Interventions that thwart mortality are needed for comatose children in malaria endemic settings. These interventions require that patients who have variable disease states be carefully stratified into an exact subgroup in order to provide therapies that impact specific mortality. New interventions may come from an in depth understanding of the in vivo physiology (define by genetics and expression profiling) of the parasite which is not only unique in these children but also not reproducible in vitro. Our field study has pioneered the use of eye exams to identify malaria retinopathy in the setting of patients with clinical cerebral malaria - these findings cleanly separate patients into "true" cerebral malaria (retinopathy positive) and coma of other causes (retinopathy negative). Moreover, our database and tissue repository of over 100 pediatric autopsies from a prospective clinicopathological correlation study of cerebral malaria is the most comprehensive available. The biology of Plasmodium falciparum in vivo in cerebral malaria includes the expression of the virulent var genes as well as a set of 800 genes which are never expressed in vitro. Using an archive and prospective collection from a stringently defined cohort of comatose patients including both cerebral malaria patients and parasitemic patients with other causes of coma, we will have the opportunity to thoroughly characterize the in vivo biology of both var and unique in vivo genes of Plasmodium falciparum and understand their control and function within human disease using a comprehensive systems biology approach. We will perform full genome sequencing of parasites from patients (including clinical cases and autopsy tissues) who meet the above definitions and have low complexity infections based on our molecular genotyping tools. We will combine this full genome information with expression data from the same parasites using our novel Nanostring approach (which allows for full imputation of gene expression by measuring only 330 marker genes from minute samples of RNA) with confirmation by RNA expression and digital gene expression. The integration of these two robust data sets will allow us to define expression quantitative trait loci within the genome both directing the overall in vivo biology and specifically elucidating control and function within the unique genes found only during human infection. At the conclusion of this study, we will have defined the network of unique in vivo expressed genes during human infection, characterized the specific expression of var genes within individual patients, and identified which of these are putative targets for intervention strategies.
Using the most stringently defined group of severe malaria patients in a coma, we will determine the biology of parasite infection by integrating full parasit genetic sequence polymorphisms with matched complete expression profiling to determine control and function of virulence and unique in vivo parasite genes.