHBE showed that these 2 candidate biomarkers were not significantly correlated among SM cases ; combining both biomarkers in a logistic regression model  resulted in a good model fit ; and using a LLR test showed that the ANGPTL4 + INHBE model was significantly better than the individual models . Collectively, these observations indicate that INHBE and ANGPTL4 are uncorrelated biomarkers and could serve as independent predictors, increasing the power when combined in a biomarker signature. A combined analysis of these 2 biomarkers selecting the highest relative value of each one (P < 0.0001).We next determined whether the candidate biomarkers could differentiate between children with CM within the group with SM. Further analysis of the 2 candidate biomarkers that were significantly associated with CM, ANGPTL4 and INHBE, showed an AUC of 0.68 , since the rupture and release of densely packed iRBC contents in close contact with endothelial cells is sufficient to cause loss of barrier integrity  transcription was upregulated in HBMEC by ignaling , 35.The previously characterized biomarkers for SM reach similar levels of sensitivity and specificity as the ones reported here, with Angiopoietin-2  at 5% hematocrit in RPMI 1640 (Corning) supplemented with 25 mM HEPES (Thermo Fisher Scientific), 25 mM sodium bicarbonate (MilliporeSigma), 0.5 mM hypoxanthine (MilliporeSigma), 0.5% Albumax II (Thermo Fisher Scientific), and 10 mg/mL gentamicin (Thermo Fisher Scientific) at 37°C in a gas mixture of 5% O2, 5% CO2, and 90% N2. Parasite cultures were synchronized using 5% sorbitol (MilliporeSigma). P. falciparum–iRBCs at the schizont stage were isolated from highly synchronous cultures using magnetic columns . Uninfected RBC lysates (RBCLs) or iRBCLs were generated by 10 freeze-thaw cycles using liquid nitrogen and a 37°C water bath.6 to 8 × 106 iRBCs/cm2, where 8 × 106 iRBCs/cm2 was estimated to be equivalent to 2 layers of iRBCs over the HBMEC monolayer .n = 120) and 2014 (n = 148). Samples were collected from children < 10 years of age diagnosed with clinical malaria , either outpatient diagnosed with UM (no further complications) or admitted to Manhiça District Hospital for SM. Cases of SM were defined as patients with a clinical diagnosis of malaria and an asexual P. falciparum parasitemia > 0 parasites/μL by microscopic examination of Giemsa-stained blood smears, and fulfilling at least 1 of the following criteria: CM (Blantyre Coma Score ≤ 2), severe anemia (packed cell volume < 15% or hemoglobin < 5 g/dL), acute respiratory distress (chest indrawing and/or deep breathing), acidosis , jaundice (bilirubin > 60 μmol/L), prostration (inability to sit or breastfeed in children old enough to do so), hypoglycemia (blood glucose < 2.2 mM), and multiple seizures (≥ 2 convulsions in the preceding 24 hours). Children with positive bacteremia were excluded. Children with SM were treated according to Mozambican national guidelines with parenteral quinine in 2006 or parenteral artesunate  in 2014, and those with UM were treated with a combination of oral amodiaquine and sulfadoxine-pyrimethamine  in 2006 or with artemether-lumefantrine  in 2014. Quantification of parasitemia was also performed by qPCR targeting the P. falciparum 18S rRNA gene  was compared between both studies, showing differences < 7% of the maximum value between both studies.Heparinized blood (10 mL) from study participants was collected and processed within 2 hours. After centrifugation at 200Power transformation on the values was performed. Based on this, the maximum value for each biomarker was selected for each patient and was used to calculate the signature. Analysis using Support Vector Classifiers, Logistic regression, and TabPFN classifier  methods P < 0.05 was considered significant. Differences in continuous variables were analyzed using the Mann-Whitney U test with Bonferonni correction to adjust for multiple testing. Differences in categorical variables were assessed using Fisher’s exact test or Pearson’s χ2 test, as appropriate.Statistical analyses were performed on Prism 9 (GraphPad Software). The Shapiro-Wilk test was used to test whether the data followed normal distribution. The specific statistical tests used are detailed in the figure legends. The exact statistical tests used are detailed in the figure legends, and Written informed consent was obtained from parents or guardians of study participants prior to participation. Ethical approval was granted by The National Mozambican Ethical Review Committee (Mozambique) and Hospital Clínic .The RNA-Seq data used in this manuscript were described before  and are CG, AR, MDF, and RV designed research studies; CG conducted experiments; AS, RB, and SM collected patient samples; RV, QB, and AM designed patient studies; CG, MDF, and AR analyzed data; and AR, CG, RV, MDF, and QB wrote the manuscript.


We investigated differences in biomarker levels, associations with mortality, blood–brain barrier integrity, neurodeficits and cognitive Z-scores in survivors up to 24-month follow-up. Admission ubiquitin C-terminal hydrolase-L1 levels were elevated >95th percentile of community children in 71 and 51%, and neurofilament-light chain levels were elevated >95th percentile of community children in 40 and 37% of children with cerebral malaria and severe malarial anaemia, respectively. Glial fibrillary acidic protein was not elevated in disease groups compared with controls. In cerebral malaria, elevated neurofilament-light chain was observed in 16 children who died in hospital compared with 166 survivors (P = 0.01); elevations in ubiquitin C-terminal hydrolase-L1 levels were associated with degree of blood–brain barrier disruption (P = 0.01); and the % predictive value for neurodeficits over follow-up  increased for ubiquitin C-terminal hydrolase-L1 , but not neurofilament-light chain . In cerebral malaria, elevated ubiquitin C-terminal hydrolase-L1 was associated with worse memory scores in children <5 years at malaria episode who crossed to over 5 years old during follow-up cognitive testing , and elevated neurofilament-light chain was associated with worse attention in children ≥5 years at malaria episode and cognitive testing . In severe malarial anaemia, elevated ubiquitin C-terminal hydrolase-L1 was associated with worse attention in children <5 years at malaria episode and cognitive testing . Ubiquitin C-terminal hydrolase-L1 and neurofilament-light chain levels are elevated in paediatric cerebral malaria and severe malarial anaemia. In cerebral malaria, elevated neurofilament-light chain is associated with mortality whereas elevated ubiquitin C-terminal hydrolase-L1 is associated with blood–brain barrier dysfunction and neurodeficits over follow-up. In cerebral malaria, both markers are associated with worse cognition, while in severe malarial anaemia, only ubiquitin C-terminal hydrolase-L1 is associated with worse cognition.Persistent neurodisability is a known complication in paediatric survivors of cerebral malaria and severe malarial anaemia. Tau, ubiquitin C-terminal hydrolase-L1, neurofilament-light chain, and glial fibrillary acidic protein have proven utility as biomarkers that predict adverse neurologic outcomes in adult and paediatric disorders. In paediatric severe malaria, elevated tau is associated with mortality and neurocognitive complications. We aimed to investigate whether a multi-analyte panel including ubiquitin C-terminal hydrolase-L1, neurofilament-light chain, and glial fibrillary acidic protein can serve as biomarkers of brain injury associated with mortality and neurodisability in cerebral malaria and severe malarial anaemia. In a prospective cohort study of Ugandan children, 18 months to 12 years of age with cerebral malaria  and neurofilament-light chain (NF-L) in paediatric severe malaria. In cerebral malaria, NF-L is associated with mortality; UCH-L1 is associated with blood–brain barrier dysfunction and neurologic deficits over follow-up. Both markers are associated with worse cognition in cerebral malaria; UCH-L1 is associated with worse cognition