Reversible host cell surface remodelling limits immune recognition and maximizes transmission of Plasmodium falciparum gametocytes.

Reducing malaria transmission has been a major pillar of control programmes and is considered crucial for achieving malaria elimination. Gametocytes, the transmissible forms of the P. falciparum parasite, arise during the blood stage of the parasite and develop through 5 morphologically distinct stages. Immature gametocytes (stage I-IV) sequester and develop in the extravascular niche of the bone marrow and possibly spleen. Only mature stage V gametocytes re-enter peripheral circulation to be taken up by mosquitoes for successful onward transmission. We have recently shown that immature, but not mature gametocytes are targets of host immune responses and identified putative target surface antigens. We hypothesize that these antigens play a role in gametocyte sequestration and contribute to acquired transmission-reducing immunity. Here we demonstrate that surface antigen expression, serum reactivity by human IgG, and opsonic phagocytosis by macrophages all show similar dynamics during gametocyte maturation, i.e., on in immature and off in mature gametocytes. Moreover, the switch in surface reactivity coincides with reversal in phosphatidylserine (PS) surface exposure, a marker for red blood cell age and clearance. PS is exposed on the surface of immature gametocytes (as well as in late asexual stages) but is removed from the surface in later gametocyte stages (IV-V). Using parasite reverse genetics and drug perturbations, we confirm that parasite protein export into the host cell and phospholipid scramblase activity are required for the observed surface modifications in asexual and sexual P. falciparum stages. These findings suggest that the dynamic surface remodelling allows (i) immature gametocyte sequestration in bone marrow and (ii) mature gametocyte release into peripheral circulation and immune evasion, therefore contributing to mature gametocyte survival in vivo and onward transmission to mosquitoes. Importantly, blocking scramblase activity during gametocyte maturation results in efficient clearance of mature gametocytes, revealing a potential path for transmission blocking interventions. Our studies have important implications for our understanding of parasite biology and form a starting point for novel intervention strategies to simultaneously reduce parasite burden and transmission. IMPORTANT: Manuscripts submitted to Review Commons are peer reviewed in a journal-agnostic way.Upon transfer of the peer reviewed preprint to a journal, the referee reports will be available in full to the handling editor.The identity of the referees will NOT be communicated to the authors unless the reviewers choose to sign their report.The identity of the referee will be confidentially disclosed to any affiliate journals to which the manuscript is transferred. GUIDELINES: For reviewers: https://www.reviewcommons.org/reviewers For authors: https://www.reviewcommons.org/authors. CONTACT: The Review Commons office can be contacted directly at: office@reviewcommons.org.

Pro-thrombotic autoantibodies targeting Platelet Factor 4/polyanion are associated with pediatric cerebral malaria.

BACKGROUND: Features of consumptive coagulopathy and thromboinflammation are prominent in cerebral malaria (CM). We hypothesized that thrombogenic autoantibodies contribute to a procoagulant state in CM. METHODS: Plasma from children with uncomplicated malaria (UM, n = 124) and CM (n = 136) was analyzed by ELISA for a panel of 8 autoantibodies including anti-Platelet Factor 4/polyanion (anti-PF4/P), anti-Phospholipid, anti-Phosphatidylserine, anti-Myeloperoxidase, anti-Proteinase 3, anti-dsDNA, anti-Beta-2-Glycoprotein I (ß2GPI), and anti-Cardiolipin. Non-malaria coma (NMC, n = 49) and healthy controls (HC, n = 56) were assayed for comparison. Associations with clinical and immune biomarkers were determined using univariate and logistic regression analyses. RESULTS: Median anti-PF4/P and anti-PS IgG levels were elevated with malaria infection relative to HC (P < 0.001) and NMC (PF4/P: P < 0.001). Anti-PF4/P IgG levels were elevated in CM (median = 0.27, IQR: 0.19-0.41) compared to UM (median = 0.19, IQR: 0.14-0.22, P ≤ 0.0001). Anti-PS IgG levels did not differ between UM and CM (P = 0.39). When CM cases were stratified by malaria retinopathy (Ret) status, levels of anti-PF4/P IgG correlated negatively with peripheral platelet count in Ret+ CM cases (Rs = 0.201, P = 0.04) and associated positively with mortality (OR = 15.2, 95% CI: 1.02-275, P = 0.048). Plasma from CM patients induced a greater platelet activation capacity in an ex-vivo assay relative to plasma from UM patients (P = 0.02). Platelet activation was associated with anti-PF4/P IgG levels (Rs = 0.293, P = 0.035). CONCLUSIONS: Thrombosis mediated by elevated anti-PF4/P autoantibodies may be one mechanism contributing to the clinical complications of CM.

