The systemic pathology of cerebral malaria in African children

Pediatric cerebral malaria carries a high mortality rate in sub-Saharan Africa. We present our systematic analysis of the descriptive and quantitative histopathology of all organs sampled from a series of 103 autopsies performed between 1996 and 2010 in Blantyre, Malawi on pediatric cerebral malaria patients and control patients (without coma, or without malaria infection) who were clinically well characterized prior to death. We found brain swelling in all cerebral malaria patients and the majority of controls. The histopathology in patients with sequestration of parasites in the brain demonstrated two patterns: (a) the “classic” appearance (i.e., ring hemorrhages, dense sequestration, and extra-erythrocytic pigment) which was associated with evidence of systemic activation of coagulation and (b) the “sequestration only” appearance associated with shorter duration of illness and higher total burden of parasites in all organs including the spleen. Sequestration of parasites was most intense in the gastrointestinal tract in all parasitemic patients (those with cerebral malarial and those without)

Plasmodium falciparum gene expression measured directly from tissue during human infection

Background: During the latter half of the natural 48-h intraerythrocytic life cycle of human Plasmodium falciparum infection, parasites sequester deep in endothelium of tissues, away from the spleen and inaccessible to peripheral blood. These late-stage parasites may cause tissue damage and likely contribute to clinical disease, and a more complete understanding of their biology is needed. Because these life cycle stages are not easily sampled due to deep tissue sequestration, measuring in vivo gene expression of parasites in the trophozoite and schizont stages has been a challenge. Methods: We developed a custom nCounter® gene expression platform and used this platform to measure malaria parasite gene expression profiles in vitro and in vivo. We also used imputation to generate global transcriptional profiles and assessed differential gene expression between parasites growing in vitro and those recovered from malaria-infected patient tissues collected at autopsy. Results: We demonstrate, for the first time, global transcriptional expression profiles from in vivo malaria parasites sequestered in human tissues. We found that parasite physiology can be correlated with in vitro data from an existing life cycle data set, and that parasites in sequestered tissues show an expected schizont-like transcriptional profile, which is conserved across tissues from the same patient. Imputation based on 60 landmark genes generated global transcriptional profiles that were highly correlated with genome-wide expression patterns from the same samples measured by microarray. Finally, differential expression revealed a limited set of in vivo upregulated transcripts, which may indicate unique parasite genes involved in human clinical infections. Conclusions: Our study highlights the utility of a custom nCounter® P. falciparum probe set, validation of imputation within Plasmodium species, and documentation of in vivo schizont-stage expression patterns from human tissues. Electronic supplementary material The online version of this article (doi:10.1186/s13073-014-0110-6) contains supplementary material, which is available to authorized users

Simulation models predict that school-age children are responsible for most human-to-mosquito Plasmodium falciparum transmission in southern Malawi

Abstract Background Malaria persists in some high-transmission areas despite extensive control efforts. Progress toward elimination may require effective targeting of specific human populations that act as key transmission reservoirs. Methods Parameterized using molecular-based Plasmodium falciparum infection data from cross-sectional community studies in southern Malawi, a simulation model was developed to predict the proportions of human-to-mosquito transmission arising from (a) children under 5 years old (U5s), (b) school-age children (SAC, 5–15 years), (c) young adults (16–30 years), and (d) adults > 30 years. The model incorporates mosquito biting heterogeneity and differential infectivity (i.e. probability that a blood-fed mosquito develops oocysts) by age and gametocyte density. Results The model predicted that SAC were responsible for more than 60% of new mosquito infections in both dry and rainy seasons, even though they comprise only 30% of this southern Malawi population. Young adults were the second largest contributors, while U5s and adults over 30 were each responsible for < 10% of transmission. While the specific predicted values are sensitive to the relative infectiousness of SAC, this group remained the most important contributor to mosquito infections under all realistic estimates. Conclusions These results suggest that U5 children play a small role compared to SAC in maintaining P. falciparum transmission in southern Malawi. Models that assume biting homogeneity overestimate the importance of U5s. To reduce transmission, interventions will need to reach more SAC and young adults. This publicly available model can be used by others to estimate age-specific transmission contributions in epidemiologically similar sites with local parameter estimates of P. falciparum prevalence and bed net use.https://deepblue.lib.umich.edu/bitstream/2027.42/143001/1/12936_2018_Article_2295.pd

