Seeking diagnostic and prognostic biomarkers for childhood bacterial pneumonia in sub-Saharan Africa: study protocol for an observational study.

INTRODUCTION: Clinically diagnosed pneumonia in children is a leading cause of paediatric hospitalisation and mortality. The aetiology is usually bacterial or viral, but malaria can cause a syndrome indistinguishable from clinical pneumonia. There is no method with high sensitivity to detect a bacterial infection in these patients and, as result, antibiotics are frequently overprescribed. Conversely, unrecognised concomitant bacterial infection in patients with malarial infections occur with omission of antibiotic therapy from patients with bacterial infections. Previously, we identified two combinations of blood proteins with 96% sensitivity and 86% specificity for detecting bacterial disease. The current project aimed to validate and improve these combinations by evaluating additional biomarkers in paediatric patients with clinical pneumonia. Our goal was to describe combinations of a limited number of proteins with high sensitivity and specificity for bacterial infection to be incorporated in future point-of-care tests. Furthermore, we seek to explore signatures to prognosticate clinical pneumonia. METHODS AND ANALYSIS: Patients (n=900) aged 2-59 months presenting with clinical pneumonia at two Gambian hospitals will be enrolled and classified according to criteria for definitive bacterial aetiology (based on microbiological tests and chest radiographs). We will measure proteins at admission using Luminex-based immunoassays in 90 children with definitive and 160 with probable bacterial aetiology, and 160 children classified according to the prognosis of their disease. Previously identified diagnostic signatures will be assessed through accuracy measures. Moreover, we will seek new diagnostic and prognostic signatures through machine learning methods, including support vector machine, penalised regression and classification trees. ETHICS AND DISSEMINATION: Ethics approval has been obtained from the Gambia Government/Medical Research Council Unit The Gambia Joint Ethics Committee (protocol 1616) and the institutional review board of Boston University Medical Centre (STUDY00000958). Study results will be disseminated to the staff of the study hospitals, in scientific seminars and meetings, and in publications. TRIAL REGISTRATION NUMBER: H-38462.

Transcriptional Categorization of the Etiology of Pneumonia Syndrome in Pediatric Patients in Malaria-Endemic Areas.

Background: Pediatric acute respiratory distress in tropical settings is very common. Bacterial pneumonia is a major contributor to morbidity and mortality rates and requires adequate diagnosis for correct treatment. A rapid test that could identify bacterial (vs other) infections would have great clinical utility. Methods and Results: We performed RNA (RNA-seq) sequencing and analyzed the transcriptomes of 68 pediatric patients with well-characterized clinical phenotype to identify transcriptional features associated with each disease class. We refined the features to predictive models (support vector machine, elastic net) and validated those models in an independent test set of 37 patients (80%-85% accuracy). Conclusions: 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.

Responses to Bacteria, Virus, and Malaria Distinguish the Etiology of Pediatric Clinical Pneumonia.

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.

Malaria. A forward genetic screen identifies erythrocyte CD55 as essential for Plasmodium falciparum invasion.

Efforts to identify host determinants for malaria have been hindered by the absence of a nucleus in erythrocytes, which precludes genetic manipulation in the cell in which the parasite replicates. We used cultured red blood cells derived from hematopoietic stem cells to carry out a forward genetic screen for Plasmodium falciparum host determinants. We found that CD55 is an essential host factor for P. falciparum invasion. CD55-null erythrocytes were refractory to invasion by all isolates of P. falciparum because parasites failed to attach properly to the erythrocyte surface. Thus, CD55 is an attractive target for the development of malaria therapeutics. Hematopoietic stem cell-based forward genetic screens may be valuable for the identification of additional host determinants of malaria pathogenesis.

Diversity-oriented synthesis probe targets Plasmodium falciparum cytochrome b ubiquinone reduction site and synergizes with oxidation site inhibitors.

BACKGROUND: The emergence and spread of drug resistance to current antimalarial therapies remains a pressing concern, escalating the need for compounds that demonstrate novel modes of action. Diversity-Oriented Synthesis (DOS) libraries bridge the gap between conventional small molecule and natural product libraries, allowing the interrogation of more diverse chemical space in efforts to identify probes of novel parasite pathways. METHODS: We screened and optimized a probe from a DOS library using whole-cell phenotypic assays. Resistance selection and whole-genome sequencing approaches were employed to identify the cellular target of the compounds. RESULTS: We identified a novel macrocyclic inhibitor of Plasmodium falciparum with nanomolar potency and identified the reduction site of cytochrome b as its cellular target. Combination experiments with reduction and oxidation site inhibitors showed synergistic inhibition of the parasite. CONCLUSIONS: The cytochrome b oxidation center is a validated antimalarial target. We show that the reduction site of cytochrome b is also a druggable target. Our results demonstrating a synergistic relationship between oxidation and reduction site inhibitors suggests a future strategy for new combination therapies in the treatment of malaria.

