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In malaria endemic areas, a high proportion of children have detectable parasitemia but show no clinical symptoms. When comatose from a cause other than malaria, this group confounds the cerebral malaria (CM) definition, making accurate diagnosis challenging. One important biomarker of CM is malarial retinopathy, a set of specific features visible in the ocular fundus. In this study, we quantified the contribution of malarial retinopathy in discriminating malaria-caused coma from non–malaria-caused coma. We estimated that 10% of our study cohort of N = 1,192 patients who met the WHO clinical definition of CM in Malawi had non-malarial coma based on a Gaussian mixture model using the parasite protein Plasmodium falciparum histidine-rich protein-2. A classification based on platelets, white blood cells, and retinopathy significantly improved the discriminative power of a previously established model including only platelets plus white blood cells (area under the receiver operating characteristic curve: 0.89 versus 0.75, P value < 0.001). We conclude that malarial retinopathy is highly predictive of malaria-caused versus non–malaria-caused coma and recommend that an ocular funduscopic examination to determine malarial retinopathy status be included in the assessment of parasitemic comatose African children.

Malaria remains a threat to public health in Malawi. It is well acknowledged that malaria research and robust evidence can have an impact on malaria policy and practice, resulting in positive population health gains. We report policy-relevant research contributions that the Malawi International Center of Excellence for Malaria Research (ICEMR) in partnership with local and international collaborators has made. Findings from our ICEMR studies have shown that long-lasting insecticide-treated bed nets (LLINs) impregnated with piperonyl butoxide reduced mosquito blood feeding more compared with conventional LLINs. On the other hand, we showed that few LLINs are maintained up to the end of their 3-year life span, and that older nets are less effective. These results support the policy change decisions by the Malawi National Malaria Control Program to switch from conventional LLINs to piperonyl butoxide LLINs, and to conduct mass LLIN distribution campaigns every 2 years. Our studies on epidemiological patterns of malaria infection showed that school-age children have higher malaria infection rates and lower use of control measures compared with younger children and adults. These findings added to the evidence base that influenced the National Malaria Control Program to endorse school-based malaria interventions as part of its national policy. Research supported by the Malawi ICEMR is contributing to in-country policy decisions and to the implementation of evidence-based interventions. Through our long-term studies we intend to continue providing practical and policy-relevant evidence necessary, ultimately, to eliminate malaria infection in Malawi.

BackgroundCerebral malaria (CM) is a rare, but severe and frequently fatal outcome of infection with Plasmodium falciparum. Pathogenetic mechanisms include endothelial activation and sequestration of parasitized erythrocytes in the cerebral microvessels. Increased concentrations of glycosaminoglycans in urine and plasma of malaria patients have been described, suggesting involvement of endothelial glycocalyx.MethodsWe used lectin histochemistry on postmortem samples to compare the distribution of multiple sugar epitopes on cerebral capillaries in children who died from CM and from nonmalarial comas.ResultsN-acetyl glucosamine residues detected by tomato lectin are generally reduced in children with CM compared to controls. We used the vascular expression of intercellular adhesion molecule 1 and mannose residues on brain capillaries of CM as evidence of local vascular inflammation, and both were expressed more highly in CM patients than controls. Sialic acid residues were found to be significantly reduced in patients with CM. By contrast, the levels of other sugar epitopes regularly detected on the cerebral vasculature were unchanged, and this suggests specific remodeling of cerebral microvessels in CM patients.ConclusionsOur findings support and expand upon earlier reports of disruptions of the endothelial glycocalyx in children with severe malaria.