in severe malarial anaemia.Datta   Severe complications leading to adverse outcomes in this multi-system disease include coma, severe anaemia, metabolic dysfunction including lactic acidosis and hypoglycaemia, coagulopathy, and acute kidney injury (AKI).6-8 Less is known about the role of brain injury biomarkers that reflect CNS damage and can differentiate between neuronal, axonal, or astroglial injury,10 although these biomarkers have been associated with adverse neurologic outcomes in paediatric disorders such as traumatic brain injury and neonatal encephalopathy.12 In severe malaria, elevated CSF and plasma levels of axonal injury marker tau have proven utility in predicting systemic complications, mortality, and cognitive impairment, though associations with worse cognition scores were observed only in cerebral malaria and not severe malarial anaemia, limiting its usefulness as a standalone biomarker in severe malaria.14 A multi-analyte panel of injury biomarkers serving different functional roles in the CNS may mitigate the limitation of a single biomarker for predicting adverse outcomes. Further, as with tau, using ultrasensitive single-molecule detection assays for quantification of low abundance CNS proteins in blood circulation can circumvent the need for invasive lumbar punctures for CSF biomarker detection in paediatric severe malaria.15Biomarkers of systemic inflammation, endothelial activation, and organ dysfunction have been identified as risk factors for mortality and cognitive impairment in severe malaria.16 NF-L is a 68-kDA microstructural protein that provides a stabilizing framework for neuronal axons to support nerve conduction and contributes towards the regeneration of damaged axons.17 GFAP (50 kDa) is a component of cytoskeletal filaments of astrocytes that are upregulated during astrocyte activation in response to neurotrauma.18 These biomarkers are in different stages of clinical development for traumatic brain injury,19 have been associated with adverse neurologic outcomes in stroke,21 and are useful in predicting cognitive impairment in adults.22-24 Recently, UCH-L1 and GFAP were approved by the U.S. FDA as point-of-care plasma markers to rule out the need for CT scans after mild traumatic brain injury in adults.25 There are, however, few studies of these markers as diagnostic or prognostic tools in paediatric traumatic brain injury26 and none in paediatric severe malaria. Children at risk of CNS injury, particularly in resource-constrained malaria-endemic countries with limited access to advanced brain imaging technologies, may benefit from access to point-of-care brain biomarker tests to identify those at risk of future cognitive impairment. A critical first step is to determine if these biomarkers are useful predictors of complications associated with CNS injury in these at-risk paediatric populations.Beyond tau, biomarkers that have proven utility as markers of acute CNS injury include ubiquitin C-terminal hydrolase-L1 (UCH-L1), neurofilament-light chain (NF-L), and glial fibrillary acidic protein (GFAP). UCH-L1, the most abundant protein in the CNS, is a 25-kDa protein expressed by neurons and involved in tagging proteins for ubiquitination or degradation in response to cellular damage.Z-scores over a 24-month follow-up. We hypothesized that plasma UCH-L1, NF-L, and GFAP levels would be elevated in children with cerebral malaria or severe malarial anaemia compared with asymptomatic community children and that the elevated levels of these biomarkers would be associated with mortality, neurologic deficits, and worse cognitive Z-scores long-term in survivors of severe malaria.To evaluate whether plasma levels of UCH-L1, NF-L, or GFAP are altered in severe malaria and associated with adverse outcomes, we measured these biomarkers in a cohort of children admitted with cerebral malaria or severe malarial anaemia, compared with asymptomatic community children, using an ultrasensitive single-molecule detection assay for measurement. We then correlated biomarker levels to BBB impairment and mortality during admission, neurologic deficits at discharge and follow-up, and cognitive outcome Plasmodium falciparum on blood smear, coma (Blantyre Coma Score <3 or Glasgow Coma Score <8), and no evidence of other known causes of coma . Severe malarial anaemia was defined as P. falciparum on blood smear and haemoglobin ≤ 5 g/dL. Children were managed according to the Ugandan Ministry of Health treatment guidelines at the time of the study. Additional sociodemographic information is as published.13The study was performed at Mulago National Referral Hospital in Kampala, Uganda from 2008 to 2015. Children were eligible if they were between 18 months and 12 years of age with cerebral malaria or severe malarial anaemia. A reference group of community children of the same age range and with no active illness were recruited from the extended family or household compounds of children with severe malaria. Cerebral malaria was defined as Written informed consent was obtained from the parents/guardians of study participants. Ethical approval was granted by the Institutional Review Boards at Makerere University School of Medicine (2008-033) and the University of Minnesota (0808M27022). The study was also approved by the Uganda National Council for Science and Technology (HS432). The study was conducted following the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelines for observational studies.13 HIV testing was performed according to the Uganda National HIV testing algorithm after obtaining consent from the parent or guardian. As part of routine testing in cerebral malaria, a lumbar puncture to collect CSF was conducted to rule out other known causes of coma in children who did not have a contraindication to a lumbar puncture and whose parents did not decline consent. Albumin was measured in CSF and plasma using a Sigma-Aldrich Bromocresol Purple Albumin assay . BBB integrity was assessed by calculating the CSF to plasma albumin index categorized by degree of disruption.27 An index value < 9 was considered consistent with an intact barrier. Values between 9 and 14 were interpreted as slight impairment, 14 and 30 as moderate impairment, and 30 and 100 as severe impairment, with moderate and severe impairment combined in this analysis.The study was conducted with samples collected at hospital admission for cerebral malaria and severe malarial anaemia and at enrolment for community children. Samples were processed and ethylenediaminetetraacetic acid (EDTA) plasma was stored at −80°C using standardized laboratory procedures, until testing. A complete blood count to enumerate haemoglobin and platelets was performed using a Beckman Coulter ACT 5 diff haematology analyzer . Biochemistries were performed on cryopreserved plasma samples for glucose, lactate, sodium, lactate dehydrogenase (LDH), creatinine, and blood urea nitrogen (BUN) using Roche Cobas Integra 400 plus Chemistry analyzer by the Advanced Research & Diagnostic Laboratory at the University of Minnesota. Creatinine was used to define the degree of AKI as previously described.Z-scores were created using scores of the Ugandan community children included in the study, and the age-range-specific cognitive assessments have been validated and used in previously published studies with Ugandan children.28Testing for gross neurologic deficits  in children with cerebral malaria, and for cognitive outcomes in children with cerebral malaria or severe malarial anaemia were performed at discharge (neurologic testing) or 1-week after discharge (cognitive testing), and at 6-, 12-, and 24-month follow-up. Neurologic testing was not done for children with severe malarial anaemia or community children. Community children underwent cognitive testing at enrolment and follow-up. Cognitive testing for children younger than 5 years included Mullen Scales of Early Learning for overall cognition, Early Childhood Vigilance Test for attention, and Color Object Association Test for associative memory, while cognitive tests for children 5 years or