Protocol for a magnetic resonance imaging study of participants in the fever RCT: Does fever control prevent brain injury in malaria?

BACKGROUND: Despite eradication efforts, ~135,000 African children sustained brain injuries as a result of central nervous system (CNS) malaria in 2021. Newer antimalarial medications rapidly clear peripheral parasitemia and improve survival, but mortality remains high with no associated decline in post-malaria neurologic injury. A randomized controlled trial of aggressive antipyretic therapy with acetaminophen and ibuprofen (Fever RCT) for malarial fevers being conducted in Malawi and Zambia began enrollment in 2019. We propose to use neuroimaging in the context of the RCT to further evaluate neuroprotective effects of aggressive antipyretic therapy. METHODS: This observational magnetic resonance imaging (MRI) ancillary study will obtain neuroimaging and neurodevelopmental and behavioral outcomes in children previously enrolled in the Fever RCT at 1- and 12-months post discharge. Analysis will compare the odds of any brain injury between the aggressive antipyretic therapy and usual care groups based upon MRI structural abnormalities. For children unable to undergo imaging without deep sedation, neurodevelopmental and behavioral outcomes will be used to identify brain injury. DISCUSSION: Neuroimaging is a well-established, valid proxy for neurological outcomes after brain injury in pediatric CNS malaria. This MRI ancillary study will add value to the Fever RCT by determining if treatment with aggressive antipyretic therapy is neuroprotective in CNS malaria. It may also help elucidate the underlying mechanism(s) of neuroprotection and expand upon FEVER RCT safety assessments.

Genotyping Plasmodium falciparum gametocytes using amplicon deep sequencing.

BACKGROUND: Understanding the dynamics of gametocyte production in polyclonal Plasmodium falciparum infections requires a genotyping method that detects distinct gametocyte clones and estimates their relative frequencies. Here, a marker was identified and evaluated to genotype P. falciparum mature gametocytes using amplicon deep sequencing. METHODS: A data set of polymorphic regions of the P. falciparum genome was mined to identify a gametocyte genotyping marker. To assess marker resolution, the number of unique haplotypes in the marker region was estimated from 95 Malawian P. falciparum whole genome sequences. Specificity of the marker for detection of mature gametocytes was evaluated using reverse transcription-polymerase chain reaction of RNA extracted from NF54 mature gametocytes and rings from a non-gametocyte-producing strain of P. falciparum. Amplicon deep sequencing was performed on experimental mixtures of mature gametocytes from two distinct parasite clones, as well as gametocyte-positive P. falciparum field isolates to evaluate the quantitative ability and determine the limit of detection of the genotyping approach. RESULTS: A 400 bp region of the pfs230 gene was identified as a gametocyte genotyping marker. A larger number of unique haplotypes was observed at the pfs230 marker (34) compared to the sera-2 (18) and ama-1 (14) markers in field isolates from Malawi. RNA and DNA genotyping accurately estimated gametocyte and total parasite clone frequencies when evaluating agreement between expected and observed haplotype frequencies in gametocyte mixtures, with concordance correlation coefficients of 0.97 [95% CI: 0.92-0.99] and 0.92 [95% CI: 0.83-0.97], respectively. The detection limit of the genotyping method for male gametocytes was 0.41 pfmget transcripts/µl [95% CI: 0.28-0.72] and for female gametocytes was 1.98 ccp4 transcripts/µl [95% CI: 1.35-3.68]. CONCLUSIONS: A region of the pfs230 gene was identified as a marker to genotype P. falciparum gametocytes. Amplicon deep sequencing of this marker can be used to estimate the number and relative frequency of parasite clones among mature gametocytes within P. falciparum infections. This gametocyte genotyping marker will be an important tool for studies aimed at understanding dynamics of gametocyte production in polyclonal P. falciparum infections.

Optic nerve sheath diameter and its association with brain swelling in pediatric cerebral malaria: a retrospective study.

