Common PIEZO1 Allele in African Populations Causes RBC Dehydration and Attenuates Plasmodium Infection.

Hereditary xerocytosis is thought to be a rare genetic condition characterized by red blood cell (RBC) dehydration with mild hemolysis. RBC dehydration is linked to reduced Plasmodium infection in vitro; however, the role of RBC dehydration in protection against malaria in vivo is unknown. Most cases of hereditary xerocytosis are associated with gain-of-function mutations in PIEZO1, a mechanically activated ion channel. We engineered a mouse model of hereditary xerocytosis and show that Plasmodium infection fails to cause experimental cerebral malaria in these mice due to the action of Piezo1 in RBCs and in T cells. Remarkably, we identified a novel human gain-of-function PIEZO1 allele, E756del, present in a third of the African population. RBCs from individuals carrying this allele are dehydrated and display reduced Plasmodium infection in vitro. The existence of a gain-of-function PIEZO1 at such high frequencies is surprising and suggests an association with malaria resistance.

Interplay between liver and blood stages of Plasmodium infection dictates malaria severity via γδ T cells and IL-17-promoted stress erythropoiesis.

Plasmodium replicates within the liver prior to reaching the bloodstream and infecting red blood cells. Because clinical manifestations of malaria only arise during the blood stage of infection, a perception exists that liver infection does not impact disease pathology. By developing a murine model where the liver and blood stages of infection are uncoupled, we showed that the integration of signals from both stages dictated mortality outcomes. This dichotomy relied on liver stage-dependent activation of Vγ4+ γδ T cells. Subsequent blood stage parasite loads dictated their cytokine profiles, where low parasite loads preferentially expanded IL-17-producing γδ T cells. IL-17 drove extra-medullary erythropoiesis and concomitant reticulocytosis, which protected mice from lethal experimental cerebral malaria (ECM). Adoptive transfer of erythroid precursors could rescue mice from ECM. Modeling of γδ T cell dynamics suggests that this protective mechanism may be key for the establishment of naturally acquired malaria immunity among frequently exposed individuals.

Interleukin-15 Complex Treatment Protects Mice from Cerebral Malaria by Inducing Interleukin-10-Producing Natural Killer Cells.

Cerebral malaria is a deadly complication of Plasmodium infection and involves blood brain barrier (BBB) disruption following infiltration of white blood cells. During experimental cerebral malaria (ECM), mice inoculated with Plasmodium berghei ANKA-infected red blood cells develop a fatal CM-like disease caused by CD8+ T cell-mediated pathology. We found that treatment with interleukin-15 complex (IL-15C) prevented ECM, whereas IL-2C treatment had no effect. IL-15C-expanded natural killer (NK) cells were necessary and sufficient for protection against ECM. IL-15C treatment also decreased CD8+ T cell activation in the brain and prevented BBB breakdown without influencing parasite load. IL-15C induced NK cells to express IL-10, which was required for IL-15C-mediated protection against ECM. Finally, we show that ALT-803, a modified human IL-15C, mediates similar induction of IL-10 in NK cells and protection against ECM. These data identify a regulatory role for cytokine-stimulated NK cells in the prevention of a pathogenic immune response.

A T Cell Receptor Locus Harbors a Malaria-Specific Immune Response Gene.

Immune response (Ir) genes, originally proposed by Baruj Benacerraf to explain differential antigen-specific responses in animal models, have become synonymous with the major histocompatibility complex (MHC). We discovered a non-MHC-linked Ir gene in a T cell receptor (TCR) locus that was required for CD8+ T cell responses to the Plasmodium berghei GAP5040-48 epitope in mice expressing the MHC class I allele H-2Db. GAP5040-48-specific CD8+ T cell responses emerged from a very large pool of naive Vß8.1+ precursors, which dictated susceptibility to cerebral malaria and conferred protection against recombinant Listeria monocytogenes infection. Structural analysis of a prototypical Vß8.1+ TCR-H-2Db-GAP5040-48 ternary complex revealed that germline-encoded complementarity-determining region 1ß residues present exclusively in the Vß8.1 segment mediated essential interactions with the GAP5040-48 peptide. Collectively, these findings demonstrated that Vß8.1 functioned as an Ir gene that was indispensable for immune reactivity against the malaria GAP5040-48 epitope.

