This approach has yet to be evaluated in children and infants. We assessed this vaccine strategy among 138 Gambian and Burkinabe children in four cohorts: 2- to 6-year olds in The Gambia, 5- to 17-month-olds in Burkina Faso, and 5- to 12-month-olds and 10-week-olds in The Gambia. We assessed induction of cellular immunity, taking into account the distinctive hematological status of young infants, and characterized the antibody response to vaccination. T cell responses peaked 7 days after boosting with modified vaccinia virus Ankara (MVA), with highest responses in infants aged 10 weeks at priming. Incorporating lymphocyte count into the calculation of T cell responses facilitated a more physiologically relevant comparison of cellular immunity across different age groups. Both CD8+ and CD4+ T cells secreted cytokines. Induced antibodies were up to 20-fold higher in all groups compared with Gambian and United Kingdom (UK) adults, with comparable or higher avidity. This immunization regimen elicited strong immune responses, particularly in young infants, supporting future evaluation of efficacy in this key target age group for a malaria vaccine.Heterologous prime-boosting with viral vectors encoding the pre-erythrocytic antigen thrombospondin-related adhesion protein fused to a multiple epitope string (ME-TRAP) induces CD8 An effective malaria vaccine is an urgent global health priority. In these studies, Ewer and colleagues describe strong T cell and antibody responses in children and infants following vaccination with a viral vectored vaccine regime encoding a pre-erythrocytic malaria antigen. This regime has previously demonstrated efficacy in adults and these data support assessment of the efficacy of this vaccine in infants. However, there is a range of diseases affecting infants, for which vaccines are yet to be developed, where an ability to induce potent CD8+ T cell responses could be important. These include respiratory syncytial virus (RSV), HIV, tuberculosis, and vaccines targeting malaria parasites at the liver stage of infection.+ T cell induction in newborn mice, suggesting that generation of such T cell responses in human infants might be difficult.+ T cells in early infancy.Vaccination is one of the most cost-effective health care interventions available, and currently used vaccines prevent an estimated 2.5 million deaths each year.Plasmodium falciparum, could help to save half a million lives each year.+ T cells; efficacy is mediated by IgG antibodies and CD4+ T cells against the circumsporozoite (CS) protein, a pre-erythrocytic antigen that is highly abundant during the sporozoite stage of the parasite life cycle.P. falciparum life cycle is also a leading target for vaccination. This stage lasts between 5.5 and 7 days in humans,+ T cells to locate and kill infected hepatocytes.A highly effective malaria vaccine against the most lethal malaria species, + and CD8+ phenotypes and IgG antibody responses in malaria-naive and semi-immune adults.P. falciparum-infected mosquitoes, ChAd63 MVA ME-TRAP elicited 21% sterile efficacy and significantly delayed the time-to-patency of malaria in a further 36% of vaccinees.+ T cells. In a recent field trial in Kenyan adults, 67% efficacy against malaria infection was induced by the same immunization regime, and again, a T cell correlate of efficacy was observed.We have previously described vaccination approaches employing the sporozoite antigen thrombospondin-related adhesion protein (TRAP) fused to a multiple epitope string (ME) in a number of delivery platforms including DNA and replication-deficient viral vectors.We present here a detailed evaluation of the high-level T cell and antibody responses induced by this regimen in young children and infants. Infants have very different hematological parameters to older children and adults: total blood volume is substantially lower and numbers of circulating lymphocytes per ml significantly higher.Here, we report T cell and antibody immunogenicity across four pediatric age strata ranging from 6 years to 10 weeks old, from three clinical vaccine studies using the ChAd63 ME-TRAP and MVA ME-TRAP regimen in malaria-exposed African children and infants. Responses are also compared to those of malaria-naive and malaria-exposed vaccinated adults. Given that each clinical trial was a small phase I study, combining the datasets together into a single analysis facilitates the observation of trends across age groups and between cohorts with differing malaria exposure. Two clinical trials were performed in the western region of The Gambia in Sukuta, where modest malaria transmission is still observed following seasonal rains, despite a substantial decline in incidence since 2003.Two hundred children were screened for eligibility across the three trials and 138 eligible children were enrolled, vaccinated, and followed up A and S1BWe report elsewhere ex vivo IFNγ ELISpot responses stratified by age and dose in group 1 and group median response data stratified by dose in groups 2, 3, and 4.+ and CD8+ T cells , and tumor necrosis factor-alpha (TNF-α) secretion from CD4 T cells . ExpressAntibody responses to vaccination in Gambian 2- to 6-year-olds (group 1) were weak, irrespective of priming or boosting dose A. IgG tiAvidity of IgG antibodies was measured at the peak time point A and wasNeutralizing antibodies to the adenovirus vector were measured prior to vaccination in group 1 only and were weaker than those measured previously in Gambian adults .We demonstrate here that immunization of children and infants with a prime-boost regimen using ChAd63 and MVA ME-TRAP induces high level T cell and antibody responses to a pre-erythrocytic malaria antigen that has previously demonstrated efficacy against CHMI in malaria naive adults and against natural infection in semi-immune adults.Higher ELISpot responses were measured in adults vaccinees compared to pediatric vaccinees using the standard ELISpot readout. However, comparisons of immunogenicity in de-escalating age groups are complex due to differences in body mass and total blood volume of participants receiving similar doses of vaccine. More specifically, variations are observed in the numbers of lymphocytes circulating per milliliter of blood between adults and children and between infants and older children. To address this, we have incorporated the lymphocyte count from the full blood count performed at the same time into the denominator of the units used to report ELISpot responses. Using this approach to take account of the much higher lymphocyte frequencies measured in young children and infants, we have quantified comparable T cell responses between adults, children, and older infants. Responses in 10-week-old infants were almost 3-fold higher compared with adults, indicating that ChAd63 and MVA ME-TRAP T cell immunogenicity is increased and not reduced with administration to young infants.While T cell responses between the two sites were comparable between 5- to 17month-olds, overall lower responses were measured in Burkina Faso, potentially due to a combined effect of age and differing malaria exposure by site. Malaria-related immunosuppression could lead to reduced vaccine immunogenicity or differences in innate immunity may exist in the early weeks of life in these children. Congruently, malaria transmission in the Gambia is markedly lower than in Burkina Faso,43Higher frequencies of antigen-specific cells per milliliter of blood trafficking through the spleen may increase the likelihood of antigen recognition from antigen-presenting cells following vaccination, giving rise to greater cellular immunity. The level of antigen-specific T cells per milliliter is likely to correlate better with vaccine efficacy than T cells per million PBMC, as the former should correlate better with the rate at which antigen-specific T cells enter the liver, which is the relevant target organ in malaria.Anti-TRAP IgG titers at the peak time point were, remarkably, ∼20-fold higher in the youngest groups of children and infants than those at the same time point post-vaccination in malaria-naive and semi-immune adults. Interestingly, titers in 2- to 6-year-old children were lower than those in adults and younger children or infants, which when assessed in combination with the similar observation in T cell frequencies, suggests vaccination of older children may be less effective at preventing malaria infection. A recent study with these viral vectors expressing TRAP and circumsporozoite protein (CS) in malaria-naive adults, proposed a role for both anti-CS and anti-TRAP antibody responses in reducing parasite density during the liver-stage of malaria infection.Avidity of antibodies measured 7 days after boosting was lower in young infants than in 5- to 12- or 5- to 17-month-olds, although the kinetics of avidity maturation were comparable between these age groups. In the Burkinabe infants, IgG1 avidity was significantly lower than in Gambian infants of the same age, perhaps again due to increased malaria exposure in Burkina Faso. Avidity of IgG antibodies alone has been shown not to be predictive of malaria vaccine efficacy for RTS,S/AS01, however, an association between the change in avidity, as well as IgG titers following the second and third dose were strongly associated with a reduction in the risk of malaria.Trypanosoma cruzi, demonstrating that under certain conditions, the immature human immune system is indeed capable of potent CTL activity.51Previous studies have demonstrated a reduction of cellular responses to vaccines in infants with reduced or defective secretion of Th1 cytokines in response to oral polio and protein-in-adjuvant vaccines, such as DTaP.In this study, we have assessed the ability of a virally vectored prime boost regime to induce immunity to a protective pre-erythrocytic malaria antigen in two populations with very different malaria endemicities. Furthermore, a novel methodology for more physiological and robust comparison between adult and pediatric T cell responses was developed and applied across several age de-escalating clinical studies. Our immunogenicity findings combined with the acceptable safety profile observed in these studiesThe primary objective of these trials was to evaluate the safety and reactogenicity of the ChAd63 ME-TRAP and MVA ME-TRAP vaccines in malaria-experienced Gambian and Burkinabe children. Secondary objectives were to evaluate the cellular and humoral immunogenicity of the vaccines in two settings of varying seasonal malaria transmission while the tertiary objective was to compare the immunogenicity of the low and high doses of MVA ME-TRAP (Gambian children only).Anopheles gambiae is the principal malaria vector. Previous studies have documented decline in incidence of malaria in The Gambia.The first clinical trial (group 1) took place from December 2010 to December 2011 in the Sukuta field site of Medical Research Council, The Gambia. Sukuta is a peri-urban Gambian village located about 30 km south of the capital Banjul. The Sukuta field site previously served as the base for the phase I trials of ChAd63 MVA ME-TRAP vaccine regimen in adults (18). In this region, malaria transmission is highly seasonal, occurring almost exclusively during the rainy season (July to December) with greatest incidence from September to November. A. gambiae is the principal malaria vector. Immunogenicity analyses of group 4 are split into two age groups: 5–12 months and 13–17 months, permitting direct analysis of 5- to 12-month-olds in the Gambia and Burkina Faso.The third clinical trial (group 4) took place from December 2012 to September 2013 in Banfora Health District in the Cascades region of South Western Burkina Faso, about 400 km southwest from the capital Ouagadougou. Malaria transmission is stable during the year, with increased levels during the rainy season from May to November, peaking from May to September.https://clinicaltrials.gov  and the Pan African Clinical Trials Registry (www.pactr.org) .An independent Data Safety and Monitoring Board (DSMB) was appointed before the trials began to provide oversight and to review the safety data reports as the trials progressed. Experienced local pediatricians served as local safety monitors (LSM) and, along with the DSMB, reviewed all safety data