older included Kaufman Assessment Battery for Children  with summary mental processing index for overall cognitive ability, the Test of Variables of Attention with D prime measure for attention, and the KABC-2 subtest for sequential processing subtest for working memory. Different cognitive testing batteries were used for children younger than 5 years and those 5 years or older; therefore, results are presented for (i) children younger than 5 years at malaria episode and follow-up malaria testing, (ii) children younger than 5 years at malaria episode who crossed to 5 years or older at follow-up testing, and (iii) children 5 years or older at time of malaria episode and follow-up testing. Age-adjusted 15 Measurement of plasma total tau, included in this study, to show the correlation between biomarkers, was similarly conducted using the HD-1 analyzer.13 Samples with values for all three biomarkers were included in this study. Samples were tested blinded to participant details and 10% of samples were run in duplicate, and for these, mean biomarker values were used for analysis. Elevated UCH-L1, NF-L, and GFAP levels were established using levels >95th percentile of community children for each marker as a reference range.Plasma UCH-L1, NF-L, and GFAP levels were measured on samples with sufficient volume for testing between August and September 2018 using the single-molecule array (Simoa) HD-1 analyzer , with a lower limit of detection at 1.74, 0.104, and 0.221 pg/mL for UCH-L1, NF-L, and GFAP, respectively.χ2 test. The a priori level of significance was 0.05 with two-tailed hypothesis tests. Spearman’s rank analysis was used to evaluate the correlation between CNS markers. The degree of BBB disruption was analysed using Cuzick's rank-based non-parametric test of trend (NP-trend).29 Logistic or linear regression, adjusting for age and sex, was used to evaluate associations of CNS marker levels with dichotomous or continuous outcomes, respectively. Continuous variables not normally distributed were log10-transformed for inclusion in regression models. To evaluate the discriminatory ability of CNS markers to predict neurologic deficits over time, non-parametric receiver operating characteristic (ROC) curve analysis was used and the area under the curve (AUC) was compared. Cognition was calculated as continuous Z-scores for three independent outcomes (one primary and two secondary) and analysed in a linear mixed effect (LME)14 model adjusted for variability in scores over time and using age-adjusted Z-scores from community children as the baseline to standardize cognitive scores for children with severe malaria. Evaluations for each cognitive outcome were not adjusted for multiple comparisons because the LMEs model incorporated scores from all time-points as a single outcome. Correction for multiple comparisons of biomarkers levels at four time-points without and with neurologic deficits, and of analysis of association with clinical laboratory factors was conducted using the Benjamini–Hochberg procedure.Analyses were done using Stata SE v17.0 . Differences in continuous measures were evaluated using Wilcoxon rank-sum tests or Kruskal–Wallis tests, where appropriate. Differences in categorical variables were measured using Pearson’s 13 Children with severe malarial anaemia were younger than children with cerebral malaria and community children, and the percentage of females with cerebral malaria, severe malarial anaemia, or community children was 41, 36, and 53, respectively  and 118 community children with sufficient plasma for testing. Children included were representative of the full cohort and baseline characteristics of children assessed for these biomarkers including a participant flow diagram, are as published.ectively . A smallectively . Clinicaectively . Most cln = 130) and 51% (n = 81) of children with cerebral malaria and severe malarial anaemia, respectively , and plasma NF-L levels were elevated in 40% (n = 73) and 37% (n = 58) of children with cerebral malaria and severe malarial anaemia, respectively  (Based on the 95th percentile of community children as the cut-off for plasma UCH-L1 (43.5 pg/mL), NF-L (7.5 pg/mL), and GFAP (284.5 pg/mL), UCH-L1 levels were elevated in 71% (< 0.001) . Median < 0.001) . Consequ30 we investigated biomarker levels in children <5 versus ≥5 years of age and in female versus male children for each study group. UCH-L1 levels were significantly elevated in children <5 years of age with cerebral malaria or severe malarial anaemia and in community children compared with children ≥5 years of age  and community children (P < 0.001) but not in children with severe malarial anaemia (P = 0.10) relative to their older counterparts (P = 0.02) and community children (P = 0.004) but not severe malarial anaemia  except GFAP and tau . Among community children, correlations were observed between UCH-L1 and tau and between NF-L and GFAP  11.12 ] compared with survivors .In 16 children with cerebral malaria who died, UCH-L1 levels were not significantly elevated [median pg/mL interquartile range (IQR) 93.5 ] compared with levels in 166 survivors , P = 0.02}. Further, UCH-L1 was associated with worse attention Z-scores over 24-month follow-up in 125 children with severe malarial anaemia who were <5 years of age at severe malarial anaemia episode and follow-up testing [β −0.42 , P = 0.02; Z-scores over 24-month follow-up in children with cerebral malaria who were ≥5 years during severe malaria episodes and testing [β −1.08 , P = 0.03; After adjusting for age and sex, admission UCH-L1 levels were associated with worse z-scores for working memory over 24-month follow-up in 65 children who were <5 years of age at cerebral malaria episode but ≥5 years of age at follow-up testing {P ≤ 0.02). NF-L levels were associated with fewer risk factors primarily in cerebral malaria, namely, elevated LDH and BUN, and AKI. In severe malarial anaemia, AKI was the only risk factor associated with NF-L  among CNS proteins,16 has been shown to complement the axon microtubule stabilization function of tau as inhibition of UCH-L1 can decrease the microtubule-binding ability of tau and promote the accumulation of abnormal aggregates of hyperphosphorylated tau,31 a critical mediator implicated in cognitive impairment in neurodegenerative disorders.32Complementary to the findings of elevated axonal microtubule marker tau,5 elevated tau was associated with worse cognition scores in cerebral malaria alone and not in severe malarial anaemia, suggesting mechanisms other than tau-mediated axonal damage are implicated in persisting cognitive impairment in severe malarial anaemia.13 This study provides evidence that UCH-L1-mediated brain injury may reflect a pathway leading to impaired cognitive functioning in young children with cerebral malaria or severe malarial anaemia, although our data warrants further investigation in a larger cohort with more children per age group. In addition, elevated NF-L was associated with worse attention scores over follow-up in children with cerebral malaria ≥5 years of age at the time of severe malaria episodes. In adults with traumatic brain injury, NF-L has been found to increase with age.33 But in this study, all biomarkers were higher in children <5 years of age compared with those ≥5 years in disease and control groups, which likely reflects age-related structural changes rather than differences in the extent of injury. Additional studies are needed to elucidate the age-related contributions of CNS markers in paediatric brain injury and associated cognitive impairment. Although GFAP was not elevated in severe malaria compared with controls and therefore excluded from additional analysis in this study, it has been implicated as a marker of impaired cognition alongside UCH-L1 in traumatic brain injury.22 The high GFAP levels observed in our control populations compared with other paediatric studies may be attributed to extracerebral production as a source of GFAP in our population, or to the varying degree to which GFAP signal is detectable in the different assays