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Find video protocols related to scientific articles indexed in Pubmed.
Pharmacokinetic interactions between primaquine and pyronaridine-artesunate in healthy adult Thai subjects.
Antimicrob. Agents Chemother.
PUBLISHED: 11-12-2014
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Pyronaridine-artesunate is a newly introduced artemisinin-based combination treatment which may be deployed together with primaquine. A single-dose randomized, three sequence cross-over study was conducted in healthy Thai volunteers to characterize potential pharmacokinetic interactions between these drugs. Seventeen healthy adults received a single oral dose of primaquine alone (30 mg base), and were then randomized to receive pyronaridine-artesunate alone (540-180 mg) or pyronaridine-artesunate plus primaquine in combination, with intervening wash-out periods between all treatments. The pharmacokinetic properties of primaquine, its metabolite carboxyprimaquine, artesunate, its metabolite dihydroartemisinin, and pyronaridine were assessed in 15 subjects using a non-compartmental approach followed by a bioequivalence evaluation. All drugs were well tolerated. The single oral dose of primaquine did not result in any clinically relevant pharmacokinetic alterations to pyronaridine, artesunate or dihydroartemisinin exposures. There were significantly higher primaquine maximum plasma drug concentrations (geometric mean ratio [90%CI]: 30% [17%-46%]) and total exposure (15% [6.4%-24%]) during co-administration with pyronaridine-artesunate compared to when given alone. Pyronaridine, like chloroquine and piperaquine, increases plasma primaquine concentrations.
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Characterization of an in vivo concentration-effect relationship for piperaquine in malaria chemoprevention.
Sci Transl Med
PUBLISHED: 10-29-2014
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A randomized, placebo-controlled trial conducted on the northwest border of Thailand compared malaria chemoprevention with monthly or bimonthly standard 3-day treatment regimens of dihydroartemisinin-piperaquine. Healthy adult male subjects (N = 1000) were followed weekly during 9 months of treatment. Using nonlinear mixed-effects modeling, the concentration-effect relationship for the malaria-preventive effect of piperaquine was best characterized with a sigmoidal Emax relationship, where plasma concentrations of 6.7 ng/ml [relative standard error (RSE), 23%] and 20 ng/ml were found to reduce the hazard of acquiring a malaria infection by 50% [that is, median inhibitory concentration (IC50)] and 95% (IC95), respectively. Simulations of monthly dosing, based on the final model and published pharmacokinetic data, suggested that the incidence of malaria infections over 1 year could be reduced by 70% with a recently suggested dosing regimen compared to the current manufacturer's recommendations for small children (8 to 12 kg). This model provides a rational framework for piperaquine dose optimization in different patient groups.
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Complex Polymorphisms in the Plasmodium falciparum Multidrug Resistance Protein 2 Gene and Its Contribution to Antimalarial Response.
Antimicrob. Agents Chemother.
PUBLISHED: 09-29-2014
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Plasmodium falciparum has the capacity to escape the actions of essentially all antimalarial drugs. ATP-binding cassette (ABC) transporter proteins are known to cause multidrug resistance in a large range of organisms, including the Apicomplexa parasites. P. falciparum genome analysis has revealed two genes coding for the multidrug resistance protein (MRP) type of ABC transporters: Pfmrp1, previously associated with decreased parasite drug susceptibility, and the poorly studied Pfmrp2. The role of Pfmrp2 polymorphisms in modulating sensitivity to antimalarial drugs has not been established. We herein report a comprehensive account of the Pfmrp2 genetic variability in 46 isolates from Thailand. A notably high frequency of 2.8 single nucleotide polymorphisms (SNPs)/kb was identified for this gene, including some novel SNPs. Additionally, we found that Pfmrp2 harbors a significant number of microindels, some previously not reported. We also investigated the potential association of the identified Pfmrp2 polymorphisms with altered in vitro susceptibility to several antimalarials used in artemisinin-based combination therapy and with parasite clearance time. Association analysis suggested Pfmrp2 polymorphisms modulate the parasite's in vitro response to quinoline antimalarials, including chloroquine, piperaquine, and mefloquine, and association with in vivo parasite clearance. In conclusion, our study reveals that the Pfmrp2 gene is the most diverse ABC transporter known in P. falciparum with a potential role in antimalarial drug resistance.
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Global extent of chloroquine-resistant Plasmodium vivax: a systematic review and meta-analysis.
Lancet Infect Dis
PUBLISHED: 09-08-2014
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Chloroquine is the first-line treatment for Plasmodium vivax malaria in most endemic countries, but resistance is increasing. Monitoring of antimalarial efficacy is essential, but in P. vivax infections the assessment of treatment efficacy is confounded by relapse from the dormant liver stages. We systematically reviewed P. vivax malaria treatment efficacy studies to establish the global extent of chloroquine resistance.
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Methylene blue inhibits the asexual development of vivax malaria parasites from a region of increasing chloroquine resistance.
