Damage-associated cellular markers in the clinical and pathogenic profile of vaccine-induced immune thrombotic thrombocytopenia.

BACKGROUND: Adenoviral vector-based COVID-19 vaccine-induced immune thrombotic thrombocytopenia (VITT) is rare but carries significant risks of mortality and long-term morbidity. The underlying pathophysiology of severe disease is still not fully understood. The objectives were to explore the pathophysiological profile and examine for clinically informative biomarkers in patients with severe VITT. METHODS: Twenty-two hospitalized patients with VITT, 9 pre- and 21 post-ChAdOx1 vaccine controls, were recruited across England, United Kingdom. Admission blood samples were analyzed for cytokine profiles, cell death markers (lactate dehydrogenase and circulating histones), neutrophil extracellular traps, and coagulation parameters. Tissue specimens from deceased patients were analyzed. RESULTS: There were strong immune responses characterized by significant elevations in proinflammatory cytokines and T helper 1 and 2 cell activation in patients with VITT. Markers of systemic endothelial activation and coagulation activation in both circulation and organ sections were also significantly elevated. About 70% (n = 15/22) of patients met the International Society for Thrombosis and Haemostasis criteria for disseminated intravascular coagulation despite negligible changes in the prothrombin time. The increased neutrophil extracellular trap formation, in conjunction with marked lymphopenia, elevated lactate dehydrogenase, and circulating histone levels, indicates systemic immune cell injury or death. Both lymphopenia and circulating histone levels independently predicted 28-day mortality in patients with VITT. CONCLUSION: The coupling of systemic cell damage and death with strong immune-inflammatory and coagulant responses are pathophysiologically dominant and clinically relevant in severe VITT.

Investigating Interactions Between Endothelial Cells and Parasitized Red Blood Cells in Skin and Subcutaneous Tissue.

In this chapter we present the methods for using biopsies of skin or subcutaneous tissue to examine the interactions between parasitized red blood cells and endothelial cells in patients with malaria infection. Punch biopsy can be used to obtain all skin layers and needle biopsy to obtain subcutaneous tissue. Smears are useful for spreading vessels on a slide for immunofluorescence staining. Specimens can be fixed and embedded for sectioning and traditional histological or immunostaining techniques or confocal microscopy with three-dimensional reconstruction. Finally, endothelium can be dissociated, allowing individual cells to be isolated for culture and ex vivo assays or used for immunophenotyping.

Host cell maturation modulates parasite invasion and sexual differentiation in Plasmodium berghei.

Malaria remains a global health problem causing more than 400,000 deaths annually. Plasmodium parasites, the causative agents of malaria, replicate asexually in red blood cells (RBCs) of their vertebrate host, while a subset differentiates into sexual stages (gametocytes) for mosquito transmission. Parasite replication and gametocyte maturation in the erythropoietic niches of the bone marrow and spleen contribute to pathogenesis and drive transmission, but the mechanisms underlying this organ enrichment remain unknown. Here, we performed a comprehensive analysis of rodent P. berghei infection by flow cytometry and single-cell RNA sequencing. We identified CD71 as a host receptor for reticulocyte invasion and found that parasites metabolically adapt to the host cell environment. Transcriptional analysis and functional assays further revealed a nutrient-dependent tropism for gametocyte formation in reticulocytes. Together, we provide a thorough characterization of host-parasite interactions in erythropoietic niches and define host cell maturation state as the key driver of parasite adaptation.

Minimally Invasive Postmortem Intestinal Tissue Sampling in Malnourished and Acutely Ill Children Is Feasible and Informative.