used.34 As few studies have concurrently examined the role of a panel of brain injury biomarkers on persisting cognitive impairment in paediatric disorders, our findings make a strong case for further exploration of multi-analyte panels of CNS biomarkers associated with cognitive impairment including in paediatric severe malaria.A novel finding of this study is that elevated admission plasma UCH-L1 levels predicted neurologic deficits at 24-month follow-up and were associated with worse associative memory scores over 24-month follow-up in children <5 years with cerebral malaria. In addition, UCH-L1 was the only CNS marker to show an association with worse attention scores in severe malarial anaemia. We noted previously that although cognitive impairment has been observed in severe malarial anaemia,9 However, a more likely explanation is physiological clearance by the glymphatic system wherein fluids and small molecules are shuttled across the CNS via aquaporin-4 water channels on glial cells.35 Furthermore, the lack of association of NF-L with a degree of BBB disruption in cerebral malaria despite being significantly elevated over levels in controls suggests that BBB dysfunction is not the only mechanism underlying brain injury in severe malaria. Indeed, the validity of NF-L as a neuronal marker whose levels in blood circulation are not influenced by BBB permeability has been observed in a mouse model of brain injury.36 Nevertheless, elevations in the highly abundant CNS protein UCH-L1 trended positively with the degree of BBB disruption as determined by increased albumin in the CSF, which was observed in 33% of the children with cerebral malaria in this cohort. BBB dysfunction is a key component in the pathogenesis of cerebral malaria37 and a hallmark of lasting damage and neurodegeneration after traumatic brain injury.38 The findings of this study reflect the need for further investigations on the different mechanisms by which falciparum malaria triggers brain injury using comprehensive in vitro 3D models of the neurovascular unit, experimental animal models, and functional brain imaging studies in humans.Detectable plasma levels of UCH-L1, NF-L, and GFAP in disease-free community children suggest ongoing blood circulation at basal levels in the absence of brain injury and BBB disruption, possibly from extracerebral sources, including oocytes and spermatogonia for UCH-L1, peripheral nerves for NF-L, and osteocytes, Leydig cells, and chondrocytes for GFAP.39-41 and may increase the risk of cognitive impairment after brain injury in survivors of severe malaria. Impaired kidney function may result in the inability to eliminate biomarkers from blood circulation.30 Hypoglycaemia is a life-threatening complication of severe malaria that is reversed by the administration of glucose in hospitalized children to prevent mortality. However, in CNS injury, increased glucose utilization by the injured brain may result in a need for glucose at a threshold higher than the current level of 2.2 mmol/L for managing hypoglycaemia in severe malaria, to prevent cognitive impairment.43 The findings of this study support further evaluation of whether prevention or early treatment of AKI or the use of a higher threshold for hypoglycemia44 may decrease brain injury and prevent long-term cognitive impairment in children with cerebral malaria or severe malarial anaemia.Our exploratory analysis of the relationships between CNS markers (UCH-L1 and NF-L) and clinical laboratory indicators of severe complications revealed important insights into the clinicopathogenesis of severe malaria. For example, elevated levels of UCH-L1 and NF-L were associated with hypoglycaemia (glucose <2.2 mmol/L), cellular injury , and kidney dysfunction . These complications are known to exacerbate the primary insult in traumatic brain injury45 Another study found that NF-L may be a useful marker for chronic traumatic brain injury detectable up to 180 days.46 Thus, the design of future prospective severe malaria studies should factor in multiple sampling time points during hospitalization and post-discharge. A recent prospective observational study was able to measure brain biomarkers pre- and post-operatively in neonates needing open-heart surgery and found GFAP and tau levels to peak at postoperative Day 2, and NF-L levels increased throughout the study period.47 Pre-injury biomarker measurements are not feasible in a study like ours where participants are seen after the onset of acute illness, further amplifying the value of a well-defined control population. Finally, brain imaging studies have elucidated key pathogenic pathways underlying severe malaria in adults and children37 and should be conducted in parallel with brain injury biomarker measurements in future cohorts when possible.Among the limitations of this study, we cannot rule out extracerebral production of UCH-L1 and NF-L in an acute inflammatory illness like severe malaria. Other confounding factors relevant to paediatric brain injury include the impact of age on circulating biomarker levels. As few studies have reported paediatric UCH-L1, NF-L, and GFAP levels and none in malaria-endemic countries, the findings of this study need to be validated in larger, multi-site paediatric studies to establish reference ranges for these biomarkers. However, the measurement of basal levels in a well-defined control population and adjustment for age mitigates concerns about non-specific elevations in the disease groups and age-related variability in biomarker levels. This study included a single admission time-point for blood sampling, collected after severely ill children were hospitalized and their condition stabilized, limiting the ability to comment on persisting elevations of these biomarkers or rule out GFAP as a relevant marker despite finding no elevation in children with severe malaria. Using the same assay for biomarker testing, a study of neonatal encephalopathy with serial sampling found that UCH-L1 levels peak between 0 and 6 h and rapidly decrease by 12 and 24 h whereas GFAP levels peak at 12, 48, and 96 h.48 While some of these preventive approaches require significant resources, it is a logical step to begin employing objective CNS biomarkers, which can be assessed using a semi-automated bench-top device, to guide the management of children with severe malaria.Malaria continues to be one of the leading causes of death and disability in Africa, disproportionately affecting children. While diagnostic and triage methods for various types of malaria  have evolved, there are no objective tools to predict children at highest risk of long-term neurologic deficits or cognitive impairment, which hinders providing timely and effective treatments or rehabilitation interventions for at-risk children. The current longitudinal study takes an important step towards reducing long-term complications due to severe malaria by analysing multimodal associations between brain injury biomarkers  detectable in systemic circulation, and clinical outcomes, such as mortality and cognitive impairment. Admission levels of UCH-L1 have proven utility as a biomarker of BBB dysfunction, neurologic deficits at 24 months, memory impairment over time in cerebral malaria, and attention deficits over time in severe malarial anaemia. NF-L showed equally valuable albeit different utility compared with UCH-L1 as a prognostic marker of death, neurological deficits at discharge, and attention impairment over time in cerebral malaria. Pending confirmation in larger, multi-site studies, these results have important implications for the clinical management of children with severe malaria. First, high levels of NF-L at admission may help prioritize interventions for children with cerebral malaria at the highest risk of death. Second, UCH-L1 and NF-L can serve as a compass to navigate who may benefit from cognitive rehabilitation, such as cognitive exercises, environmental enrichment, nutritional supplementation, physical therapy, and speech therapy.fcad323_Supplementary_DataClick here for additional data file.


