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Human African Trypanosomiasis (HAT) is a vector-borne disease caused by kinetoplastid parasites of the Trypanosoma genus. The disease proceeds in two stages, with a hemolymphatic blood stage and a meningo-encephalic brain stage. In the latter stage, the parasite causes irreversible damage to the brain leading to sleep cycle disruption and is fatal if untreated. An orally bioavailable treatment is highly desirable. In this study, we present a brain-penetrant, parasite-selective 20S proteasome inhibitor that was rapidly optimized from an HTS singleton hit to drug candidate compound 7 that showed cure in a stage II mouse efficacy model. Here, we describe hit expansion and lead optimization campaign guided by cryo-electron microscopy and an in silico model to predict the brain-to-plasma partition coefficient Kp as an important parameter to prioritize compounds for synthesis. The model combined with in vitro and in vivo experiments allowed us to advance compounds with favorable unbound brain-to-plasma ratios (Kp,uu) to cure a CNS disease such as HAT.

Trypanosoma bruceiis the causative agent of the Human and Animal African Trypanosomiases. Mammalian stage parasites infect the bloodstream, central nervous system, but also skin, adipose tissue or lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose which is readily available in mammalian tissues. In addition to glucose, glycerol can be utilised as a carbon and ATP source and also as a substrate for gluconeogenesis. The physiological relevance of glycerol-fed gluconeogenesis for mammalian-infective trypanosome life-cycle stages, however, remins elusive. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase, transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data renders unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and precludes the participation of a novel biochemical pathway involving transaldolase in the process. Sustained metabolic flux in gluconeogenesis in our mutants, including in a triple-null strain, provides new insights into gluconeogenesis and the pentose phosphate pathway, and improves the current understanding of carbon metabolism of the mammalian-infective stages ofT. brucei.

Venous thromboembolism (VTE) refers to a blood clot that starts in a vein. The risk of developing VTE is highest after major surgery or a major injury, or when someone has heart failure, cancer, or infectious disease (e.g., COVID-19). Without prompt treatment to break up clots and prevent more from forming, VTE can restrict or block blood flow and oxygen, which can damage the body tissue or organs. VTE can occur without any obvious signs, and imaging technologies are used. Alternatively rapid measurement of thrombin generation (TG) and D-dimer could be used to make a fast, portable, and easy-to-use diagnostic platform for VTE. Here, we have demonstrated a diagnostic sensing platform with the ability of simultaneous detection of TG and D-dimer in human plasma. Modifications were made to both the assay protocols to eliminate the need for sample dilution and incubation steps. Using a substantially reduced sample volume, the measurement results show comparable performance to the gold standard method. Our platform is able to deliver accurate and cost-effective results for both TG and D-dimer assays when using undiluted plasma in under 15 min. The assays presented are therefore a good candidate technology for use in a point-of-care platform to diagnose VTE.

Control of African animal trypanosomiasis (AAT) is hampered by limited diagnostics, inappropriate trypanocide use, poor drug quality, and drug resistance.\ud The scope and quality of current literature on AAT incidence, control, and resistance does not allow for robust comparisons or assessment of the validity of extrapolating to other populations.\ud A united effort is needed to address AAT at local, national, and international settings to ensure a greater chance for success.\ud AAT control programmes must be sustainable through funding, cross-sectoral engagement, and fostering sustainable behavioural change through incentives and accountability.\ud Trypanocides are a key control component of African animal trypanosomiasis (AAT) in tsetse-infested areas of sub-Saharan Africa. While farmers are dependent upon trypanocides, recent research highlights their inappropriate and ineffective use, problems with drug quality, and treatment failure. There are currently gaps in knowledge and investment in inexpensive AAT diagnostics, understanding of drug resistance, and the effective use of trypanocides in the field. Without this important knowledge it is difficult to develop best practice and policy for existing drugs or to inform development and use of new drugs. There needs to be better understanding of the drivers and behavioural practices around trypanocide use so that they can be incorporated into sustainable solutions needed for the development of effective control of AAT.