BACKGROUND: Intestinal disorders such as environmental enteric dysfunction (EED) are prevalent in low- and middle-income countries (LMICs) and important contributors to childhood undernutrition and mortality. Autopsies are rarely performed in LMICs but minimally invasive tissue sampling is increasingly deployed as a more feasible and acceptable procedure, although protocols have been devoid of intestinal sampling to date. We sought to determine (1) the feasibility of postmortem intestinal sampling, (2) whether autolysis precludes enteric biopsies' utility, and (3) histopathologic features among children who died during hospitalization with acute illness or undernutrition. METHODS: Transabdominal needle and endoscopic forceps upper and lower intestinal sampling were conducted among children aged 1 week to 59 months who died while hospitalized in Blantyre, Malawi. Autolysis ratings were determined for each hematoxylin and eosin slide, and upper and lower intestinal scoring systems were adapted to assess histopathologic features and their severity. RESULTS: Endoscopic and transabdominal sampling procedures were attempted in 28 and 14 cases, respectively, with >90% success obtaining targeted tissue. Varying degrees of autolysis were present in all samples and precluded histopathologic scoring of 6% of 122 biopsies. Greater autolysis in duodenal samples was seen with longer postmortem interval (Beta = 0.06, 95% confidence interval, 0.02-0.11). Histopathologic features identified included duodenal Paneth and goblet cell depletion. Acute inflammation was absent but chronic inflammation was prevalent in both upper and lower enteric samples. Severe chronic rectal inflammation was identified in children as young as 5.5 weeks. CONCLUSIONS: Minimally invasive postmortem intestinal sampling is feasible and identifies histopathology that can inform mortality contributors.

Localised release of matrix metallopeptidase 8 in fatal cerebral malaria.

OBJECTIVE: Cerebral malaria (CM) is a complication of Plasmodium falciparum malaria, in which progressive brain swelling is associated with sequestration of parasites and impaired barrier function of the cerebral microvascular endothelium. To test the hypothesis that localised release of matrix metallopeptidase 8 (MMP8) within the retina is implicated in microvascular leak in CM, we examined its expression and association with extravascular fibrinogen leak in a case-control study of post-mortem retinal samples from 13 Malawian children who met the clinical case definition of CM during life. Cases were seven children who were found on post-mortem examination to have 'true-CM' (parasite sequestration in brain blood vessels), whilst controls were six children who had alternative causes of death ('faux-CM', no parasite sequestration in blood vessels). METHODS: We used immunofluorescence microscopy and independent scoring, by two assessors blinded to the CM status, to assess MMP8 expression, extravascular fibrinogen as an indicator of vascular leak and their co-localisation in the retinal microvasculature. RESULTS: In 'true-CM' subjects, MMP8 staining was invariably associated with sequestered parasites and a median of 88% (IQR = 74-91%) of capillaries showed MMP8 staining, compared with 14% (IQR = 3.8-24%) in 'faux-CM' (P-value = 0.001). 41% (IQR = 28-49%) of capillaries in 'true-CM' subjects showed co-localisation of extravascular fibrinogen leak and MMP8 staining, compared with 1.8% of capillaries in 'faux-CM' (IQR = 0-3.9%, P-value = 0.01). Vascular leak was rare in the absence of MMP8 staining. CONCLUSION: Matrix metallopeptidase 8 was extensively expressed in retinal capillaries of Malawian children with malarial retinopathy and strongly associated with vascular leak. Our findings implicate MMP8 as a cause of the vascular endothelial barrier disruption in CM, which may precipitate fatal brain swelling.

Plasmodium falciparum Gametocyte Density and Infectivity in Peripheral Blood and Skin Tissue of Naturally Infected Parasite Carriers in Burkina Faso.

BACKGROUND: Plasmodium falciparum transmission depends on mature gametocytes that can be ingested by mosquitoes taking a blood meal on human skin. Although gametocyte skin sequestration has long been hypothesized as important contributor to efficient malaria transmission, this has never been formally tested. METHODS: In naturally infected gametocyte carriers from Burkina Faso, we assessed infectivity to mosquitoes by direct skin feeding and membrane feeding. We directly quantified male and female gametocytes and asexual parasites in finger-prick and venous blood samples, skin biopsy samples, and in of mosquitoes that fed on venous blood or directly on skin. Gametocytes were visualized in skin tissue with confocal microscopy. RESULTS: Although more mosquitoes became infected when feeding directly on skin then when feeding on venous blood (odds ratio, 2.01; 95% confidence interval, 1.21-3.33; P = .007), concentrations of gametocytes were not higher in the subdermal skin vasculature than in other blood compartments; only sparse gametocytes were observed in skin tissue. DISCUSSION: Our data strongly suggest that there is no significant skin sequestration of P. falciparum gametocytes. Gametocyte densities in peripheral blood are thus informative for predicting onward transmission potential to mosquitoes and can be used to target and monitor malaria elimination initiatives.

Testing the effect of PAR1 inhibitors on Plasmodium falciparum-induced loss of endothelial cell barrier function.

