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Publication Open Access Genetic loci associated with delayed clearance of Plasmodium falciparum following artemisinin treatment in Southeast Asia.(2013-01-02) Takala-Harrisona, Shannon; Clark, Taane G.; Cummings, Michael P.; Miotto,Olivo; Dondorp, Arjen M.; Fukudaf, Mark M.; Nosten, Francois; Noedl, Harald; Mallika Imwong; มัลลิกา อิ่มวงศ์; Bethell, Delia; Se, Youry; Lon, Chanthap; Tyner, Stuart D.; Saunders, David L.; Socheat, Duong; Ariey, Frederic; Phyo, Aung Pyae; Starzengruber, Peter; Fuehrer, Hans-Peter; Swoboda, Paul; Stepniewska, Kasia; Flegg, Jennifer; Arze, Cesar; Cerqueira, Gustavo C.; Silva, Joana C.; Ricklefs, Stacy M.; Porcella, Stephen F.; Stephens, Robert M.; Adams, Matthew; Kenefic, Leo J.; Campino, Susana; Auburn, Sarah; MacInnis, Bronwyn; Kwiatkowski, Dominic P.; Su, Xin-zhuan; White, Nicholas J.; Ringwald, Pascal; Plowe, Christopher V.; Plowe, Christopher V.; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Research Unit.; Mahidol University. Faculty of Tropical Medicine. Department of Molecular Tropical Medicine and Genetics.; Mahidol University. Faculty of Tropical Medicine. Shoklo Malaria Research Unit.The recent emergence of artemisinin-resistant Plasmodium falciparum malaria in western Cambodia could threaten prospects for malaria elimination. Identification of the genetic basis of resistance would provide tools for molecular surveillance, aiding efforts to contain resistance. Clinical trials of artesunate efficacy were conducted in Bangladesh, in northwestern Thailand near the Myanmar border, and at two sites in western Cambodia. Parasites collected from trial participants were genotyped at 8,079 single nucleotide polymorphisms (SNPs) using a P. falciparum-specific SNP array. Parasite genotypes were examined for signatures of recent positive selection and association with parasite clearance phenotypes to identify regions of the genome associated with artemisinin resistance. Four SNPs on chromosomes 10 (one), 13 (two), and 14 (one) were significantly associated with delayed parasite clearance. The two SNPs on chromosome 13 are in a region of the genome that appears to be under strong recent positive selection in Cambodia. The SNPs on chromosomes 10 and 13 lie in or near genes involved in postreplication repair, a DNA damage-tolerance pathway. Replication and validation studies are needed to refine the location of loci responsible for artemisinin resistance and to understand the mechanism behind it; however, two SNPs on chromosomes 10 and 13 may be useful markers of delayed parasite clearance in surveillance for artemisinin resistance in Southeast Asia.Publication Open Access The role of previously unmeasured organic acids in the pathogenesis of severe malaria(2015) Herdman, M. Trent; Natthida Sriboonvorakul; Leopold, Stije J.; Sam Douthwaite; Sanjib Mohanty; M. Mahtab Uddin Hassan; Maude, Richard J.; Kingston, Hugh WF; Katherine Plewes; Prakaykaew Charunwatthana; Kamolrat Silamut; Woodrow, Charles J.; Kesinee Chotinavich; Hossain, Md. Amir; Faiz, M. Abul; Saroj Mishra; Natchanun Leepipatpiboon; White, Nicholas J.; Day, Nicholas PJ; Joel Tarning; Dondorp, Arjen M.; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research UnitIntroduction: Severe falciparum malaria is commonly complicated by metabolic acidosis. Together with lactic acid (LA), other previously unmeasured acids have been implicated in the pathogenesis of falciparum malaria. Methods: In this prospective study, we characterised organic acids in adults with severe falciparum malaria in India and Bangladesh. Liquid chromatography-mass spectrometry was used to measure organic acids in plasma and urine. Patients were followed until recovery or death. Results: Patients with severe malaria (n=138), uncomplicated malaria (n=102), sepsis (n=32) and febrile encephalopathy (n=35) were included. Strong ion gap (mean±SD) was elevated in severe malaria (8.2 mEq/L±4.5) and severe sepsis (8.6 mEq/L±7.7) compared with uncomplicated malaria (6.0 mEq/L±5.1) and encephalopathy (6.6 mEq/L±4.7). Compared with uncomplicated malaria, severe malaria was characterised by elevated plasma LA, hydroxyphenyllactic acid (HPLA), α-hydroxybutyric acid and β-hydroxybutyric acid (all P<0.05). In urine, concentrations of methylmalonic, ethylmalonic and α-ketoglutaric acids were also elevated. Multivariate logistic regression showed that plasma HPLA was a strong independent predictor of death (odds ratio [OR] 3.5, 95 % confidence interval [CI] 1.6–7.5, P=0.001), comparable to LA (OR 3.5, 95 % CI 1.5–7.8, P=0.003) (combined area under the receiver operating characteristic curve 0.81). Conclusions: Newly identified acids, in addition to LA, are elevated in patients with severe malaria and are highly predictive of fatal outcome. Further characterisation of their sources and metabolic pathways is now needed.Publication Open Access Pharmacokinetic properties of intramuscular versus oral syrup paracetamol in Plasmodium falciparum malaria(2016) Thanaporn Wattanakul; Pramote Teerapong; Plewes, Katherine; Newton, Paul N.; Wirongrong Chierakul; Kamolrat Silamut; Kesinee Chotivanich; Ronnatrai Ruengweerayut; White, Nicholas J.; Dondorp, Arjen M.; Tarning, Joel; Mahidol University. Faculty of Tropical Medicine. Mahidol‑Oxford Tropical Medicine Research Unit.Background: Fever is an inherent symptom of malaria in both adults and children. Paracetamol (acetaminophen) is the recommended antipyretic as it is inexpensive, widely available and has a good safety profile, but patients may not be able to take the oral drug reliably. A comparison between the pharmacokinetics of oral syrup and intramuscular paracetamol given to patients with acute falciparum malaria and high body temperature was performed. Methods: A randomized, open-label, two-treatment, crossover, pharmacokinetic study of paracetamol dosed orally and intramuscularly was conducted. Twenty-one adult patients with uncomplicated falciparum malaria were randomized to receive a single 600 mg dose of paracetamol either as syrup or intramuscular injection on day 0 followed by a single dose administered by the alternative route on day 1. Paracetamol plasma concentrations were quantified frequently and modelled simultaneously using nonlinear mixed-effects modelling. The final population pharmacokinetic model was used for dose optimization simulations. Relationships between paracetamol concentrations with temperature and parasite half-life were investigated using linear and non-linear regression analyses. Results: The population pharmacokinetic properties of paracetamol were best described by a two-compartment disposition model, with zero-order and first-order absorption for intramuscular and oral syrup administration, respectively. The relative bioavailability of oral syrup was 84.4 % (95 % CI 68.2–95.1 %) compared to intramuscular administration. Dosing simulations showed that 1000 mg of intramuscular or oral syrup administered six-hourly reached therapeutic steady state concentrations for antipyresis, but more favourable concentration–time profiles were achieved with a loading dose of 1500 mg, followed by a 1000 mg maintenance dose. This ensured that maximum therapeutic concentrations were reached rapidly during the first 6 h. No significant relationships between paracetamol concentrations and temperature or parasite half-life were found. Conclusions: Paracetamol plasma concentrations after oral syrup and intramuscular administration in patients with acute falciparum malaria were described successfully by a two-compartment disposition model. Relative oral bioavailability compared to intramuscular dosing was estimated as 84.4 % (95 % CI 68.2–95.1 %). Dosing simulations showed that a loading dose followed by six-hourly dosing intervals reduced the time delay to reach therapeutic drug levels after both routes of administration. The safety and efficacy of loading dose paracetamol antipyretic regimens now needs to be established in larger studies.Publication Open Access Comparative genome‑wide analysis and evolutionary history of haemoglobin‑processing and haem detoxification enzymes in malarial parasites(2016) Patrath Ponsuwanna; Theerarat Kochakarn; Duangkamon Bunditvorapoom; Krittikorn Kümpornsin; Otto, Thomas D.; Chase Ridenour; Kesinee Chotivanich; Prapon Wilairat; White, Nicholas J.; Olivo Miotto; Thanat Chookajorn; Mahidol University. Faculty of Tropical Medicine. Genomic and Evolutionary Medicine Unit, Centre of Excellence in MalariaBackground: Malaria parasites have evolved a series of intricate mechanisms to survive and propagate within host red blood cells. Intra-erythrocytic parasitism requires these organisms to digest haemoglobin and detoxify ironbound haem. These tasks are executed by haemoglobin-specific proteases and haem biocrystallization factors that are components of a large multi-subunit complex. Since haemoglobin processing machineries are functionally and genetically linked to the modes of action and resistance mechanisms