J. Antimicrob. Chemother.
PUBLISHED: 08-21-2014
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Methylene blue, once discarded due to its unsettling yet mild side effects, has now found a renewed place in the pharmacopoeia of modern medicine. The continued spread of drug-resistant Plasmodium vivax and Plasmodium falciparum has also led to a recent re-examination of methylene blue's potent antimalarial properties. Here we examine the ex vivo susceptibility profile of Plasmodium spp. isolates to methylene blue; the isolates were from a region on the Thai-Myanmar border where there are increasing rates of failure when treating vivax malaria with chloroquine.
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Efficacy and day 7 plasma piperaquine concentrations in African children treated for uncomplicated malaria with dihydroartemisinin-piperaquine.
PLoS ONE
PUBLISHED: 08-18-2014
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One promising new Artemisinin-based combination therapies (ACTs) is dihydroartemisinin-piperaquine (DHA-PQ). However, the pharmacokinetics of piperaquine and the relationship between drug levels and clinical efficacy are incompletely characterized, particularly in children.
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Comprehensive identification of single nucleotide polymorphisms associated with beta-lactam resistance within pneumococcal mosaic genes.
PLoS Genet.
PUBLISHED: 08-01-2014
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Traditional genetic association studies are very difficult in bacteria, as the generally limited recombination leads to large linked haplotype blocks, confounding the identification of causative variants. Beta-lactam antibiotic resistance in Streptococcus pneumoniae arises readily as the bacteria can quickly incorporate DNA fragments encompassing variants that make the transformed strains resistant. However, the causative mutations themselves are embedded within larger recombined blocks, and previous studies have only analysed a limited number of isolates, leading to the description of "mosaic genes" as being responsible for resistance. By comparing a large number of genomes of beta-lactam susceptible and non-susceptible strains, the high frequency of recombination should break up these haplotype blocks and allow the use of genetic association approaches to identify individual causative variants. Here, we performed a genome-wide association study to identify single nucleotide polymorphisms (SNPs) and indels that could confer beta-lactam non-susceptibility using 3,085 Thai and 616 USA pneumococcal isolates as independent datasets for the variant discovery. The large sample sizes allowed us to narrow the source of beta-lactam non-susceptibility from long recombinant fragments down to much smaller loci comprised of discrete or linked SNPs. While some loci appear to be universal resistance determinants, contributing equally to non-susceptibility for at least two classes of beta-lactam antibiotics, some play a larger role in resistance to particular antibiotics. All of the identified loci have a highly non-uniform distribution in the populations. They are enriched not only in vaccine-targeted, but also non-vaccine-targeted lineages, which may raise clinical concerns. Identification of single nucleotide polymorphisms underlying resistance will be essential for future use of genome sequencing to predict antibiotic sensitivity in clinical microbiology.
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Spread of artemisinin resistance in Plasmodium falciparum malaria.
N. Engl. J. Med.
PUBLISHED: 07-31-2014
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Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and now poses a threat to the control and elimination of malaria. Mapping the geographic extent of resistance is essential for planning containment and elimination strategies.
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Spiroindolone KAE609 for falciparum and vivax malaria.
N. Engl. J. Med.
PUBLISHED: 07-31-2014
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KAE609 (cipargamin; formerly NITD609, Novartis Institute for Tropical Diseases) is a new synthetic antimalarial spiroindolone analogue with potent, dose-dependent antimalarial activity against asexual and sexual stages of Plasmodium falciparum.
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Polymorphisms in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes: parasite risk factors that affect treatment outcomes for P. falciparum malaria after artemether-lumefantrine and artesunate-amodiaquine.
Am. J. Trop. Med. Hyg.
PUBLISHED: 07-21-2014
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Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 - 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36-17.97, P < 0.001 : were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine.
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High-throughput ultrasensitive molecular techniques for quantifying low-density malaria parasitemias.
J. Clin. Microbiol.
PUBLISHED: 07-02-2014
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The epidemiology of malaria in "low-transmission" areas has been underestimated. Molecular detection methods have revealed higher prevalences of malaria than conventional microscopy or rapid diagnostic tests, but these typically evaluate finger-prick capillary blood samples (?5 ?l) and therefore cannot detect parasite densities of <200/ml. Their use underestimates true parasite carriage rates. To characterize the epidemiology of malaria in low-transmission settings and plan elimination strategies, more sensitive quantitative PCR (qPCR) is needed to identify and quantify low-density malaria parasitemias. A highly sensitive "high-volume" quantitative PCR (qPCR) method based on Plasmodium sp. 18S RNA was adapted for blood sample volumes of ?250 ?l and scaled for high throughput. The methods were validated by assessment of the analytical sensitivity and specificity, diagnostic sensitivity, and specificity, efficiency, precision, analytical and diagnostic accuracies, limit of detection, root cause analysis of false positives, and robustness. The high-volume qPCR method based on Plasmodium sp. 18S RNA gave high PCR efficiency of 90 to 105%. Concentrations of parasite DNA from large volumes of blood gave a consistent analytical detection limit (LOD) of 22 parasites/ml (95% CI, 21.79 to 74.9), which is some 2,500 times more sensitive than conventional microscopy and 50 times more sensitive than currently used PCR methods from filter paper blood spots. The diagnostic specificity was 99.75%. Using automated procedures it was possible to process 700 blood samples per week. A very sensitive and specific high-throughput high-volume qPCR method for the detection of low-density parasitemias (>20 parasites/ml) was developed and validated.