between dose escalations. In addition, trials were conducted according to ICH Good Clinical Practice guidelines and were monitored by an external organization . The Gambian Government/Medical Research Council Joint Ethics Committee, The Gambia Medicines Board, the Burkina Faso Ministry of Health and Institutional Bioethics Committee, the UK Medicines and Healthcare products Regulatory Authority, and Oxford Tropical Research Ethics Committee  granted approval of the study protocol. All three trials were registered with 10vp) followed by low dose MVA ME-TRAP (1 × 108 plaque-forming units [ PFU]); group 1b: low dose ChAd63 ME-TRAP (1 × 1010vp) followed by high dose MVA ME-TRAP (2 × 108 PFU); group 1c: control HDCRV 1 mL followed by HDCRV 1 mL; group 1d: high dose ChAd63 ME-TRAP (5 × 1010vp) followed by low dose MVA ME-TRAP (1 × 108 PFU); group 1e: high dose ChAd63 ME-TRAP (5 × 1010vp) followed by high dose MVA ME-TRAP (2 × 108 PFU); group 1f: Control HDCRV 1 mL followed by HDCRV 1 mL intramuscular (IM). All vaccinations were separated by an 8-week interval.We conducted three phase Ib studies. The first study  in The Gambia was the pediatric arm of a phase Ib single-blind, randomized controlled, dose-escalation study in adults that has been reported previously.10vp) followed by low dose MVA ME-TRAP (1 × 108 PFU); group b: high dose ChAd63 ME-TRAP (5 × 1010vp) followed by low dose MVA ME-TRAP (1 × 108 PFU); group c: no vaccine. For group 4, all children received high dose ChAd63 ME-TRAP (5 × 1010vp) followed by low dose MVA ME-TRAP (1 × 108 PFU).For groups 2 and 3, 36 eligible children in each group were randomized to receive either group a: low dose ChAd63 ME-TRAP  ChAd63 ME-TRAP and 2 × 108 PFU MVA ME-TRAP.Pediatric groups are compared to malaria naive adult vaccinees in the UK and semi-immune adult vaccinees the Gambia, who all received 5 × 10An independent statistician at the Centre for Statistics in Medicine, Oxford performed a stratified randomization of participants . The list of eligible children after screening was sent to the statistician who carried out the randomization. The statistician had no knowledge of the participants, except the age, as this was required for the stratification. The children were randomly allocated to six groups in dose-escalated fashion. This was done to determine the tolerable doses as this was the first time ChAd63 and MVA ME-TRAP vaccines were being administered in Gambian children. The investigators and the vaccinators were un-blinded to the group allocations. However, the study children’s parents/carers and field workers who conducted post-vaccination assessment of reactogenicity and solicited symptoms were blinded to the group allocations.These phase Ib trials were not powered to detect differences between groups. The sample size was based on general acceptance of this size for initial assessment of safety, tolerability, and immunogenicity of the investigational vaccines in a malaria endemic area and this size balances the need to avoid exposing a large group of study participants to an unknown risk with the need for useful safety and immunogenicity data from an adequate sample size.The Clinical Biomanufacturing Facility  and IDT (Germany) manufactured ChAd63 ME-TRAP and MVA ME-TRAP respectively under Good Manufacturing Practice conditions, respectively as previously described.Blood samples were stored at room temperature prior to processing, which was completed within 6 hr of venepuncture. PBMC were separated by density centrifugation from heparinized whole blood and resuspended in RPMI containing 10% heat-inactivated, batch-tested, sterile-filtered fetal bovine serum (FBS) previously screened for low reactivity , 1% L-glutamine, and 1% penicillin/streptomycin. Cell counts were performed using trypan blue staining and a microscope according to an established standard operating procedure (SOP) in the lab.6 PBMC, with a median of 18 SFC/106 PBMC. Pools were considered positive if the response was greater than the median plus 2 SDs (59 SFC/106) of all negative control wells after subtraction of the autologous background. The lower limit of detection for the assay was 28 SFC for ME-TRAP. Staphylococcal enterotoxin B (0.02 μg/mL) and phytohemmagglutinin-L (10 μg/mL) were used as a positive control, whereby responses of >800 SFC/106 passed quality control (QC). Reagents and methods were standardized between the two trial sites. Lymphocyte counts per milliliter of blood were taken directly from the hematology breakdown or calculated by multiplying the number of white blood cells per milliliter of blood by the lymphocyte differential percentage. These data were routinely obtained at post-vaccination time points as part of the safety assessments. This value was then multiplied by the number of SFC per million PBMC to produce the number of SFC per milliliter of blood.Ex vivo (18 hr stimulation) ELISpot assays were performed using Multiscreen IP ELISpot plates (Millipore), human IFNγ SA-ALP antibody kits (Mabtech), and BCIP NBT-plus chromogenic substrate (Moss Inc.). Cells were cultured in RPMI (Sigma) containing 10% heat-inactivated, sterile-filtered fetal calf serum, previously screened for low reactivity  supplemented with 1% L-glutamine and 1% penicillin/streptomycin. Antigens were tested in duplicate with either 200,000 or 250,000 PBMC added to each well of the ELISpot plate. TRAP peptides were 20 amino acids in length, overlapping by 10 amino acids (NeoBioLab), assayed in six pools of seven to ten peptides at 10 μg/mL. Plates were counted using an AID automated ELISpot counter , using identical settings for all plates, and counts were adjusted only to remove artifacts. Responses were averaged across duplicate wells, responses in unstimulated (negative control) wells were subtracted from each individual pool, then responses to individual pools were summed for each strain of the TRAP antigen. Responses to the negative control were always <142 SFC/10PBMC were frozen in FBS containing 10% DMSO and stored in the vapor phase of liquid nitrogen. Of the 30 PBMC samples available in group 4, we selected samples where the ELISpot on fresh PBMC at day gave a response over 250 SFC per million PBMC, as responses lower than this would not be detectable by flow cytometry.6 PBMC (Novagen) before stimulation overnight at 37°C with 5% CO2, either with Staphylococcal enterotoxin B, a pool of 56 peptides at 2 μg/mL spanning the entire length of the TRAP protein from the T9/96 strain of P. falciparum (Neopeptide) or an unstimulated control. Brefeldin A (BD Biosciences) 1 μg/mL and monensin (eBioscience) 1 μg/mL were added after 2 hr into the incubation and left to incubate for a further 16 hr. Cells were then washed in fluorescence-activated cell sorting (FACS) buffer  for 20 min at room temperature in the dark. Cells were then washed in FACS buffer and permeabilized for 20 min with Cytofix/Cytoperm (BD Biosciences) then washed in 1:10 permeabilization buffer (BD Biosciences). A cocktail of antibodies for surface and intracellular staining was added and incubated for 30 min at room temperature in the dark. The antibody cocktail is described in Thawing was performed rapidly in a water bath and cells were rested for 2 hr with benzonase at 25 U/10+ cells were analyzed per sample. Data were prepared and analyzed using FlowJo v9.6.2 (Treestar) with a hierarchical gating strategy. A sample gating strategy is shown in + or CD8+ T cells after subtraction of the unstimulated control could not be detected were excluded from the analysis. All samples had a minimum of 31,000 CD4+ or 8,000 CD8+ T cells in the parent population, thus the lower limit of detection for the assay was 0.0042% for CD4+ T cells and 0.022% for CD8+ T cells. A response was classified as positive if the response to peptide was greater than the medium control for the corresponding sample. Analysis of polyfunctionality was not undertaken due to low event numbers acquired from small pediatric samples.At least 29,000 live CD34 cells/well). Heat inactivated test samples were diluted 4-fold from 1:9 to 1:2,304 in 10% FBS in DMEM and incubated 1:1 with ChAd63 expressing the secreted alkaline phosphatase gene (8 × 107 vp/mL) for 1 hr at 37°C. Serum and virus were then added to 293 cells in a volume of 200 μL in duplicate for 1 hr, after which sample and virus were aspirated and replaced with fresh 10% FB DMEM. A virus-only control was included. After 22–26 hr at 37°C, 50 μL of medium was assayed for SEAP activity using a Phospha-Light TROPIX phosphatase assay (Applied Biosystems) in black assay plates, and luminescence was measured after 45 min on a Thermo-Fisher Varioskan Flash Luminometer. Anti-vector neutralization titers were defined as the dilution of serum showing 50% reduction in SEAP activity, based on observed % inhibition values relative to SEAP activity from virus alone. For trial A, anti-vector antibodies were measured in serum and for trial B plasma was used, however, we have determined that these sample types are equivalent for this assay.One day prior to performing the assay, GripTite 293 cells (Invitrogen) were seeded in 96-well plates , 0.166 (IgG2), 0.2002 (IgG3), 0.151 (IgG4), 0.362 (IgM), and 0.374 (IgA).50) was used as a measure of avidity. This was calculated from the intercept of the curve for each sample with the line of 50% reduction of the OD405 in the NaSCN-free well for each sample.IgG antibody avidity was assessed by sodium thiocyanate (NaSCN)-displacement ELISA. The assays were conducted as for total IgG ELISAs except that sera were individually diluted in dilution buffer to a level calculated to reach an OD405 of 1.0  and plated at 50 μL/well in 16 wells of a 96-well plate. Plates were incubated for 2 hr at room temperature (RT) before chaotropic agent NaSCN was added in duplicate at increasing concentrations down the plate (0–7 Molar [M]). Plates were incubated for 15 min at RT before washing, incubation with secondary antibody and development as for the total IgG assay. The concentration of NaSCN required to reduce the OD405 to 50% of that without NaSCN  and added to two plates, one of which was incubated with anti-IgG1 and the other with anti-IgG3. Secondary antibodies used were the same as used for the Isotype ELISAs.Group data display geometric mean or median. Matched pairs analyses were conducted where time points within a group were compared and excludes volunteers with missing data at any time point. UK and Gambian adult data were combined for several ELISpot and ELISA analyses due to no statistically significant differences between the two populations. A Kruskal-Wallis test was used to compare ELISpot and ELISA data in children against the adult control group, with Dunn’s multiple comparisons post-test. Kruskal-Wallis tests for comparisons between all groups were also used, with Dunn’s multiple comparisons post-test. For statistical analyses, an alpha-level of 0.05 was considered significant and all p values are two-tailed. All analyses were performed in GraphPad Prism, Mac version 6. (GraphPad Software).Study Design, M.O.A., A.B.T., J.B.Y., S.H.H., N.A.A., C.J.A.D., K.L.F., B.K., S.B.S., K.B., A.V.S.H., I.N., and K.J.E.; Project Management and Regulatory Affairs, R.R., N.K.V., O.L., A.M.L., and E.B.I.; Provided Clinical Care to Participants, M.O.A., A.B.T., J.B.Y., and U.J.A.; Immunological Assay Design, C.M.B., G.B., N.J.E., K.L.F., A.V.S.H., I.N., and K.J.E.; Performed Experiments, C.M.B., A.D., G.B., G.S.S., Y.J.J., O.O., N.J.E., C.T., N.O., M.O., J.N.-J., A.D., I.N., K.J.E.; Data Analysis, C.M.B., G.B., N.J.E., I.N., and K.J.E.; Data Interpretation, C.M.B., G.B., A.V.S.H., I.N., and K.J.E.; Preparation of Figures, C.M.B., G.B., and K.J.E.; Manuscript Preparation, M.O.A., C.M.B., G.B., A.V.S.H., and K.J.E.The following authors have declared that no conflicts of interest exist: C.M.B., A.D., G.B., G.S.S., Y.J.J., O.O., N.J.E., C.T., N.O., M.O., J.N.-J., A.D., M.A.O., A.B.T., J.B.Y., J.U.A., S.H.H., N.A.A., R.R., C.J.A.D., R.C., N.K.V., O.L., A.M.L. K.L.F., B.K., E.B.I., S.B.S., K.B., I.N., and K.J.E. A.V.S.H. is a named inventor on patent applications on malaria vectored vaccines and immunization regimens. Authors from ReiThera are employees of and/or shareholders in ReiThera, which is developing vectored vaccines for malaria and other diseases.