of several anti-malarial drugs, an understanding of their evolutionary history is important for drug development and drug resistance prevention. Methods: Maximum likelihood trees of genetic repertoires encoding haemoglobin processing machineries within Plasmodium species, and with the representatives of Apicomplexan species with various host tropisms, were created. Genetic variants were mapped onto existing three-dimensional structures. Genome-wide single nucleotide polymorphism data were used to analyse the selective pressure and the effect of these mutations at the structural level. Results: Recent expansions in the falcipain and plasmepsin repertoires are unique to human malaria parasites especially in the Plasmodium falciparum and P. reichenowi lineage. Expansion of haemoglobin-specific plasmepsins occurred after the separation event of Plasmodium species, but the other members of the plasmepsin family were evolutionarily conserved with one copy for each sub-group in every Apicomplexan species. Haemoglobin-specific falcipains are separated from invasion-related falcipain, and their expansions within one specific locus arose independently in both P. falciparum and P. vivax lineages. Gene conversion between P. falciparum falcipain 2A and 2B was observed in artemisinin-resistant strains. Comparison between the numbers of non-synonymous and synonymous mutations suggests a strong selective pressure at falcipain and plasmepsin genes. The locations of amino acid changes from non-synonymous mutations mapped onto protein structures revealed clusters of amino acid residues in close proximity or near the active sites of proteases. Conclusion: A high degree of polymorphism at the haemoglobin processing genes implicates an imposition of selective pressure. The identification in recent years of functional redundancy of haemoglobin-specific proteases makes them less appealing as potential drug targets, but their expansions, especially in the human malaria parasite lineages, unequivocally point toward their functional significance during the independent and repetitive adaptation events in malaria parasite evolutionary history.Publication Open Access Motivations and perceptions of community advisory boards in the ethics of medical research: the case of the Thai-Myanmar border.(2014-02-17) Maung, Lwin K.; Cheah, Phaik Yeong; Cheah, Phaik Kin; White, Nicholas J.; Day, Nicholas P.; Nosten, Francois; Parker, Michael; Parker, Michael; Mahidol University. Faculty of Tropical Medicine. Mahidol Oxford Research Unit.or of approaches adopted. Justifications given for its use also vary. Community engagement is, for example, variously seen to be of value in: the development of more effective and appropriate consent processes; improved understanding of the aims and formsPublication Open Access Intervals to Plasmodium falciparum recurrence after anti-malarial treatment in pregnancy: a longitudinal prospective cohort(2015) Natthapon Laochan; Zaloumis, Sophie G.; Mallika Imwong; Usa Lek-Uthai; Alan Brockman; Kanlaya Sriprawat; Jacher Wiladphaingern; White, Nicholas J.; François Nosten; McGready, Rose; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research Unit (MORU)Background: Plasmodium falciparum infections adversely affect pregnancy. Anti-malarial treatment failure is common. The objective of this study was to examine the duration of persistent parasite carriage following anti-malarial treatment in pregnancy. Methods: The data presented here are a collation from previous studies carried out since 1994 in the Shoklo Malaria Research Unit (SMRU) on the Thailand-Myanmar border and performed using the same unique methodology detailed in the Materials and Methods section. Screening for malaria by microscopy is a routine part of weekly antenatal care (ANC) visits and therapeutic responses to anti-malarials were assessed in P. falciparum malaria cases. Women with microscopy confirmed P. falciparum malaria had a PCR blood spot from a finger-prick sample collected. Parasite DNA was extracted from the blood-spot samples using saponin lysis/Chelex extraction method and genotyped using polymorphic segments of MSP1, MSP2 and GLURP. Recurrent infections were classified by genotyping as novel, recrudescent or indeterminate. Factors associated with time to microscopy-detected recrudescence were analysed using multivariable regression techniques. Results: From December 1994 to November 2009, 700 women were treated for P. falciparum and there were 909 recurrent episodes (481 novel