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An integrated lab-on-chip for rapid identification and simultaneous differentiation of tropical pathogens.
PLoS Negl Trop Dis
PUBLISHED: 07-01-2014
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Tropical pathogens often cause febrile illnesses in humans and are responsible for considerable morbidity and mortality. The similarities in clinical symptoms provoked by these pathogens make diagnosis difficult. Thus, early, rapid and accurate diagnosis will be crucial in patient management and in the control of these diseases. In this study, a microfluidic lab-on-chip integrating multiplex molecular amplification and DNA microarray hybridization was developed for simultaneous detection and species differentiation of 26 globally important tropical pathogens. The analytical performance of the lab-on-chip for each pathogen ranged from 102 to 103 DNA or RNA copies. Assay performance was further verified with human whole blood spiked with Plasmodium falciparum and Chikungunya virus that yielded a range of detection from 200 to 4×105 parasites, and from 250 to 4×107 PFU respectively. This lab-on-chip was subsequently assessed and evaluated using 170 retrospective patient specimens in Singapore and Thailand. The lab-on-chip had a detection sensitivity of 83.1% and a specificity of 100% for P. falciparum; a sensitivity of 91.3% and a specificity of 99.3% for P. vivax; a positive 90.0% agreement and a specificity of 100% for Chikungunya virus; and a positive 85.0% agreement and a specificity of 100% for Dengue virus serotype 3 with reference methods conducted on the samples. Results suggested the practicality of an amplification microarray-based approach in a field setting for high-throughput detection and identification of tropical pathogens.
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Population pharmacokinetics of quinine in pregnant women with uncomplicated Plasmodium falciparum malaria in Uganda.
J. Antimicrob. Chemother.
PUBLISHED: 06-25-2014
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Oral quinine is used for the treatment of uncomplicated malaria during pregnancy, but few pharmacokinetic data are available for this population. Previous studies have reported a substantial effect of malaria on the pharmacokinetics of quinine resulting from increased ?-1-acid glycoprotein levels and decreased cytochrome P450 3A4 activity. The aim of this study was to investigate the pharmacokinetic properties of oral quinine in pregnant women with uncomplicated malaria in Uganda using a population approach.
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Linking international clinical research with stateless populations to justice in global health.
BMC Med Ethics
PUBLISHED: 06-20-2014
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In response to calls to expand the scope of research ethics to address justice in global health, recent scholarship has sought to clarify how external research actors from high-income countries might discharge their obligation to reduce health disparities between and within countries. An ethical framework-'research for health justice'-was derived from a theory of justice (the health capability paradigm) and specifies how international clinical research might contribute to improved health and research capacity in host communities. This paper examines whether and how external funders, sponsors, and researchers can fulfill their obligations under the framework.
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KAF156 is an antimalarial clinical candidate with potential for use in prophylaxis, treatment, and prevention of disease transmission.
Antimicrob. Agents Chemother.
PUBLISHED: 06-09-2014
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Renewed global efforts toward malaria eradication have highlighted the need for novel antimalarial agents with activity against multiple stages of the parasite life cycle. We have previously reported the discovery of a novel class of antimalarial compounds in the imidazolopiperazine series that have activity in the prevention and treatment of blood stage infection in a mouse model of malaria. Consistent with the previously reported activity profile of this series, the clinical candidate KAF156 shows blood schizonticidal activity with 50% inhibitory concentrations of 6 to 17.4 nM against P. falciparum drug-sensitive and drug-resistant strains, as well as potent therapeutic activity in a mouse models of malaria with 50, 90, and 99% effective doses of 0.6, 0.9, and 1.4 mg/kg, respectively. When administered prophylactically in a sporozoite challenge mouse model, KAF156 is completely protective as a single oral dose of 10 mg/kg. Finally, KAF156 displays potent Plasmodium transmission blocking activities both in vitro and in vivo. Collectively, our data suggest that KAF156, currently under evaluation in clinical trials, has the potential to treat, prevent, and block the transmission of malaria.
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Small Molecule Targeting Malaria Merozoite Surface Protein-1 (MSP-1) Prevents Host Invasion of Divergent Plasmodial Species.
