The Consortium on Newborn Screening in Africa for sickle cell disease: study rationale and methodology.

Sickle cell disease (SCD) is a common condition within sub-Saharan Africa and associated with high under-5 mortality (U5M). The American Society of Hematology instituted the Consortium on Newborn Screening in Africa (CONSA) for SCD, a 7-country network of sites to implement standardized newborn hemoglobinopathy screening and early intervention for children with SCD in sub-Saharan Africa. CONSA's overall hypothesis is that early infant SCD screening and entry into standardized, continuous care will reduce U5M compared with historical estimates in the region. Primary trial objectives are to determine the population-based birth incidence of SCD and effectiveness of early standardized care for preventing early mortality consortium-wide at each country's site(s). Secondary objectives are to establish universal screening and early interventions for SCD within clinical networks of CONSA partners and assess trial implementation. Outcomes will be evaluated from data collected using a shared patient registry. Standardized trial procedures will be implemented among designated birth populations in 7 African countries whose programs met eligibility criteria. Treatment protocol includes administering antibacterial and antimalarial prophylaxis and standard childhood vaccinations against infections commonly affecting children with SCD. Infants with a positive screen and confirmation of SCD within the catchment areas defined by each consortium partner will be enrolled in the clinical intervention protocol and followed regularly until age of 5 years. Effectiveness of these early interventions, along with culturally appropriate family education and counseling, will be evaluated by comparing U5M in the enrolled cohort to estimated preprogram data. Here, we describe the methodology planned for this trial.

Automated measurement of malaria parasitaemia among asymptomatic blood donors in Malawi using the Sysmex XN-31 analyser: could such data be used to complement national malaria surveillance in real time?

BACKGROUND: The recent worldwide increase in malaria cases highlights the need for renewed efforts to eliminate malaria. The World Health Organization advocates that malaria surveillance becomes a core intervention. Current methods to estimate the malaria burden rely on clinical malaria case reports and surveys of asymptomatic parasite infection mainly from children < 5 years. In this study the hypothesis was that screening blood donors for malaria parasites would provide real-time information on the asymptomatic reservoir of parasites in the adult population and mirror other surveillance data. METHODS: This study was conducted in Malawi, a high malaria burden country, at the Malawi Blood Transfusion Service, which collects blood units at donation sites countrywide. A secondary analysis was conducted on data obtained from a prior Sysmex XN-31 analyser malaria diagnostic evaluation study utilizing residual donor blood samples. XN-31 malaria results, donor age, sex, geographical location, and collection date, were analysed using standard statistical methods. RESULTS: The malaria parasite prevalence in blood donors was 11.6% (614/5281 samples) increasing seasonally from December (8.6%) to April (18.3%). The median age was 21 years and 45.9% of donors were from urban areas, which showed a lower prevalence compared to non-urban regions. The Central administrative region had the highest and the Northern region the lowest malaria parasite prevalence. The donors were predominantly male (80.2%), 13.1% of whom had malaria parasites, which was significantly higher (p < 0.0001) than for female donors (7.4%). Multivariable logistic regression analysis showed that age, location, and collection month were significant predictors of malaria positivity in males, whereas in females only location was significant. There was no gender difference in parasite density nor gametocyte carriage. CONCLUSIONS: This study demonstrates the powerful utility of screening blood donors for malaria parasites using the XN-31, which not only improves the safety of blood transfusion, but provides valuable complementary surveillance data for malaria control, especially targeting males, who are generally excluded from periodic household surveys. Blood donations are sourced countrywide, year-round, and thus provide dynamic, real-time information on the malaria burden. Furthermore, the XN-31 identifies the asymptomatic human reservoir of infectious gametocytes, which must be targeted to eliminate malaria.

Identification and Profiling of a Novel Diazaspiro[3.4]octane Chemical Series Active against Multiple Stages of the Human Malaria Parasite Plasmodium falciparum and Optimization Efforts.