and 428 recrudescent) confirmed by PCR genotyping. Most of the recurrences, 85 % (770/909), occurred after treatment with quinine monotherapy, artesunate monotherapy or artesunate-clindamycin. The geometric mean number of days to recurrence was significantly shorter in women with recrudescent infection, 24.5 (95 %: 23.4-25.8), compared to re-infection, 49.7 (95 %: 46.9-52.7), P <0.001. The proportion of recrudescent P. falciparum infections that occurred after days 28, 42 and 63 from the start of treatment was 29.1 % (124/428), 13.3 % (57/428) and 5.6 % (24/428). Recrudescent infections ≥100 days after treatment occurred with quinine and mefloquine monotherapy, and quinine + clindamycin and artesunate + atovaquone-proguanil combination therapy. Treatments containing an artemisinin derivative or an intercalated Plasmodium vivax infection increased the geometric mean interval to recrudescence by 1.28-fold (95 % CI: 1.09-1.51) and 2.19-fold (1.77-2.72), respectively. Intervals to recrudescence were decreased 0.83-fold (0.73-0.95) if treatment was not fully supervised (suggesting incomplete adherence) and 0.98-fold (0.96-0.99) for each doubling in baseline parasitaemia. Conclusions: Prolonged time to recrudescence may occur in pregnancy, regardless of anti-malarial treatment. Long intervals to recrudescence are more likely with the use of artemisinin-containing treatments and also observed with intercalated P. vivax infections treated with chloroquine. Accurate determination of drug efficacy in pregnancy requires longer duration of follow-up, preferably until delivery or day 63, whichever occurs last.Publication Open Access Protein-based signatures of functional evolution in Plasmodium falciparum(2011-09-14) Gardner, Kate B.; Sinha, Ipsita; Bustamante, Leyla Y.; Day, Nicholas P.J.; White, Nicholas J.; Woodrow, Charles J.; Woodrow, Charles J.; Mahidol University. Faculty of Tropical Medicine. Wellcome Trust Mahidol University-Oxford Tropical Medicine Research Unit (MORU).BACKGROUND: It has been known for over a decade that Plasmodium falciparum proteins are enriched in non-globular domains of unknown function. The potential for these regions of protein sequence to undergo high levels of genetic drift provides a fundamental challenge to attempts to identify the molecular basis of adaptive change in malaria parasites. RESULTS: Evolutionary comparisons were undertaken using a set of forty P. falciparum metabolic enzyme genes, both within the hominid malaria clade (P. reichenowi) and across the genus (P. chabaudi). All genes contained coding elements highly conserved across the genus, but there were also a large number of regions of weakly or non-aligning coding sequence. These displayed remarkable levels of non-synonymous fixed differences within the hominid malaria clade indicating near complete release from purifying selection (dN/dS ratio at residues non-aligning across genus: 0.64, dN/dS ratio at residues identical across genus: 0.03). Regions of low conservation also possessed high levels of hydrophilicity, a marker of non-globularity. The propensity for such regions to act as potent sources of non-synonymous genetic drift within extant P. falciparum isolates was confirmed at chromosomal regions containing genes known to mediate drug resistance in field isolates, where 150 of 153 amino acid variants were located in poorly conserved regions. In contrast, all 22 amino acid variants associated with drug resistance were restricted to highly conserved regions. Additional mutations associated with laboratory-selected drug resistance, such as those in PfATPase4 selected by spiroindolone, were similarly restricted while mutations in another calcium ATPase (PfSERCA, a gene proposed to mediate artemisinin resistance) that reach significant frequencies in field isolates were located exclusively in poorly conserved regions consistent with genetic drift. CONCLUSION: Coding sequences of malaria parasites contain prospectively definable domains subject to neutral or nearly neutral evolution on a scale that appears unrivalled in biology. This distinct evolutionary landscape has potential to confound analytical methods developed for other genera. Against this tide of genetic drift, polymorphisms mediating functional change stand out to such an extent that evolutionary context provides a useful signal for identifying the molecular basis of drug resistance in malaria parasites, a finding that is of relevance to both genome-wide and candidate gene studies in this genus.