Characterizing the mode of action of antimalarial compounds that emerge from high-throughput phenotypic screens is central to understanding how parasite resistance to these drugs can emerge. Here, we have employed untargeted metabolomics to inform on the mechanism of action of antimalarial leads with different speed of kill profiles being developed by the Novartis Institute of Tropical Diseases (NITD). Time-resolved global changes in malaria parasite metabolite profiles upon drug treatment were quantified using liquid chromatography-based mass spectrometry (LC-MS) and compared to untreated controls. Using this approach, we confirmed previously reported metabolomics profiles of the fast-killing (2.5h) drug dihydroartemisinin (DHA) and the slower killing atovaquone (ATQ). A slow acting antimalarial lead from NITD of imidazolopiperazine (IZP) class, GNF179, elicited little or no discernable metabolic change in malaria parasites in the same 2.5h window of drug exposure. In contrast, fast killing drugs, DHA and the spiroindolone (NITD246) elicited similar metabolomic profiles both in terms of kinetics and content. DHA and NITD246 induced peptide losses consistent with disruption of haemoglobin catabolism and also interfered with the pyrimidine biosynthesis pathway. Two members of the recently described novel class of antimalarial agents of the 5-aryl-2-amino-imidazothiadiazole (ITD) class also exhibited a fast-acting profile that also featured peptide losses indicative of disrupted haemoglobin catabolism. Our screen demonstrates that structurally unrelated, fast acting antimalarial compounds generate similar biochemical signatures in Plasmodium pointing to a common mechanism associated with rapid parasite death. Our study describes a potential biochemical signature that may serve to identify other fast acting drug candidates.ImportanceIn malaria drug discovery, understanding the mode of action of lead compounds is important as it helps in predicting the potential emergence of drug resistance in the field when these drugs are eventually deployed. In this study, we have employed metabolomics technologies to characterize the potential targets of antimalarial drug candidates in the developmental pipeline at NITD. We show that NITD fast acting leads belonging to spiroindolone and imidazothiadiazole class induce a common biochemical theme in drug exposed malaria parasites which is similar to another fast acting, clinically available drug, DHA. These biochemical features which are absent in a slower acting NITD lead (GNF17) point to haemoglobin digestion and inhibition of the pyrimidine pathway as potential action points for these drugs. These biochemical themes can be used to identify fast drug candidates of similar profiles in future drug discovery programs.

This paper describes the design and synthesis of Strathclyde minor groove binders (S-MGBs) that have been truncated by the removal of a pyrrole ring in order to mimic the structure of the natural product, disgocidine. S-MGBs have been found to be active against many different organisms, however, selective antiparasitic activity is required. A panel of seven truncated S-MGBs was prepared and the activities examined against a number of clinically relevant organisms including several bacteria and parasites. The effect of the truncation strategy on S-MGB aggregation in aqueous environment was also investigated using 1H inspection and DOSY experiments. A lead compound, a truncated S-MGB, which possesses significant activity only against trypanosomes and Leishmania has been identified for further study and was also found to be less affected by aggregation compared to its full-length analogue., This paper describes the design and synthesis of Strathclyde minor groove binders (S-MGBs) that have been truncated by the removal of a pyrrole ring in order to mimic the structure of the natural product, disgocidine.

Graphical abstract, Highlights • Apicoplast LipB deletion leads to changed antioxidant expression that precedes and coincides with accelerated differentiation. • 3D7 Plasmodium exhibits changes in glycolysis and tricarboxylic acid cycle activity after deletion of apicoplast LipB. • When LipB is deleted from NF54 Plasmodium, the resulting parasites cannot complete their development in mosquitoes., Malaria is still one of the most important global infectious diseases. Emergence of drug resistance and a shortage of new efficient antimalarials continue to hamper a malaria eradication agenda. Malaria parasites are highly sensitive to changes in the redox environment. Understanding the mechanisms regulating parasite redox could contribute to the design of new drugs. Malaria parasites have a complex network of redox regulatory systems housed in their cytosol, in their mitochondrion and in their plastid (apicoplast). While the roles of enzymes of the thioredoxin and glutathione pathways in parasite survival have been explored, the antioxidant role of α-lipoic acid (LA) produced in the apicoplast has not been tested. To take a first step in teasing a putative role of LA in redox regulation, we analysed a mutant Plasmodium falciparum (3D7 strain) lacking the apicoplast lipoic acid protein ligase B (lipB) known to be depleted of LA. Our results showed a change in expression of redox regulators in the apicoplast and the cytosol. We further detected a change in parasite central carbon metabolism, with lipB deletion resulting in changes to glycolysis and tricarboxylic acid cycle activity. Further, in another Plasmodium cell line (NF54), deletion of lipB impacted development in the mosquito, preventing the detection of infectious sporozoite stages. While it is not clear at this point if the observed phenotypes are linked, these findings flag LA biosynthesis as an important subject for further study in the context of redox regulation in asexual stages, and point to LipB as a potential target for the development of new transmission drugs.