Objectives: To assess whether viral, bacterial, metabolic, and autoimmune diseases are missed by conventional diagnostics among children with severe acute encephalopathy in sub-Saharan Africa. Study design: One hundred thirty-four children (6 months to 18 years) presenting with nontraumatic coma or convulsive status epilepticus to 1 of 4 medical referral centers in Uganda, Malawi, and Rwanda were enrolled between 2015 and 2016. Locally available diagnostic tests could be supplemented in 117 patients by viral, bacterial, and 16s quantitative polymerase chain reaction testing, metagenomics, untargeted metabolomics, and autoimmune immunohistochemistry screening. Results: Fourteen (12%) cases of viral encephalopathies, 8 (7%) cases of bacterial central nervous system (CNS) infections, and 4 (4%) cases of inherited metabolic disorders (IMDs) were newly identified by additional diagnostic testing as the most likely cause of encephalopathy. No confirmed cases of autoimmune encephalitis were found. Patients for whom additional diagnostic testing aided causal evaluation (aOR 3.59, 90% CI 1.57-8.36), patients with a viral CNS infection (aOR 7.91, 90% CI 2.49-30.07), and patients with an IMD (aOR 9.10, 90% CI 1.37-110.45) were at increased risk for poor outcome of disease. Conclusions: Viral and bacterial CNS infections and IMDs are prevalent causes of severe acute encephalopathy in children in Uganda, Malawi, and Rwanda that are missed by conventional diagnostics and are associated with poor outcome of disease. Improved diagnostic capacity may increase diagnostic yield and might improve outcome of disease.

Background Cerebral malaria is still a major cause of death in children in sub-Saharan Africa. Among survivors, debilitating neurological sequelae can leave children with permanent cognitive impairments and societal stigma, resulting in taxing repercussions for their families. This study investigated the effect of delay in presentation to medical care on outcome in children with cerebral malaria in Malawi. Methods This retrospective study included participants enrolled in a longstanding study of cerebral malaria between 2001 and 2021 and considered coma duration prior to arrival at hospital (with or without anti-malarial treatment), HIV status, blood lactate levels at admission and age as factors that could affect clinical outcome. Outcomes were categorized as full recovery, sequelae at the time of discharge, or death. A multinomial regression was fit and run controlling for coma duration, HIV status, lactate levels and age, to determine the association between each explanatory variable and outcome. Results A total of 1663 children with cerebral malaria, aged 6 months to 14 years were included. Longer coma duration (in hours) was associated with greater odds of developing sequelae (OR = 1.023, 95% CI 1.007–1.039, p = 0.006) but not death (OR = 1.00, 95% CI 0.986–1.015, p = 0.961). Younger age (in months) was also correlated with higher rates of sequelae, (OR = 0.990, 95% CI 0.983–0.997, p = 0.004) but not with increased mortality (OR = 0.998, 95% CI 0.993–1.003, p = 0.335). Blood lactate levels on admission were correlated with mortality (OR = 1.125, 95% CI 1.090–1.161, p Conclusions In Malawian children with cerebral malaria, higher rates of sequelae were significantly associated with extended coma duration prior to admission and younger age. Mortality rates were correlated with increased lactate levels on admission. The differential effects of variables on clinical outcomes suggest that there may be different pathogenic pathways leading to sequelae and death. Actions taken by parents and health care professionals are critical in defining when patients arrive at hospital and determining their ultimate outcome.

Malaria resulted in an estimated 627,000 deaths in 2020, the majority of which occurred in children under 5 years of age. Cerebral malaria (CM) is a severe manifestation of the disease with case fatality rates of up to 40%. Autopsies in children with CM have demonstrated sequestration of Plasmodium falciparum parasites in the brain as well as multiple other organs. Thus, multiple organ dysfunction syndrome (MODS) may be present in pediatric patients with CM, but its frequency and association with mortality have not been evaluated. This is a retrospective study of data collected prospectively from children with CM admitted in Blantyre, Malawi. Physical examination findings and laboratory values necessary to calculate a Pediatric Logistic Organ Dysfunction–2 (PELOD-2) score, a validated method that quantifies organ dysfunction in critically ill children, were abstracted. A total of 145 patients were included. Mortality was 15% (n = 22). Ten patients (7%) had single organ dysfunction, 36 (25%) had two organs involved, 68 (47%) had dysfunction of three organs, and 31 (21%) patients had four organs affected. Beyond neurologic dysfunction, other organ systems involved included hematologic (77%), renal (61%), cardiovascular (44%), and respiratory (1%). The median PELOD-2 score on admission was 4 (interquartile range [IQR] = 3–6) in survivors and 6.5 (IQR = 5–10) in the nonsurvivors (P