Despite the widespread use and availability of rapidly acting anti-malarials, the fatality rate of severe malaria in sub-Saharan Africa remains high. Adjunctive therapies that target the host response to malaria infection may further decrease mortality over that of anti-malarial agents alone. Peroxisome proliferator-activated receptor-gamma agonists (e.g. rosiglitazone) have been shown to act on several pathways implicated in the pathogenesis of severe malaria and may improve clinical outcome as an adjunctive intervention.®) in children with uncomplicated malaria. The primary outcomes were tolerability and safety, including clinical, haematological, biochemical, and electrocardiographic evaluations.In this study, the safety and tolerability of adjunctive rosiglitazone in paediatric uncomplicated malaria infection was evaluated in Mozambique, as a prelude to its evaluation in a randomized controlled trial in paediatric severe malaria. The study was a prospective, randomized, double-blind, placebo-controlled, phase IIa trial of rosiglitazone (0.045 mg/kg/dose) twice daily for 4 days versus placebo as adjunctive treatment in addition to Mozambican standard of care (artemisinin combination therapy CoartemThirty children were enrolled: 20 were assigned to rosiglitazone and 10 to placebo. Rosiglitazone treatment did not induce hypoglycaemia nor significantly alter clinical, biochemical, haematological, or electrocardiographic parameters.Adjunctive rosiglitazone was safe and well-tolerated in children with uncomplicated malaria, permitting the extension of its evaluation as adjunctive therapy for severe malaria.The trial is registered with Clinicaltrials.gov, NCT02694874 Malaria causes an estimated 212 million infections and 429,000 deaths annually . HoweverPeroxisome proliferator-activated receptor-gamma (PPARγ) is a member of the family of nuclear hormone receptors that function as ligand-activated transcription factors via their heterodimerization with another nuclear receptor, retinoic X receptor (RXR) –19. PPARPlasmodium falciparum parasitized erythrocytes, and to reduce parasite-induced pro-inflammatory cytokine secretion from monocytes and macrophages in vitro [Rosiglitazone has been shown to enhance macrophage phagocytosis of in vitro . In a prin vitro , 31. In in vitro . In thisin vitro , 32.Together, these results support the hypothesis that adjunctive rosiglitazone may improve outcomes in patients with severe malaria. Since the majority of severe malaria and associated deaths occur in children under 5 years of age in sub-Saharan Africa, we conducted a phase IIa safety and tolerability trial in Mozambican children with uncomplicated malaria, as a prelude to undertaking a randomized trial in children with severe malaria.® Dispersible; artemether–lumefantrine 20 mg/120 mg, Novartis) with dosage determined by body weight, twice daily as recommended by National guidelines [https://www.sealedenvelope.com/). Randomization codes were placed inside individual sealed envelopes that were opened only by the nursing staff responsible for the administration of the drug. The remaining investigators were blind to the allocated intervention. All laboratory tests and statistical analyses were performed blinded to treatment group. Enrollment took place between February and March 2016.This was a prospective, parallel arm, unequally randomized, placebo-controlled, double-blind trial of rosiglitazone versus placebo, in 30 Mozambican children with uncomplicated malaria. All children received the Mozambican standard of care for uncomplicated malaria  (Ref. 230/CNBS/15), the pharmaceutical department of the Mozambican Ministry of Health (Ref. 374/380/DF2016), the Clinical Research Ethics Committee of the Hospital Clínic, Barcelona, Spain (Ref. HCB/2015/0981), and the University Health Network Research Ethics Committee, Toronto, Canada (UHN REB Number 15-9013-AE). All research was conducted according to the principles expressed in the Declaration of Helsinki. The trial was registered with ClinicalTrials.gov on 9 December 2015, NCT02694874. All participants and their parents/legal guardians were given detailed oral and written information about the trial, and children were recruited only after a written informed consent was signed by their parents/legal guardians. Verbal assent was obtained from children over the age of 8.Centro de Investigação em Saúde de Manhiça (CISM) at the Manhiça District Hospital (MDH), in southern Mozambique. A detailed description of CISM may be found elsewhere [P. falciparum by microscopy, whereby a thick blood film confirmed malaria infection with parasitaemia >2500 parasites/μL. Children were excluded if they were known to have any known pre-existing illness , presented any reason for hospitalization, or if they had clinical or laboratory evidence of severe malaria . Children receiving any therapy with potential anti-malarial activity (including cotrimoxazole), or treatment with a TZD were also excluded. Patients were approached after voluntarily presenting to MDH as part of routine care, and no financial incentives were provided.The trial was conducted by the lsewhere . In Mozalsewhere . Parents®, GlaxoSmithKline) and an identical looking placebo manufactured at the Hospital Clínic’s pharmacology department in Barcelona, Spain, were packaged and labelled to ensure blinding of study staff and hospital personnel. Children received either rosiglitazone (0.045 mg/kg/dose) or placebo twice daily for 4 days [Rosiglitazone  and serious adverse events (SAEs) were recorded and monitored throughout the study.Statistical analyses were performed with SPSS v.24 and Graph Pad Prism v.7. Differences between groups were assessed using the Fisher’s exact test for categorical demographic values, and by t test or Mann–Whitney U test (two-tailed) for clinical laboratory data based on the distribution of the data. Glucose measures at each time point were compared using a Mann–Whitney U test (two-tailed).Mann–Whitney U tests were used to compare the median changes from baseline for glucose, AST, ALT, haemoglobin and haematocrit between the two treatment arms. Mean haematocrit and haemoglobin values were compared using ANOVA. A p value <0.05 was considered as statistically significant.The trial’s flow diagram is shown in Fig. Treatment with rosiglitazone did not induce hypoglycaemia, and all glucose were above the lower blood glucose threshold of 2.5 mmol/L . These were observed at baseline in two children receiving placebo. They spontaneously normalized and were not associated with any clinical symptoms or signs. Median changes in AST levels between baseline and day 7 did not significantly differ between placebo and treatment arms . Nor was there a significant difference in median changes in ALT between placebo and rosiglitazone treated participants  between baseline and day 7. For the remaining biochemical parameters, including urea, creatinine, LDH, direct or indirect bilirubin, no significant differences or trending abnormalities were observed.Haematological adverse events were uncommon. Three children had a haemoglobin decrease >2 g/dL from their baseline values, and further two had a haemoglobin value below the 7 g/dL threshold; none were associated with clinical manifestations. Median changes in haemoglobin levels between baseline and day 14 did not significantly differ between the placebo and rosiglitazone arms . Median changes in haematocrit levels between baseline and day 14 did not significantly differ between the placebo and rosiglitazone arms . There was no significant difference between the treatment groups on haematocrit or haemoglobin levels (p = 0.13 and p = 0.12, respectively, Fig. Table Mean parasite densities at baseline and throughout the study were similar between groups, Fig. . Median In this study, the safety and tolerability of rosiglitazone as an adjunctive therapy for the treatment of uncomplicated malaria was investigated, as a first step before evaluating its efficacy in the treatment of severe paediatric malaria and prevention of malaria-associated neurocognitive complications. Although rosiglitazone has a well-established safety profile, and millions of doses have been administered to adults with type II diabetes mellitus , this waHypoglycaemia is a common and life-threatening complication of malaria , and druA second important aspect of this trial was to assess potential adverse cardiac events. Initial case–control studies of long-term use in elderly high-risk diabetic patients reported a potential increase in acute cardiac events ; howeverRosiglitazone has been reported to decrease mean haemoglobin and haematocrit in a dose-related fashion in adults, particularly when it is taken on a daily basis . This poRosiglitazone, malaria, and many anti-malarial drugs can be associated with increases in hepatic transaminases –44. In tThis study was not powered to evaluate efficacy endpoints of rosiglitazone. As such, the outcomes of the uncomplicated malaria participants recruited to evaluate the safety and tolerability of the drug, may not be sufficiently informative of the potential that the drug has to impact the course of disease. Although increase parasite clearance times were previously observed in adult patients randomized to rosiglitazone in a previous randomized control trial these patients received atovaquone/proguanil . Even wiThis is the first report of rosiglitazone use in African children with an acute uncomplicated malaria infection. The safety and tolerability results, including no vomiting, no idiosyncratic drug reactions, and no serious adverse events support its continued evaluation as an adjuvant therapy in the treatment of severe paediatric malaria.