Objective: Early stage diagnosis of sepsis without overburdening health services is essential to improving patient outcomes. Methods: A fast and simple-to-use platform that combines an integrated circuit with paper microfluidics for simultaneous detection of multiple-metabolites appropriate for diagnostics was presented. Paper based sensors are a primary candidate for widespread deployment of diagnostic or test devices. However, the majority of devices today use a simple paper strip to detect a single marker using the reflectance of light. However, for many diseases such as sepsis, one biomarker is not sufficient to make a unique diagnosis. In this work multiple measurements are made on patterned paper simultaneously. Using laser ablation to fabricate microfluidic channels on paper provides a flexible and direct approach for mass manufacture of disposable paper strips. A reusable photodiode array on a complementary metal oxide semiconductor chip is used as the transducer. Results: The system measures changes in optical absorbance in the paper to achieve a cost-effective and easily implemented system that is capable of multiple simultaneous assays. Potential sepsis metabolite biomarkers glucose and lactate have been studied and quantified with the platform, achieving sensitivity within the physiological range in human serum. Conclusion: We have detailed a disposable paper-based CMOS photodiode sensor platform for real-time simultaneous detection of metabolites for diseases such as sepsis. Significance: A combination of a low-cost paper strip with microfluidic channels and a sensitive CMOS photodiode sensor array makes our platform a robust portable and inexpensive biosensing device for multiple diagnostic tests in many different applications.

The kinetochore is a macromolecular structure that assembles on the centromeres of chromosomes and provides the major attachment point for spindle microtubules during mitosis. In Trypanosoma brucei the proteins that make up the kinetochore are highly divergent, with the inner kinetochore comprising at least 20 distinct and essential proteins (KKT1-20) that include four protein kinases, CLK1 (KKT10), CLK2 (KKT19), KKT2 and KKT3. We performed a phenotypic screen of T. brucei bloodstream forms with a Novartis kinase-focused inhibitor library, which identified a number of selective inhibitors with potent pan-kinetoplastid activity. Deconvolution of an amidobenzimidazole series using a selection of 37 T. brucei mutants that over-express known essential protein kinases identified CLK1 as the primary target. Biochemical studies show that the irreversible competitive inhibition of CLK1 is dependent on a Michael acceptor forming an irreversible bond with C215 in the ATP binding pocket, a residue that is not present in human CLK1, thereby providing selectivity. Chemical inhibition of CLK1 impairs inner kinetochore recruitment and compromises cell cycle progression, leading to cell death. We show that KKT2 is a substrate for CLK1 and identify phosphorylation of S508 to be essential for KKT2 function and for kinetochore assembly. We propose that CLK1 is part of a novel signalling cascade that controls kinetochore function via phosphorylation of the inner kinetochore protein kinase KKT2. This work highlights a novel drug target for trypanosomatid parasitic protozoa and a new chemical tool for investigating the function of their divergent kinetochores.

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each resistant line, the predominant wild-type sterol, ergosta-5,7,24-trienol, was replaced by other sterol intermediates. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter gene was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for their ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several AmB resistant lines showed reduced virulence, at least two lines displayed heightened virulence in mice whilst retaining their resistance phenotype, emphasising the risks of resistance emerging to this critical drug.

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each line, the predominant ergostane-type sterol of wild-type cells was replaced by other sterol species. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). These differences appeared to relate to the presence of lipids in the former. Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several lines showed reduced virulence, at least one AmB resistant line displayed heightened virulence in mice whilst retaining its resistance phenotype, emphasising the risks of resistance emerging to this critical drug.