Background: Sequestration and cytoadherence of Plasmodium falciparum-infected erythrocytes (IE) to microvascular endothelium alters endothelial barrier function and plays a role in the pathogenesis of severe malaria. Binding of IE is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1) and the PfEMP1 variants that binds to endothelial protein C receptor (EPCR) have, in particular, been associated with the dysregulation of the coagulation/inflammation pathways in endothelial cells. This has prompted speculation about the role of protease-activated receptor-1 (PAR1) activation and signalling in causing endothelial activation and loss of barrier function in cerebral malaria. Methods: We used a co-culture of primary human brain microvascular endothelial cells (HBMEC) with P. falciparum material, recombinant PfEMP1 or lysates from IE, and measured barrier function by trans endothelial electrical resistance (TEER).  A selection of PAR1 inhibitors was tested for their ability to reverse the P. falciparum and thrombin induced decrease in barrier function. Results: An initial screen in the presence of recombinant PfEMP1 identified a few inhibitors that were able to reduce the rapid thrombin-induced barrier disruption even when activated protein C (aPC) was unable to do so. However, in the IE lysate co-culture system we identified a mechanism that slowly reduces barrier function and which is insensitive to PAR1 inhibitors. Conclusions: The selected PAR1 inhibitors were able to reverse the disruption of barrier function by thrombin but did not reverse the IE lysate induced disruption of barrier function, implicating a different PAR1-independent mechanism.  These findings have implications for the design of adjunct therapies to reduce brain swelling in cerebral malaria.

Parasite histones are toxic to brain endothelium and link blood barrier breakdown and thrombosis in cerebral malaria.

Microvascular thrombosis and blood-brain barrier (BBB) breakdown are key components of cerebral malaria (CM) pathogenesis in African children and are implicated in fatal brain swelling. How Plasmodium falciparum infection causes this endothelial disruption and why this occurs, particularly in the brain, is not fully understood. In this study, we have demonstrated that circulating extracellular histones, equally of host and parasite origin, are significantly elevated in CM patients. Higher histone levels are associated with brain swelling on magnetic resonance imaging. On postmortem brain sections of CM patients, we found that histones are colocalized with P falciparum-infected erythrocytes sequestered inside small blood vessels, suggesting that histones might be expelled locally during parasite schizont rupture. Histone staining on the luminal vascular surface colocalized with thrombosis and leakage, indicating a possible link between endothelial surface accumulation of histones and coagulation activation and BBB breakdown. Supporting this, patient sera or purified P falciparum histones caused disruption of barrier function and were toxic to cultured human brain endothelial cells, which were abrogated with antihistone antibody and nonanticoagulant heparin. Overall, our data support a role for histones of parasite and host origin in thrombosis, BBB breakdown, and brain swelling in CM, processes implicated in the causal pathway to death. Neutralizing histones with agents such as nonanticoagulant heparin warrant exploration to prevent brain swelling in the development or progression of CM and thereby to improve outcomes.

A More Granular View of Neutrophils in Malaria.

Neutrophils are abundant innate immune cells with crucial roles in immunity and vascular inflammation. Recent evidence indicates that neutrophils have a dual role in malaria, contributing to both pathogenesis and control of Plasmodium. We discuss emerging mechanisms behind these opposing functions and identify key outstanding questions.

New Insights into Malaria Pathogenesis.

Malaria remains a major public health threat in tropical and subtropical regions across the world. Even though less than 1% of malaria infections are fatal, this leads to about 430,000 deaths per year, predominantly in young children in sub-Saharan Africa. Therefore, it is imperative to understand why a subset of infected individuals develop severe syndromes and some of them die and what differentiates these cases from the majority that recovers. Here, we discuss progress made during the past decade in our understanding of malaria pathogenesis, focusing on the major human parasite Plasmodium falciparum.

Neutrophil extracellular traps drive inflammatory pathogenesis in malaria.

Neutrophils are essential innate immune cells that extrude chromatin in the form of neutrophil extracellular traps (NETs) when they die. This form of cell death has potent immunostimulatory activity. We show that heme-induced NETs are essential for malaria pathogenesis. Using patient samples and a mouse model, we define two mechanisms of NET-mediated inflammation of the vasculature: activation of emergency granulopoiesis via granulocyte colony-stimulating factor production and induction of the endothelial cytoadhesion receptor intercellular adhesion molecule-1. Soluble NET components facilitate parasite sequestration and mediate tissue destruction. We demonstrate that neutrophils have a key role in malaria immunopathology and propose inhibition of NETs as a treatment strategy in vascular infections.