Microscopic analysis of stained blood smear is the most suitable method of malaria diagnosis. However, gaps were observed among clinical laboratory professionals in microscopic diagnosis of malaria. A cross-sectional study was conducted in December 2015 among 46 laboratory professionals. Data was collected via on-site assessment and panel testing. The slide panel testing was composed of positive and negative slides. The kappa score was used to estimate the agreement between participants and reference reader. The overall agreement between the study participants and the reference reader in malaria detection was 79% (kappa = 0.62). Participating in refresher training on malaria microscopy ) and malaria epidemic investigation (AOR = 4.1 CI = 1.1–14.5) had statistical significant association with detection rate of malaria parasites. Laboratory professionals showed low performance in malaria microscopy. Most of the study participants were graded “in-training” in laboratory diagnosis of malaria. Malaria is the major cause of morbidity and mortality in the developing world in which 80% cases and 90% deaths reside in the African continent . In EthiOne of the main approaches of malaria control relays on early diagnosis followed by timely treatment. Owing to lack of specificity of signs and symptoms of malaria, the application of clinical diagnosis is only limited to areas lacking laboratory facilities for malaria diagnosis , 5. GiemHowever, little efforts were made so far to precisely determine and identify sources of error in microscopic diagnosis and quantification of parasitemia and to secure quality control program for malaria microscopy . ConsequTherefore, the current study was aimed at assessing the general laboratory logistics and performance of laboratory professionals on malaria microscopy in Tigray, North Ethiopia.Institution based cross-sectional study was conducted among selected laboratory service providers of Tigray from December 2015 to August 2016. The study area is located 586 kilometers far from the capital Addis Ababa. The mean annual temperature ranges from 16°C to 22°C and the altitude ranges from 1200 to 2400 meters above the sea level. The annual rain fall is 668 mm. During the time of data collection, there were a total of 61 laboratory professionals working in thirty-two governmental health centers and three hospitals. Most of the laboratories provide microscopic examination of urine sediment, blood film, stool, acid fast bacilli staining, and gram staining routinely. Malaria diagnosis is mainly based on blood film examination and Rapid Diagnostic Tests (RDTs) in the health posts. Laboratory professionals working at the selected health institutions on malaria microscopic diagnosis were the study population.z2p(1 − p)/d2) considering 95% confidence level, 0.5 proportion, assuming that malaria microscopy in 50% of the health care laboratories is below the standard and marginal error of (5%). Thus, a sample size of 384 was calculated. However, the expected total sample size in the selected target areas is less than the calculated sample size (N = 35); we adjusted our sample size based on the correction formula; n = n0/1 + (n0 − 1)/N. Therefore, a total sample size of 22 was calculated and all permanent/stable laboratory professionals engaged in malaria microscopy in the selected health institutions were involved.The sample size was determined using single population proportion estimate , and stained (using 3% Giemsa stain solution for 30 minutes). The stained blood film was washed with distilled water and air dried. Detection and species identification of the plasmodium parasites were made via thick and thin blood films, respectively.For the purpose of slide panel preparation, about 5 ml of venous blood was collected from confirmed malaria outpatients at Maytsebri Health Center. Negative slides on the other hand were prepared from apparently healthy persons who were also negative for malaria. The panel slides were prepared and validated by malaria microscopists at Tigray regional laboratory. Both thick and thin blood films were prepared. In the thick film preparation, three drops of blood were distributed over an area of 1 cm P. falciparum ++++, P. vivax +, P. falciparum +, and P. vivax +++ in thin thick blood films, respectively. Slide panel E possessed both P. falciparum and P. vivax. Slide panel G possessed malaria negative blood films. A total of 276 blood films (6 for each participant) were distributed. Study participants were then requested to report the detection of malaria , Plasmodium species identification , and grade parasitemia (semiquantitatively).The panel testing comprised six samples. Slide panels A, B, C, and D contained kappa score. Chi-square and logistic regression tests were employed to compare the performance of professionals with demographic variables and other factors. P value of <0.05 was considered statistical significant.Data were entered and analyzed using Statistical Package for Social Sciences (SPSS) version 20. Descriptive statistics were used to get summary measures. Performance of laboratory professionals on malaria microscopy was tested by computing the specificity, sensitivity, and negative and positive predictive values of their reports. Percent of agreement was determined byIt includes participants who scored ≥90% agreement with reference reader in the detection, speciation, and quantification of malaria parasites.It icludes participants who scored ≥80% but <90% agreement with reference reader in the detection, speciation, and quantification of malaria parasites.It includes participants who scored ≥70% but <80% agreement with reference reader in the detection, speciation, and quantification of malaria parasites.It includes participants who scored <70% agreement with reference reader in the detection, speciation, and quantification of malaria parasites based on the recommendation of the World Health Organization . P. falciparum .Major errors include (i) incorrect diagnosis of malaria, that is, reporting false positive or false negative results, (ii) not mentioning the presence of P. vivax and of a mixed infection (reporting single infection in case of mixed parasites) and (ii) reporting a parasite density differing one “+” from the reference result [Minor errors include (i) identification error ofe result .Ethical clearance was sought from the Ethics Review Committee of the College of Health Sciences at Mekelle University with the reference number (ERC055/2015). Informed written consent was secured from the participants and blood donors for slide panel preparation. Codes were provided for participants to maintain confidentiality of their report on malaria slides.A total of 46 clinical laboratory professionals were enrolled in the study. The mean age of the participants was 28.9 years. Males accounted for 32 (69.6%) of the participants. Majority of the participants served for less than five years in the diagnostic centers (50%). About 39% of the participants took refreshment trainings on malaria microscopy whereas more than half (58.7%) of the participants had participated in malaria epidemic investigations .Out of the total 22 laboratories inspected, 7 (30.4%), 10 (43.5%), and 5 (22.7%) had one, two, and three functional microscopes, respectively. Majority (52.2%) of laboratories employed Binocular with external light type of microscope.Most (87%) of the laboratories had guideline for preparation of working reagents. Giemsa stain was the only kind of stain being utilized for malaria microscopy. Similarly, all the clinical laboratories were utilizing new microscopic slides for individual patients . Most of the inspected laboratories (91.3%) were using semiquantitative (plus system) of quantification to count parasites . Drying The overall sensitivity, specificity, PPV and NPV of laboratory professionals in detection, and species identification of malaria parasites were 63% and 95.7%, 93.6% and 72.1%, and 78.3%, 95.7%, 94.4%, and 81.5%, respectively . Out of  Plasmodium falciparum (+) and only 6.5% and 2.2% of the participants correctly reported the species and parasite density, respectively. Mixed infections of Pf and Pv (Slide E) were correctly reported only by 15.2% of the participants. About 10% of laboratory professionals have reported negative slides (slide G) as false positive P. falciparum. Nearly 35% participants committed major error for falsely reporting P. vivax species in the case of P. falciparum infection. Similarly, about half of the participants in our study committed minor error in reporting mixed infections (Sixty-three percent of the participants correctly reported the species of slide A but only 10.9% of them correctly reported the parasite density (++++). Similarly 30.4% of the participants correctly reported the species of slide B; however, only 15.2% were able to report the parasites density. Slide C containsfections .k = 0.62), 87% (k = 0.58), and 65% (k = 0.55), respectively. The lowest sensitivity (21.7%) and agreement (59%) were observed on mixed infections. The detection rate of laboratory professionals was better for P. vivax (63%) than P. falciparum (56.5%).As depicted in On regression analysis, taking refresher training on malaria microscopy and participation in malaria epidemic investigation had significant association with proficiency of laboratory personnel on malaria microscopy. Laboratory personnel who took refresher training were 7 times better in detection and identification of malaria parasites compared to those who did not take refresher training . Similarly those laboratory personnel who participated in malaria epidemic investigation were 4.1 times better than those who did not participate in epidemic investigation in detection and identification of malaria parasites  .In Ethiopia diagnostic and treatment health facilities employed microscopic diagnosis of malaria using Giemsa stain. However, there is disparity in detection rate and speciation of malaria parasites among different health institutions which might be related with the qualification and experience of medical laboratory professionals. Therefore, this study showed the first performance evaluation of laboratory professionals on malaria microscopy in diagnostic centers of Tigray and needs to be considered by Federal Ministry of Health for future national study and action.This study showed the presence of adequate staining reagents, microscopic slides, drying racks, and guidelines for reagent preparation. However, only 58% and 39.1% of the participants participated in epidemic investigation and refresher trainings. This finding is in sharp contrast with previous study done in Northwest Ethiopia .The overall sensitivity and specificity of laboratory professional reports in detecting malaria parasites were 63% and 95.7%, respectively. The sensitivity was lower than findings from Hawassa (82%) , Addis A P. falciparum positive slides (43.5%) than P. vivax positive slides (37%). This contradicted a study from Hawassa, Ethiopia, where false negativity was higher in P. vivax [ P. falciparum in this study (43.5%) was higher than a report from Ethiopia (40.4%) [ P. falciparum identification could be due to the predominance of P. vivax in the area. The overall agreement between participants and reference readers in detection (K = 0.62), species identification (K = 0.58), and quantification of the plasmodium (K = 0.55) in the present study was inconsistent