Background We explored a metabolic etiology of cerebral malaria (CM) coma. Methods Plasma metabolites were compared between Malawian children with CM and mild Plasmodium falciparum malaria. A candidate molecule was further studied in animal models of malaria. Results Clinically abnormal concentrations of pipecolic acid (PA) were present in CM plasma, and nearly normal in mild malaria samples. PA is renally cleared and the elevated PA blood levels were associated with renal insufficiency, which was present only in CM subjects. Prior studies demonstrate that PA has neuromodulatory effects and is generated by malaria parasites. PA brain levels in Plasmodium berghei ANKA–infected animals in the experimental cerebral malaria (ECM) model inversely correlated with normal behavior and correlated with blood-brain barrier (BBB) permeability. Mice infected with malaria species that do not induce neurological abnormalities or manifest BBB permeability had elevated plasma PA levels similar to ECM plasma at 7 days postinfection; however, they had low PA levels in the brain compared to ECM mice brains at 7 days postinfection. Conclusions Our model suggests that malaria-generated PA induces coma in CM and in ECM. The role of BBB permeability and the mechanisms of PA neuromodulation in CM will require additional investigation.

Background: Non-traumatic coma is a common acute childhood presentation to healthcare facilities in Africa and is associated with high morbidity and mortality. Historically, the majority of cases were attributed to cerebral malaria (CM). With the recent drastic reduction in malaria incidence, non-malarial coma is becoming a larger proportion of cases and determining the aetiology is diagnostically challenging, particularly in resource-limited settings. The purpose of this study will be to evaluate the aetiology and prognosis of non-traumatic coma in African children.Methods: From inception onwards, systematic searches of MEDLINE, Embase, and Scopus, will identify prospective and retrospective studies (including randomised controlled trials, cluster randomised trials, cohort studies, cross-sectional, and case-control studies) recruiting children (1 month-16 years) with non-traumatic coma (defined by Blantyre Coma Score ≤2 or comparable alternative) from any African country. Disease-specific studies will be included given that coma is associated and reported. The primary outcome is to determine the aetiology (infectious and non-infectious) of non-traumatic coma in African children, with pooled prevalence estimates of causes (e.g. malaria). Secondary outcomes are to determine overall estimates of morbidity and mortality of all cause non-traumatic coma and disease-specific states of non-traumatic coma, where available. Random effects meta-analysis will summarize aetiology data and in-hospital and post-discharge mortality. Heterogeneity will be quantified with τ2, I2, and Cochran’s Q test.Discussion: This systematic review will provide a summary of the best available evidence on the aetiology and outcome of non-traumatic coma in African children. Systematic Review Registration: This review is registered in PROSPERO International Prospective Register of Systematic Reviews (CRD42020141937).

The recent decline in global malaria burden has stimulated efforts toward Plasmodium falciparum elimination. Understanding the biology of malaria transmission stages may provide opportunities to reduce or prevent onward transmission to mosquitoes. Immature P. falciparum transmission stages, termed stages I to IV gametocytes, sequester in human bone marrow before release into the circulation as mature stage V gametocytes. This process likely involves interactions between host receptors and potentially immunogenic adhesins on the infected red blood cell (iRBC) surface. Here, we developed a flow cytometry assay to examine immune recognition of live gametocytes of different developmental stages by naturally exposed Malawians. We identified strong antibody recognition of the earliest immature gametocyte-iRBCs (giRBCs) but not mature stage V giRBCs. Candidate surface antigens (n = 30), most of them shared between asexual- and gametocyte-iRBCs, were identified by mass spectrometry and mouse immunizations, as well as correlations between responses by protein microarray and flow cytometry. Naturally acquired responses to a subset of candidate antigens were associated with reduced asexual and gametocyte density, and plasma samples from malaria-infected individuals were able to induce immune clearance of giRBCs in vitro. Infected RBC surface expression of select candidate antigens was validated using specific antibodies, and genetic analysis revealed a subset with minimal variation across strains. Our data demonstrate that humoral immune responses to immature giRBCs and shared iRBC antigens are naturally acquired after malaria exposure. These humoral immune responses may have consequences for malaria transmission potential by clearing developing gametocytes, which could be leveraged for malaria intervention.

