BACKGROUND The RTS,S/AS01 vaccine targets the circumsporozoite protein of Plasmodium falciparum and has partial protective efficacy against clinical and severe malaria disease in infants and children. We investigated whether the vaccine efficacy was specific to certain parasite genotypes at the circumsporozoite protein locus. METHODS We used polymerase chain reaction–based next-generation sequencing of DNA extracted from samples from 4985 participants to survey circumsporozoite protein polymorphisms. We evaluated the effect that polymorphic positions and haplotypic regions within the circumsporozoite protein had on vaccine efficacy against first episodes of clinical malaria within 1 year after vaccination. RESULTS In the per-protocol group of 4577 RTS,S/AS01-vaccinated participants and 2335 control-vaccinated participants who were 5 to 17 months of age, the 1-year cumulative vaccine efficacy was 50.3% (95% confidence interval [CI], 34.6 to 62.3) against clinical malaria in which parasites matched the vaccine in the entire circumsporozoite protein C-terminal (139 infections), as compared with 33.4% (95% CI, 29.3 to 37.2) against mismatched malaria (1951 infections) (P = 0.04 for differential vaccine efficacy). The vaccine efficacy based on the hazard ratio was 62.7% (95% CI, 51.6 to 71.3) against matched infections versus 54.2% (95% CI, 49.9 to 58.1) against mismatched infections (P = 0.06). In the group of infants 6 to 12 weeks of age, there was no evidence of differential allele-specific vaccine efficacy. CONCLUSIONS These results suggest that among children 5 to 17 months of age, the RTS,S vaccine has greater activity against malaria parasites with the matched circumsporozoite protein allele than against mismatched malaria. The overall vaccine efficacy in this age category will depend on the proportion of matched alleles in the local parasite population; in this trial, less than 10% of parasites had matched alleles. (Funded by the National Institutes of Health and others.)

BackgroundDuring intra-erythrocytic development, late asexually replicating Plasmodium falciparum parasites sequester from peripheral circulation. This facilitates chronic infection and is linked to severe disease and organ-specific pathology including cerebral and placental malaria. Immature gametocytes - sexual stage precursor cells - likewise disappear from circulation. Recent work has demonstrated that these sexual stage parasites are located in the hematopoietic system of the bone marrow before mature gametocytes are released into the bloodstream to facilitate mosquito transmission. However, as sequestration occurs only in vivo and not during in vitro culture, the mechanisms by which it is regulated and enacted (particularly by the gametocyte stage) remain poorly understood.ResultsWe generated the most comprehensive P. falciparum functional gene network to date by integrating global transcriptional data from a large set of asexual and sexual in vitro samples, patient-derived in vivo samples, and a new set of in vitro samples profiling sexual commitment. We defined more than 250 functional modules (clusters) of genes that are co-expressed primarily during the intra-erythrocytic parasite cycle, including 35 during sexual commitment and gametocyte development. Comparing the in vivo and in vitro datasets allowed us, for the first time, to map the time point of asexual parasite sequestration in patients to 22 hours post-invasion, confirming previous in vitro observations on the dynamics of host cell modification and cytoadherence. Moreover, we were able to define the properties of gametocyte sequestration, demonstrating the presence of two circulating gametocyte populations: gametocyte rings between 0 and approximately 30 hours post-invasion and mature gametocytes after around 7 days post-invasion.ConclusionsThis study provides a bioinformatics resource for the functional elucidation of parasite life cycle dynamics and specifically demonstrates the presence of the gametocyte ring stages in circulation, adding significantly to our understanding of the dynamics of gametocyte sequestration in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0133-7) contains supplementary material, which is available to authorized users.

SummaryApicomplexans are a diverse group of obligate parasites occupying different intracellular niches that require modification to meet the needs of the parasite. To efficiently manipulate their environment, apicomplexans translocate numerous parasite proteins into the host cell. Whereas some parasites remain contained within a parasitophorous vacuole membrane (PVM) throughout their developmental cycle, others do not, a difference that affects the machinery needed for protein export. A signal-mediated pathway for protein export into the host cell has been characterized in Plasmodium parasites, which maintain the PVM. Here, we functionally demonstrate an analogous host-targeting pathway involving organellar staging prior to secretion in the related bovine parasite, Babesia bovis, a parasite that destroys the PVM shortly after invasion. Taking into account recent identification of a similar signal-mediated pathway in the coccidian parasite Toxoplasma gondii, we suggest a model in which this conserved pathway has evolved in multiple steps from signal-mediated trafficking to specific secretory organelles for controlled secretion to a complex protein translocation process across the PVM.

Rationale: Plasma-detectable biomarkers that rapidly and accurately diagnose bacterial infections in children with suspected pneumonia could reduce the morbidity of respiratory disease and decrease the unnecessary use of antibiotic therapy.Objectives: Using 56 markers measured in a multiplexed immunoassay, we sought to identify proteins and protein combinations that could discriminate bacterial from viral or malarial diagnoses. Methods:We selected 80 patients with clinically diagnosed pneumonia (as defined by the World Health Organization) who also met criteria for bacterial, viral, or malarial infection based on clinical, radiographic, and laboratory results. Ten healthy community control subjects were enrolled to assess marker reliability. Patients were subdivided into two sets: one for identifying potential markers and another for validating them.Measurements and Main Results: Three proteins (haptoglobin, tumor necrosis factor receptor 2 or IL-10, and tissue inhibitor of metalloproteinases 1) were identified that, when combined through a classification tree signature, accurately classified patients into bacterial, malarial, and viral etiologies and misclassified only one patient with bacterial pneumonia from the validation set. The overall sensitivity and specificity of this signature for the bacterial diagnosis were 96 and 86%, respectively. Alternative combinations of markers with comparable accuracy were selected by support vector machine and regression models and included haptoglobin, IL-10, and creatine kinase-MB.Conclusions: Combinations of plasma proteins accurately identified children with a respiratory syndrome who were likely to have bacterial infections and who would benefit from antibiotic therapy. When used in conjunction with malaria diagnostic tests, they may improve diagnostic specificity and simplify treatment decisions for clinicians.