J. Infect. Dis.
PUBLISHED: 05-26-2014
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Malaria causes nearly 1 million deaths annually. Recent emergence of multidrug resistance highlights the need to develop novel therapeutic interventions against human malaria. Given the involvement of sugar binding plasmodial proteins in host invasion, we set out to identify such proteins as targets of small glycans. Combining multidisciplinary approaches, we report the discovery of a small molecule inhibitor, NIC, capable of inhibiting host invasion through interacting with a major invasion-related protein, merozoite surface protein-1 (MSP-1). This interaction was validated through computational, biochemical, and biophysical tools. Importantly, treatment with NIC prevented host invasion by Plasmodium falciparum and Plasmodium vivax-major causative organisms of human malaria. MSP-1, an indispensable antigen critical for invasion and suitably localized in abundance on the merozoite surface represents an ideal target for antimalarial development. The ability to target merozoite invasion proteins with specific small inhibitors opens up a new avenue to target this important pathogen.
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Single-cell genomics for dissection of complex malaria infections.
Genome Res.
PUBLISHED: 05-08-2014
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Most malaria infections contain complex mixtures of distinct parasite lineages. These multiple-genotype infections (MGIs) impact virulence evolution, drug resistance, intra-host dynamics, and recombination, but are poorly understood. To address this we have developed a single-cell genomics approach to dissect MGIs. By combining cell sorting and whole-genome amplification (WGA), we are able to generate high-quality material from parasite-infected red blood cells (RBCs) for genotyping and next-generation sequencing. We optimized our approach through analysis of >260 single-cell assays. To quantify accuracy, we decomposed mixtures of known parasite genotypes and obtained highly accurate (>99%) single-cell genotypes. We applied this validated approach directly to infections of two major malaria species, Plasmodium falciparum, for which long term culture is possible, and Plasmodium vivax, for which no long-term culture is feasible. We demonstrate that our single-cell genomics approach can be used to generate parasite genome sequences directly from patient blood in order to unravel the complexity of P. vivax and P. falciparum infections. These methods open the door for large-scale analysis of within-host variation of malaria infections, and reveal information on relatedness and drug resistance haplotypes that is inaccessible through conventional sequencing of infections.
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Selection of drug resistance-mediating Plasmodium falciparum genetic polymorphisms by seasonal malaria chemoprevention in Burkina Faso.
Antimicrob. Agents Chemother.
PUBLISHED: 04-14-2014
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Seasonal malaria chemoprevention (SMC), with regular use of amodiaquine plus sulfadoxine-pyrimethamine (AQ/SP) during the transmission season, is now a standard malaria control measure in the Sahel subregion of Africa. Another strategy under study is SMC with dihydroartemisinin plus piperaquine (DP). Plasmodium falciparum single nucleotide polymorphisms (SNPs) in P. falciparum crt (pfcrt), pfmdr1, pfdhfr, and pfdhps are associated with decreased response to aminoquinoline and antifolate antimalarials and are selected by use of these drugs. To characterize selection by SMC of key polymorphisms, we assessed 13 SNPs in P. falciparum isolated from children aged 3 to 59 months living in southwestern Burkina Faso and randomized to receive monthly DP or AQ/SP for 3 months in 2009. We compared SNP prevalence before the onset of SMC and 1 month after the third treatment in P. falciparum PCR-positive samples from 120 randomly selected children from each treatment arm and an additional 120 randomly selected children from a control group that did not receive SMC. The prevalence of relevant mutations was increased after SMC with AQ/SP. Significant selection was seen for pfcrt 76T (68.5% to 83.0%, P = 0.04), pfdhfr 59R (54.8% to 83.3%, P = 0.0002), and pfdhfr 108N (55.0% to 87.2%, P = 0.0001), with trends toward selection of pfmdr1 86Y, pfdhfr 51I, and pfdhps 437G. After SMC with DP, only borderline selection of wild-type pfmdr1 D1246 (mutant; 7.7% to 0%, P = 0.05) was seen. In contrast to AQ/SP, SMC with DP did not clearly select for known resistance-mediating polymorphisms. SMC with AQ/SP, but not DP, may hasten the development of resistance to components of this regimen. (This study has been registered at ClinicalTrials.gov under registration no. NCT00941785.).
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Uncomplicated Plasmodium vivax malaria in pregnancy associated with mortality from acute respiratory distress syndrome.
Malar. J.