In rural African settings, most of the children under the coverage of Seasonal Malaria Chemoprevention (SMC) are also undernourished at the time of SMC delivery, justifying the need for packaging malarial and nutritional interventions. This study aimed at assessing the impact of SMC by coupling the intervention with nutrients supplementation for preventing malaria in children less than 5 years old in Burkina Faso.A randomized trial was carried out between July 2020 and June 2021 in the health district of Nanoro, Burkina Faso. Children (n = 1059) under SMC coverage were randomly assigned to one of the three study arms SMC + Vitamin A  or SMC + Vitamin A + Zinc  or SMC + Vitamin A + PlumpyDoz(tm) -a medium quantity—lipid-based nutrient supplement (MQ-LNS). Children were followed up for one year that included an active follow-up period of 6 months with scheduled monthly home visits followed by 6 months passive follow-up. At each visit, capillary blood sample was collected for malaria diagnosis by rapid diagnosis test (RDT).Adding nutritional supplements to SMC had an effect on the incidence of malaria. A reduction of 23% (adjusted IRR = 0.77 (95%CI 0.61–0.97) in the odds of having uncomplicated malaria in SMC-APd arm but not with SMC-AZc arm adjusted IRR = 0.82 (95%CI 0.65–1.04) compare to control arm was observed. A reduction of 52%, adjusted IRR = 0.48 (95%CI 0.23–0.98) in the odds of having severe malaria was observed in SMC-APd arm compared to control arm. Besides the effect on malaria, this combined strategy had an effect on all-cause morbidity. More specifically, a reduction of morbidity odds of 24%, adjusted IRR = 0.76 (95%CI 0.60–0.94) in SMC-APd arm compared to control arm was observed. Unlike clinical episodes, no effect of nutrient supplementation on cross sectional asymptomatic infections was observed.Adding nutritional supplements to SMC significantly increases the impact of this intervention for preventing children from malaria and other childhood infections.Trial registration: NCT04238845. Malaria remains a life-threatening infectious disease causing about 619,000 deaths annually in the world, with children under 5 years of age accounting for about 80% of all malaria related deaths . SMC is Although SMC intervention has been shown to be very effective , 4, the Plasmodium falciparum. The baseline assessment showed a high prevalence of child malnutrition during the rainy season in the area [The study was carried out in a rural setting in the Central-western part of Burkina Faso, at the department of Soaw, one of the five departments of the health district of Nanoro. The study site included the coverage area of the health centre of Soaw  and the coverage area of the health and social promotion centre of Zoetgomdé . According to the health and demographic surveillance system (HDSS) of Nanoro, the population in 2020 was estimated at 7293 inhabitants for the village of Soaw, 2179 inhabitant for the village Rakalo, 931 inhabitants for the village of Zoetgomdé and 2262 inhabitants for the village of Kalwaka . MalariaThis was a randomized open label trial carried out between July 2020 and June 2021. The study protocol was published previously . ChildreIncluded children were randomly assigned following a computer-generated randomization list to one of the three study arms SMC + Vitamin A (SMC-A) or SMC + Vitamin A + Zinc (SMC-AZc) or SMC + Vitamin A + PlumpyDoz™, (SMC-APd) a Medium Quantity Lipid-based Nutrient Supplement. Single dose of 200000UI of Vitamin A per child was administered within 3 weeks preceding the start of SMC campaign. One sachet of PlumpyDoz™  was offered daily over a period of 3 months while 10 mg of elemental Zinc was administered daily in 6 days per week for the same period as for PlumpyDoz™ (3 months). The supply of nutritional supplements was done every two weeks and at each contact during monthly home visits. The first dose was administered under supervision by the field worker. Children were followed up for one year including an active follow-up period of 6 months with scheduled monthly home visits followed by a 6 months passive follow-up period i.e. no scheduled home visit during that period. Besides the monthly home visits, parents were advised to bring back the children to the health centres when they were sick. The research nurses team was posted at each health centre to collect all the study related information during participants’ visits.At each visit, physical examination was performed. Body temperature was determined using a contactless thermometer and reported directly on degree Celsius. Direct interview was performed for assessing any occurrences of apparent and common symptoms regarding a pre-defined list of symptoms. Capillary blood sample was collected for malaria diagnosis by RDT and for haemoglobin measurement. Histidine Rich Protein-2 (HRP-2) RDT was used. Haemoglobin was measured using HemoCue® 201 + and Hb level was directly recorded in g/dL. Any participant requiring medical care detected during the home visit was immediately referred to the health centre.versus control arm (SMC-A). Clinical malaria was defined as any case of medical issue that led to a consultation at the health centre and for which malaria was diagnosed after confirmation by RDT. Clinical malaria was either uncomplicated or severe regarding the absence of presence of severity criteria as defined by the national guidelines for malaria cases management. Secondary outcomes included RDT or microscopy positivity rates during the monthly home visits which were considered as asymptomatic cross-sectional parasitaemia. Participants in which no outcome was recorded and who missed at least 3 consecutive scheduled visits were considered as loss to follow-up.The main outcome was the incidence of clinical malaria during the follow-up period for a pair-wise comparison between any of the intervention arms i.e. SMC-AZc or SMC-APd Data were captured using eCRF designed on REDCap software and analysed with Stata 14 software (StataCorp). Statistical analysis was undertaken as outlined in the detailed study protocol published previously . To thisThe effect of the intervention on uncomplicated malaria and on all-cause morbidity was assessed through the diagnosis at each attendance to health facilities (unscheduled visits), while asymptomatic infections were detected during monthly home visits using negative binomial regression, with results expressed as incidence risk ratio (IRR). For this, baseline adjustments were made for age, gender and the use of insecticide-treated bed nets, as they generally improve the precision of the treatment contrasts.The risk of severe malaria was investigated using logistic regression. Similar predictors were used, as aforementioned. Variables with a P < 0.20 in univariate analyses were included in multivariable analyses.In accordance with the protocol, 1059 children were enrolled at a rate of 353 per arm. Out of them, 795 children were recruited in the area covered by Soaw medical centre, while 264 children were from area covered by the health and social promotion centre of Zoetgomdé. Figure After in-depth interview with the parents and verification of document, some children  were over the age of 59 months but were considered eligible because they were still included in the SMC target according to information from the health centres in each locality and were actually covered by the SMC campaign.From the start of the intervention until the end of the follow-up, 774 uncomplicated malaria cases were diagnosed in 1037 children. A total of 297 cases were observed in SMC-A arm while 245 and 232 cases were observed in SMC-AZc and SMC-APd arms, respectively. A representation of the temporal dynamic of uncomplicated malaria cases was provided in Fig. A reduction of 18% and 23% in the risk of having uncomplicated malaria was observed in SMC-AZc and SMC-APd arms respectively compared to the control arm  cases of severe malaria were observed. Almost all cases resulted in severe anaemia (low Hb level). Except one fatal case which occurred in SMC-A arm , the rest of severe malaria cases were successfully managed until recovery. A reduction of 52%, adjusted OR = 0.48 (0.23–0.98) in the risk of having severe malaria was observed in SMC-APd arm compared to control arm (Table RDT positivity rate (RPR) during monthly home visits was similar in the three study arms. RPR was higher in October regardless the study arm. Unlike clinical episodes, no effect of nutrient supplementation on cross sectional asymptomatic infections was observed. Figure versus 291 in SMC-AZc and 262 in SMC-APd. Figure Disease burden was measured through the number of attendances to healthcare facilities from the start of the supplementation until the end of the study home visit regardless the type of infection. Overall, 895 attendances to healthcare facilities were observed. Out of them, 342 were registered in SMC-A All cause morbidity was reduced in children receiving nutrient supplementation compared to control group. This reduction was more important and significant with children receiving Plumpy’Doz™ supplementation Table .Table 4ECurrently, there is a growing interest of combining health interventions for efficient management of resources to ensure sustainability while improving health outcomes . This stThough previous studies reported the protective effect of AZc supplementation , no signInterestingly, a synergetic action was observed with the MQ-LNS Plumpy’Doz™ enhancing the protective effect of SMC against clinical malaria while no effect on asymptomatic carriage was observed. This would mean that the addition of nutritional supplements would not protect the infection but would contribute to preventing the evolution of asymptomatic cases to clinical forms. Two plausible reasons initially evoked which could justify this result resides on the one hand in the reinforcement of the immune system by the nutritional supplements and on the other hand in the improvement of the absorption of the drug AQSP given during SMC campaign. The latter case seems less likely due to the lack of difference in asymptomatic carriage because increased drug absorption in children who received supplementation would necessarily lead to a difference not only in clinical cases but also in asymptomatic carriage. The persistence of the HRP2 protein in the absence of the parasite , 28 can Beyond investigating the mechanisms underlying this synergistic effect, the most important relies in the extend of the reduction of the incidence of malaria as well as all cause morbidity by this combined strategy. In view of the level of reduction in the incidence of clinical malaria, especially severe malaria, this study demonstrates the need for this combined strategy in malaria control in endemic settings. Similarly, previous study conducted in Nigeria reported lower odds of clinical malaria in children who received a MQ-LNS in addition to SMC compared to children receiving SMC alone .In terms of implementation, the door-to-door strategy for SMC offered an excellent opportunity for nutritional supplementation, which effectively reduces costs for implementation at scale. Acceptability of this combined strategy was not assessed, which represents a limitation for this study, but MQ-LNS are usually liked by children and their addition could also contribute to improving adherence to SMC. Another limit of this study relies in the duration of the supplementation, which was 3 months (delay in the acquisition of the Plumpy’Doz™ due to the closing of the borders during the Covid 19 pandemic) not covering the entire period of the SMC, which may reduce the expected effect.Finally, the cross-sectional home visits during the follow-up allowed smooth characterization of the high transmission peak which was stretched towards the end of the year suggesting the need for an extension of SMC during this period in the area.Adding nutritional supplements to SMC significantly increases the impact of this intervention for preventing children from malaria and other childhood infections.