Introduction: Mortality in pediatric cerebral malaria (CM) in low- and middle-income countries (LMICs) is associated with brain swelling on magnetic resonance imaging (MRI); however, MRI is unavailable in most LMICs. Optic nerve sheath diameter (ONSD) measurement is an inexpensive method of detecting increased intracranial pressure compared with the invasive opening pressure (OP). Our primary objective was to determine if increased ONSD correlated with brain swelling on MRI in pediatric CM. Our secondary objective was to determine if increased ONSD correlated with increased OP and/or poor neurological outcome in pediatric CM. We hypothesized that increased ONSD would correlate with brain swelling on MRI and increased OP and that ONSD would be higher in survivors with sequelae and non-survivors. Methods: We performed a retrospective chart review of children aged 0-12 years in Blantyre, Malawi, from 2013 to 2022 with CM as defined by the World Health Organization. Brain swelling on admission MRI was characterized by brain volume scores (BVS); severe swelling was scored as 7-8, mild-to-moderate as 4-6, normal as 3. The admission ONSD was measured via ultrasound; it was defined as abnormal if it was >4.5 mm in children >1 year and >4 mm in children <1 year. Favorable outcome was defined as a normal neurological exam on discharge in survivors. The primary and secondary objectives were evaluated using Spearman's correlation; and the demographics were compared using chi-square and the Kruskal-Wallis test (Stata, College Station, TX, USA). Results: Median age of the 207-patients cohort was 50 months [interquartile range (IQR) 35-75]; 49% (n = 102) were female. Of those, 73% (n = 152) had a favorable outcome, and 14% (n = 30) died. Twenty-nine (14%) had a normal BVS, 134 (65%) had mild-to-moderate swelling, and 44 (21%) had severe swelling. ONSD was elevated in 86% (n = 178) of patients, while 12% of patients had increased OP. There was a weakly positive correlation between BVS and ONSD (r = 0.14, p = 0.05). The median ONSD was not significantly different compared by discharge outcome (p = 0.11) or by BVS (p = 0.18). Conclusion: ONSD was not a reliable tool to correlate with BVS, neurological outcome, or OP in children with CM. Future studies to identify alternative methods of early identification of CM patients at highest risk for morbidity and mortality are urgently needed.

SARS-CoV-2 Seroprevalence and Vaccine Uptake among Pregnant Women at First Antenatal Care Visits in Malawi.

Many SARS-CoV-2 infections are asymptomatic, thus reported cases underestimate actual cases. To improve estimates, we conducted surveillance for SARS-CoV-2 seroprevalence among pregnant women attending their first antenatal care visit (ANC1) from June 2021 through May 2022. We administered a questionnaire to collect demographic, risk factors, and COVID-19 vaccine status information and tested dried blood spots for SARS-CoV-2 antibodies. Although <1% of ANC1 participants reported having had COVID-19, monthly SARS-CoV-2 seroprevalence increased from 15.4% (95% CI: 10.5-21.5) in June 2021 to 65.5% (95% CI: 55.5-73.7) in May 2022. Although COVID-19 vaccination was available in March 2021, uptake remained low, reaching a maximum of 9.5% (95% CI: 5.7-14.8) in May 2022. Results of ANC1 serosurveillance provided prevalence estimates helpful in understanding this population case burden that was available through self-report and national case reports. To improve vaccine uptake, efforts to address fears and misconceptions regarding COVID-19 vaccines are needed.

Rapid diagnosis and prognosis of malaria infection using a microfluidic point-of-care immunoassay.

Malaria is a major cause of illness and death worldwide. Rapid diagnostic tests are the most widely used tool for detecting malaria infection, however, they only provide binary results and lack the sensitivity needed to detect many asymptomatic infections. Molecular assays for quantifying malaria biomarkers offer higher detection sensitivity, however, they are time-consuming, and require expert training and expensive equipment, making them unsuitable for use in most of Africa. To address the need for simple, accurate and field-deployable malaria diagnostic tests, we have developed a microfluidic point-of-care (mPOC) immunoassay for rapid quantification of Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a malaria parasite biomarker, in whole blood. This device features two diagnostic modes for detecting PfHRP2 at low (100's pg/mL) and high (1,000's ng/mL) concentrations, making it useful for multiple diagnostic applications, including the detection of asymptomatic infection, prediction of disease outcomes and diagnosis of cerebral malaria. Measurements of PfHRP2 in blood samples from malaria patients demonstrates that this platform offers similar accuracy as an ultra-sensitive PfHRP2 enzyme-linked immunosorbent assay (ELISA) test, while being 12× faster and simpler to use. This mPOC immunoassay can be deployed in rural health centers to assist clinicians in diagnosing and triaging malaria patients, ultimately improving patient outcomes.