Diversity-oriented synthesis-facilitated medicinal chemistry: toward the development of novel antimalarial agents.

Here, we describe medicinal chemistry that was accelerated by a diversity-oriented synthesis (DOS) pathway, and in vivo studies of our previously reported macrocyclic antimalarial agent that derived from the synthetic pathway. Structure-activity relationships that focused on both appendage and skeletal features yielded a nanomolar inhibitor of P. falciparum asexual blood-stage growth with improved solubility and microsomal stability and reduced hERG binding. The build/couple/pair (B/C/P) synthetic strategy, used in the preparation of the original screening library, facilitated medicinal chemistry optimization of the antimalarial lead.

In vitro resistance selections for Plasmodium falciparum dihydroorotate dehydrogenase inhibitors give mutants with multiple point mutations in the drug-binding site and altered growth.

Malaria is a preventable and treatable disease; yet half of the world's population lives at risk of infection, and an estimated 660,000 people die of malaria-related causes every year. Rising drug resistance threatens to make malaria untreatable, necessitating both the discovery of new antimalarial agents and the development of strategies to identify and suppress the emergence and spread of drug resistance. We focused on in-development dihydroorotate dehydrogenase (DHODH) inhibitors. Characterizing resistance pathways for antimalarial agents not yet in clinical use will increase our understanding of the potential for resistance. We identified resistance mechanisms of Plasmodium falciparum (Pf) DHODH inhibitors via in vitro resistance selections. We found 11 point mutations in the PfDHODH target. Target gene amplification and unknown mechanisms also contributed to resistance, albeit to a lesser extent. These mutant parasites were often hypersensitive to other PfDHODH inhibitors, which immediately suggested a novel combination therapy approach to preventing resistance. Indeed, a combination of wild-type and mutant-type selective inhibitors led to resistance far less often than either drug alone. The effects of point mutations in PfDHODH were corroborated with purified recombinant wild-type and mutant-type PfDHODH proteins, which showed the same trends in drug response as the cognate cell lines. Comparative growth assays demonstrated that two mutant parasites grew less robustly than their wild-type parent, and the purified protein of those mutants showed a decrease in catalytic efficiency, thereby suggesting a reason for the diminished growth rate. Co-crystallography of PfDHODH with three inhibitors suggested that hydrophobic interactions are important for drug binding and selectivity.

Harnessing evolutionary fitness in Plasmodium falciparum for drug discovery and suppressing resistance.

Drug resistance emerges in an ecological context where fitness costs restrict the diversity of escape pathways. These pathways are targets for drug discovery, and here we demonstrate that we can identify small-molecule inhibitors that differentially target resistant parasites. Combining wild-type and mutant-type inhibitors may prevent the emergence of competitively viable resistance. We tested this hypothesis with a clinically derived chloroquine-resistant (CQ(r)) malaria parasite and with parasites derived by in vitro selection with Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors. We screened a chemical library against CQ(s) and CQ(r) lines and discovered a drug-like compound (IDI-3783) that was potent only in the CQ(r) line. Surprisingly, in vitro selection of Plasmodium falciparum resistant to IDI-3783 restored CQ sensitivity, thereby indicating that CQ might once again be useful as a malaria therapy. In parallel experiments, we selected P. falciparum lines resistant to structurally unrelated PfDHODH inhibitors (Genz-666136 and DSM74). Both selections yielded resistant lines with the same point mutation in PfDHODH:E182D. We discovered a compound (IDI-6273) more potent against E182D than wild-type parasites. Selection of the E182D mutant with IDI-6273 yielded a reversion to the wild-type protein sequence and phenotype although the nucleotide sequence was different. Importantly, selection with a combination of Genz-669178, a wild-type PfDHODH inhibitor, and IDI-6273, a mutant-selective PfDHODH inhibitor, did not yield resistant parasites. These two examples demonstrate that the compromise between resistance and evolutionary fitness can be exploited to design therapies that prevent the emergence and spread of resistant organisms.