Plasmodium falciparum inside those erythrocytes. Our results indicate an ancient Mediterranean presence of P. falciparum, which remains responsible for most malaria deaths in Africa.Microscopy of mummified visceral tissue from a Medici family member in Italy identified a potential blood vessel containing erythrocytes. Giemsa staining, atomic force microscopy, and immunohistochemistry confirmed  The Medici family was a powerful family from Florence, Italy, that gained prominence under Cosimo de’ Medici in the early 15th century (Plasmodium spp.; members of this genus are the causative agent of different types of human malaria (P. falciparum that is dominant in peripheral blood of infected patients and a diagnostic hallmark (P. falciparum by using immunohistochemistry with polyclonal mouse antiserum against Plasmodium spp.–specific aldolase (P. falciparum–specific histidine-rich protein HRPII (P. falciparum endoplasmic reticulum resident protein Pf39 (In 2011, selected jars of organs from Medici family members were opened centuries after burial to examine their contents, revealing that multiple tissue pieces were still present . The Inshallmark , panel CP. falciparum (P. falciparum developmental stages (We determined that parasitemia was 38% in the Medici tissue, which appeared high (P. falciparum (Plasmodium spp. (P. falciparum infections, Maurer’s clefts are crucial for initiating host-parasite interactions; they are responsible for severe disease and patient death by enabling protein trafficking that causes cytoadherence within organs (The most striking parasite-derived erythrocyte modification is the establishment of secretory organelles, known as Maurer’s clefts, that reside within the cytoplasm of terminally differentiated host erythrocytes infected with P. falciparum (We performed glycan analysis by using mass spectrometry and molecular analyses . We idenPlasmodium spp. parasites (P. falciparum was detected (Plasmodium resistance to standard treatments further hampers positive therapeutic outcomes. Medici family members were known to hunt in marshlands around Florence and in Tuscany that served as breeding grounds for mosquito vectors capable of transmitting Additional information for microscopic evidence of malaria infection in visceral tissue from Medici family, Italy.


Accurate diagnosis and timely treatment are crucial in combating malaria.We evaluated the diagnostic performance of three Rapid Diagnostic Tests (RDTs) in diagnosing febrile patients, namely: Abbott NxTek Eliminate Malaria Ag Pf (detecting HRP2), Rapigen Biocredit Malaria Ag Pf (detecting HRP2 and LDH on separate bands), and SD Bioline Malaria Ag Pf (detecting HRP2). Results were compared to qPCR.P. falciparum with a mean parasite density of 12.5parasites/μL. The sensitivity of the Biocredit RDT was 52.2% (107/205), NxTek RDT was 49.3% (101/205), and Bioline RDT was 40.5% (83/205). When samples with parasite densities lower than 20 parasites/uL were excluded (n=116), a sensitivity of 88.8% , 89.9% , and 78.7%  was obtained. All three RDTs demonstrated specificity above 95%. The limits of detection was 84 parasites/μL (NxTek), 56 parasites/μL , and 331 parasites/μL (Bioline). None of the three qPCR-confirmed P. falciparum positive samples, identified solely through the LDH target, carried hrp2/3 deletions.Among 449 clinical patients, 45.7% (205/449) tested positive by qPCR for The Biocredit and NxTek RDTs demonstrated comparable diagnostic efficacies and both RDTs performed better than Bioline RDT. Malaria remains a significant public health concern in sub-Saharan Africa, including Ghana, where an estimated 5.3 million cases and 12,500 deaths were reported in 2021 [Plasmodium falciparum rely on detecting specific proteins such as histidine-rich proteins 2 and 3 (HRP2 and HRP3), parasite lactate dehydrogenase (LDH), or aldolase [hrp2 and hrp3 genes will lead to false-negative RDT results, even in patients with high parasite density infections [RDTs for aldolase . HRP2-baaldolase –8. Howevfections , 9. Thesfections –12, but fections .Numerous studies have investigated the diagnostic performance of RDTs and found varying sensitivities –9, 14–18hrp2/3 deletion and enables surveillance of deletion status, as samples positive for LDH but negative for HRP2/3 can be selected for molecular confirmation of deletion status.Here, we assess the performance of the NxTek Eliminate Malaria Ag Pf, SD Bioline Malaria Ag Pf, and Biocredit Malaria Ag Pf RDTs in diagnosing clinical patients in Ghana. The NxTek and Biocredit test are considered next-generation ultra-sensitive tests. The NxTek and Bioline have one test band for HRP2. The Biocredit Malaria Ag Pf. (LDH/HRP2) has two separate test bands for HRP2 and LDH. Having both targets as separate bands allow diagnosis in the case of Prior to sample collection, informed written consent was obtained from each individual. For minors, assent was obtained in addition to consent obtained from legal guardians. This study was approved by the Committee on Human Research, Publications, and Ethics of the School of Medical Sciences, KNUST (CHRPE/AP/030/20), the University of Notre Dame Institutional Review Board (19–04-5321), and The Ohio State University Institutional Review Board (2020H0539).Samples were collected from health centers in Mankranso  and Agona  in the Ashanti region of Ghana. The Ashanti region has a reported malaria prevalence of 22% by microscopy . Blood sThree different RDT kits were compared, the RDT, NxTek Eliminate Malaria Ag Pf. ((lot no. 05LDG008B), manufactured by Abbott, the Bioline Malaria Ag Pf. (lot no. 05CDH037C), also manufactured by Abbott, and the Biocredit Malaria Ag Pf. (LDH/HRP2) (lot no. H052BSA002), manufactured by Rapigen. Test were conducted according to manufaturer’s instructions. Tests were considered invalid and repeated if the control band was not positive.P. falciparum varATS multi-copy gene was carried out using a previously described protocol with 4 μL DNA as target resulting in a 95% limit of detection of 0.3 parasties/μL blood [hrp2/3 deletion typing for samples that were negative for HRP2 on RDTs but positive for LDH and that were confirmed positive by qPCR was done by multiplexed digital PCR as previously described [DNA was extracted from 100 μL blood and eluted in 100 μL elution buffer using the Macherey-Nagel NucleoMag extraction kit. qPCR of the μL blood . hrp2/3 escribed .We assumed a test positivity of 50% and obtained a minimum sample size of 384 required to detect a 5% difference in sensitivity with a 95% confidence level  using a previously described method . A totalSensitivity was calculated as the number of infections detected by an RDT divided by the number of infections detected by qPCR, and against thresholds of 2000, 200, and 20 parasites/μL (by qPCR). This was done to comparability with other studies, as different methods for sample collection, DNA extraction, and qPCR result in different sensitivities and, thus, different numbers of positive samples . Specifip values to compare groups for qPCR test positivity and RDT sensitivity were calculated by Chi-square test, while Welch’s ANOVA was used for parasite density.The area under the receiver operating characteristic curve (AUC) was calculated with a nonparametric analysis using 1000 bootstrap replications. As for PPV, a threshold of 20 parasites/μL by qPCR was set to count a sample as true positive, and samples with >0 to 20 parasites/μL were exluded from the calculation. As parasite density distributions were skewed, geometric mean densities are given whenever densities are reported. CI95 stands for the 95% confidence interval. The A total of 449 clinical samples were collected and analyzed. P. falciparum by varATS qPCR, with a