We have previously demonstrated the efficacy of 6-diazo-5-oxo-L-norleucine (DON), a glutamine analog, as a possible adjunctive therapy to mitigate the clinical severity of human cerebral malaria (HCM) (Gordon et al. PNAS 2016). The pathology of HCM is heterogeneous, but generally includes the accumulation of infected red blood cells in the brain vascular endothelium, cerebral microhemorrhages, compromised blood-brain barrier and brain swelling. These changes manifest in severe neurologic symptoms including headache, fever, ataxia, seizures, retinopathy, and coma. The mouse model of CM, experimental cerebral malaria (ECM), approximates the main characteristics of HCM when susceptible C57BL/6 mice are infected with Plasmodium berghei ANKA (PbA) parasites. Studies in these mice found that treatment with DON, even in late stage ECM, rescued animals and increased survival while mitigating and reversing blood-brain barrier dysfunction and brain swelling. Recently, Seydel et al. (NEJM 2015) used magnetic resonance imaging (MRI) to characterize HCM pathology and correlate it to disease severity in children in Malawi. They observed increased brain volume in children that died from HCM that was uncommon in children that survived HCM. Herein, we perform MRI studies in the mouse model to determine if the disease course effectively recapitulates that of HCM, based on morphological changes in MRI. These studies may help elucidate the mechanisms underlying CM pathology, aiding the development of effective treatments. Furthermore, we utilize MRI to probe the mechanisms of action of recovery with DON treatment in order to evaluate the drug for possible clinical trials in HCM.

We studied the effects of anti-CD4 treatment of diabetic ACI rats on the induction of tolerance to allogeneic (Lewis) islet allografts. When given as a 4-day treatment regimen, OX38, a mouse anti-rat CD4 antibody, caused depletion of greater than 80% of CD4+ cells from the peripheral blood of treated rats. After induction of diabetes (a single high-dose bolus of streptozocin) and 3 days after the initiation of anti-CD4 immunotherapy, recipient ACI rats were transplanted with fully allogeneic (Lewis) islets of Langerhans via the portal circulation. These transplanted islets were capable of returning the anti-CD4-treated ACI recipients to normoglycemia, which was maintained indefinitely in the absence of further immunosuppression. In contrast, treatment of recipient rats with OX8, an anti-CD8 monoclonal antibody (MoAb), induced only a slight prolongation of graft survival (less than or equal to 30 days). Further characterization of the cellular requirements for the induction of long-term transplantation survival revealed that successful pretransplantation anti-CD4 therapy could be ablated by the coincident treatment of recipient rats with depleting levels of anti-CD8 MoAb. These data point to the necessity of a regulator CD8+ cell in the induction of anti-CD4-mediated transplantation survival in this rat model of islet transplantation.

The penetration of rifampicin into CSF was studied in seven patients who had undergone external ventriculostomy for occlusive hydrocephalus without major disturbance of the blood-CSF barrier. After the first dose of rifampicin 600 mg i.v. over 3 h, blood and CSF concentrations were determined serially by HPLC. Peak CSF concentrations obtained 0-8 h (median = 1 h) after the end of the infusion ranged from 0.57 to 1.24 mg/L (median = 0.73 mg/L). Elimination from CSF was slower than from serum (T1/2 beta CSF: 9.1-21.0 h (median = 14.5 h, n = 5); T1/2 beta serum: 2.2-5.8 h (median = 3.6 h, n = 7)). Based on the ratios of the areas under the concentration-time curves in CSF and serum, the overall penetration of rifampicin into CSF was 0.13-0.42 (median = 0.22). These results demonstrate effective CSF penetration and favourable pharmacokinetics of rifampicin in the absence of meningeal inflammation. They support the use of rifampicin as part of a combination therapy not only for tuberculosis of the central nervous system (CNS), but also for staphylococcal and listerial infections of the CNS in which there may be little meningeal inflammation.

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