A novel diazaspiro[3.4]octane series was identified from a Plasmodium falciparum whole-cell high-throughput screening campaign. Hits displayed activity against multiple stages of the parasite lifecycle, which together with a novel sp3-rich scaffold provided an attractive starting point for a hit-to-lead medicinal chemistry optimization and biological profiling program. Structure-activity-relationship studies led to the identification of compounds that showed low nanomolar asexual blood-stage activity (<50 nM) together with strong gametocyte sterilizing properties that translated to transmission-blocking activity in the standard membrane feeding assay. Mechanistic studies through resistance selection with one of the analogues followed by whole-genome sequencing implicated the P. falciparum cyclic amine resistance locus in the mode of resistance.

Multistage and transmission-blocking targeted antimalarials discovered from the open-source MMV Pandemic Response Box.

Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H+-ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages.

Evaluation of automated malaria diagnosis using the Sysmex XN-30 analyser in a clinical setting.

BACKGROUND: Early and accurate diagnosis of malaria is a critical aspect of efforts to control the disease, and several diagnostic tools are available. Microscopic assessment of a peripheral blood smear enables direct visualization of parasites in infected red blood cells and is the clinical diagnostic gold standard. However, it is subjective and requires a high level of skill. Numerous indirect detection methods are in use, but are not ideal since surrogate markers of infection are measured. This study describes the first clinical performance evaluation of the automated Sysmex XN-30 analyser, which utilizes fluorescence flow cytometry to directly detect and quantitate parasite-infected red blood cells. RESULTS: Residual EDTA blood samples from suspected malaria cases referred for routine diagnosis were analysed on the XN-30. Parasitaemia was reported as a percentage, as well as absolute numbers of infected red blood cells, and scattergrams provided a visual image of the parasitized red blood cell clusters. The results reported by the XN-30 correlated with microscopy and the analyser demonstrated 100% sensitivity and specificity. Measurements were reproducible and storage of samples at room temperature did not affect the parameters. Several Plasmodium species were detected, including Plasmodium falciparum, Plasmodium vivax and Plasmodium ovale. The XN-30 also identified the transmissible gametocytes as a separate cluster on the scattergrams. Abnormal red blood cell indices (low haemoglobin and raised reticulocyte counts), haemoglobinopathies and thrombocytopenia did not interfere with the detection of parasites. The XN-30 also generated a concurrent full blood count for each sample. CONCLUSIONS: The novel technology of the Sysmex XN-30 provides a robust, rapid, automated and accurate platform for diagnosing malaria in a clinical setting. The objective enumeration of red blood cells infected with Plasmodium species makes it suitable for global use and allows monitoring of the parasite load once therapy has been initiated, thereby providing an early marker of drug resistance. The automated generation of a full blood count for each sample provides an opportunity for detecting unsuspected cases. Asymptomatic carriers can also be identified, which will be useful in blood transfusion centres, and will enable treatment of these individuals to prevent the spread of the disease.

Multistage Antiplasmodium Activity of Astemizole Analogues and Inhibition of Hemozoin Formation as a Contributor to Their Mode of Action.

A drug repositioning approach was leveraged to derivatize astemizole (AST), an antihistamine drug whose antimalarial activity was previously identified in a high-throughput screen. The multistage activity potential against the Plasmodium parasite's life cycle of the subsequent analogues was examined by evaluating against the parasite asexual blood, liver, and sexual gametocytic stages. In addition, the previously reported contribution of heme detoxification to the compound's mode of action was interrogated. Ten of the 17 derivatives showed half-maximal inhibitory concentrations (IC50s) of <0.1 µM against the chloroquine (CQ)-sensitive Plasmodium falciparum NF54 ( PfNF54) strain while maintaining submicromolar potency against the multidrug-resistant strain, PfK1, with most showing low likelihood of cross-resistance with CQ. Selected analogues ( PfNF54-IC50 < 0.1 µM) were tested for cytotoxicity on Chinese hamster ovarian (CHO) cells and found to be highly selective (selectivity index > 100). Screening of AST and its analogues against gametocytes revealed their moderate activity (IC50: 1-5 µM) against late stage P. falciparum gametocytes, while the evaluation of activity against P. berghei liver stages identified one compound (3) with 3-fold greater activity than the parent AST compound. Mechanistic studies showed a strong correlation between in vitro inhibition of ß-hematin formation by the AST derivatives and their antiplasmodium IC50s. Analyses of intracellular inhibition of hemozoin formation within the parasite further yielded signatures attributable to a possible perturbation of the heme detoxification machinery.