Sequence-based association and selection scans identify drug resistance loci in the Plasmodium falciparum malaria parasite.

Through rapid genetic adaptation and natural selection, the Plasmodium falciparum parasite--the deadliest of those that cause malaria--is able to develop resistance to antimalarial drugs, thwarting present efforts to control it. Genome-wide association studies (GWAS) provide a critical hypothesis-generating tool for understanding how this occurs. However, in P. falciparum, the limited amount of linkage disequilibrium hinders the power of traditional array-based GWAS. Here, we demonstrate the feasibility and power improvements gained by using whole-genome sequencing for association studies. We analyzed data from 45 Senegalese parasites and identified genetic changes associated with the parasites' in vitro response to 12 different antimalarials. To further increase statistical power, we adapted a common test for natural selection, XP-EHH (cross-population extended haplotype homozygosity), and used it to identify genomic regions associated with resistance to drugs. Using this sequence-based approach and the combination of association and selection-based tests, we detected several loci associated with drug resistance. These loci included the previously known signals at pfcrt, dhfr, and pfmdr1, as well as many genes not previously implicated in drug-resistance roles, including genes in the ubiquitination pathway. Based on the success of the analysis presented in this study, and on the demonstrated shortcomings of array-based approaches, we argue for a complete transition to sequence-based GWAS for small, low linkage-disequilibrium genomes like that of P. falciparum.

Genomic sequencing of Plasmodium falciparum malaria parasites from Senegal reveals the demographic history of the population.

Malaria is a deadly disease that causes nearly one million deaths each year. To develop methods to control and eradicate malaria, it is important to understand the genetic basis of Plasmodium falciparum adaptations to antimalarial treatments and the human immune system while taking into account its demographic history. To study the demographic history and identify genes under selection more efficiently, we sequenced the complete genomes of 25 culture-adapted P. falciparum isolates from three sites in Senegal. We show that there is no significant population structure among these Senegal sampling sites. By fitting demographic models to the synonymous allele-frequency spectrum, we also estimated a major 60-fold population expansion of this parasite population ∼20,000-40,000 years ago. Using inferred demographic history as a null model for coalescent simulation, we identified candidate genes under selection, including genes identified before, such as pfcrt and PfAMA1, as well as new candidate genes. Interestingly, we also found selection against G/C to A/T changes that offsets the large mutational bias toward A/T, and two unusual patterns: similar synonymous and nonsynonymous allele-frequency spectra, and 18% of genes having a nonsynonymous-to-synonymous polymorphism ratio >1.

Diversity-Oriented Synthesis Yields a Novel Lead for the Treatment of Malaria.

Here, we describe the discovery of a novel antimalarial agent using phenotypic screening of Plasmodium falciparum asexual blood-stage parasites. Screening a novel compound collection created using diversity-oriented synthesis (DOS) led to the initial hit. Structure-activity relationships guided the synthesis of compounds having improved potency and water solubility, yielding a subnanomolar inhibitor of parasite asexual blood-stage growth. Optimized compound 27 has an excellent off-target activity profile in erythrocyte lysis and HepG2 assays and is stable in human plasma. This compound is available via the molecular libraries probe production centers network (MLPCN) and is designated ML238.

Human cerebral malaria and Plasmodium falciparum genotypes in Malawi.

BACKGROUND: Cerebral malaria, a severe form of Plasmodium falciparum infection, is an important cause of mortality in sub-Saharan African children. A Taqman 24 Single Nucleotide Polymorphisms (SNP) molecular barcode assay was developed for use in laboratory parasites which estimates genotype number and identifies the predominant genotype. METHODS: The 24 SNP assay was used to determine predominant genotypes in blood and tissues from autopsy and clinical patients with cerebral malaria. RESULTS: Single genotypes were shared between the peripheral blood, the brain, and other tissues of cerebral malaria patients, while malaria-infected patients who died of non-malarial causes had mixed genetic signatures in tissues examined. Children with retinopathy-positive cerebral malaria had significantly less complex infections than those without retinopathy (OR = 3.7, 95% CI [1.51-9.10]).The complexity of infections significantly decreased over the malaria season in retinopathy-positive patients compared to retinopathy-negative patients. CONCLUSIONS: Cerebral malaria patients harbour a single or small set of predominant parasites; patients with incidental parasitaemia sustain infections involving diverse genotypes. Limited diversity in the peripheral blood of cerebral malaria patients and correlation with tissues supports peripheral blood samples as appropriate for genome-wide association studies of parasite determinants of pathogenicity.