Health workers in low-resource settings often lack the support and tools to follow evidence-based clinical recommendations for diagnosing, treating and managing sick patients. Digital technologies, by combining patient health information and point-of-care diagnostics with evidence-based clinical protocols, can help improve the quality of care and the rational use of resources, and save patient lives. A growing number of electronic clinical decision support algorithms (CDSAs) on mobile devices are being developed and piloted without evidence of safety or impact. Here, we present a target product profile (TPP) for CDSAs aimed at guiding preventive or curative consultations in low-resource settings. This document will help align developer and implementer processes and product specifications with the needs of end users, in terms of quality, safety, performance and operational functionality. To identify the characteristics of CDSAs, a multidisciplinary group of experts (academia, industry and policy makers) with expertise in diagnostic and CDSA development and implementation in low-income and middle-income countries were convened to discuss a draft TPP. The TPP was finalised through a Delphi process to facilitate consensus building. An agreement greater than 75% was reached for all 40 TPP characteristics. In general, experts were in overwhelming agreement that, given that CDSAs provide patient management recommendations, the underlying clinical algorithms should be human-interpretable and evidence-based. Whenever possible, the algorithm’s patient management output should take into account pretest disease probabilities and likelihood ratios of clinical and diagnostic predictors. In addition, validation processes should at a minimum show that CDSAs are implementing faithfully the evidence they are based on, and ideally the impact on patient health outcomes. In terms of operational needs, CDSAs should be designed to fit within clinic workflows and function in connectivity-challenged and high-volume settings. Data collected through the tool should conform to local patient privacy regulations and international data standards.

We have identified sets of genes that are differentially expressed in pediatric patients with pneumonia syndrome attributable to different infections and requiring different therapeutic interventions. Findings of this study demonstrate that human transcription signatures in infected patients recapitulate the underlying biology and provide models for predicting a bacterial diagnosis to inform treatment.

Background In the absence of proper guidelines and algorithms, available rapid diagnostic tests (RDTs) for common acute undifferentiated febrile illnesses are often used inappropriately. Methods Using prevalence data of 5 common febrile illnesses from India and Cambodia, and performance characteristics (sensitivity and specificity) of relevant pathogen-specific RDTs, we used a mathematical model to predict the probability of correct identification of each disease when diagnostic testing occurs either simultaneously or sequentially in various algorithms. We developed a web-based application of the model so as to visualize and compare output diagnostic algorithms when different disease prevalence and test performance characteristics are introduced. Results Diagnostic algorithms with appropriate sequential testing predicted correct identification of etiology in 74% and 89% of patients in India and Cambodia, respectively, compared with 46% and 49% with simultaneous testing. The optimally performing sequential diagnostic algorithms differed in India and Cambodia due to varying disease prevalence. Conclusions Simultaneous testing is not appropriate for the diagnosis of acute undifferentiated febrile illnesses with presently available tests, which should deter the unsupervised use of multiplex diagnostic tests. The implementation of adaptive algorithms can predict better diagnosis and add value to the available RDTs. The web application of the model can serve as a tool to identify the optimal diagnostic algorithm in different epidemiological settings, while taking into account the local epidemiological variables and accuracy of available tests.

BACKGROUND:Differentiating the etiology of acute febrile respiratory illness in children is a challenge in lowincome, malaria-endemic settings because the main pathogens responsible (viruses, bacteria, and malaria parasites) overlap in clinical presentation and frequently occur together as mixed infections. The critical task is to rapidly identify bacterial pneumonia to enable appropriate antibiotic treatment, ideally at point of care. Current diagnostic tests are insufficient and there is a need for the discovery and development of new tools. Here we report the identification of a unique biomarker signature that can be identified in blood samples. METHODS:Blood samples from 195 pediatric Mozambican patients with clinical pneumonia were analyzed with an aptamer-based high dynamic range assay to quantify ~1200 proteins. For discovery of new biomarkers, we identified a training set of patient samples in which the underlying etiology of the pneumonia was established as bacterial, viral or malaria. Proteins whose abundances varied significantly between patients with verified etiologies (FDR<0.01) formed the basis for predictive diagnostic models that were created using machine learning techniques (Random Forest, Elastic Net). These models were validated on a dedicated test set of samples. RESULTS:219 proteins had significantly different abundances between bacterial and viral infections, and 151 differed between bacterial infections and a mixed pool of viral and malaria infections. Predictive diagnostic models achieved >90% sensitivity and >80% specificity, regardless of whether one or two pathogen classes were present. Bacterial pneumonia was strongly associated with markers of neutrophil activity, in particular neutrophil degranulation. Degranulation markers included HP, LCN2, LTF, MPO, MMP8, PGLYRP1, RETN, SERPINA1, S100A9, and SLPI. CONCLUSION:Blood protein signatures highly associated with neutrophil biology reliably differentiated bacterial pneumonia from other causes. With appropriate technology, these markers could provide the basis for a rapid diagnostic for field-based triage for antibiotic treatment of pediatric pneumonia.