Post hospital admission blood lactate measurements are associated with mortality but not neurologic morbidity in children with cerebral malaria.

BACKGROUND: In children with cerebral malaria (CM) admission blood lactate has previously guided intravenous fluid therapy and been validated as a prognostic biomarker associated with death. The usefulness of post-admission measurements of blood lactate in children with CM is less clear. The strength of association between blood lactate and neurological sequelae in CM survivors, as well as the optimal duration of post-admission measurements of blood lactate to identify children at higher risk of adverse outcomes is unknown. METHODS: A retrospective cohort study of 1674 Malawian children with CM hospitalized from 2000 to 2018 who had blood lactate measurements every 6 h for the first 24 h after admission was performed. The strength of association between admission lactate or values measured at any time point in the first 24 h post-admission and outcomes (mortality and neurological morbidity in survivors) was estimated. The duration of time after admission that lactate remained a valid prognostic biomarker was assessed. RESULTS: When lactate is analysed as a continuous variable, children with CM who have higher values at admission have a 1.05-fold higher odds (95% CI 0.99-1.11) of death compared to those with lower lactate values. Children with higher blood lactate at 6 h have 1.16-fold higher odds (95% CI 1.09-1.23) of death, compared to those with lower values. If lactate levels are dichotomized into hyperlactataemic (lactate > 5.0 mmol/L) or not, the strength of association between admission lactate and mortality increases (OR = 2.49, 95% CI 1.47-4.22). Blood lactate levels obtained after 18 h post-admission are not associated with outcomes. Similarly, the change in lactate concentrations through time during the first 24 h of hospital admission is not associated with outcomes. Blood lactate during hospitalization is not associated with adverse neurologic outcomes in CM survivors. CONCLUSIONS: In children with CM, blood lactate is associated with death but not neurologic morbidity in survivors. To comprehensively estimate prognosis, blood lactate in children with CM should be assessed at admission and for 18 h afterwards.

MRI-Based Brain Volume Scoring in Cerebral Malaria Is Externally Valid and Applicable to Lower-Resolution Images.

BACKGROUND AND PURPOSE: Children with cerebral malaria have an elevated risk of mortality and neurologic morbidity. Both mortality and morbidity are associated with initially increased brain volume on MR imaging, as graded by the Brain Volume Score, a subjective ordinal rating scale created specifically for brain MRIs in children with cerebral malaria. For the Brain Volume Score to be more widely clinically useful, we aimed to determine its independent reproducibility and whether it can be applicable to lower-resolution MRIs. MATERIALS AND METHODS: To assess the independent reproducibility of the Brain Volume Score, radiologists not associated with the initial study were trained to score MRIs from a new cohort of patients with cerebral malaria. These scores were then compared with survival and neurologic outcomes. To assess the applicability to lower-resolution MRI, we assigned Brain Volume Scores to brain MRIs degraded to simulate a very-low-field (64 mT) portable scanner and compared these with the original scores assigned to the original nondegraded MRIs. RESULTS: Brain Volume Scores on the new cohort of patients with cerebral malaria were highly associated with outcomes (OR for mortality = 16, P < .001). Scoring of the simulated degraded images remained consistent with the Brain Volume Scores assigned to the original higher-quality (0.35 T) images (intraclass coefficients > 0.86). CONCLUSIONS: Our findings demonstrate that the Brain Volume Score is externally valid in reproducibly predicting outcomes and can be reliably assigned to lower-resolution images.

Cytolytic circumsporozoite-specific memory CD4+ T cell clones are expanded during Plasmodium falciparum infection.

Clinical immunity against Plasmodium falciparum infection develops in residents of malaria endemic regions, manifesting in reduced clinical symptoms during infection and in protection against severe disease but the mechanisms are not fully understood. Here, we compare the cellular and humoral immune response of clinically immune (0-1 episode over 18 months) and susceptible (at least 3 episodes) during a mild episode of Pf malaria infection in a malaria endemic region of Malawi, by analysing peripheral blood samples using high dimensional mass cytometry (CyTOF), spectral flow cytometry and single-cell transcriptomic analyses. In the clinically immune, we find increased proportions of circulating follicular helper T cells and classical monocytes, while the humoral immune response shows characteristic age-related differences in the protected. Presence of memory CD4+ T cell clones with a strong cytolytic ZEB2+ T helper 1 effector signature, sharing identical T cell receptor clonotypes and recognizing the Pf-derived circumsporozoite protein (CSP) antigen are found in the blood of the Pf-infected participants gaining protection. Moreover, in clinically protected participants, ZEB2+ memory CD4+ T cells express lower level of inhibitory and chemotactic receptors. We thus propose that clonally expanded ZEB2+ CSP-specific cytolytic memory CD4+ Th1 cells may contribute to clinical immunity against the sporozoite and liver-stage Pf malaria.