with previous reports from south Ethiopia [In this study, the proportion of false negativity was higher inP. vivax . Failure (40.4%) , Canada  (40.4%) , and USA (40.4%) . This hiEthiopia , Addis AEthiopia , and HonEthiopia . P. vivax monoinfection. It was in sharp contrast with previous studies [ P. falciparum monoinfection. This higher rate of failure in reporting mixed infections might be due to the fact that most study participants were not taking sufficient refreshment training on malaria microscopy, similar to that reported somewhere else in Ethiopia [Nearly half of the participants in the present study reported mixed infections wrongly as studies , 12 in wEthiopia , 12. TheEthiopia . P. falciparum identification was better at high density parasitemia (66.7%) slides than the lower density (50%). This was coherent with previous studies in other parts of Ethiopia [ P. falciparum parasites on high density parasitemia was lower than previous reports elsewhere [In this study lower agreement on parasite detection and identification of slides with low parasitic density and mixed infection were observed. Moreover, the performance of laboratory professionals inEthiopia , 12. Howlsewhere , 16.Participating in malaria epidemic investigation and refresher training on malaria microscopy were independently associated with a better performance of malaria microscopic diagnosis in our study. It is in line with study from Congo .The current study addressed the performance of clinical laboratory professionals on malaria microscopy using stained malaria slides. Hence the extent to which professionals stain malaria blood films was not assessed and it was taken as the main limitation of this study. P. falciparum parasitemia. Hence refreshing trainings should be delivered for the professionals periodically on malaria microscopy.Despite availability of consumables for malaria microscopy, professionals showed low performance in malaria microscopy. Based on WHO recommendation most of them in this study were graded “in-training” both in detection and in species identification of malaria parasites. Participants were better in species identification than in parasite detection. They showed the poorest agreement in the detection of mixed infections and low density


Plasmodium falciparum genetic diversity and multiplicity of infection (MOI) are parasite features that have been suggested to influence the acquisition of protective immunity against malaria. This study sought to assess the relationship between MOI and parasite density (PD) in malaria patients living in the Central Region of Ghana and to determine whether naturally occurring antibody levels against P. falciparum GLURP (PF3D7_1035300) and MSP3 (PF3D7_1035400) antigens are associated with decreased parasite load.P. falciparum malaria. Microscopy was used to estimate P. falciparum parasite density and polymerase chain reaction (PCR) amplification of the polymorphic regions of msp1 (PF3D7_0930300) and msp2 (PF3D7_0206800) was used for parasite genotyping and MOI determination. ELISA was used to measure the serum IgG concentration of R0 fragment of GLURP (GLURP(R0)) and MSP3 antibodies.Dried filter paper blood blots were obtained from children and adults diagnosed with uncomplicated P. falciparum by PCR using either the msp1 or msp2 genotyping primer sets. The most prevalent msp1 and msp2 alleles were KI and 3D7, respectively. The geometric mean (GM) for MOI determined by both msp1 and msp2 genotyping was 1.3 for the entire population and was generally higher in children than in adults. Seropositivity was estimated at 67 and 63% for GLURP(R0) and MSP3 antibodies, respectively, and antibody titers were negatively correlated with parasite density.All 115 samples were positive for The negative correlation between naturally occurring GLURP(R0) and MSP3 antibody levels and parasite density observed in this study suggest that augmenting the antibody response with the GMZ2 vaccine could enhance protection in the Central Region of Ghana.The online version of this article (doi:10.1186/s13071-017-2338-7) contains supplementary material, which is available to authorized users. P. falciparum parasites is likely to complicate disease control. This diversity within P. falciparum has served as a key survival mechanism for the parasite, as it allows for immune evasion as well as drug resistance [P. falciparum parasite genotypes, such as PfEMP1-DBL1α, have also been implicated with severe malaria [Plasmodium falciparum merozoite surface proteins 1 (MSP1: PF3D7_0930300) and 2 (MSP2: PF3D7_0206800) are widely used in the field for determining parasite diversity and multiplicity of infection. The msp1 gene has been divided into three allelic families namely K1, MAD20, and RO33 type based on the diversity within the Block 2 region of the gene sequence [msp2 gene is dimorphic [P. falciparum parasites [The 2015 annual report of the Ghana National Malaria Control Program reported that 39.2% of all the Outpatient Department cases recorded in the Central Region were due to malaria [sistance , 5. Spec malaria  and the  malaria , again s malaria . Merozoiimorphic  based onarasites .P. falciparum have also been implicated in the maintenance of strain-specific anti-merozoite antibodies. An increase in the number of FC27 alleles in an infection has been associated with increased antibody titres to the MSP2-Dd2  but not MSP2-Ch150/9  antigen [Infections of  antigen . To cove antigen . Multicl antigen , 14. It  antigen , as anti antigen .In addition to merozoite surface proteins, parasite antigens released when mature schizont-infected erythrocytes burst are also exposed to host immune defenses and one of these, GLURP(R0), is included in a current vaccine candidate GMZ2 –20. GMZ2Peripheral whole blood samples used for dried filter paper blood blots, thick and thin blood smears and harvesting of serum were collected from each participant as part of a larger study from three districts in the Central Region of Ghana . The hemTwo 3 mm punches (discs) of dried blood blots from the filter paper were used for gDNA extraction using the Saponin-Chelex extraction method . Brieflymsp1 allelic families  and the two msp2 allelic families (FC27 and 3D7), nested PCR was performed using family-specific primers [2, 133 nM of each forward and reverse primer, 0.5 units of One Taq DNA polymerase  and 4 μl (about 0.25 μl of whole blood) of genomic DNA (gDNA) template. The inner nested reaction mixture contained 200 nM dNTP, 1.8 mM MgCl2, 200 nM of each forward and reverse primer and 0.5 unit of One Taq DNA polymerase supplemented with 0.5 μl of the primary PCR product. The cycling parameters for the nested PCR consisted of an initial denaturation at 94 °C for 3 min, followed by 30 cycles at 94 °C for 1 min; 50–59 °C (optimized for each primer pair) for 35 s, and 68 °C for 2 min 30 s; with a final extension at 68 °C for 5 min. The PCR reactions were all run on a Biometra TAdvance thermal cycler . Positive gDNA controls for each allelic family  and no template negative control were included in each set of PCR reactions. PCR products were separated on 2% ethidium bromide-stained agarose gels and visualized under UV illumination.To distinguish between the three major  primers  shown inmsp1 or msp2 genotype reactions. Samples with more than one PCR fragment per marker (infecting genotype) were considered as containing multiclonal infections, whereas samples with only one infecting genotype for either marker were considered as clonal with respect to that marker. The geometric mean (GM) of MOI for both msp1 and msp2 genotyping was calculated by determining the GM for the sum of the MOI identified for each sample divided by the total number of samples positive for the same marker.The number of concurrent infecting genotypes contained in each sample, which represents the multiplicity of infection (MOI) for that sample, was calculated as the highest number of alleles obtained in the sample after either One hundred microliters of affinity purified GLURP(R0) , 29 or Mmsp1 and msp2 allelic family as well as the geometric mean and corresponding 95% confidence interval were calculated using frequency distribution in GraphPad Prism. The correlation between age and PD, GLURP(R0) and MSP3 IgG concentrations was performed using GraphPad Prism. SPSS descriptive statistics (cross tabs) was used to determine the descriptive statistics for the participants per study site  and analyzed using GraphPad Prism v7 and SPSS v.22 (IBM). The frequency of each P. falciparum parasite density (PD) was 7715  parasites/μl blood with a standard error of the mean of 3230. The GM of the hemoglobin concentrations within the population was 9.4 (95% CI: 9.0–9.8)  were from Assin Fosu, 36 (31%) from Twifo Praso and 32 (28%) from Abura Dunkwa , followed by MAD20, 38 (30.1%), and RO33 at 33 (26.2%). Similarly, from the 99 samples that yielded positive PCR fragments for the msp2 gene, a total of 52 samples contained parasites belonging to the FC27 allele family, of which 10 (19.2%) harbored double infections and thus resulted in a total of 62 PCR fragments . Seventy-six samples contained parasites belonging to the 3D7 allele family, with 9/76 (11.8%) of the samples harboring two distinct parasite populations thereby producing a total frequency of 85 PCR fragments (57.8%)  and 99 (86.1%) were successfully amplified for msp1 revealed that 62 samples produced a single PCR fragment, two PCR fragments were obtained from 29 samples and 6 samples produced three different PCR fragments   , but positively correlated with PD . The GM of MOI determined by both msp1 and msp2 genotyping generally decreased with increasing age .Combining the number of independent PCR fragments obtained for the three families, specific PCR amplifications for nts Fig. . The GM age Fig. . The over = 0.588, P < 0.0001). A significantly positive correlation was found to exist between age and IgG concentrations of both GLURP(R0)  and MSP3  antibodies (Table IgG concentrations of antibodies against MSP3 were significantly higher than those against GLURP(R0)  as well as GLURP(R0)  IgG concentrations. Seropositivity in this study was defined as serum antibody levels higher than the cutoff value, which is the mean of the antibody concentrations of the negative control plus 2 standard deviations. Applying the cutoff of 589 ng/μl and 3249 ng/μl for GLURP(R0) and MSP3, respectively; 67% of the participants were seropositive for GLURP(R0) and 63% for MSP3.In contrast, PD was negatively correlated with MSP3 (Spearman’s P = 0.008), but not GLURP(R0) antibodies , across the varying msp2 MOI categories. No significant association was found between either GLURP(R0)  or MSP3  IgG concentrations and MOI determined by msp1 genotyping.Although the GM of the MOI in this study was relatively low, a significant association was identified with IgG concentrations of MSP3  and MSP3) in a cohort of patients presenting to the hospital with uncomplicated malaria. The geometric mean (GM) of the parasite density (PD) in the study participants was 7715 parasites/μl  Fig. , previouThe negative correlation between naturally occurring GLURP(R0) and MSP3 antibody levels and parasite density observed in this study suggest that augmenting the antibody response with the GMZ2 vaccine, or other vaccines that stimulates an increased production of antibodies against GLURP(R0) and MSP3, could enhance protection in the Central Region of Ghana. However, diversity in candidate vaccine antigens could influence the effect and will have to be monitored prior to and during vaccine trials.Additional file 1: Table S1.Abbreviations: n, number of patients; GM, geometric mean; 95% CI, 95% confidence interval of the GM. Numbers in parenthesis represent the total number of patients. (DOCX 79 kb) Demographic features of the study participants per site and overall. Additional file 2: Figure S1.a) and GLURP(R0) (b) IgG seropositivity and Age (left y-axis) and PD (right y-axis). SPSS derived graphs showing the relationship between the mean Age (left y-axis) and PD/μl blood  of the study participants and IgG seropositivity to GLURP(R0) and MSP3. Error bars represent the standard error of the mean. 0, seronegative; 1, seropositive for MSP3 (a) and GLURP(R0) (b) antibodies. (DOCX 278 kb) Relationship between MSP3  and stratified patient age (b). (DOCX 131 kb) A graphical representation of the correlation between log transformed PD and patient age (