Structure-Activity Relationship Studies and Plasmodium Life Cycle Profiling Identifies Pan-Active N-Aryl-3-trifluoromethyl Pyrido[1,2- a]benzimidazoles Which Are Efficacious in an in Vivo Mouse Model of Malaria.

Structure-activity relationship studies involving N-aryl-3-trifluoromethyl pyrido[1,2- a]benzimidazoles (PBI) identified several compounds possessing potent in vitro activities against the asexual blood, liver, and gametocyte stages of the Plasmodium parasite with no cross-resistance to chloroquine. Frontrunner lead compounds with good in vitro absorption, distribution, metabolism, and excretion (ADME) profiles were subjected to in vivo proof-of-concept studies in NMRI mice harboring the rodent P. berghei infection. This led to the identification of compounds 10 and 49, effecting 98% and 99.93% reduction in parasitemia with mean survival days of 12 and 14, respectively, at an oral dose of 4 × 50 mg/kg. In vivo pharmacokinetics studies on 10 revealed slow absorption, low volume of distribution, and low clearance profiles. Furthermore, this series displayed a low propensity to inhibit the human ether-a-go-go-related gene (hERG) potassium ion channel whose inhibition is associated with cardiotoxicity.

UCT943, a Next-Generation Plasmodium falciparum PI4K Inhibitor Preclinical Candidate for the Treatment of Malaria.

The 2-aminopyridine MMV048 was the first drug candidate inhibiting Plasmodium phosphatidylinositol 4-kinase (PI4K), a novel drug target for malaria, to enter clinical development. In an effort to identify the next generation of PI4K inhibitors, the series was optimized to improve properties such as solubility and antiplasmodial potency across the parasite life cycle, leading to the 2-aminopyrazine UCT943. The compound displayed higher asexual blood stage, transmission-blocking, and liver stage activities than MMV048 and was more potent against resistant Plasmodium falciparum and Plasmodium vivax clinical isolates. Excellent in vitro antiplasmodial activity translated into high efficacy in Plasmodium berghei and humanized P. falciparum NOD-scid IL-2Rγ null mouse models. The high passive permeability and high aqueous solubility of UCT943, combined with low to moderate in vivo intrinsic clearance, resulted in sustained exposure and high bioavailability in preclinical species. In addition, the predicted human dose for a curative single administration using monkey and dog pharmacokinetics was low, ranging from 50 to 80 mg. As a next-generation Plasmodium PI4K inhibitor, UCT943, based on the combined preclinical data, has the potential to form part of a single-exposure radical cure and prophylaxis (SERCaP) to treat, prevent, and block the transmission of malaria.

Potent Plasmodium falciparum gametocytocidal compounds identified by exploring the kinase inhibitor chemical space for dual active antimalarials.

Objectives: Novel chemical tools to eliminate malaria should ideally target both the asexual parasites and transmissible gametocytes. Several imidazopyridazines (IMPs) and 2-aminopyridines (2-APs) have been described as potent antimalarial candidates targeting lipid kinases. However, these have not been extensively explored for stage-specific inhibition of gametocytes in Plasmodium falciparum parasites. Here we provide an in-depth evaluation of the gametocytocidal activity of compounds from these chemotypes and identify novel starting points for dual-acting antimalarials. Methods: We evaluated compounds against P. falciparum gametocytes using several assay platforms for cross-validation and stringently identified hits that were further profiled for stage specificity, speed of action and ex vivo efficacy. Physicochemical feature extraction and chemogenomic fingerprinting were applied to explore the kinase inhibition susceptibility profile. Results: We identified 34 compounds with submicromolar activity against late stage gametocytes, validated across several assay platforms. Of these, 12 were potent at <100 nM (8 were IMPs and 4 were 2-APs) and were also active against early stage gametocytes and asexual parasites, with >1000-fold selectivity towards the parasite over mammalian cells. Front-runner compounds targeted mature gametocytes within 48 h and blocked transmission to mosquitoes. The resultant chemogenomic fingerprint of parasites treated with the lead compounds revealed the importance of targeting kinases in asexual parasites and gametocytes. Conclusions: This study encompasses an in-depth evaluation of the kinase inhibitor space for gametocytocidal activity. Potent lead compounds have enticing dual activities and highlight the importance of targeting the kinase superfamily in malaria elimination strategies.