Cerebral Metabolic Crisis in Pediatric Cerebral Malaria.

Cerebral metabolic energy crisis (CMEC), often defined as a cerebrospinal fluid (CSF) lactate: pyruvate ratio (LPR) >40, occurs in various diseases and is associated with poor neurologic outcomes. Cerebral malaria (CM) causes significant mortality and neurodisability in children worldwide. Multiple factors that could lead to CMEC are plausible in these patients, but its frequency has not been explored. Fifty-three children with CM were enrolled and underwent analysis of CSF lactate and pyruvate levels. All 53 patients met criteria for a CMEC (median CSF LPR of 72.9 [interquartile range [IQR]: 58.5-93.3]). Half of children met criteria for an ischemic CMEC (median LPR of 85 [IQR: 73-184]) and half met criteria for a nonischemic CMEC (median LPR of 60 [IQR: 54-79]. Children also underwent transcranial doppler ultrasound investigation. Cerebral blood flow velocities were more likely to meet diagnostic criteria for low flow (<2 standard deviation from normal) or vasospasm in children with an ischemic CMEC (73%) than in children with a nonischemic CMEC (20%, p = 0.04). Children with an ischemic CMEC had poorer outcomes (pediatric cerebral performance category of 3-6) than those with a nonischemic CMEC (46 vs. 22%, p = 0.03). CMEC was ubiquitous in this patient population and the processes underlying the two subtypes (ischemic and nonischemic) may represent targets for future adjunctive therapies.

Protocol for a Magnetic Resonance Imaging (MRI) Study of Participants in the Fever Randomized Controlled Trial: Does fever control prevent brain injury in malaria?

Background: Despite eradication efforts, ~135,000 African children sustained brain injuries as a result of central nervous system (CNS) malaria in 2021. Newer antimalarial medications rapidly clear peripheral parasitemia and improve survival, but mortality remains high with no associated decline in post-malaria neurologic injury. A randomized controlled trial of aggressive antipyretic therapy with acetaminophen and ibuprofen (Fever RCT) for malarial fevers being conducted in Malawi and Zambia began enrollment in 2019. We propose to use neuroimaging in the context of the RCT to further evaluate neuroprotective effects of aggressive antipyretic therapy. Methods: This observational magnetic resonance imaging (MRI) ancillary study will obtain neuroimaging and neurodevelopmental and behavioral outcomes in children previously enrolled in the Fever RCT at 1- and 12-months post discharge. Analysis will compare the odds of any brain injury between the aggressive antipyretic therapy and usual care groups based upon MRI structural abnormalities. For children unable to undergo imaging without deep sedation, neurodevelopmental and behavioral outcomes will be used to identify brain injury. Discussion: Neuroimaging is a well-established, valid proxy for neurological outcomes after brain injury in pediatric CNS malaria. This MRI ancillary study will add value to the Fever RCT by determining if treatment with aggressive antipyretic therapy is neuroprotective in CNS malaria. It may also help elucidate the underlying mechanism(s) of neuroprotection and expand upon FEVER RCT safety assessments.

Predicting Acute and Post-Recovery Outcomes in Cerebral Malaria and Other Comas by Optical Coherence Tomography (OCT in CM) - A protocol for an observational cohort study of Malawian children.

Cerebral malaria (CM) remains a significant global health challenge with high morbidity and mortality. Malarial retinopathy has been shown to be diagnostically and prognostically significant in the assessment of CM. The major mechanism of death in paediatric CM is brain swelling. Long term morbidity is typically characterised by neurological and neurodevelopmental sequelae. Optical coherence tomography can be used to quantify papilloedema and macular ischaemia, identified as hyperreflectivity. Here we describe a protocol to test the hypotheses that quantification of optic nerve head swelling using optical coherence tomography can identify severe brain swelling in CM, and that quantification of hyperreflectivity in the macula predicts neurodevelopmental outcomes post-recovery. Additionally, our protocol includes the development of a novel, low-cost, handheld optical coherence tomography machine and artificial intelligence tools to assist in image analysis.