Falciparum malaria is most prevalent in the sub-Saharan African region, which harbors about 90% of all malaria cases and fatalities globally. Infection by the falciparum species often manifests with a spectrum of multi-organ complications , some of which are life-threatening. Spontaneous subdural empyema is a very rare complication of cerebral malaria that portends a very poor prognosis unless diagnosed and treated promptly. We report a case of spontaneous subdural empyema in a 58-year-old woman from Tanzania who presented with high-grade fever, decreased urine output, and altered sensorium.Malaria remains a significant public health problem of the tropical world.  Spontaneous subdural empyema (SDE), which refers to a collection of pus between the dura and arachnoid mater, is one of the unusual but highly fatal complication of Plasmodium falciparum malaria.2Malaria is a mosquito-borne disease of public health importance that threatens nearly one half of the global population.4 In this antibiotic era, SDE-related mortality is reportedly between 14% and 18%; however, in underprivileged nations mortality rate approaches 100%, similar to the pre-antibiotic era.5 Moreover, clinical presentation during admission is a good prognostic indicator, that is, the mortality rate is <10% in alert patients and rises to 75% in patients presenting with coma.6 We report a case of a 58-year-old woman from Tanzania who developed a spontaneous SDE following a high-parasitemia falciparum infection.Diagnosing spontaneous SDE in patients with cerebral malaria is often a dilemma, and if diagnosis and management is delayed, the risk of long-term neurological sequelae and death is very high.falciparum malaria , and the sequestered red cells contained the mature forms of the parasite (trophozoites and meronts). She was started on intravenous artesunate 240 mg at 0, 12, 24, and 48 hours, and intravenous clindamycin 600 mg twice daily for 3 days was also administered as per the current local guidelines for the management of severe falciparum malaria. However, due to clinical deterioration, she was referred to us on the fourth day of hospitalization in the private facility for expert management. There was no prior history of head injury or fall from a height, convulsions, or anticoagulant use. Furthermore, there was no history suggestive of recent sinusitis, rhinitis, or mastoiditis.A 58-year-old woman was referred to us from a private facility with a 6-day history of altered sensorium and high-grade fever. She has history of systemic hypertension for 8 years with poor medication adherence. She was diagnosed with high-parasitemia 4V2M3). Her blood pressure was 156/109 mm Hg, pulse rate was 116 beats/minute, random blood glucose was 12.2 mmol/L, and she weighed 90.3 kg. She had a reduced urine output (300 mL/24 hours) with elevation of urea  and creatinine . On her blood count, leukocyte concentration was 15.4 × 109/L (range = 4.5-10.5 × 109/L) with neutrophilia (75%), hemoglobin was 9.5g/dL (range = 12-16 g/dL), and she had thrombocytopenia . In liver panel, she had elevated aspartate transaminase  but normal alanine transaminase (38 IU/L), and both direct  and total bilirubin  were elevated. She tested negative for HIV and had normal electrolytes. Electrocardiogram revealed sinus tachycardia (rate 124) and ECHO showed mild left ventricular hypertrophy with good systolic function (ejection fraction 62%). Intravenous artesunate 240 mg was reinstated, and she received 3 units of platelet concentrate and was resumed on her antihypertensives . Hemodialysis was also started, and after 5 sessions, her renal functions returned to normal ; however, her altered mentation did not improve.On admission, she was febrile (39.2°C) with a Glasgow Coma Score (GCS) of 9/15 (EA brain magnetic resonance imaging (MRI) was then ordered, which revealed a bilaterally thin but widely spread subdural collection in the frontoparietal region  and 2. Afalciparum malaria is associated with numerous complications, including cerebral malaria, pulmonary edema, acute renal failure, severe anemia, and metabolic acidosis. Although these complications may have variable presentations depending on age and geographical location, their potential for rapid development and progression to death remain high.7 In majority of patients with severe malaria such complications coexist or rather evolve rapidly and thus the presence of one of them should warrant both searching for others and aggressive management.Severe 8 Hemorrhagic complications including subarachnoid hemorrhage, cerebral hemorrhage, subdural empyema, and subdural hematoma are potential but extremely rare sequelae of falciparum malaria.913 Furthermore, although focal neurological signs are not a typical feature in malaria presentation, its presence should raise an index of suspicion for a spontaneous SDE diagnosis. On extensive literature search we did not find any case of bilateral SDE associated with falciparum malaria. Nevertheless, prompt diagnosis coupled by initiation of broad-spectrum antibiotics is one single indicator of high importance for favorable outcome in SDE. In conclusion, the presence of focal neurological deficits in patients with severe falciparum malaria should be regarded as a neurosurgical emergency unless brain imaging indicates otherwise.Despite its rarity, subdural empyema accounts for about a third of focal intracranial infections with frontal sinusitis as the leading etiology.


The impact of malaria on global health has continually prompted the need to develop effective diagnostic strategies. In malaria endemic regions, routine diagnosis is hampered by technical and infrastructural challenges to laboratories. These laboratories lack standard facilities, expertise or diagnostic supplies; thus, therapy is administered based on clinical or self-diagnosis. There is the need for accurate diagnosis of malaria due to the continuous increase in the cost of medication, and the emergence and spread of drug resistant strains. However, the widely utilized Giemsa-stained microscopy and immunochromatographic tests for malaria are liable to several drawbacks, including inadequate sensitivity and false-positive outcomes. Alternative methods that offer improvements in performance are either expensive, have longer turnaround time or require a level of expertise that makes them unsuitable for point-of-care (POC) applications. These gaps necessitate exploration of more efficient detection techniques with the potential of POC applications, especially in resource-limited settings. This minireview discusses some of the recent trends and new approaches that are seeking to improve the clinical diagnosis of malaria. Plasmodium parasites or antigens in the peripheral blood of febrile patients and asymptomatic carriers. However, current techniques including microscopy and rapid diagnostic tests (RDTs) do not have satisfactory sensitivity for parasitemia. Alternative methods with superior performance are relatively expensive with low throughput; thus, rendering them unsuitable for routine use. Therefore, there is the need for the development of effective diagnostic strategies for field application, where diagnostic expertise in malaria is often lacking [The launch of several initiatives to eradicate malaria  has resu lacking ,7. New tThis minireview highlights some of the efforts seeking to improve or develop new diagnostic techniques for the detection and diagnosis of malaria. These include high throughput immunochemical assays, nucleic acid detection techniques, biomarker identification and biosensing approaches with or without microfluidic channels; these techniques allow for the identification of disease-specific biomarkers rather than the simple documentation of the presence of the pathogen.Plasmodium spp. include light and fluorescence microscopy, RDTs such as immunochromatographic lateral flow assays [Plasmodium spp. in Giemsa-stained microscopy or its antigens with an RDT.The diagnostic tools currently available for the identification of w assays , serologw assays . The algNew insights into disease specific markers have also been the hallmark of clinical malaria diagnostics. Diagnostic biomarkers can be genomic, transcriptomic, proteomic or metabolomic markers . In malaPlasmodium infections can be more accurately investigated with minimal inconvenience to patients. Trace amounts of malaria parasite DNA have been detected in urine and saliva from some malaria-infected individuals, however, it is unclear whether the amount of parasite DNA present in these body fluids is representative of the parasite load in peripheral blood [Almost all malaria diagnostic methods rely on blood or its products for the detection of the disease. In cases where repeated sampling is required, the invasive processes involved in specimen collection are frequently associated with poor compliance . Where sal blood .b gene (cytb) of parasites in matched blood, saliva and urine from malaria patients demonstrated a significant improvement in the diagnostic performance of cytb in saliva and urine over blood-film microscopy [According to Nwakanma et al., saliva is a promising non-invasive approach for detecting malaria infection . They recroscopy .P. falciparum Histidine-Rich Protein 2 (PfHRP2) showed moderate level of sensitivity when compared with blood-film microscopy in a normal field setting with a sensitivity of 83.75% and specificity of 83.48% [PfHRP2 concentration in the urine at the time of sampling [A recently developed Urine Malaria Test™ (UMT) dipstick that detects f 83.48% . The UMTf 83.48%  with a dsampling ,34,35. Tsampling .New molecular approaches to parasite detection seek to reduce cost, Therapeutic Turnaround Time (TTAT) and labor intensity, where the conventional nested 18S rRNA PCR assay has fallen short in clinical application. Despite being highly sensitive and specific, the nPCR assay is laborious, relatively time consuming and provides only qualitative data. These drawbacks make it unsuitable for routine first-line screening or field application especially in resource-limited settings. An ideal molecular assay must be low-cost and have the capacity to screen large number of samples with ease and within a short TTAT. Quantitative molecular methods such as real-time PCR have relatively short assay preparation and analysis time and as such, better suited for large-scale studies than nPCR assays. Moreover, they