Effects of saline or albumin fluid bolus in resuscitation: evidence from re-analysis of the FEAST trial.

BACKGROUND: Fluid resuscitation is the recommended management of shock, but increased mortality in febrile African children in the FEAST trial. We hypothesised that fluid bolus-induced deaths in FEAST would be associated with detectable changes in cardiovascular, neurological, or respiratory function, oxygen carrying capacity, and blood biochemistry. METHODS: We developed composite scores for respiratory, cardiovascular, and neurological function using vital sign data from the FEAST trial, and used them to compare participants from FEAST with those from four other cohorts and to identify differences between the bolus (n=2097) and no bolus (n=1044) groups of FEAST. We calculated the odds of adverse outcome for each ten-unit increase in baseline score using logistic regression for each cohort. Within FEAST participants, we also compared haemoglobin and plasma biochemistry between bolus and non-bolus patients, assessed the effects of these factors along with the vital sign scores on the contribution of bolus to mortality using Cox proportional hazard models, and used Bayesian clustering to identify subgroups that differed in response to bolus. The FEAST trial is registered with ISRCTN, number ISRCTN69856593. FINDINGS: Increasing respiratory (odds ratio 1·09, 95% CI 1·07-1·11), neurological (1·26, 1·21-1·31), and cardiovascular scores (1·09, 1·05-1·14) were associated with death in FEAST (all p<0·0001), and with adverse outcomes for specific scores in the four other cohorts. In FEAST, fluid bolus increased respiratory and neurological scores and decreased cardiovascular score at 1 h after commencement of the infusion. Fluid bolus recipients had mean 0·33 g/dL (95% CI 0·20-0·46) reduction in haemoglobin concentration after 8 h (p<0·0001), and at 24 h had a decrease of 1·41 mEq/L (95% CI 0·76-2·06; p=0·0002) in mean base excess and increase of 1·65 mmol/L (0·47-2·8; p=0·0070) in mean chloride, and a decrease of 0·96 mmol/L (0·45 to 1·47; p=0·0003) in bicarbonate. There were similar effects of fluid bolus in three patient subgroups, identified on the basis of their baseline characteristics. Hyperchloraemic acidosis and respiratory and neurological dysfunction induced by saline or albumin bolus explained the excess mortality due to bolus in Cox survival models. INTERPRETATION: In the resuscitation of febrile children, albumin and saline boluses can cause respiratory and neurological dysfunction, hyperchloraemic acidosis, and reduction in haemoglobin concentration. The findings support the notion that fluid resuscitation with unbuffered electrolyte solutions may cause harm and their use should be cautioned. The effects of lower volumes of buffered solutions should be evaluated further. FUNDING: Medical Research Council, Department for International Development, National Institute for Health Research, Imperial College Biomedical Research Centre.

Cerebral malaria is associated with differential cytoadherence to brain endothelial cells.

Sequestration of Plasmodium falciparum-infected erythrocytes (IE) within the brain microvasculature is a hallmark of cerebral malaria (CM). Using a microchannel flow adhesion assay with TNF-activated primary human microvascular endothelial cells, we demonstrate that IE isolated from Malawian paediatric CM cases showed increased binding to brain microvascular endothelial cells compared to IE from uncomplicated malaria (UM) cases. Further, UM isolates showed significantly greater adhesion to dermal than to brain microvascular endothelial cells. The major mediator of parasite adhesion is P. falciparum erythrocyte membrane protein 1, encoded by var genes. Higher levels of var gene transcripts predicted to bind host endothelial protein C receptor (EPCR) and ICAM-1 were detected in CM isolates. These data provide further evidence for differential tissue binding in severe and uncomplicated malaria syndromes, and give additional support to the hypothesis that CM pathology is based on increased cytoadherence of IE in the brain microvasculature.

Safety of lumbar puncture in comatose children with clinical features of cerebral malaria.