Discrete class I molecules on brain endothelium differentially regulate neuropathology in experimental cerebral malaria.

Cerebral malaria is the deadliest complication that can arise from Plasmodium infection. CD8 T-cell engagement of brain vasculature is a putative mechanism of neuropathology in cerebral malaria. To define contributions of brain endothelial cell major histocompatibility complex (MHC) class I antigen-presentation to CD8 T cells in establishing cerebral malaria pathology, we developed novel H-2Kb LoxP and H-2Db LoxP mice crossed with Cdh5-Cre mice to achieve targeted deletion of discrete class I molecules, specifically from brain endothelium. This strategy allowed us to avoid off-target effects on iron homeostasis and class I-like molecules, which are known to perturb Plasmodium infection. This is the first endothelial-specific ablation of individual class-I molecules enabling us to interrogate these molecular interactions. In these studies, we interrogated human and mouse transcriptomics data to compare antigen presentation capacity during cerebral malaria. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM), we observed that H-2Kb and H-2Db class I molecules regulate distinct patterns of disease onset, CD8 T-cell infiltration, targeted cell death and regional blood-brain barrier disruption. Strikingly, ablation of either molecule from brain endothelial cells resulted in reduced CD8 T-cell activation, attenuated T-cell interaction with brain vasculature, lessened targeted cell death, preserved blood-brain barrier integrity and prevention of ECM and the death of the animal. We were able to show that these events were brain-specific through the use of parabiosis and created the novel technique of dual small animal MRI to simultaneously scan conjoined parabionts during infection. These data demonstrate that interactions of CD8 T cells with discrete MHC class I molecules on brain endothelium differentially regulate development of ECM neuropathology. Therefore, targeting MHC class I interactions therapeutically may hold potential for treatment of cases of severe malaria.

Brain endothelial STING1 activation by Plasmodium-sequestered heme promotes cerebral malaria via type I IFN response.

Cerebral malaria (CM) is a life-threatening form of Plasmodium falciparum infection caused by brain inflammation. Brain endothelium dysfunction is a hallmark of CM pathology, which is also associated with the activation of the type I interferon (IFN) inflammatory pathway. The molecular triggers and sensors eliciting brain type I IFN cellular responses during CM remain largely unknown. We herein identified the stimulator of interferon response cGAMP interactor 1 (STING1) as the key innate immune sensor that induces Ifnß1 transcription in the brain of mice infected with Plasmodium berghei ANKA (Pba). This STING1/IFNß-mediated response increases brain CXCL10 governing the extent of brain leukocyte infiltration and blood-brain barrier (BBB) breakdown, and determining CM lethality. The critical role of brain endothelial cells (BECs) in fueling type I IFN-driven brain inflammation was demonstrated in brain endothelial-specific IFNß-reporter and STING1-deficient Pba-infected mice, which were significantly protected from CM lethality. Moreover, extracellular particles (EPs) released from Pba-infected erythrocytes activated the STING1-dependent type I IFN response in BECs, a response requiring intracellular acidification. Fractionation of the EPs enabled us to identify a defined fraction carrying hemoglobin degradation remnants that activates STING1/IFNß in the brain endothelium, a process correlated with heme content. Notably, stimulation of STING1-deficient BECs with heme, docking experiments, and in vitro binding assays unveiled that heme is a putative STING1 ligand. This work shows that heme resultant from the parasite heterotrophic activity operates as an alarmin, triggering brain endothelial inflammatory responses via the STING1/IFNß/CXCL10 axis crucial to CM pathogenesis and lethality.

Host Transcriptional Meta-signatures Reveal Diagnostic Biomarkers for Plasmodium falciparum Malaria.