PUBLISHED: 04-03-2014
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The association between severe malaria and Plasmodium vivax species is contentious. On the Thai-Myanmar border, all pregnant women are followed systematically with active weekly malaria screening. Over a 27-year period of providing antenatal care, 48,983 have been prospectively followed until pregnancy outcome (miscarriage or delivery) and 4,298 women have had P. vivax detected at least once. Reported here is the first known P. vivax-associated death amongst these women. The initial patient presentation was of uncomplicated P. vivax (0.5% parasitaemia) in a term, multigravida woman who responded rapidly to oral artesunate and mefloquine treatment, clearing her blood stage parasites within 48 hours. The patient appeared well, was ambulatory and due to be discharged but became unwell with acute respiratory distress syndrome (ARDS) requiring ventilation three days (67 hours) into treatment. Despite induction and delivery of a stillborn foetus, ventilatory requirements increased and the patient died on day 7. The patient had a low body mass index. Sensitive detection with nested PCR confirmed only the presence of P. vivax species and concomitant infections such as tuberculosis and human immunodeficiency virus (HIV) were also ruled out. The contemporaneous treatment of acute uncomplicated P. vivax and the onset of ARDS on day 3 in this patient implies a possible but unconfirmed association with death in this patient. Assuming this death was caused by P. vivax, the risk of ARDS-related maternal mortality in this setting did not differ significantly between Plasmodium falciparum and P. vivax (0.24 per 1,000 (1/4,158) versus 0.23 per 1,000 (1/4,298), contrary to the increased risk of maternal mortality from P. falciparum compared to P. vivax, 2.89 per 1,000 (12/4,158) versus 0.23 per 1,000 (1/4,298), P?=?0.003.
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Glycophorin C (CD236R) mediates vivax malaria parasite rosetting to normocytes.
Blood
PUBLISHED: 03-20-2014
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Rosetting phenomenon has been linked to malaria pathogenesis. Although rosetting occurs in all causes of human malaria, most data on this subject has been derived from Plasmodium falciparum. Here, we investigate the function and factors affecting rosette formation in Plasmodium vivax. To achieve this, we used a range of novel ex vivo protocols to study fresh and cryopreserved P vivax (n = 135) and P falciparum (n = 77) isolates from Thailand. Rosetting is more common in vivax than falciparum malaria, both in terms of incidence in patient samples and percentage of infected erythrocytes forming rosettes. Rosetting to P vivax asexual and sexual stages was evident 20 hours postreticulocyte invasion, reaching a plateau after 30 hours. Host ABO blood group, reticulocyte count, and parasitemia were not correlated with P vivax rosetting. Importantly, mature erythrocytes (normocytes), rather than reticulocytes, preferentially form rosetting complexes, indicating that this process is unlikely to directly facilitate merozoite invasion. Although antibodies against host erythrocyte receptors CD235a and CD35 had no effect, Ag-binding fragment against the BRIC 4 region of CD236R significantly inhibited rosette formation. Rosetting assays using CD236R knockdown normocytes derived from hematopoietic stem cells further supports the role of glycophorin C as a receptor in P vivax rosette formation.
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Treatment of suspected hyper-reactive malarial splenomegaly (HMS) in pregnancy with mefloquine.
Am. J. Trop. Med. Hyg.
PUBLISHED: 03-03-2014
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Malaria infections in pregnancy are associated with adverse outcomes for both mother and child. There are few data on hyper-reactive malarial splenomegaly, an aberrant immunological response to chronic or recurrent malaria in pregnancy. This retrospective assessment reviewed the impact of mefloquine treatment on pregnant women with suspected hyper-reactive malarial splenomegaly in an area of low malaria transmission in the 1990s, showing significant reductions in spleen size and anemia and anti-malarial antibody titers without any notable negative effect on treated women or their newborns.
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Single nucleotide polymorphisms in Plasmodium falciparum V type H(+) pyrophosphatase gene (pfvp2) and their associations with pfcrt and pfmdr1 polymorphisms.
Infect. Genet. Evol.
PUBLISHED: 03-02-2014
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Chloroquine resistance in Plasmodium falciparum malaria has been associated with pfcrt 76T (chloroquine resistance transporter gene) and pfmdr1 86Y (multidrug resistance gene 1) alleles. Pfcrt 76T enables transport of protonated chloroquine out of the parasites digestive vacuole resulting in a loss of hydrogen ions (H(+)). V type H(+) pyrophosphatase (PfVP2) is thought to pump H(+) into the digestive vacuole. This study aimed to describe the geographic distribution of single nucleotide polymorphisms in pfvp2 and their possible associations with pfcrt and pfmdr1 polymorphisms.
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A barcode of organellar genome polymorphisms identifies the geographic origin of Plasmodium falciparum strains.
Nat Commun
PUBLISHED: 02-20-2014
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Malaria is a major public health problem that is actively being addressed in a global eradication campaign. Increased population mobility through international air travel has elevated the risk of re-introducing parasites to elimination areas and dispersing drug-resistant parasites to new regions. A simple genetic marker that quickly and accurately identifies the geographic origin of infections would be a valuable public health tool for locating the source of imported outbreaks. Here we analyse the mitochondrion and apicoplast genomes of 711 Plasmodium falciparum isolates from 14 countries, and find evidence that they are non-recombining and co-inherited. The high degree of linkage produces a panel of relatively few single-nucleotide polymorphisms (SNPs) that is geographically informative. We design a 23-SNP barcode that is highly predictive (~92%) and easily adapted to aid case management in the field and survey parasite migration worldwide.