OBJECTIVE: We assessed the independent association of lumbar puncture (LP) and death in Malawian children admitted to the hospital with the clinical features of cerebral malaria (CM). METHODS: This was a retrospective cohort study in Malawian children with clinical features of CM. Allocation to LP was nonrandom and was associated with severity of illness. Propensity score-based analyses were used to adjust for this bias and assess the independent association between LP and mortality. RESULTS: Data were available for 1,075 children: 866 (80.6%) underwent LP and 209 (19.4%) did not. Unadjusted mortality rates were lower in children who underwent LP (15.3% vs 26.7% in the no-LP group) but differences in covariates between the 2 groups suggested bias in LP allocation. After propensity score matching, all covariates were balanced. Propensity score-based analyses showed no change in mortality rate associated with LP: by inverse probability weighting, the average risk reduction was 2.0% at 12 hours (95% confidence interval -1.5% to 5.5%, p = 0.27) and 1.7% during hospital admission (95% confidence interval -4.5% to 7.9%, p = 0.60). Undergoing LP did not change the risk of mortality in subanalyses of children with severe brain swelling on MRI or in those with papilledema. CONCLUSION: In comatose children with suspected CM who were clinically stable, we found no evidence that LP increases mortality, even in children with objective signs of raised intracranial pressure.

HIV coinfection influences the inflammatory response but not the outcome of cerebral malaria in Malawian children.

OBJECTIVES: Study of the effect of HIV on disease progression in heterogeneous severe malaria syndromes with imprecise diagnostic criteria has led to varying results. Characteristic retinopathy refines cerebral malaria (CM) diagnosis, enabling more precise exploration of the hypothesis that HIV decreases the cytokine response in CM, leading to higher parasite density and a poor outcome. METHODS: We retrospectively reviewed data on clinical progression and laboratory parameters in 877 retinopathy-positive CM cases admitted 1996-2011 (14.4% HIV-infected) to a large hospital in Malawi. Admission plasma levels of TNF, interleukin-10, and soluble intercellular adhesion molecule (sICAM-1) were measured by ELISA in 135 retinopathy-positive CM cases. RESULTS: HIV-infected CM cases had lower median plasma levels of TNF (p = 0.008), interleukin-10 (p = 0.045) and sICAM-1 (p = 0.04) than HIV-uninfected cases. Although HIV-infected children were older and more likely to have co-morbidities, HIV-status did not significantly affect parasite density (p = 0.90) or outcome (24.8% infected, vs. 18.5% uninfected; p = 0.13). CONCLUSION: In this well-characterised CM cohort, HIV-coinfection was associated with marked blunting of the inflammatory response but did not affect parasite density or outcome. These data highlight the complex influence of HIV on severe malaria and bring into question systemic inflammation as a primary driver of pathogenesis in human CM.

Beta-lactamases in Enterobacteriaceae infections in children.

Multi-drug resistance in Gram negative bacteria, particularly in Enterobacteriaceae, is a major clinical and public health challenge. The main mechanism of resistance in Enterobacteriaceae is linked to the production of beta-lactamase hydrolysing enzymes such as extended spectrum beta-lactamases (ESBL), AmpC beta-lactamases and carbapenemases (Carbapenemase Producing Enterobacteriaceae (CPE)). ESBL and CPE resistance genes are located on plasmids, which can be transmitted between Enterobacteriaceae, facilitating their spread in hospitals and communities. These plasmids usually harbour multiple additional co-resistance genes, including to trimethoprim-sulfamethoxazole, aminoglycosides, and fluoroquinolones, making these infections challenging to treat. Asymptomatic carriage in healthy children as well as community acquired infections are increasingly reported, particularly with ESBL. Therapeutic options are limited and previously little used antimicrobials such as fosfomycin and colistin have been re-introduced in clinical practice. Paediatric experience with these agents is limited hence there is a need to further examine their clinical efficacy, dosage and toxicity in children. Antimicrobial stewardship along with strict infection prevention and control practices need to be adopted widely in order to preserve currently available antimicrobials. The future development of novel agents effective against beta-lactamases producers and their applicability in children is urgently needed to address the challenge of multi-resistant Gram negative infections.

Persistent endothelial activation and inflammation after Plasmodium falciparum Infection in Malawian children.

Endothelial dysregulation is central to the pathogenesis of acute Plasmodium falciparum infection. It has been assumed that this dysregulation resolves rapidly after treatment, but this return to normality has been neither demonstrated nor quantified. We therefore measured a panel of plasma endothelial markers acutely and in convalescence in Malawian children with uncomplicated or cerebral malaria. Evidence of persistent endothelial activation and inflammation, indicated by increased plasma levels of soluble intracellular adhesion molecule 1, angiopoetin 2, and C-reactive protein, were observed at 1 month follow-up visits. These vascular changes may represent a previously unrecognized contributor to ongoing malaria-associated morbidity and mortality.