mean parasite density of 12.5 parasites/μL. There were no statistically significant differences in positivity by qPCR based on participant’s age or gender (205/449 (45.7%) clinical samples tested positive for r gender . Of the r gender . Parasitr gender .hrp2 or hrp3 deletions. The parasite densities of these samples ranged from 2–5 parasites/μL.The Biocredit RDT demonstrated higher sensitivity for the HRP2 target (88.8% at densities >20 parasites/μL), compared to the LDH target (75.3%). Three qPCR-confirmed infections were detected by LDH only  thus, seThis study showed similarly high sensitivities of the NxTek and the Biocredit RDT. These tests are considered ultra-sensitive, i.e. substantially more sensitive than previous tests such as the SB Bioline. They detected around 50% of all qPCR-positive infections, compared to around 40% by the SD Bioline. At densities >200 parasites/μL, the NxTek and Biocredit sensitivity was >93%, and 89% for the SD Bioline. The LOD of the NxTek and Biocredit is approximately four-fold lower than the LOD for the SD Bioline, and the LOD of the Biocredit confirmed values obtained from a previous study in Burundi applying the same methodology [P. falciparum malaria-positive samples detected via the LDH target alone carried hrp2/3 deletions. The current data thus corroborated recent findings of very low frequency of hrp2/3 deletions in Ghana [hrp2 deletion is prevalent. While to-date in Ghana HRP2-based RDTs remain effective, the Biocredit RDT will be a reliable option for malaria diagnosis shall deletions ever spread in the country.None of the three in Ghana , 29. TheIn conclusion, the NxTek and Biocredit RDTs showed higher sensitivity and a lower LOD compared to the SD Bioline RDT, which has been in use for clinical diagnosis by the Ghana National Malaria Control Programme . Such mo


Intermittent Preventive Treatment of malaria in infants (IPTi) is a malaria control strategy consisting of the administration of an anti-malarial drug alongside routine immunizations. So far, this is being implemented nationwide in Sierra Leone only. IPTi has been renamed as Perennial Malaria Chemoprevention -PMC-, accounting for its recently recommended expansion into the second year of life. Before starting a pilot implementation on PMC, the currently implemented strategy and malaria prevalence were assessed in young children in selected areas of Sierra Leone.A cross-sectional, community-based, multi-stage cluster household survey was conducted from November to December 2021 in selected districts of the Northern and northwestern provinces of Sierra Leone among 10–23 months old children, whose caretakers gave written informed consent to participate in the survey. Coverage of IPTi and malaria prevalence—assessed with rapid diagnostic tests—were calculated using percentages and 95% confidence intervals weighted for the sampling design and adjusted for non-response within clusters. Factors associated with RDT + and iPTi coverage were also assessed.A total of 720 children were recruited. Coverage of three IPTi doses was 50.57% , while prevalence of malaria infection was 28.19% (95% CI 24.81–31.84). Most children had received IPTi1 , and IPTi2  and over half of the children also received IPTi3 . The uptake of each IPTi dose was lower than that of the vaccines administered at the same timepoint at all contacts.In Sierra Leone, half of the children received the three recommended doses of IPTi indicating an increase in its uptake compared to previous data of just a third of children receiving the intervention. However, efforts need to be made in improving IPTi coverage, especially in the planned expansion of the strategy into the second year of life following recent WHO guidelines.The online version contains supplementary material available at 10.1186/s12936-023-04575-6. Despite global efforts for malaria control, the infection continues to have a devastating impact on people’s health in endemic areas with a global estimate of 247 million malaria cases and 619,000 malaria deaths in 2021 compared to 245 million malaria cases and 625,000 malaria deaths in 2020 . I. I6]. InAs part a of a multi-country pilot project , to promote and expand IPTi implementation that includes Sierra Leone  a househ.IPTi has been recently renamed by the WHO as Perennial Malaria Chemoprevention (PMC), accounting for its expansion into the second year of life given the added EPI contacts during this period . MozambiChildren aged 10 to 23 months inclusive at the moment of the interview, whose caretaker agreed to participate in the survey by signing an informed consent and who lived in MULTIPLY project districts of Sierra Leone  were the targeted population for the HHS. Malaria prevalence in children U5 in 2016 was 38%, 59%, and 56%, in these three districts, respectively [According to the latest available estimate of 32.2% IPTi coverage , it was This was a cross-sectional, community-based, multi-stage cluster HHS. Sample selection was undertaken in three stages as follows  by the number of children to be interviewed in each cluster. For feasibility reasons, the cluster size was 12 children per day per cluster . TherefoInformation on IPTi administration was recorded directly from the EPI card except when this was not available in which case it was obtained by asking the child’s caretaker. A malaria rapid diagnostic test (RDT) was performed in all children regardless of symptoms or signs of malaria; if the RDT was positive anti-malarial treatment was provided and children were referred following national guidelines for malaria treatment and referral.Electronic devices were used for data collection onto the REDCap Mobile Android App , 13. OncOnline data quality rules were executed on mobile devices during the interview and data entry process, and a set of data quality rules executed once new data were uploaded in the server. Periodic data quality and progress reports were produced with the last data received. Reports computed survey profiles for each district based on uploaded data were crossed-checked with the paper-based survey profile completed manually to identify inconsistencies or missing records.Statistical analyses were conducted with weighted data to account for the complex sampling design  or interquartile range (IQR), respectively.Malaria prevalence and IPTi coverage were determined using weighted percentages and 95% CIs. Malaria prevalence was defined as the proportion of children with a positive malaria RDT result; Prevalence of clinical malaria was defined as the proportion of children with positive malaria RDT result plus fever (axillary temperature ≥ 37.5 °C) or history of fever in the last 24 h.IPTi coverage was defined under two scenarios: (i) IPTi administered irrespective of the EPI contact with complete IPTi coverage being defined as the proportion of children who received the recommended three doses of IPTi.; and scenario (ii) whereby IPTi is administered at the recommended EPI contacts, i.e. at 10 weeks (IPTi 1), 14 weeks (IPTi 2), and 9 months (IPTi 3) of age, respectively. In this case the number of children who received IPTi3 does not necessarily represent complete coverage. Thus, some children may have received IPTi at nine months, being recorded as IPTi 3 for registration purposes, but actually they had missed the first and/or the second IPTi doses.Multilevel mixed-effects logistic regression accounting for nesting of participants within clusters and households was used to identify variables associated with a positive RDT result as well as having received the three IPTi doses. Univariable and multivariable-adjusted three-level random-intercept models with households nested within clusters were estimated using variables selected a