Subspecies of the protozoan parasite Trypanosoma brucei are the causative agents of Human African Trypanosomiasis (HAT), a debilitating neglected tropical disease prevalent across sub-Saharan Africa. HAT case numbers have steadily decreased since the start of the century, and sustainable elimination of one form of the disease is in sight. However, key to this is the development of novel drugs to combat the disease. Acoziborole is a recently developed benzoxaborole, currently in advanced clinical trials, for treatment of stage 1 and stage 2 HAT. Importantly, acoziborole is orally bioavailable, and curative with one dose. Recent studies have made significant progress in determining the molecular mode of action of acoziborole. However, less is known about the potential mechanisms leading to acoziborole resistance in trypanosomes. In this study, an in vitro-derived acoziborole-resistant cell line was generated and characterised. The AcoR line exhibited significant cross-resistance with the methyltransferase inhibitor sinefungin as well as hypersensitisation to known trypanocides. Interestingly, transcriptomics analysis of AcoR cells indicated the parasites had obtained a procyclic- or stumpy-like transcriptome profile, with upregulation of procyclin surface proteins as well as differential regulation of key metabolic genes known to be expressed in a life cycle-specific manner, even in the absence of major morphological changes. However, no changes were observed in transcripts encoding CPSF3, the recently identified protein target of acoziborole. The results suggest that generation of resistance to this novel compound in vitro can be accompanied by transcriptomic switches resembling a procyclic- or stumpy-type phenotype., Author summary Human African Trypanosomiasis (HAT), also known as Sleeping Sickness, is a parasitic disease prevalent across sub-Saharan Africa, where it is transmitted by blood-sucking Tsetse flies. The disease is caused by a single-celled protozoan parasite called Trypanosoma brucei. HAT case numbers have decreased over the past two decades, and elimination is in sight. However, to meet the WHO objective of sustainable HAT elimination, newer and safer drugs are needed. Acoziborole is a new drug that has proven safe and effective against HAT and, unlike previous drugs, it can be orally administered. Whilst the mode of action of acoziborole was recently identified, less is known regarding the mechanisms by which T. brucei could become resistant to acoziborole. To address this, we generated a drug resistant cell line under laboratory conditions, in order to analyse the differences between resistant and sensitive cells. By sequencing the transcriptome, we observed that many genes associated with mammalian-infective parasites are down-regulated, whilst genes associated with the insect stage are up-regulated. As a result of these differences, the metabolic effects of acoziborole on parasites are nullified. These data suggest that parasite differentiation, albeit on a gene expression level, is potentially a mechanism of drug resistance in T. brucei, although it is doubtful whether this mechanism could occur in the field where mammalian immune effectors would destroy parasites differentiating this way.

Acute febrile illnesses are still a major cause of mortality and morbidity globally, particularly in low to middle income countries. The aim of this study was to determine any possible metabolic commonalities of patients infected with disparate pathogens that cause fever. Three liquid chromatography-mass spectrometry (LC-MS) datasets investigating the metabolic effects of malaria, leishmaniasis and Zika virus infection were used. The retention time (RT) drift between the datasets was determined using landmarks obtained from the standard reference mixtures generally used in the quality control of the LC-MS experiments. We used fitted Gaussian Process models (GPs) to perform a high level correction of the RT drift between the experiments, followed by standard peakset alignment between the samples with corrected RTs of the three LC-MS datasets. Statistical analysis, annotation and pathway analysis of the integrated peaksets were subsequently performed. Metabolic dysregulation patterns common across the datasets were identified, with kynurenine pathway being the most affected pathway between all three fever-associated datasets.

Human African trypanosomiasis caused by Trypanosoma brucei gambiense is a parasitic infection that usually progresses to coma and death unless treated. WHO has updated its guidelines for the treatment of this infection on the basis of independent literature reviews and using the Grading of Recommendations Assessment, Development and Evaluation methodology. The first-line treatment options, pentamidine and nifurtimox-eflornithine combination therapy, have been expanded to include fexinidazole, an oral monotherapy given a positive opinion from the European Medicines Agency. Fexinidazole is recommended for individuals who are aged 6 years and older with a bodyweight of 20 kg or more, who have first-stage or second-stage gambiense human African trypanosomiasis and a cerebrospinal fluid leucocyte count less than 100 per mu L. Nifurtimox-eflornithine combination therapy remains recommended for patients with 100 leucocytes per mu L or more. Without clinical suspicion of severe second-stage disease, lumbar puncture can be avoided and fexinidazole can be given. Fexinidazole should only be administered under supervision of trained health staff. Because these recommendations are expected to change clinical practice considerably, health professionals should consult the detailed WHO guidelines. These guidelines will be updated as evidence accrues.