Absence of Acanthocytosis in Huntington's Disease-like 2: A Prospective Comparison with Huntington's Disease.

Background: Huntington's Disease-like 2 (HDL2) is classified as a neuroacanthocytosis; however, this remains unverified. We aim to determine if acanthocytes are present in HDL2 and whether acanthocytes can differentiate HDL2 from Huntington's disease (HD). Methods: We prospectively compared 13 HD and 12 HDL2 cases against 21 unaffected controls in Johannesburg. Blood smears were prepared using international standards and reviewed by at least two blinded reviewers. An acanthocytosis rate of greater than 1.2% in the dry smear or greater than 3.7% in the wet smear was designated a priori as the threshold for clinical significance based on previously established standards. Flow cytometry was performed on all but four of the cases. Red cell membrane protein analysis was performed on all participants. Results: There were 12 HDL2, 13 HD, and 21 controls enrolled. None of the HD or HDL2 participants had defined acanthocytosis or other morphological abnormalities. None of the HD or HDL2 cases had evidence of an abnormal band 3. Discussion: Acanthocytosis was not identified in either HDL2 or HD in our patient population. Our results, based on the first prospective study of acanthocytes in HDL2 or HD, suggest that screening for acanthocytes will not help establish the diagnosis of HD or HDL2, nor differentiate between the two disorders and raises the question if HDL2 should be placed within the neuroacanthocytosis syndromes.

Antimalarial Pyrido[1,2-a]benzimidazoles: Lead Optimization, Parasite Life Cycle Stage Profile, Mechanistic Evaluation, Killing Kinetics, and in Vivo Oral Efficacy in a Mouse Model.

Further structure-activity relationship (SAR) studies on the recently identified pyrido[1,2-a]benzimidazole (PBI) antimalarials have led to the identification of potent, metabolically stable compounds with improved in vivo oral efficacy in the P. berghei mouse model and additional activity against parasite liver and gametocyte stages, making them potential candidates for preclinical development. Inhibition of hemozoin formation possibly contributes to the mechanism of action.

Discovering New Transmission-Blocking Antimalarial Compounds: Challenges and Opportunities.

The ability to target human-mosquito parasite transmission challenges global malaria elimination. However, it is not obvious what a transmission-blocking drug will look like; should it target only parasite transmission stages; be combined with a partner drug killing the pathogenic asexual stages; or kill both the sexual and asexual blood stages, preferably displaying polypharmacology? The development of transmission-blocking antimalarials requires objective analyses of the current strategies. Here, pertinent issues and questions regarding the target candidate profile of a transmission-blocking compound, and its role in malaria elimination strategies, are highlighted and novel perspectives proposed. The essential role of a test cascade that integrates screening and validation strategies to identify next-generation transmission-blocking antimalarials is emphasised.

Localization and interactions of Plasmodium falciparum SWIB/MDM2 homologues.

BACKGROUND: Malaria remains a global health problem and the majority of deaths are caused by Plasmodium falciparum parasites. Due to the rapid emergence of drug-resistant strains, novel avenues of research on the biology of the parasite are needed. The massive proliferation of asexual, intra-erythrocytic parasites every 48 h could kill the human host prior to transmission of slow-developing gametocytes to the mosquito vector. A self-induced P. falciparum programmed cell death mechanism has been hypothesized to maintain this balance between the parasite and its two hosts, but molecular participants of the cell death pathway in P. falciparum have not been characterized. Proteins with SWIB/MDM2 domains play a key role in metazoan programmed cell death and this study provides the first evaluation of two parasite SWIB/MDM2 homologues, PF3D7_0518200 (PfMDM2) and PF3D7_0611400 (PfSWIB). METHODS: The function of these proteins was assessed by predicting their structural topology with the aid of bioinformatics and determining their location within live transgenic parasites, expressing green fluorescent protein-tagged PfMDM2 and PfSWIB under normal and elevated temperatures, which mimic fever and which are known to induce a programmed cell death response. Additionally, P. falciparum phage display library technology was used to identify binding partners for the two parasite SWIB/MDM2 domains. RESULTS: Structural features of the SWIB/MDM2 domains of PfMDM2 and PfSWIB, suggested that they are chromatin remodelling factors. The N-terminal signal sequence of PfMDM2 directed the protein to the mitochondrion under both normal and heat stress conditions. Plasmodium falciparum phage display library technology revealed that the C-terminal SWIB/MDM2 domain of PfMDM2 interacted with a conserved protein containing a LisH domain. PfSWIB localized to the cytoplasm under normal growth conditions, while approximately 10% of the heat-stressed trophozoite-stage parasites presented a rapid but short-lived nuclear localization pattern. Two PfSWIB binding partners, a putative Aurora-related kinase and a member of the inner membrane complex, were identified. CONCLUSION: These novel data provide insight into the function of two parasite SWIB/MDM2 homologues and suggest that PfMDM2 plays a role within the mitochondrion and that PfSWIB is involved in a stage-specific, heat-stress, response pathway.