Increased brain microvascular hemoglobin concentrations in children with cerebral malaria.

Brain swelling is associated with death from cerebral malaria, but it is unclear whether brain swelling is caused by cerebral edema or vascular congestion-two pathological conditions with distinct effects on tissue hemoglobin concentrations. We used near-infrared spectroscopy (NIRS) to noninvasively study cerebral microvascular hemoglobin concentrations in 46 Malawian children with cerebral malaria. Cerebral malaria was defined by the presence of the malaria parasite Plasmodium falciparum on a blood smear, a Blantyre coma score of 2 or less, and retinopathy. Children with uncomplicated malaria (n = 33) and healthy children (n = 29) were enrolled as comparators. Cerebral microvascular hemoglobin concentrations were higher among children with cerebral malaria compared with those with uncomplicated malaria [median (25th, 75th): 145.2 (95.2, 190.0) µM versus 82.9 (65.7, 105.4) µM, P = 0.008]. Cerebral microvascular hemoglobin concentrations correlated with brain swelling score determined by MRI (r = 0.37, P = 0.03). Fluctuations in cerebral microvascular hemoglobin concentrations over a 30-min time period were characterized using detrended fluctuation analysis (DFA). DFA determined self-similarity of the cerebral microvascular hemoglobin concentration signal to be lower among children with cerebral malaria compared with those with uncomplicated malaria [0.63 (0.54, 0.70) versus 0.91 (0.82, 0.94), P < 0.0001]. The lower self-similarity of the hemoglobin concentration signal in children with cerebral malaria suggested impaired regulation of cerebral blood flow. The elevated cerebral tissue hemoglobin concentration and its correlation with brain swelling suggested that excess blood volume, potentially due to vascular congestion, may contribute to brain swelling in cerebral malaria.

Differential Effects of Antimalarial Drugs on Parasite Clearance Rates Are Reflected by Plasmodium falciparum Ring Ratio.

Background: The location of Plasmodium falciparum within the body is determined by the life cycle of the parasite; young rings are in the peripheral blood, whereas mature parasites are sequestered in deep tissues. We can calculate a "ring ratio," the proportion of parasites in the periphery to the total number of parasites in the body. Artesunate acts on all parasite life stages, whereas quinine is effective only on sequestered parasites. Children with cerebral malaria (CM) treated with artesunate clear parasites faster than those treated with quinine. In this study, we established the relationship between ring ratio and parasite clearance rate and used the ring ratio to determine if the benefit derived from artesunate treatment could be attributed to its broader effect on life cycle stages. Methods: Ring ratios were calculated for 400 hospitalized children with CM in Blantyre, Malawi between 2010 and 2019 (quinine: 2010-2013, artesunate: 2014-2019). Results: In both treatment groups, parasite clearance rates were positively associated with the ring ratios, with a stronger association in the artesunate era than the quinine era. In the quinine era, an increase of 1-unit log10 difference between parasitemia and plasma P falciparum histidine-rich protein 2 (a proxy for ring ratio) resulted in a 0.27-unit increase in the parasite clearance rate, whereas in the artesunate era an equal increase resulted in a 0.41-unit increase (P = .04 for the difference). Conclusions: This analysis provides in vivo evidence supporting the hypothesis that more rapid parasite clearance rates in artesunate recipients are due to its superiority over quinine in killing ring-stage parasites.

Temporal Trends of Blood Glucose in Children with Cerebral Malaria.

Hypoglycemia, defined as a blood glucose < 2.2 mmol/L, is associated with death in pediatric cerebral malaria (CM). The optimal duration of glucose monitoring in CM is unknown. We collected data from 1,674 hospitalized Malawian children with CM to evaluate the association between hypoglycemia and death or neurologic disability in survivors. We assessed the optimal duration of routine periodic measurements of blood glucose. Children with hypoglycemia at admission had a 2.87-fold higher odds (95% CI: 1.35-6.09) of death and, if they survived, a 3.21-fold greater odds (95% CI: 1.51-6.86) of sequelae at hospital discharge. If hypoglycemia was detected at 6 hours but not at admission, there was a 7.27-fold higher odds of death (95% CI: 1.85-8.56). The presence of newly developed hypoglycemia after admission was not independently associated with neurological sequelae in CM survivors. Among all new episodes of blood sugar below a treatment threshold of 3.0 mmol/L, 94.7% occurred within 24 hours of admission. In those with blood sugar below 3.0 mmol/L in the first 24 hours, low blood sugar persisted or recurred for up to 42 hours. Hypoglycemia at admission or 6 hours afterward is strongly associated with mortality in CM. Children with CM should have 24 hours of post-admission blood glucose measurements. If a blood glucose less than the treatment threshold of 3.0 mmol/L is not detected, routine assessments may cease. Children who have blood sugar values below the treatment threshold detected within the first 24 hours should continue to have periodic glucose measurements for 48 hours post-admission.