A histological method for quantifying Plasmodium falciparum in the brain in fatal paediatric cerebral malaria

Background: The sequestration of Plasmodium falciparum-infected erythrocytes in brain microvasculature through cytoadherence to endothelium, is the hallmark of the definitive diagnosis of cerebral malaria and plays a critical role in malaria pathogenesis. The complex pathophysiology, which leads each patient to the final outcome of cerebral malaria, is multifaceted and thus, metrics to delineate specific patterns within cerebral malaria are needed to further parse patients. Methods: A method was developed for quantification utilizing counts of capillary contents (early-stage parasites, late-stage parasites and fibrin) from histological preparations of brain tissue after death, and compared it to the standard approach, in which the percentage of parasitized vessels in cross-section is determined. Results: Within the initial cohort of 50 patients, two different observers agreed closely on the percentage of vessels parasitized, pigmented parasites and pigment globules (ICC = 0.795-0.970). Correlations between observers for correct diagnostic classification were high (Kendall’s tau-b = 0.8779, Kappa = 0.8413). When these methods were applied prospectively to a second set of 50 autopsy samples, they revealed a heterogeneous distribution of sequestered parasites in the brain with pigmented parasites and pigment globules present in the cerebellum > cortex > brainstem. There was no difference in the distribution of early stages of parasites or in the percentage of vessels parasitized across the same sites. The second cohort of cases was also used to test a previously published classification and regression tree (CART) analysis; the quantitative data alone were able to accurately classify and distinguish cerebral malaria from non-cerebral malaria. Classification errors occurred within a subclassification of cerebral malaria (CM1 vs CM2). A repeat CART analysis for the second cohort generated slightly different classification rules with more accurate subclassification, although misclassification still occurred. Conclusions: The traditional measure of parasite sequestration in falciparum malaria, the percentage of vessels parasitized, is the most reliable and consistent for the general diagnosis of cerebral malaria. Methods that involve quantitative measures of different life cycle stages are useful for distinguishing patterns within the cerebral malaria population; these subclassifications may be important for studies of disease pathogenesis and ancillary treatment

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

This work was supported by grants from the NIH (T.E.T., 5R01AI034969-14) and a Clinical Fellowship from The Wellcome Trust, United Kingdom (C.A.M, 88758). The Malawi-Liverpool-Wellcome Clinical Research Programme is supported by core funding from The Wellcome Trust, UK.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). Conclusions. 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.Publisher PDFPeer reviewe

High prevalence of Plasmodium falciparum gametocyte infections in school-age children using molecular detection: patterns and predictors of risk from a cross-sectional study in southern Malawi

Abstract Background In endemic areas, many people experience asymptomatic Plasmodium infections, particularly older children and adults, but their transmission contribution is unknown. Though not the exclusive determinant of infectiousness, transmission from humans to mosquitoes requires blood meals containing gametocytes. Gametocytes often occur at submicroscopic densities, challenging measurement in human populations. More sensitive molecular techniques allow better characterization of gametocyte epidemiologic patterns. Methods Approximately 30 households were selected from each of eight sites in southern Malawi during two cross-sectional surveys. Blood was sampled from 623 people during the dry season and 896 the following rainy season. Among people PCR-positive for Plasmodium falciparum, mature gametocytes were detected by qRT-PCR. Regression models evaluated predictors of gametocyte carriage and density in the total population and among those with PCR-positive infections. Results The prevalence of gametocyte carriage by molecular testing was 3.5% during the dry season and 8.6% during the rainy season, and by microscopy 0.8 and 3.3%, respectively. Nearly half of PCR-positive infections carried gametocytes, regardless of recent symptom status. Among P. falciparum-infected people, only living in unfinished houses and age were significantly associated with gametocyte presence. Infected people in unfinished houses had higher odds of carrying gametocytes (OR 2.24, 95% CI 1.16–4.31), and 31% (95% CI 3–65%) higher gametocyte density than those in finished houses. School-age children (5–15 years), had higher odds than adults (≥16 years) of having gametocytes when infected (OR 2.77, 95% CI 1.47–5.19), but 31% (95% CI 11–47%) lower gametocyte density. Children <5 years did not have significantly higher odds of gametocyte carriage or density when infected than adults. Conclusions School-age children frequently carry gametocytes in communities of southern Malawi and represent an under-recognized reservoir of infection. Malaria elimination strategies should address these frequently asymptomatic reservoirs, especially in highly endemic areas. Improved household construction may also reduce the infectious reservoir.http://deepblue.lib.umich.edu/bitstream/2027.42/134670/1/12936_2016_Article_1587.pd

Mechanisms of Transcranial Doppler Ultrasound phenotypes in paediatric cerebral malaria remain elusive.