Identification and functional validation of the novel antimalarial resistance locus PF10_0355 in Plasmodium falciparum.

The Plasmodium falciparum parasite's ability to adapt to environmental pressures, such as the human immune system and antimalarial drugs, makes malaria an enduring burden to public health. Understanding the genetic basis of these adaptations is critical to intervening successfully against malaria. To that end, we created a high-density genotyping array that assays over 17,000 single nucleotide polymorphisms (∼ 1 SNP/kb), and applied it to 57 culture-adapted parasites from three continents. We characterized genome-wide genetic diversity within and between populations and identified numerous loci with signals of natural selection, suggesting their role in recent adaptation. In addition, we performed a genome-wide association study (GWAS), searching for loci correlated with resistance to thirteen antimalarials; we detected both known and novel resistance loci, including a new halofantrine resistance locus, PF10_0355. Through functional testing we demonstrated that PF10_0355 overexpression decreases sensitivity to halofantrine, mefloquine, and lumefantrine, but not to structurally unrelated antimalarials, and that increased gene copy number mediates resistance. Our GWAS and follow-on functional validation demonstrate the potential of genome-wide studies to elucidate functionally important loci in the malaria parasite genome.

A high-throughput screen targeting malaria transmission stages opens new avenues for drug development.

A major goal of the worldwide malaria eradication program is the reduction and eventual elimination of malaria transmission. All currently available antimalarial compounds were discovered on the basis of their activity against the asexually reproducing red blood cell stages of the parasite, which are responsible for the morbidity and mortality of human malaria. Resistance against these compounds is widespread, and there is an urgent need for novel approaches to reduce the emergence of resistance to new antimalarials as they are introduced. We have established and validated the first high-throughput assay targeting the red blood cell parasite stage required for transmission, the sexually reproducing gametocyte. This assay will permit identification of compounds specifically targeting the transmission stages in addition to the asexual stage parasites. Such stage-specific compounds may be used in a combination therapy, reducing the emergence of resistance by blocking transmission of resistant parasites that may be selected in a patient.

Aminoindoles, a novel scaffold with potent activity against Plasmodium falciparum.

This study characterizes aminoindole molecules that are analogs of Genz-644442. Genz-644442 was identified as a hit in a screen of ~70,000 compounds in the Broad Institute's small-molecule library and the ICCB-L compound collection at Harvard Medical School. Genz-644442 is a potent inhibitor of Plasmodium falciparum in vitro (50% inhibitory concentrations [IC50s], 200 to 285 nM) and inhibits P. berghei in vivo with an efficacy of > 99% in an adapted version of Peters' 4-day suppressive test (W. Peters, Ann. Trop. Med. Parasitol. 69:155-171, 1975). Genz-644442 became the focus of medicinal chemistry optimization; 321 analogs were synthesized and were tested for in vitro potency against P. falciparum and for in vitro absorption, distribution, metabolism, and excretion (ADME) properties. This yielded compounds with IC50s of approximately 30 nM. The lead compound, Genz-668764, has been characterized in more detail. It is a single enantiomer with IC50s of 28 to 65 nM against P. falciparum in vitro. In the 4-day P. berghei model, when it was dosed at 100 mg/kg of body weight/day, no parasites were detected on day 4 postinfection. However, parasites recrudesced by day 9. Dosing at 200 mg/kg/day twice a day resulted in cures of 3/5 animals. The compound had comparable activity against P. falciparum blood stages in a human-engrafted NOD-scid mouse model. Genz-668764 had a terminal half-life of 2.8 h and plasma trough levels of 41 ng/ml when it was dosed twice a day orally at 55 mg/kg/day. Seven-day rat safety studies showed a no-observable-adverse-effect level (NOAEL) at 200 mg/kg/day; the compound was not mutagenic in Ames tests, did not inhibit the hERG channel, and did not have potent activity against a broad panel of receptors and enzymes. Employing allometric scaling and using in vitro ADME data, the predicted human minimum efficacious dose of Genz-668764 in a 3-day once-daily dosing regimen was 421 mg/day/70 kg, which would maintain plasma trough levels above the IC90 against P. falciparum for at least 96 h after the last dose. The predicted human therapeutic index was approximately 3, on the basis of the exposure in rats at the NOAEL. We were unable to select for parasites with >2-fold decreased sensitivity to the parent compound, Genz-644442, over 270 days of in vitro culture under drug pressure. These characteristics make Genz-668764 a good candidate for preclinical development.