A Novel Pyrazolopyridine with in Vivo Activity in Plasmodium berghei- and Plasmodium falciparum-Infected Mouse Models from Structure-Activity Relationship Studies around the Core of Recently Identified Antimalarial Imidazopyridazines.

Toward improving pharmacokinetics, in vivo efficacy, and selectivity over hERG, structure-activity relationship studies around the central core of antimalarial imidazopyridazines were conducted. This study led to the identification of potent pyrazolopyridines, which showed good in vivo efficacy and pharmacokinetics profiles. The lead compounds also proved to be very potent in the parasite liver and gametocyte stages, which makes them of high interest.

Nowhere to hide: interrogating different metabolic parameters of Plasmodium falciparum gametocytes in a transmission blocking drug discovery pipeline towards malaria elimination.

BACKGROUND: The discovery of malaria transmission-blocking compounds is seen as key to malaria elimination strategies and gametocyte-screening platforms are critical filters to identify active molecules. However, unlike asexual parasite assays measuring parasite proliferation, greater variability in end-point readout exists between different gametocytocidal assays. This is compounded by difficulties in routinely producing viable, functional and stage-specific gametocyte populations. Here, a parallel evaluation of four assay platforms on the same gametocyte populations was performed for the first time. This allowed the direct comparison of the ability of different assay platforms to detect compounds with gametocytocidal activity and revealed caveats in some assay readouts that interrogate different parasite biological functions. METHODS: Gametocytogenesis from Plasmodium falciparum (NF54) was optimized with a robust and standardized protocol. ATP, pLDH, luciferase reporter and PrestoBlue® assays were compared in context of a set of 10 reference compounds. The assays were performed in parallel on the same gametocyte preparation (except for luciferase reporter lines) using the same drug preparations (48 h). The remaining parameters for each assay were all comparable. RESULTS: A highly robust method for generating viable and functional gametocytes was developed and comprehensively validated resulting in an average gametocytaemia of 4%. Subsequent parallel assays for gametocytocidal activity indicated that different assay platforms were not able to screen compounds with variant chemical scaffolds similarly. Luciferase reporter assays revealed that synchronized stage-specific gametocyte production is essential for drug discovery, as differential susceptibility in various gametocyte developmental populations is evident. CONCLUSIONS: With this study, the key parameters for assays aiming at testing the gametocytocidal activity of potential transmission blocking molecules against Plasmodium gametocytes were accurately dissected. This first and uniquely comparative study emphasizes differential effects seen with the use of different assay platforms interrogating variant biological systems. Whilst this data is informative from a biological perspective and may provide indications of the drug mode of action, it does highlight the care that must be taken when screening broad-diversity chemotypes with a single assay platform against gametocytes for which the biology is not clearly understood.

Intrinsic disorder within the erythrocyte binding-like proteins from Plasmodium falciparum.

