Inferring Developmental Stage Composition from Gene Expression in Human Malaria

Abstract

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.

Transcriptional profiling defines dynamics of parasite tissue sequestration during malaria infection

“…Similar observations were made in a comparison between five field strains and three laboratory-adapted strains [27]. We obtained independent evidence for the estimated circulation time of asexual parasites by directly estimating asexual parasite stage for each field sample, using a previously developed linear regression model [29]. Interestingly, variation in asexual parasite stage across samples correlated with variant expression patterns of individual stage-specific clusters, suggesting that parasite populations show highly synchronized cell cycle patterns during infection.…”

Comparison of molecular quantification of Plasmodium falciparum gametocytes by Pfs25 qRT-PCR and QT-NASBA in relation to mosquito infectivity

“…For all of these targets, transcript detection is possible both by the more convenient QT-NASBA and by the more laborious but more reproducible qRT-PCR. Design of intron-spanning primers may close the gap between the attractiveness of both methods and retain the advantage of the more reproducible qRT-PCR [41]. …”

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

“…qRT-PCR was conducted on the preserved RNA of all PCR-positive and ~1% of PCR-negative individuals to test for stage-specific P. falciparum mRNA. qRT-PCR used a multiplex assay developed by Joice et al [46] and validated in a clinical trial in Uganda by Chang et al [47] that distinguishes mature (stage IV–V) gametocytes from other stages.…”