are relatively less expensive due to the use of small volumes of “low-cost” primer/fluorophore . A summaPlasmodium spp. is robust and much more cost-effective than nPCR [More recently, a transcription-based amplification system was developed for quantitative nucleic acid sequence-based amplification (QT-NASBA)  that tarhan nPCR ,37,46. TPlasmodium spp. [P. falciparum [P. vivax using a combination of pan-genus and P. falciparum specific LAMP primers [P. falciparum as a high-throughput technique [Plasmodium genus is 2.5 parasites/μL from whole blood and 25.0 parasites/μL from dried blood spots and is relatively less expensive per test. This offers a cost-effective molecular diagnostic platform in resource-limited settings. A combination of LAMP with a lateral flow device has been developed to target dihydrofolate reductase thymidylate synthase (dhfr-ts) genes of P. falciparum and P. vivax in blood or extracted DNA [Plasmodium spp. in blood samples from malaria-suspected patients [falciparum species. These performance characteristics have a diagnostic accuracy superior to microscopy and are comparable to nPCR [P. vivax α-tubulin was also developed using six primers that recognize the targeted gene at different regions [LAMP is the most widely researched isothermal diagnostic technology that has been used to identify all the human ium spp. ,53. A colciparum  and enab primers . Optimiz primers  has beenechnique . Its limcted DNA . It conscted DNA . Howeverpatients . It usespatients . The NIN to nPCR . The sel regions . The senBiosensors are self-contained analytical devices which can analyze complex matrices such as blood and urine without the need for additional processing steps or reagent addition, in contrast to other bioanalytical systems .P. falciparum infected red blood cells was immobilized onto the surface of a gold nanoparticle-modified screen-printed electrode. The assay showed good sensitivity with a linearity ranging from 102 cells/mL to 108 cells/mL [A cell based label-free electrochemical biosensor that detects parasitized red blood cells has been developed by Kumar et al. . In thiscells/mL . The usecells/mL .P. falciparum and P. vivax in infected blood [Ittarrat et al. developed a sensitive and specific DNA biosensor based on Quartz Crystal Microbalance (QCM) technology. The assay could significantly differentiate between ed blood . In theied blood .P. vivax lactate dehydrogenase (PvLDH) and P. falciparum lactate dehydrogenase (PfLDH). In the study, a single-stranded DNA aptamer for pLDH was used as a probe to selectively detect recombinant PvLDH and PfLDH in vitro with detection limits of 108.5 fM and 120.1 fM, respectively. In addition, the aptasensor could detect infected blood samples from non-infected blood with detection limit of 1 parasite/µL [A class of nucleic acid molecules called aptamers have also been utilized to recognize and bind target molecules with high specificity . Single-asite/µL . ClearlyThe use of a single disease marker is clearly of little use; thus, there is a continuing demand for diagnostic platforms that offer the potential of improved diagnostic accuracy and high-throughput capability. Multiplexed biomarker analysis enables TTAT, saves labor and offers the possibility of further miniaturization of the devices. For malaria, it is especially relevant in speciation of the infection which would guide treatment course , as wellPfHRP2 and pan-malarial aldolase to differentiate species in malaria infection. Nash et al. [N-isopropylacrylamide) (pNIPAm) could enrich and detect recombinant PfHRP2 and pan-malarial aldolase antigens [P. falciparum in blood donors with suspected malaria. Their findings established a high diagnostic performance with 95.8% specificity and 90.4% sensitivity of [Multiplexed detection systems have commonly used h et al.  have demantigens . A multiantigens , using aivity of . Given tAn attempt to ease the laborious burden of screening large samples against multiple antigens by enzyme immunoassays (EIA) saw Fouda and colleagues  implemenP. falciparum saliva antigens with the aim of assessing the exposure to parasites or bites from the vector [P. falciparum 11.1 gene (Pf11.1) were successfully implemented. Although such assays are more popular for the evaluation of immune responses to malarial antigens, their diagnostic application in non-endemic regions where antibody titers may be much lower in exposed persons merits further research.Ambrosino et al.  developee vector . In totaP. falciparum), AMA-1 gene (P. vivax) and the plasmepsin genes . The assay showed sensitivities of 95.8%, 89.5%, 94.1%, 100% and 100% for P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi, respectively, versus microscopically confirmed malaria cases. The assay had detection limits for the Plasmodium spp. in the range of 1 to 6 parasites/μL of blood with specificities from 98.6% to 100% [A high-throughput multiplex 5′ nuclease qPCR assay was developed by Reller et al.  to targe to 100% . Further to 100% .A significant challenge to the efforts towards the global elimination of malaria is the lack of POC diagnostic methods in endemic areas for detecting parasites in asymptomatic individuals, who are the reservoirs for transmission. Despite the evolution of diagnostic methods over the past few decades, microscopy and RDTs are still the most widely used techniques even though they have low sensitivity and specificity for malaria. These challenges associated with microscopy and RDTs warrants the exploration and implementation of molecular techniques as well as biosensing-based methods for more accurate detection, quantitation and POC application. Nucleic acid-based detection methods are highly sensitive but remain confined to research laboratories because they are expensive to run and maintain; thus, rendering them unsuitable for routine applications. The justifications for a multiplexed approach, in conjunction with biosensors, for malaria diagnosis is compelling, given the high fatality rates associated with mixed infections. Multiplexed testing strategies will improve detection rates and reduce the risk of outbreaks, as well as guide treatments. Biosensing technology has an additional advantage due its suitability for off-lab situations and is ideal for POC application. It is imperative that this technology is further exploited for the development of inexpensive, ultrasensitive and field-ready assays for malaria diagnosis.


Hypoglycaemia is a frequent complication among admitted children, particularly in malaria-endemic areas. This study aimed to estimate the occurrence of hypoglycaemia not only upon admission but throughout the first 72 h of hospitalization in children admitted with malaria.A simple pilot study to continuously monitor glycaemia in children aged 0–10 years, admitted with malaria in a rural hospital was conducted in Southern Mozambique by inserting continuous glucose monitors (CGMs) in subcutaneous tissue of the abdominal area, producing glycaemia readings every 5 min.Glucose was continuously monitored during a mean of 48 h, in 74 children. Continuous measurements of blood glucose were available for 72/74 children (97.3%). Sixty-five of them were admitted with density-specific malaria diagnosis criteria . Five children (7.7%) had hypoglycaemia (<54 mg/dL) on admission as detected by routine capillary determination. Analysing the data collected by the CGMs, hypoglycaemia episodes (<54 mg/dL) were detected in 10/65 (15.4%) of the children, of which 7 (10.8%) could be classified as severe (≤45 mg/dL). No risk factors were independently associated with the presence of at least one episode of hypoglycaemia (<54 mg/dL) during hospitalization. Only one death occurred among a normoglycaemic child. All episodes of hypoglycaemia detected by CGMs were subclinical episodes or not perceived by caregivers or clinical staff.Hypoglycaemia beyond admission in children with malaria appears to be much more frequent than what had been previously described. The clinical relevance of these episodes of hypoglycaemia in the medium or long term remains to be determined. Hypoglycaemia is a common problem in paediatric emergency admissions in Africa, and is associated with a wide variety of disorders and diseases. In descriptive studies conducted in different countries of Africa, the prevalence of hypoglycaemia on admission ranged between 3.2 and 7.3% of the paediatric patients –6. The pPlasmodium parasite, hyperinsulinism caused by quinine (whenever used), and lack of adequate supplementation/oral intake in cases of severe malaria, especially in cerebral malaria [In patients with malaria, decreased levels of glycaemia are common and secondary to the consumption of glucose by the  malaria , 12, 13. malaria , 14, 15.The studies conducted to date in children admitted with malaria performed their occasional glycaemia assessments using glucometers that need a finger prick each time the glucose level must be assessed, or relied on the analysis of venous blood samples. These methods only allow limited number of glycaemia assessments during the day. However the evolution of blood glucose is dynamic and hypoglycaemia may occur and be undetected in severely ill patients in the time lapse between two assessments, even after presenting normal blood glucose levels on admission.To improve monitoring of blood glucose, continuous glucose monitors (CGMs), initially developed for a more timely control of adult and paediatric patients suffering from type 1 diabetes, , 17, havUsing the CGMs during the initial and most critical days of admission, could allow a better detection of hypoglycaemia episodes, and a comprehensive description of the dynamics and evolution of blood glucose. This would help establishing some preliminary risk factors for hypoglycaemia during the first days of illness, and help to detect those patients who could most benefit from additional supplementation and a more thorough control to improve prognosis during hospitalization.A pilot study was conducted using CGMs to continuously assess blood glycaemia and aimed to determine the occurrence of hypoglycaemia episodes detected by these devices in children admitted with malaria, trying to identify risk factors associated with the development of intra-admission hypoglycaemic episodes.This study was conducted in Manhiça, southern Mozambique. Manhiça is a semi-rural, savannah setting with a predominantly young population (19.1% are <5 years of age) . SeasonaThe Manhiça Health Research Center (CISM) is a leading research centre in Africa with a well-developed clinical laboratory supporting the Manhiça District Hospital (MDH). The CISM runs a demographic surveillance system linked to a morbidity surveillance platform established at the Manhiça district Hospital and five additional primary health centres. A detailed description of CISM and the study area can be found elsewhere .This prospective pilot study, conducted at the Manhiça District Hospital during a period of 9 months (1st September 2013 to 31st May 2014), recruited paediatric patients diagnosed with malaria in order to estimate the real occurrence of hypoglycaemia not only upon admission but also throughout the first 72 h of hospitalization. Children aged ≤10 years and fulfilling criteria for admission because of malaria diagnosis and living in the study area, were eligible to be included in the study if guardians agreed.Plasmodium falciparum parasitaemia, thick