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Clinical audit to enhance safe practice of skilled birth attendants for the fetus with nuchal cord: evidence from a refugee and migrant cohort.
BMC Pregnancy Childbirth
PUBLISHED: 02-13-2014
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Current evidence for optimal management of fetal nuchal cord detected after the head has birthed supports techniques that avoid ligation of the umbilical cord circulation. Routine audit found frequent unsafe management of nuchal cord by skilled birth attendants (SBAs) in migrant and refugee birth centres on the Thai-Burmese border.
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Motivations and perceptions of community advisory boards in the ethics of medical research: the case of the Thai-Myanmar border.
BMC Med Ethics
PUBLISHED: 01-31-2014
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Community engagement is increasingly promoted as a marker of good, ethical practice in the context of international collaborative research in low-income countries. There is, however, no widely agreed definition of community engagement 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 forms of research; higher recruitment rates; the identification of important ethical issues; the building of better relationships between the community and researchers; the obtaining of community permission to approach potential research participants; and, the provision of better health care. Despite these diverse and potentially competing claims made for the importance of community engagement, there is very little published evidence on effective models of engagement or their evaluation.
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Population pharmacokinetic assessment of the effect of food on piperaquine bioavailability in patients with uncomplicated malaria.
Antimicrob. Agents Chemother.
PUBLISHED: 01-21-2014
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Previously published literature reports various impacts of food on the oral bioavailability of piperaquine. The aim of this study was to use a population modeling approach to investigate the impact of concomitant intake of a small amount of food on piperaquine pharmacokinetics. This was an open, randomized comparison of piperaquine pharmacokinetics when administered as a fixed oral formulation once daily for 3 days with (n=15) and without (n=15) concomitant food to patients with uncomplicated Plasmodium falciparum malaria in Thailand. Nonlinear mixed-effects modeling was used to characterize the pharmacokinetics of piperaquine and the influence of concomitant food intake. A modified Monte Carlo mapped power approach was applied to evaluate the relationship between statistical power and various degrees of covariate effect sizes of the given study design. Piperaquine population pharmacokinetics were described well in fasting and fed patients by a three-compartment distribution model with flexible absorption. The final model showed a 25% increase in relative bioavailability per dose occasion during recovery from malaria but demonstrated no clinical impact of concomitant intake of a low-fat meal. Body weight and age were both significant covariates in the final model. The novel power approach concluded that the study was adequately powered to detect a food effect of at least 35%. This modified Monte Carlo mapped power approach may be a useful tool for evaluating the power to detect true covariate effects in mixed-effects modeling and a given study design. A small amount of food does not affect piperaquine absorption significantly in acute malaria.
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Dense genomic sampling identifies highways of pneumococcal recombination.
Nat. Genet.
PUBLISHED: 01-15-2014
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Evasion of clinical interventions by Streptococcus pneumoniae occurs through selection of non-susceptible genomic variants. We report whole-genome sequencing of 3,085 pneumococcal carriage isolates from a 2.4-km(2) refugee camp. This sequencing provides unprecedented resolution of the process of recombination and its impact on population evolution. Genomic recombination hotspots show remarkable consistency between lineages, indicating common selective pressures acting at certain loci, particularly those associated with antibiotic resistance. Temporal changes in antibiotic consumption are reflected in changes in recombination trends, demonstrating rapid spread of resistance when selective pressure is high. The highest frequencies of receipt and donation of recombined DNA fragments were observed in non-encapsulated lineages, implying that this largely overlooked pneumococcal group, which is beyond the reach of current vaccines, may have a major role in genetic exchange and the adaptation of the species as a whole. These findings advance understanding of pneumococcal population dynamics and provide information for the design of future intervention strategies.
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A morphometric and histological study of placental malaria shows significant changes to villous architecture in both Plasmodium falciparum and Plasmodium vivax infection.
Malar. J.
PUBLISHED: 01-04-2014
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Malaria in pregnancy remains a major health problem. Placental malaria infection may cause pathophysiological changes in pregnancy and result in morphological changes to placental villi. Quantitative histomorphological image analysis of placental biopsies was performed to compare placental villous architecture between active or treated placental malaria cases and controls.
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Characterization of the Commercially-Available Fluorescent Chloroquine-BODIPY Conjugate, LynxTag-CQGREEN, as a Marker for Chloroquine Resistance and Uptake in a 96-Well Plate Assay.