BACKGROUND: Transcriptomics has been used to evaluate immune responses during malaria in diverse cohorts worldwide. However, the high heterogeneity of cohorts and poor generalization of transcriptional signatures reported in each study limit their potential clinical applications. METHODS: We compiled 28 public data sets containing 1556 whole-blood or peripheral blood mononuclear cell transcriptome samples. We estimated effect sizes with Hedge's g value and the DerSimonian-Laird random-effects model for meta-analyses of uncomplicated malaria. Random forest models identified gene signatures that discriminate malaria from bacterial infections or malaria severity. Parasitological, hematological, immunological, and metabolomics data were used for validation. RESULTS: We identified 3 gene signatures: the uncomplicated Malaria Meta-Signature, which discriminates Plasmodium falciparum malaria from uninfected controls; the Malaria or Bacteria Signature, which distinguishes malaria from sepsis and enteric fever; and the cerebral Malaria Meta-Signature, which characterizes individuals with cerebral malaria. These signatures correlate with clinical hallmark features of malaria. Blood transcription modules indicate immune regulation by glucocorticoids, whereas cell development and adhesion are associated with cerebral malaria. CONCLUSIONS: Transcriptional meta-signatures reflecting immune cell responses provide potential biomarkers for translational innovation and suggest critical roles for metabolic regulators of inflammation during malaria.

Plasma from older children in Malawi inhibits Plasmodium falciparum binding in 3D brain microvessels.

A hallmark of cerebral malaria is sequestration of Plasmodium falciparum-infected erythrocytes (IEs) in the brain microcirculation. Antibodies contribute to malaria immunity, but it remains unclear whether functional antibodies targeting parasite-expressed ligand can block cytoadhesion in the brain. Here, we screened the plasma of older children and young adults in Malawi to characterize the antibody response against the P. falciparum-IE surface and used a bioengineered 3D human brain microvessel model incorporating variable flow dynamics to measure adhesion blocking responses. We found a strong correlation between surface antibody reactivity by flow cytometry and reduced P. falciparum-IE binding in 3D microvessels. Moreover, there was a threshold of surface antibody reactivity necessary to achieve robust inhibitory activity. Our findings provide evidence of the acquisition of adhesion blocking antibodies against cerebral binding variants in people exposed to stable P. falciparum transmission and suggest the quality of the inhibitory response can be influenced by flow dynamics.

Adult Cerebral Malaria: Acute and Subacute Imaging Findings, Long-term Clinical Consequences.

Cerebral malaria is an important cause of mortality and neurodisability in endemic regions. We show magnetic resonance imaging (MRI) features suggestive of cytotoxic and vasogenic cerebral edema followed by microhemorrhages in 2 adult UK cases, comparing them with an Indian cohort. Long-term follow-up images correlate ongoing changes with residual functional impairment.

Neurons upregulate PD-L1 via IFN/STAT1/IRF1 to alleviate damage by CD8+ T cells in cerebral malaria.

BACKGROUND: Cerebral malaria (CM) is the most lethal complication of malaria, and survivors usually endure neurological sequelae. Notably, the cytotoxic effect of infiltrating Plasmodium-activated CD8+ T cells on cerebral microvasculature endothelial cells is a prominent feature of the experimental CM (ECM) model with blood-brain barrier disruption. However, the damage effect of CD8+ T cells infiltrating the brain parenchyma on neurons remains unclear. Based on the immunosuppressive effect of the PD-1/PD-L1 pathway on T cells, our previous study demonstrated that the systemic upregulation of PD-L1 to inhibit CD8+ T cell function could effectively alleviate the symptoms of ECM mice. However, it has not been reported whether neurons can suppress the pathogenic effect of CD8+ T cells through the PD-1/PD-L1 negative immunomodulatory pathway. As the important inflammatory factor of CM, interferons can induce the expression of PD-L1 via different molecular mechanisms according to the neuro-immune microenvironment. Therefore, this study aimed to investigate the direct interaction between CD8+ T cells and neurons, as well as the mechanism of neurons to alleviate the pathogenic effect of CD8+ T cells through up-regulating PD-L1 induced by IFNs. METHODS: Using the ECM model of C57BL/6J mice infected with Plasmodium berghei ANKA (PbA), morphological observations were conducted in vivo by electron microscope and IF staining. The interaction between the ECM CD8+ T cells (immune magnetic bead sorting from spleen of ECM mice) and primary cultured cortical neurons in vitro was observed by IF staining and time-lapse photography. RNA-seq was performed to analyze the signaling pathway of PD-L1 upregulation in neurons induced by IFNß or IFNγ, and verified through q-PCR, WB, IF staining, and flow cytometry both in vitro and in vivo using IFNAR or IFNGR gene knockout mice. The protective effect of adenovirus-mediated PD-L1 IgGFc fusion protein expression was verified in ECM mice with brain stereotaxic injection in vivo and in primary cultured neurons via viral infection in vitro. RESULTS: In vivo, ECM mice showed infiltration of activated CD8+ T cells and neuronal injury in the brain parenchyma. In vitro, ECM CD8+ T cells were in direct contact with neurons and induced axonal damage, as an active behavior. The PD-L1 protein level was elevated in neurons of ECM mice and in primary cultured neurons induced by IFNß, IFNγ, or ECM CD8+ T cells in vitro. Furthermore, the IFNß or IFNγ induced neuronal expression of PD-L1 was mediated by increasing STAT1/IRF1 pathway via IFN receptors. The increase of PD-L1 expression in neurons during PbA infection was weakened after deleting the IFNAR or IFNGR. Increased PD-L1 expression by adenovirus partially protected neurons from CD8+ T cell-mediated damage both in vitro and in vivo. CONCLUSION: Our study demonstrates that both type I and type II IFNs can induce neurons to upregulate PD-L1 via the STAT1/IRF1 pathway mediated by IFN receptors to protect against activated CD8+ T cell-mediated damage, providing a targeted pathway to alleviate neuroinflammation during ECM.