and thin blood films are prepared, processed and double read in the CISM laboratory according to standard procedures [Hospital surveillance data are routinely collected for all children less than 15 years old visiting the outpatient clinics in the study area and those admitted to the MDH. Clinical data, including medical history, physical examination, routine laboratory basic investigations, ICD-10 based diagnosis, outcome and medication prescribed are collected on questionnaires on a round the clock basis and reviewed daily by senior medical staff before being entered in specific databases. On admission, a finger-prick blood sample is collected to determine PCV and blood glucose concentration. In order to quantify ocedures . Slides ocedures  to guideHIV status information is not routinely collected. An HIV rapid diagnostic test or other molecular diagnostic methods for those <18 months of age is performed to those children with suspected HIV infection.Malaria is defined at MDH as a positive slide regardless of parasitaemia. In order to increase sensitivity and specificity of malaria case definition, we used an age cut-off for the level of parasitaemia based on previous malariometric indicators published for the Manhiça District , 25. ThuInternamento de curta duração, ICD), where trained people were working. Recruitment was based on the first reading of the blood slide and all children with presence of parasites, fulfilling inclusion criteria when the devices were available were asked to participate in the study. A standardized study specific questionnaire was completed at the moment of recruitment. Demographic, nutritional and clinical data such as vital signs and anthropometry were also recorded. Medication, food and liquids other than water intakes, and events such as vomiting and diarrhoea were daily recorded during the time the CGM was inserted.Recruitment occurred during the working hours (07.00 am–07.00 pm) in the “Short stay admission” ward  for continuous glucose monitoring for 72 h starting upon admission. Children enrolled were connected to the Medtronic® devices after the signature of an informed consent (IC) by the child’s guardian. The CGMS is composed by the ENLITE sensor (glucose sensor) and the Mini Link transmitter (wireless transmitter), which does not offer real-time information.We aimed to use the CGMs  every 6 h (4 times a day). Correction of glucose levels was performed when necessary following WHO guidelines with an intravenous bolus of 5 mL/kg of 10% dextrose which has been the standard of care at MDH(7).Values recorded through the continuous monitoring software were downloaded via Care Link™ after sensor removal. Data obtained with the CGMs were compared with those obtained by capillary blood glucometer (CBG). We preferred a finger-prick method, as a comparison to CBG, rather than the more accurate laboratory method because the first is the most used in clinical practice at the MDH.Statistical analyses were performed using StataCorp. 2015. Stata Statistical Software: Release 14. College Station, TX: StataCorp LP. Descriptive statistics were provided for all variables in the dataset, including frequency tabulations for binary/categorical variables and mean and standard deviation for normal distributed continuous variables and median and interquartile range (IQR) for non-normal continuous variables.p value of <0.05 and Confidence intervals (CI) at 95% level.Firth logistic regression was used in order to address issues of separability, small sample sizes and bias of the parameter estimates. Only children fulfilling malaria criteria according to our study definition were included in the analysis. A multivariate logistic regression with penalized likelihood analysis was performed among children fulfilling the age and density-specific malaria diagnosis criteria to assess risk factors for having hypoglycaemia during the admission measured by the CGM, including those variables which showed an association with having hypoglycaemia in the univariate analysis, based on their size of effect (OR <0.5 or >1.5). Statistical significance was set at a Paired glucose readings from the continuous glucose monitor (when available) and the standard capillary assessment  were compared using a modified Bland–Altman analysis to measure agreement with repeated measures and a Clarke error grid analysis (CEG). The CEG identified five areas with different error in accuracy combined with the severity of clinical consequences:Region A: values within 20% of the reference sensor.Region B: values outside 20% of the reference sensor, but that would not lead to an inappropriate treatment.Region C: values leading to an unnecessary treatment.Region D: values indicating a potentially dangerous failure to detect hypo- or hyper-glycaemia.Region E: values that would confuse treatment of hypoglycaemia for hyperglycaemia, and vice versa.These graphs were produced using R .A total of 74 children with a positive blood slide in the first reading and admitted because of malaria at MDH were enrolled in the study from September 2013 to May 2014  age on admission was 31.5 (5–84) months. Thirty-three children of 74 were male (44.6%) and 22/74 (29.7%) fulfilled the criteria for being considered severe malaria cases. Causes of admission of children recruited in the study are summarized in Table The devices were well tolerated in all patients and no adverse skin reactions, infections, or bleeding in the insertion site occurred during the study period Fig. . No patiOn admission, blood glycaemia was measured by capillary assessment. Five of sixty-five (7.7%) children presented hypoglycaemia <54 mg/dL and three of these episodes (4.6%) were categorized as severe (<45 mg/dL). Hyperglycaemia was detected in 1/65 (1.5%) patients.Analysing the data collected by the CGM, hypoglycaemia (<54 mg/dL) was detected in 10 of the 65 children (15.4%). Seven of these ten hypoglycaemia episodes (70.0%) were categorized as severe (<45 mg/dL). Six of seventy-two (8.3%) had recurrent (more than one) hypoglycaemia (<54 mg/dL) episodes detected by the CGM. All episodes of hypoglycaemia <54 mg/dL detected by CGMs were subclinical or “non-witnessed” episodes with no development of further complications. Mean (SD) time between CGMs insertion and sensor activation was 82.1 (9.3) minutes. Hypoglycaemic episodes (<54 mg/dL) were nocturne in 5/10 children with hypoglycaemia (50.0%) and the mean (SD) duration of fasting preceding the hypoglycaemic episode was 5.5 h (1.2). Hyperglycaemia was detected in 11/65 (15.9%) of the patients, occurring in 4/10 (36.4%) in the first 2 h after food ingestion.Table The multivariate analysis showed no risk factors independently associated with the presence of at least one episode of any level of hypoglycaemia during the admission Table . Four riWe used data from 72 patients where CGM measurements were available to assess agreement between both methods of measuring blood glucose. It was possible to obtain 474 paired CGM-CBG measurements. Figure Figure This is the first study conducted in a low-income country using CGMs in a rural district hospital. Additionally, it is the first study assessing glucose homeostasis thorough CGMs in children with malaria. Continuous glucose monitoring has been advocated for many years to improve glycaemic management in critical care settings , 20, amoA previous study conducted in the same hospital demonstrated that malaria was a strong predictor of hypoglycaemia  . Since tUsing CGMs, at least one episode of hypoglycaemia <54 mg/dL) was detected in 10/65 children 15.4%) and in 7/65 10.8%) using <45 mg/dL threshold, confirming our pre-defined hypothesis that hypoglycaemia in patients with malaria occurs much more frequently than previously described. Only a few studies have explored blood glucose levels beyond admission and throughout hospitalization .4% and i, 37 and 4 mg/dL w% using <P. falciparum malaria during a 16-h period of controlled fasting. During the following 8 h after controlled fasting, plasma glucose concentrations decreased and no episodes of hypoglycaemia were described. In the same study, a linear regression analysis using a mixed linear model was performed and showed children with non-severe malaria would develop hypoglycaemia after 26 h of controlled fasting and the children with severe malaria would develop hypoglycemia after 33 h of controlled fasting (p 0.036) [CGMs were not useful to measure glycaemia at the time of admission, since the sensors started, as expected , to detep 0.036) . It is lSurprisingly, higher axillary temperature was more common among those children whose glycaemia remained normal in the univariate analysis. This finding needs to be interpreted with caution, as no adjustment for multiplicity of testing has been performed. No risk factors independently associated with having at least one episode of any level of hypoglycaemia were found in the multivariate analysis. Caution is however needed when interpreting these results, since sample size was small and likely insufficient to detect significant differences among both glycaemia groups.Only one child died in this cohort. It has been demonstrated that malaria is a risk factor to develop hypoglycaemia but it is not a predictor of death because of hypoglycaemia . A previversus children with malaria) and/or the use of different glucometers.The CEG and modified Bland–Altman analyses for all glucose levels demonstrated that the CGM provides similar readings to the CBG. Indeed, 98% of all readings in the CEG were included in the safe A or B regions and there was no evidence of an increase of the measurements into zones , that would lead to inadequate management of hyper- or hypo-glycaemia, as has been previously reported in a study conducted in preterm newborns . The Bla® device with values obtained from different blood glucose tests and found great correlation between the two methods. However, the interpretation of results in a different population and with different interventional glucose levels needs to be cautious [The CEG was initially used to test the accuracy of blood glucose measurement in adults or children with diabetes . Two precautious .This study has several limitations. First, the study lacked sufficient statistical power to detect significant associations between hypoglycaemia and its determinants, given the small sample size. Second, as the number of devices was limited, recruitment could not be performed continuously, jeopardizing the representativeness of these results. Third, the CGM system has proven adequate and robust for hyperglycaemia determination , but itsP. falciparum malaria, because many more hypoglycaemia episodes seem to occur, in spite of their poor clinical translation. A higher sample size would be needed to help establishing some preliminary risk factors for hypoglycaemia during the first days of illness, and help detecting those patients who could most benefit from additional supplementation and a more thorough control to improve prognosis during hospitalization.Although the use of CGM appears insufficiently cost-effective in resource-constrained settings, these data suggest that the CGM is a clinically accurate instrument to evaluate blood glucose levels in children with malaria. Moreover, these data suggest that hypoglycaemic events detected by conventional glucometers may be only the tip of the iceberg of the real burden of hypoglycaemia in children admitted with