Genome-wide SNP genotyping highlights the role of natural selection in Plasmodium falciparum population divergence.

BACKGROUND: The malaria parasite Plasmodium falciparum exhibits abundant genetic diversity, and this diversity is key to its success as a pathogen. Previous efforts to study genetic diversity in P. falciparum have begun to elucidate the demographic history of the species, as well as patterns of population structure and patterns of linkage disequilibrium within its genome. Such studies will be greatly enhanced by new genomic tools and recent large-scale efforts to map genomic variation. To that end, we have developed a high throughput single nucleotide polymorphism (SNP) genotyping platform for P. falciparum. RESULTS: Using an Affymetrix 3,000 SNP assay array, we found roughly half the assays (1,638) yielded high quality, 100% accurate genotyping calls for both major and minor SNP alleles. Genotype data from 76 global isolates confirm significant genetic differentiation among continental populations and varying levels of SNP diversity and linkage disequilibrium according to geographic location and local epidemiological factors. We further discovered that nonsynonymous and silent (synonymous or noncoding) SNPs differ with respect to within-population diversity, inter-population differentiation, and the degree to which allele frequencies are correlated between populations. CONCLUSIONS: The distinct population profile of nonsynonymous variants indicates that natural selection has a significant influence on genomic diversity in P. falciparum, and that many of these changes may reflect functional variants deserving of follow-up study. Our analysis demonstrates the potential for new high-throughput genotyping technologies to enhance studies of population structure, natural selection, and ultimately enable genome-wide association studies in P. falciparum to find genes underlying key phenotypic traits.

A general SNP-based molecular barcode for Plasmodium falciparum identification and tracking.

BACKGROUND: Single nucleotide polymorphism (SNP) genotyping provides the means to develop a practical, rapid, inexpensive assay that will uniquely identify any Plasmodium falciparum parasite using a small amount of DNA. Such an assay could be used to distinguish recrudescence from re-infection in drug trials, to monitor the frequency and distribution of specific parasites in a patient population undergoing drug treatment or vaccine challenge, or for tracking samples and determining purity of isolates in the laboratory during culture adaptation and sub-cloning, as well as routine passage. METHODS: A panel of twenty-four SNP markers has been identified that exhibit a high minor allele frequency (average MAF > 35%), for which robust TaqMan genotyping assays were constructed. All SNPs were identified through whole genome sequencing and MAF was estimated through Affymetrix array-based genotyping of a worldwide collection of parasites. These assays create a "molecular barcode" to uniquely identify a parasite genome. RESULTS: Using 24 such markers no two parasites known to be of independent origin have yet been found to have the same allele signature. The TaqMan genotyping assays can be performed on a variety of samples including cultured parasites, frozen whole blood, or whole blood spotted onto filter paper with a success rate > 99%. Less than 5 ng of parasite DNA is needed to complete a panel of 24 markers. The ability of this SNP panel to detect and identify parasites was compared to the standard molecular methods, MSP-1 and MSP-2 typing. CONCLUSION: This work provides a facile field-deployable genotyping tool that can be used without special skills with standard lab equipment, and at reasonable cost that will unambiguously identify and track P. falciparum parasites both from patient samples and in the laboratory.

A genome-wide map of diversity in Plasmodium falciparum.

Genetic variation allows the malaria parasite Plasmodium falciparum to overcome chemotherapeutic agents, vaccines and vector control strategies and remain a leading cause of global morbidity and mortality. Here we describe an initial survey of genetic variation across the P. falciparum genome. We performed extensive sequencing of 16 geographically diverse parasites and identified 46,937 SNPs, demonstrating rich diversity among P. falciparum parasites (pi = 1.16 x 10(-3)) and strong correlation with gene function. We identified multiple regions with signatures of selective sweeps in drug-resistant parasites, including a previously unidentified 160-kb region with extremely low polymorphism in pyrimethamine-resistant parasites. We further characterized 54 worldwide isolates by genotyping SNPs across 20 genomic regions. These data begin to define population structure among African, Asian and American groups and illustrate the degree of linkage disequilibrium, which extends over relatively short distances in African parasites but over longer distances in Asian parasites. We provide an initial map of genetic diversity in P. falciparum and demonstrate its potential utility in identifying genes subject to recent natural selection and in understanding the population genetics of this parasite.