Role of astrocyte senescence regulated by the non- canonical autophagy in the neuroinflammation associated to cerebral malaria.

BACKGROUND: Cerebral malaria (CM) is a fatal neuroinflammatory syndrome caused (in humans) by the protozoa Plasmodium (P.) falciparum. Glial cell activation is one of the mechanisms that contributes to neuroinflammation in CM. RESULT: By studying a mouse model of CM (caused by P. berghei ANKA), we describe that the induction of autophagy promoted p21-dependent senescence in astrocytes and that CXCL-10 was part of the senescence-associated secretory phenotype. Furthermore, p21 expression was observed in post-mortem brain and peripheral blood samples from patients with CM. Lastly, we found that the depletion of senescent astrocytes with senolytic drugs abrogated inflammation and protected mice from CM. CONCLUSION: Our data provide evidence for a novel mechanism through which astrocytes could be involved in the neuropathophysiology of CM. p21 gene expression in blood cell and an elevated plasma CXCL-10 concentration could be valuable biomarkers of CM in humans. In the end, we believe senolytic drugs shall open up new avenues to develop newer treatment options.

Neutrophils impose strong immune pressure against PfEMP1 variants implicated in cerebral malaria.

Plasmodium falciparum, the deadliest form of human malaria, remains one of the major threats to human health in endemic regions. Its virulence is attributed to its ability to modify infected red blood cells (iRBC) to adhere to endothelial receptors by placing variable antigens known as PfEMP1 on the iRBC surface. PfEMP1 expression determines the cytoadhesive properties of the iRBCs and is implicated in severe malaria. To evade antibody-mediated responses, the parasite undergoes continuous switches of expression between different PfEMP1 variants. Recently, it became clear that in addition to antibody-mediated responses, PfEMP1 triggers innate immune responses; however, the role of neutrophils, the most abundant white blood cells in the human circulation, in malaria remains elusive. Here, we show that neutrophils recognize and kill blood-stage P. falciparum isolates. We identify neutrophil ICAM-1 and specific PfEMP1 implicated in cerebral malaria as the key molecules involved in this killing. Our data provide mechanistic insight into the interactions between neutrophils and iRBCs and demonstrate the important influence of PfEMP1 on the selective innate response to cerebral malaria.

Cerebral malaria presenting as nonconvulsive status epilepticus: a case report.