priori. Poststratification adjustment were not applied to the sampling-weights for the model estimations since multilevel models require separate weights for each stage-level. Therefore, these regression models were estimated by complete-case analysis accounting for the complex multistage sample design. The assumption that data were missing completely at random (MCAR) was assessed using the regression-based approach suggested by Rouzinov S. and Berchtold A . Odds rap-value < 0.05 was considered statistically significant.All statistical analyses were performed using Stata, version 17  ; all staFrom November to December 2021, 9,104 households were listed from 60 randomly selected clusters in project areas . Information on IPTi and vaccine administration was either retrieved from the child’s vaccination card  or self-reported by the child’s caretaker .A total of 53.42% children had received all vaccines scheduled from birth to 9 months of age (fully immunized) as per the national EPI schedule, while 64.03% of the surveyed children were fully immunized, as per WHO definition . The majOf the 720 children, 13 of them did not provide information regarding the IPTi intake. Among the remaining 707 children, 368 received three doses of IPTi , 197 received two doses of IPTi , and 73 received one dose only of IPTi , regardless of administration timepoints. Overall, 638 children received one or more IPTi doses , 565 received two or more doses  , 577 children received IPTi2 at 14 weeks of age , and 420 children received IPTi3 at 9 months of age  . By district, the prevalence was 23.26% (95% CI 17.51–30.21), 29.17% (24.44–34.39), and 35.71% (95% CI 28.62–43.49) in Bombali, Port Loko and Tonkolili, respectively. Overall, the prevalence of clinical malaria (fever or history of fever plus a positive RDT) was 17.10% (95% CI 14.08–20.62)  . Compared with Bombali district, children residing in Tonkolili district were more likely to test positive for malaria .A substantial amount of variation across clusters was found; the variance of the random effects for cluster is 0.8330 (95%CI 0.1735–3.9996) in the adjusted model. Adjusted for all other studied covariates, older children were more likely to have malaria infection , those who slept under a mosquito net the previous night  and those whose caretaker was paid-employed  were more likely to have received the complete three IPTi doses , children whose caretakers were males  and of Muslim religion  and children residing in Port Loko district  were less likely to have received complete three doses of IPTi.Similar to the adjusted model with malaria infection as outcome variable, a substantial amount of variation across clusters was found; the variance of the random effects for cluster is 0.8965 (95%CI 0.6140–1.3092). Adjusted for all other studied covariates, older children  are lower than that (59%) found in the same districts and nation-wide in the latest malaria indicator survey (MIS) undertaken nation-wide by the Ministry of Health and Sanitation during the peak of the malaria season (July–September 2021) . Both figures are also higher than the 32.2% coverage of complete IPTi reported in 2018. Regarding malaria prevalence it was 43.65% by RDT in these districts, reflecting the higher transmission intensity during the MIS 2021 compared to that when this survey was carried-out at the beginning of the dry season.Alike the observation on coverage of vaccines and IPTi doses at each EPI contact in this study, in the 2018 survey the proportion of children who received IPTi at each timepoint was also lower than vaccination rates at the same EPI contact. These findings indicate that although IPTi has been successfully integrated into the EPI program in Sierra Leone, some limitations still exist that represent missed opportunities for some children attending the EPI scheme who do not receive the recommended malaria prevention at the same visit. Similarly, regarding malaria control in pregnancy it has been also reported missed opportunities in the proportion of pregnant women receiving intermittent preventive treatment in pregnancy (IPTp), being systematically lower than the proportion of women attending the antenatal clinic visit . As withIn this survey, infants who had received three doses of IPTi were less likely to have malaria infection, indicating the effectiveness of the intervention in preventing malaria. On the other hand, older age was associated with a higher risk of the child having malaria infection at the time of the interview. This may be explained by the fact that the last IPTi dose is usually administered at around 9 months of age. Since the post-prophylactic effect of SP is about 5 weeks, older infants and children may not be protected against malaria compared to younger children. For this reason, the WHO has released new guidelines recommending the expansion of IPTi or PMC into the second year of life . The EPIOlder age was associated with increased probability of having received the three IPTi doses. This may be due to the fact that older children have had more time to attend the EPI program and, albeit delayed, received all intervention doses. Children who slept under a mosquito net the night before were also more likely to have received the three IPTi doses, probably reflecting a positive health behaviour. On the other hand, children in Port Loko district were less likely to have received the complete course of IPTi. This finding coincides with the results from the 2019 Sierra Leone’s Demographic and Health Survey, showing Port Loko district to have the lowest immunization coverage in the country . The WHOThis study has some limitations. First, the list of selected clusters was based on the 2015 census with two of the clusters being outside project areas, for which they had to be replaced by backup clusters, leading to potential underrepresentation of part of the area, and thus to potential non-response bias. Secondly, although information on child’s IPTi and immunization status was obtained from the child’s vaccination card in most cases, there is a potential for recall bias when this information was reported by the caretaker.The strengths of this study are the use of a robust multi-stage cluster sampling approach to select a representative sample of children U2 from areas where the MULTIPLY project will be implemented. Field teams underwent a rigorous training, a 2 day pre-test, and a feedback session before the start of the HHS. In addition, data collection was carefully supervised by the investigators team. This maximized compliance with the sampling methodology, standard operating procedures, and reduced the potential for selection bias and protocol deviations. The use of electronic devices for direct data entry in the field ensured data quality by eliminating errors in data transfer from paper forms while quality rules were automatically executed in mobile devices. Lastly, the rate of non-response among eligible children was 1.1%, much lower than the 10% estimated during sample size calculation, thus minimizing the potential for non-response bias at individual level.The results of this study show an increase in IPTi coverage since the pilot evaluation carried out in 2018. Although these findings are encouraging, just half of the surveyed children had received the recommended full IPTi course. In addition, uptake misalignment between IPTi and concomitant vaccines given at the same EPI contacts persist. Improving young children’s health by reducing their malaria burden requires increasing efforts to promote and expand malaria prevention coverage into the second year of life.Additional file 1. MULTIPLY community-based household survey standard operating procedure.Additional file 2. Malaria prevalence.