PLoS ONE
PUBLISHED: 01-01-2014
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Chloroquine was a cheap, extremely effective drug against Plasmodium falciparum until resistance arose. One approach to reversing resistance is the inhibition of chloroquine efflux from its site of action, the parasite digestive vacuole. Chloroquine accumulation studies have traditionally relied on radiolabelled chloroquine, which poses several challenges. There is a need for development of a safe and biologically relevant substitute. We report here a commercially-available green fluorescent chloroquine-BODIPY conjugate, LynxTag-CQGREEN, as a proxy for chloroquine accumulation. This compound localized to the digestive vacuole of the parasite as observed under confocal microscopy, and inhibited growth of chloroquine-sensitive strain 3D7 more extensively than in the resistant strains 7G8 and K1. Microplate reader measurements indicated suppression of LynxTag-CQGREEN efflux after pretreatment of parasites with known reversal agents. Microsomes carrying either sensitive- or resistant-type PfCRT were assayed for uptake; resistant-type PfCRT exhibited increased accumulation of LynxTag-CQGREEN, which was suppressed by pretreatment with known chemosensitizers. Eight laboratory strains and twelve clinical isolates were sequenced for PfCRT and Pgh1 haplotypes previously reported to contribute to drug resistance, and pfmdr1 copy number and chloroquine IC50s were determined. These data were compared with LynxTag-CQGREEN uptake/fluorescence by multiple linear regression to identify genetic correlates of uptake. Uptake of the compound correlated with the logIC50 of chloroquine and, more weakly, a mutation in Pgh1, F1226Y.
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Quantifying low birth weight, preterm birth and small-for-gestational-age effects of malaria in pregnancy: a population cohort study.
PLoS ONE
PUBLISHED: 01-01-2014
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The association between malaria during pregnancy and low birth weight (LBW) is well described. This manuscript aims to quantify the relative contribution of malaria to small-for-gestational-age (SGA) infants and preterm birth (PTB) in pregnancies accurately dated by ultrasound on the Thai-Myanmar border at the Shoklo Malaria Research Unit.
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Gestational diabetes mellitus prevalence in Maela refugee camp on the Thai-Myanmar border: a clinical report.
Glob Health Action
PUBLISHED: 01-01-2014
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Individuals in conflict-affected areas rarely get appropriate care for chronic or non-infectious diseases. The prevalence of gestational diabetes mellitus (GDM) is increasing worldwide, and new evidence shows conclusively that the negative effects of hyperglycemia occur even at mild glucose elevations and that these negative effects can be attenuated by treatment. Scientific literature on gestational diabetes in refugee camp settings is critically limited.
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Two fatal cases of melioidosis on the Thai-Myanmar border.
F1000Res
PUBLISHED: 01-01-2014
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Melioidosis is endemic in areas of Southeast Asia, however, there are no published reports from the Thai-Myanmar border.  We report the first two cases of fatal melioidosis in this region. This is of great public health importance and highlights the need to increase clinical awareness of melioidosis on the Thai-Myanmar border and to assess the true burden of disease in the area through improved case detection and Burkholderia pseudomallei prevalence studies.
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Genetic variability of Plasmodium malariae dihydropteroate synthase (dhps) in four Asian countries.
PLoS ONE
PUBLISHED: 01-01-2014
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The dihydropteroate synthase (dhps) genes of 44 P. malariae strains from four Asian countries were isolated. Only a limited number of polymorphisms were observed. Comparison with homologous mutations in other Plasmodium species showed that these polymorphisms are unlikely to be associated with sulfadoxine resistance.
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Rapid clinical assessment to facilitate the triage of adults with falciparum malaria, a retrospective analysis.
PLoS ONE
PUBLISHED: 01-01-2014
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Most adults dying from falciparum malaria will die within 48 hours of their hospitalisation. An essential component of early supportive care is the rapid identification of patients at greatest risk. In resource-poor settings, where most patients with falciparum malaria are managed, decisions regarding patient care must frequently be made using clinical evaluation alone.
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Invasion-inhibitory antibodies elicited by immunization with Plasmodium vivax apical membrane antigen-1 expressed in Pichia pastoris yeast.
Infect. Immun.
PUBLISHED: 12-30-2013
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In a recent vaccine trial performed with African children, immunization with a recombinant protein based on Plasmodium falciparum apical membrane antigen 1 (AMA-1) conferred a significant degree of strain-specific resistance against malaria. To contribute to the efforts of generating a vaccine against P. vivax malaria, we expressed the ectodomain of P. vivax AMA-1 (PvAMA-1) as a secreted soluble protein in the methylotrophic yeast Pichia pastoris. Recognized by a high percentage of sera from individuals infected by P. vivax, this recombinant protein was found to have maintained its antigenicity. The immunogenicity of this protein was evaluated in mice using immunization protocols that included homologous and heterologous prime/boost strategies with plasmid DNA and recombinant protein. We used the following formulations containing different adjuvants: aluminum salts (Alum), Bordetella pertussis monophosphoryl lipid A (MPLA), flagellin FliC from Salmonella Typhimurium, saponin Quil A, or Incomplete Freund Adjuvant (IFA). The formulations containing the adjuvants Quil A or IFA elicited the highest IgG antibody titers. Significant antibody titers were also obtained using a formulation developed for human use containing MPLA or Alum plus MPLA. Recombinant PvAMA-1 produced under conditions of good laboratory practice provided a good yield, high purity, low endotoxin levels, no microbial contaminants, and reproduced the experimental immunizations. Most relevant for vaccine development was the fact that immunization with PvAMA-1 elicited invasion-inhibitory antibodies against different Asian isolates of P. vivax. Our results show that AMA-1 expressed in P. pastoris is a promising antigen for use in future pre-clinical and clinical studies.