BACKGROUND: Malaria is an infectious malady caused by Plasmodium parasites, cerebral malaria standing out as one of its most severe complications. Clinical manifestation include elevated body temperature, loss of consciousness, and seizures. However, reports of cerebral malaria presenting as nonconvulsive status epilepticus are extremely rare. The case presented involves psychiatric symptoms, with the electroencephalogram indicated nonconvulsive status epilepticus associated with cerebral malaria. CASE PRESENTATION: A 53-year-old male, was urgently admitted, due to confusion and abnormal behaviour for 10 h. The patient returned to China after developing a fever while working in Tanzania two months ago. The blood smear revealed Plasmodium vivax and Plasmodium falciparum, and he was diagnosed with malaria. He recovered following anti-malarial treatment. After admission, the patient was confused, unable to communicate normally, and unwilling to cooperate with the physical examination. Plasmodium was not found in the blood smear, but the DNA sequence of P. falciparum was discovered using metagenomic next-generation sequencing of cerebrospinal fluid. Brain MRI revealed no significant abnormalities. Continuous electroencephalogram monitoring revealed that the patient had non-convulsive status epilepticus, which was treated with diazepam and levetiracetam. The patient had normal consciousness and behaviour. He received anti-malarial treatment for two weeks and fully recovered. CONCLUSIONS: This case demonstrates that nonconvulsive status epilepticus can be a manifestation of cerebral malaria. It is imperative for attending physicians to heighten vigilance when encountering patients with a history of travel to malaria-endemic regions or a prior malaria infection, especially in the presence of unusual clinical presentations.

Longitudinal associations of plasma amino acid levels with recovery from malarial coma.

BACKGROUND: Disordered amino acid metabolism is observed in cerebral malaria (CM). This study sought to determine whether abnormal amino acid concentrations were associated with level of consciousness in children recovering from coma. Twenty-one amino acids and coma scores were quantified longitudinally and the data were analysed for associations. METHODS: In a prospective observational study, 42 children with CM were enrolled. Amino acid levels were measured at entry and at frequent intervals thereafter and consciousness was assessed by Blantyre Coma Scores (BCS). Thirty-six healthy children served as controls for in-country normal amino acid ranges. Logistic regression was employed using a generalized linear mixed-effects model to assess associations between out-of-range amino acid levels and BCS. RESULTS: At entry 16/21 amino acid levels were out-of-range. Longitudinal analysis revealed 10/21 out-of-range amino acids were significantly associated with BCS. Elevated phenylalanine levels showed the highest association with low BCS. This finding held when out-of-normal-range data were analysed at each sampling time. CONCLUSION: Longitudinal data is provided for associations between abnormal amino acid levels and recovery from CM. Of 10 amino acids significantly associated with BCS, elevated phenylalanine may be a surrogate for impaired clearance of ether lipid mediators of inflammation and may contribute to CM pathogenesis.

An imported malaria case with repeated episodes of neurological syndromes resulting from different Plasmodium species.

BACKGROUND: Imported cerebral malaria (CM) cases in non-endemic areas are often misdiagnosed, which delays treatment. Post-malaria neurological syndrome (PMNS) after recovery from severe malaria can also complicate diagnosis. CASE: We report an imported malaria case from West Africa with two sequential episodes with neurological syndromes within about a month. The first episode was diagnosed as CM with microscopy-positive Plasmodium falciparum infection. The second episode, occurring a month after the recovery from the first CM episode, was consistent with PMNS, since malaria parasites were not detected by microscopy in peripheral blood smears. However, this diagnosis was complicated by the detection of Plasmodium vivax in peripheral blood by PCR, suggesting a potential cause of the second episode by P. vivax. CONCLUSION: This study suggests that PMNS often occurs after severe falciparum malaria. Concurrent P. vivax infection with pathogenic biomass being predominantly extravascular further complicates accurate diagnosis.

CD47 blockade reduces the pathologic features of experimental cerebral malaria and promotes survival of hosts with Plasmodium infection.

CD47 is an antiphagocytic "don't eat me" signal that inhibits programmed cell removal of self. As red blood cells (RBCs) age they lose CD47 expression and become susceptible to programmed cell removal by macrophages. CD47-/- mice infected with Plasmodium yoelii, which exhibits an age-based preference for young RBCs, were previously demonstrated to be highly resistant to malaria infection. Our study sought to test the therapeutic benefit of CD47 blockade on ameliorating the clinical syndromes of experimental cerebral malaria (ECM), using the Plasmodium berghei ANKA (Pb-A) murine model. In vitro we tested the effect of anti-CD47 mAb on Plasmodium-infected RBC phagocytosis and found that anti-CD47 treatment significantly increased clearance of Plasmodium-infected RBCs. Infection of C57BL/6 mice with Pb-A is lethal and mice succumb to the clinical syndromes of CM between days 6 and 10 postinfection. Strikingly, treatment with anti-CD47 resulted in increased survival during the cerebral phase of Pb-A infection. Anti-CD47-treated mice had increased lymphocyte counts in the peripheral blood and increased circulating levels of IFN-γ, TNF-α, and IL-22. Despite increased circulating levels of inflammatory cytokines, anti-CD47-treated mice had reduced pathological features in the brain. Survival of ECM in anti-CD47-treated mice was correlated with reduced cellular accumulation in the cerebral vasculature, improved blood-brain barrier integrity, and reduced cytotoxic activity of infiltrating CD8+ T cells. These results demonstrate the therapeutic benefit of anti-CD47 to reduce morbidity in a lethal model of ECM, which may have implications for preventing mortality in young African children who are the highest casualties of CM.