The ability of Interleukin-10 to negate haemozoin-related pro-inflammatory effects has the potential to restore impaired macrophage function associated with malaria infection.

BACKGROUND: Although pro-inflammatory cytokines are involved in the clearance of Plasmodium falciparum during the early stages of the infection, increased levels of these cytokines have been implicated in the pathogenesis of severe malaria. Amongst various parasite-derived inducers of inflammation, the malarial pigment haemozoin (Hz), which accumulates in monocytes, macrophages and other immune cells during infection, has been shown to significantly contribute to dysregulation of the normal inflammatory cascades. METHODS: The direct effect of Hz-loading on cytokine production by monocytes and the indirect effect of Hz on cytokine production by myeloid cells was investigated during acute malaria and convalescence using archived plasma samples from studies investigating P. falciparum malaria pathogenesis in Malawian subjects. Further, the possible inhibitory effect of IL-10 on Hz-loaded cells was examined, and the proportion of cytokine-producing T-cells and monocytes during acute malaria and in convalescence was characterized. RESULTS: Hz contributed towards an increase in the production of inflammatory cytokines, such as Interferon Gamma (IFN-γ), Tumor Necrosis Factor (TNF) and Interleukin 2 (IL-2) by various cells. In contrast, the cytokine IL-10 was observed to have a dose-dependent suppressive effect on the production of TNF among other cytokines. Cerebral malaria (CM) was characterized by impaired monocyte functions, which normalized in convalescence. CM was also characterized by reduced levels of IFN-γ-producing T cell subsets, and reduced expression of immune recognition receptors HLA-DR and CD 86, which also normalized in convalescence. However, CM and other clinical malaria groups were characterized by significantly higher plasma levels of pro-inflammatory cytokines than healthy controls, implicating anti-inflammatory cytokines in balancing the immune response. CONCLUSIONS: Acute CM was characterized by elevated plasma levels of pro-inflammatory cytokines and chemokines but lower proportions of cytokine-producing T-cells and monocytes that normalize during convalescence. IL-10 is also shown to have the potential to indirectly prevent excessive inflammation. Cytokine production dysregulated by the accumulation of Hz appears to impair the balance of the immune response to malaria and exacerbates pathology.

Retinal imaging technologies in cerebral malaria: a systematic review.

BACKGROUND: Cerebral malaria (CM) continues to present a major health challenge, particularly in sub-Saharan Africa. CM is associated with a characteristic malarial retinopathy (MR) with diagnostic and prognostic significance. Advances in retinal imaging have allowed researchers to better characterize the changes seen in MR and to make inferences about the pathophysiology of the disease. The study aimed to explore the role of retinal imaging in diagnosis and prognostication in CM; establish insights into pathophysiology of CM from retinal imaging; establish future research directions. METHODS: The literature was systematically reviewed using the African Index Medicus, MEDLINE, Scopus and Web of Science databases. A total of 35 full texts were included in the final analysis. The descriptive nature of the included studies and heterogeneity precluded meta-analysis. RESULTS: Available research clearly shows retinal imaging is useful both as a clinical tool for the assessment of CM and as a scientific instrument to aid the understanding of the condition. Modalities which can be performed at the bedside, such as fundus photography and optical coherence tomography, are best positioned to take advantage of artificial intelligence-assisted image analysis, unlocking the clinical potential of retinal imaging for real-time diagnosis in low-resource environments where extensively trained clinicians may be few in number, and for guiding adjunctive therapies as they develop. CONCLUSIONS: Further research into retinal imaging technologies in CM is justified. In particular, co-ordinated interdisciplinary work shows promise in unpicking the pathophysiology of a complex disease.

Viral, Bacterial, Metabolic, and Autoimmune Causes of Severe Acute Encephalopathy in Sub-Saharan Africa: A Multicenter Cohort Study.

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.