The ability of the malaria parasite, Plasmodium falciparum, to proliferate within the human host depends on its invasion of erythrocytes. Erythrocyte binding-like (EBL) proteins play crucial roles in the attachment of merozoites to human erythrocytes by binding to specific receptors on the cell surface. In this study, we have carried out a bioinformatics analysis of the three EBL proteins EBA-140, EBA-175 and EBA-181 and show that they contain a large amount of intrinsic disorder in particular within the RIII-V domains. The functional role of these domains has so far not been identified, although antibodies raised against these regions were shown to inhibit parasite invasion. Here, we obtain a more complete structural and dynamic view of the EBL proteins by focusing on the biophysical characterization of a smaller construct of the RIII-V regions of EBA-181 (EBA-181945-1097). We show using a number of techniques that EBA-181945-1097 is intrinsically disordered, and we obtain a detailed structural and dynamic characterization of the protein at atomic resolution using nuclear magnetic resonance (NMR) spectroscopy. Our results show that EBA-181945-1097 is essentially a statistical coil with the presence of several turn motifs and does not possess transiently populated secondary structures as is common for many intrinsically disordered proteins that fold via specific, pre-formed molecular recognition elements.

Novel exon 2 α spectrin mutation and intragenic crossover: three morphological phenotypes associated with four distinct α spectrin defects.

Hereditary pyropoikilocytosis is a severe hemolytic anemia caused by spectrin deficiency and defective spectrin dimer self-association, typically found in African populations. We describe two Utah families of northern European ancestry including 2 propositi with atypical non-microcytic hereditary pyropoikilocytosis, 7 hereditary elliptocytosis members and one asymptomatic carrier. The underlying molecular defect is a novel mutation in the alpha(α) spectrin gene, SPTA(R34P) that impairs spectrin tetramer formation. It is inherited in trans to the hypomorphic SPTA(αLELY) in the 2 propositi and 5 of 7 hereditary elliptocytosis individuals indicating that SPTA(αLELY) is not the sole determinant of the variable clinical expression. α Spectrin mRNA was mildly decreased in all hereditary elliptocytosis subjects, whereas both hereditary pyropoikilocytosis propositi had a severe decrease to ~10% of normal. Genotyping identified a unique SPTA intragenic crossover and uniparental disomy in one hereditary elliptocytosis individual. Two additional crossover events demonstrated the susceptibility of SPTA gene to rearrangement and revealed a novel segregation of the two SPTA(αLELY) mutations. We conclude that the profound phenotypic heterogeneity in these families can be attributed to the SPTA(R34P) mutation in combination with: 1) inheritance in trans of either SPTA(αLELY); or 2) the wild-type SPTA; 3) a decrease of α spectrin mRNA; and 4) SPTA intragenic crossover.

Reduced glycerol incorporation into phospholipids contributes to impaired intra-erythrocytic growth of glycerol kinase knockout Plasmodium falciparum parasites.

BACKGROUND: Malaria is a devastating disease and Plasmodium falciparum is the most lethal parasite infecting humans. Understanding the biology of this parasite is vital in identifying potential novel drug targets. During every 48-hour intra-erythrocytic asexual replication cycle, a single parasite can produce up to 32 progeny. This extensive proliferation implies that parasites require substantial amounts of lipid precursors for membrane biogenesis. Glycerol kinase is a highly conserved enzyme that functions at the interface of lipid synthesis and carbohydrate metabolism. P. falciparum glycerol kinase catalyzes the ATP-dependent phosphorylation of glycerol to glycerol-3-phosphate, a major phospholipid precursor. METHODS: The P. falciparum glycerol kinase gene was disrupted using double crossover homologous DNA recombination to generate a knockout parasite line. Southern hybridization and mRNA analysis were used to verify gene disruption. Parasite growth rates were monitored by flow cytometry. Radiolabelling studies were used to assess incorporation of glycerol into parasite phospholipids. RESULTS: Disruption of the P. falciparum glycerol kinase gene produced viable parasites, but their growth was significantly reduced to 56.5±1.8% when compared to wild type parasites. (14)C-glycerol incorporation into the major phospholipids of the parasite membrane, phosphatidylcholine and phosphatidylethanolamine, was 48.4±10.8% and 53.1±5.7% relative to an equivalent number of wild type parasites. CONCLUSIONS: P. falciparum glycerol kinase is required for optimal intra-erythrocytic asexual parasite development. Exogenous glycerol may be used as an alternative carbon source for P. falciparum phospholipid biogenesis, despite the lack of glycerol kinase to generate glycerol-3-phosphate. GENERAL SIGNIFICANCE: These studies provide new insight into glycerolipid metabolism in P. falciparum.