BACKGROUND: Cerebral malaria (CM) results in significant paediatric death and neurodisability in sub-Saharan Africa. Several different alterations to typical Transcranial Doppler Ultrasound (TCD) flow velocities and waveforms in CM have been described, but mechanistic contributors to these abnormalities are unknown. If identified, targeted, TCD-guided adjunctive therapy in CM may improve outcomes. METHODS: This was a prospective, observational study of children 6 months to 12 years with CM in Blantyre, Malawi recruited between January 2018 and June 2021. Medical history, physical examination, laboratory analysis, electroencephalogram, and magnetic resonance imaging were undertaken on presentation. Admission TCD results determined phenotypic grouping following a priori definitions. Evaluation of the relationship between haemodynamic, metabolic, or intracranial perturbations that lead to these observed phenotypes in other diseases was undertaken. Neurological outcomes at hospital discharge were evaluated using the Paediatric Cerebral Performance Categorization (PCPC) score. RESULTS: One hundred seventy-four patients were enrolled. Seven (4%) had a normal TCD examination, 57 (33%) met criteria for hyperaemia, 50 (29%) for low flow, 14 (8%) for microvascular obstruction, 11 (6%) for vasospasm, and 35 (20%) for isolated posterior circulation high flow. A lower cardiac index (CI) and higher systemic vascular resistive index (SVRI) were present in those with low flow than other groups (p \u3c 0.003), though these values are normal for age (CI 4.4 [3.7,5] l/min/m2, SVRI 1552 [1197,1961] dscm-5m2). Other parameters were largely not significantly different between phenotypes. Overall, 118 children (68%) had a good neurological outcome. Twenty-three (13%) died, and 33 (19%) had neurological deficits. Outcomes were best for participants with hyperaemia and isolated posterior high flow (PCPC 1-2 in 77 and 89% respectively). Participants with low flow had the least likelihood of a good outcome (PCPC 1-2 in 42%) (p \u3c 0.001). Cerebral autoregulation was significantly better in children with good outcome (transient hyperemic response ratio (THRR) 1.12 [1.04,1.2]) compared to a poor outcome (THRR 1.05 [0.98,1.02], p = 0.05). CONCLUSIONS: Common pathophysiological mechanisms leading to TCD phenotypes in non-malarial illness are not causative in children with CM. Alternative mechanistic contributors, including mechanical factors of the cerebrovasculature and biologically active regulators of vascular tone should be explored

Inferring Developmental Stage Composition from Gene Expression in Human Malaria

In the current era of malaria eradication, reducing transmission is critical. Assessment of transmissibility requires tools that can accurately identify the various developmental stages of the malaria parasite, particularly those required for transmission (sexual stages). Here, we present a method for estimating relative amounts of Plasmodium falciparum asexual and sexual stages from gene expression measurements. These are modeled using constrained linear regression to characterize stage-specific expression profiles within mixed-stage populations. The resulting profiles were analyzed functionally by gene set enrichment analysis (GSEA), confirming differentially active pathways such as increased mitochondrial activity and lipid metabolism during sexual development. We validated model predictions both from microarrays and from quantitative RT-PCR (qRT-PCR) measurements, based on the expression of a small set of key transcriptional markers. This sufficient marker set was identified by backward selection from the whole genome as available from expression arrays, targeting one sentinel marker per stage. The model as learned can be applied to any new microarray or qRT-PCR transcriptional measurement. We illustrate its use in vitro in inferring changes in stage distribution following stress and drug treatment and in vivo in identifying immature and mature sexual stage carriers within patient cohorts. We believe this approach will be a valuable resource for staging lab and field samples alike and will have wide applicability in epidemiological studies of malaria transmission