Objectives Fatty acid oxidation (FAO) and glycolysis have been implicated in immune regulation and activation of macrophages. However, investigation of human monocyte intracellular metabolism in the context of the hypoxic and inflammatory rheumatoid arthritis (RA) synovium is lacking. We hypothesized that exposure of monocytes to the hypoxic and inflammatory RA environment would have a profound impact on their metabolic state, and potential to contribute to disease pathology. Methods Human monocytes were isolated from buffy coats and exposed to hypoxia. Metabolic profiling of monocytes was carried out by LC-MS metabolomics. Inflammatory mediator release after LPS or RA-synovial fluid (RA-SF) stimulation was analysed by ELISA. FAO was inhibited by etomoxir or enhanced with exogenous carnitine supplementation. Transcriptomics of RA blood monocytes and RA-SF macrophages was carried out by microarray. Results Hypoxia exacerbated monocyte-derived CCL20 and IL-1β release in response to LPS, and increased glycolytic intermediates at the expense of carnitines. Modulation of carnitine identified a novel role for FAO in the production of CCL20 in response to LPS. Transcriptional analysis of RA blood monocytes and RA-SF macrophages revealed that fatty acid metabolism was altered and CCL20 increased when monocytes enter the synovial environment. In vitro analysis of monocytes showed that RA-SF increases carnitine abundance and CCL20 production in hypoxia, which was exacerbated by exogenous carnitine. Conclusion This work has revealed a novel inflammatory mechanism in RA that links FAO to CCL20 production in human monocytes, which could subsequently contribute to RA disease pathogenesis by promoting the recruitment of Th17 cells and osteoclastogenesis.

Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon sensitivity to metabolic inhibition. Like the bloodstream stage of T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly lower in bloodstream stage T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Transcriptomics analysis showed higher levels of transcripts associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation, and the glycosomal succinate shunt in T. congolense, compared to T. brucei. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and saturated fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with metabolic inhibitors, confirming that electron transport chain activity is not essential in T. congolense. However, the parasite exhibits increased sensitivity to inhibition of mitochondrial pyruvate import, compared to T. brucei. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold higher EC50 for the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions., Author summary Animal African Trypanosomiasis (AAT), also known as Nagana, is a devastating disease affecting livestock across sub-Saharan Africa. AAT is primarily caused by the parasite Trypanosoma congolense, yet our biological knowledge about this pathogen is poor, especially compared to the related species T. brucei, subspecies of which cause the human disease Sleeping Sickness. Understanding the core metabolism of T. congolense is crucial in order to gain insights into the infection biology of this important pathogen, as well as providing the potential to identify new drug targets. In this work, we addressed the lack of knowledge concerning T. congolense by carrying out a comprehensive analysis of core metabolism, and comparing the data to T. brucei. We then used the findings of metabolic differences to predict differential sensitivity to inhibitors of metabolic function. We show that unlike T. brucei, where glucose metabolism leads to high levels of pyruvate excretion, T. congolense metabolises glucose to other end-products, namely succinate, malate and acetate. Moreover, there are pronounced differences in the way T. congolense uses glucose to feed into other areas of metabolism. Further analysis also suggests that T. congolense mostly scavenges lipids and fatty acids, rather than synthesising them de novo. To validate these findings, we confirm that T. congolense is differentially susceptible to metabolic inhibitors compared to T. brucei, and that, in particular, T. congolense is significantly less sensitive to inhibitors of fatty acid synthesis. Our study provides a foundation of functional metabolic knowledge on T. congolense, with insights into how this parasite fundamentally differs from T. brucei.

Falciparum malaria is clinically heterogeneous and the relative contribution of parasite and host in shaping disease severity remains unclear. We explored the interaction between inflammation and parasite variant surface antigen (VSA) expression, asking whether this relationship underpins the variation observed in controlled human malaria infection (CHMI). We uncovered marked heterogeneity in the host response to blood challenge; some volunteers remained quiescent, others triggered interferon-stimulated inflammation and some showed transcriptional evidence of myeloid cell suppression. Significantly, only inflammatory volunteers experienced hallmark symptoms of malaria. When we tracked temporal changes in parasite VSA expression to ask whether variants associated with severe disease rapidly expand in naive hosts, we found no transcriptional evidence to support this hypothesis. These data indicate that parasite variants that dominate severe malaria do not have an intrinsic growth or survival advantage; instead, they presumably rely upon infection-induced changes in their within-host environment for selection.