Cerebral Plasmodium falciparum malaria: The role of PfEMP1 in its pathogenesis and immunity, and PfEMP1-based vaccines to prevent it.

Malaria, a mosquito-borne infectious disease caused by parasites of the genus Plasmodium continues to be a major health problem worldwide. The unicellular Plasmodium-parasites have the unique capacity to infect and replicate within host erythrocytes. By expressing variant surface antigens Plasmodium falciparum has evolved to avoid protective immune responses; as a result in endemic areas anti-malaria immunity develops gradually over many years of multiple and repeated infections. We are studying the role of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) expressed by asexual stages of P. falciparum responsible for the pathogenicity of severe malaria. The immunopathology of falciparum malaria has been linked to cyto-adhesion of infected erythrocytes to specific host receptors. A greater appreciation of the PfEMP1 molecules important for the development of protective immunity and immunopathology is a prerequisite for the rational discovery and development of a safe and protective anti-disease malaria vaccine. Here we review the role of ICAM-1 and EPCR receptor adhering falciparum-parasites in the development of severe malaria; we discuss our current research to understand the factors involved in the pathogenesis of cerebral malaria and the feasibility of developing a vaccine targeted specifically to prevent this disease.

Association of Endothelial Protein C Receptor (EPCR) rs867186 Gene Polymorphism With Increased Levels of Soluble EPCR and High Risk of Severe Malaria and Fatality in Beninese Children.

The endothelial protein C receptor (EPCR)-rs867186 G allele has been linked to high plasma levels of soluble EPCR (sEPCR) and controversially associated with either susceptibility or resistance to severe and cerebral malaria. In this study, quantitative enzyme-linked immunosorbent assay and sequencing were used to assess sEPCR levels and EPCR-rs867186 polymorphism in blood samples from Beninese children with different clinical presentations of malaria. Our findings show that sEPCR levels were higher at hospital admission than during convalescence and that EPCR-rs867186 G allele was associated with increased sEPCR plasma levels, malaria severity, and mortality rate (P < .001, P = .03, and P = .04, respectively), suggesting a role of sEPCR in the pathogenesis of severe malaria.

Identification of disease-related genes in Plasmodium berghei by network module analysis.

BACKGROUND: Plasmodium berghei has been used as a preferred model for studying human malaria, but only a limited number of disease-associated genes of P. berghei have been reported to date. Identification of new disease-related genes as many as possible will provide a landscape for better understanding the pathogenesis of P. berghei. METHODS: Network module analysis method was developed and applied to identify disease-related genes in P. berghei genome. Sequence feature identification, gene ontology annotation, and T-cell epitope analysis were performed on these genes to illustrate their functions in the pathogenesis of P. berghei. RESULTS: 33,314 genes were classified into 4,693 clusters. 4,127 genes shared by six malaria parasites were identified and are involved in many aspects of biological processes. Most of the known essential genes belong to shared genes. A total of 63 clusters consisting of 405 P. berghei genes were enriched in rodent malaria parasites. These genes participate in various stages of parasites such as liver stage development and immune evasion. Combination of these genes might be responsible for P. berghei infecting mice. Comparing with P. chabaudi, none of the clusters were specific to P. berghei. P. berghei lacks some proteins belonging to P. chabaudi and possesses some specific T-cell epitopes binding by class-I MHC, which might together contribute to the occurrence of experimental cerebral malaria (ECM). CONCLUSIONS: We successfully identified disease-associated P. berghei genes by network module analysis. These results will deepen understanding of the pathogenesis of P. berghei and provide candidate parasite genes for further ECM investigation.

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.