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PlasmoView: A web-based resource to visualise global Plasmodium falciparum genomic variation.
J. Infect. Dis.
PUBLISHED: 12-12-2013
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Malaria is a global public health challenge, with drug resistance a major barrier to disease control and elimination. To meet the urgent need for better treatments and vaccines, a deeper knowledge of Plasmodium biology and malaria epidemiology is required. An improved understanding of the genomic variation of malaria parasites, especially the most virulent Plasmodium falciparum (Pf) species, has the potential to yield new insights in these areas. High-throughput sequencing and genotyping is generating large amounts of genomic data across multiple parasite populations. The resulting ability to identify informative variants, particularly single-nucleotide polymorphisms (SNPs), will lead to the discovery of intra- and inter-population differences and thus enable the development of genetic barcodes for diagnostic assays and clinical studies. Knowledge of genetic variability underlying drug resistance and other differential phenotypes will also facilitate the identification of novel mutations and contribute to surveillance and stratified medicine applications. The PlasmoView interactive web-browsing tool enables the research community to visualise genomic variation and annotation (e.g. biological function) in a geographic setting. The first release contains over 600,000 high-quality SNPs in 631 Pf isolates from laboratory strains and four malaria-endemic regions (West Africa, East Africa, Southeast Asia and Oceania).
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A High-Content Phenotypic Screen Reveals the Disruptive Potency of Quinacrine and 3,4-Dichlorobenzamil on the Digestive Vacuole of Plasmodium falciparum.
Antimicrob. Agents Chemother.
PUBLISHED: 11-11-2013
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Plasmodium falciparum is the etiological agent of malignant malaria and has been shown to exhibit features resembling programmed cell death. This is triggered upon treatment with low micromolar doses of chloroquine or other lysosomotrophic compounds and is associated with leakage of the digestive vacuole contents. In order to exploit this cell death pathway, we developed a high-content screening method to select compounds that can disrupt the parasite vacuole, as measured by the leakage of intravacuolar Ca(2+). This assay uses the ImageStream 100, an imaging-capable flow cytometer, to assess the distribution of the fluorescent calcium probe Fluo-4. We obtained two hits from a small library of 25 test compounds, quinacrine and 3,4-dichlorobenzamil. The ability of these compounds to permeabilize the digestive vacuole in laboratory strains and clinical isolates was validated by confocal microscopy. The hits could induce programmed cell death features in both chloroquine-sensitive and -resistant laboratory strains. Quinacrine was effective at inhibiting field isolates in a 48-h reinvasion assay regardless of artemisinin clearance status. We therefore present as proof of concept a phenotypic screening method with the potential to provide mechanistic insights to the activity of antimalarial drugs.
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Genetic marker suitable for identification and genotyping of Plasmodium ovale curtisi and Plasmodium ovale wallikeri.
J. Clin. Microbiol.
PUBLISHED: 09-25-2013
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We present a seminested PCR method that specifically discriminates between Plasmodium ovale curtisi and P. ovale wallikeri with high sensitivity. The test is based on species-specific amplification of a size-polymorphic fragment of the tryptophan-rich antigen gene, potra, which also permits discrimination of intraspecific sequence variants at this locus.
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G6PD testing in support of treatment and elimination of malaria: recommendations for evaluation of G6PD tests.
Malar. J.
PUBLISHED: 09-11-2013
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Malaria elimination will be possible only with serious attempts to address asymptomatic infection and chronic infection by both Plasmodium falciparum and Plasmodium vivax. Currently available drugs that can completely clear a human of P. vivax (known as "radical cure"), and that can reduce transmission of malaria parasites, are those in the 8-aminoquinoline drug family, such as primaquine. Unfortunately, people with glucose-6-phosphate dehydrogenase (G6PD) deficiency risk having severe adverse reactions if exposed to these drugs at certain doses. G6PD deficiency is the most common human enzyme defect, affecting approximately 400 million people worldwide.Scaling up radical cure regimens will require testing for G6PD deficiency, in at two levels: 1) individual level to ensure safe case management, and 2) population level to understand the risk in the local population to guide Plasmodium vivax treatment policy. Several technical and operational knowledge gaps must be addressed to expand access to G6PD deficiency testing and to ensure that a patients G6PD status is known before deciding to administer an 8-aminoquinoline-based drug.In this report from a stakeholder meeting held in Thailand on October 4 and 5, 2012, G6PD testing in support of radical cure is discussed in detail. The focus is on challenges to the development and evaluation of G6PD diagnostic tests, and on challenges related to the operational aspects of implementing G6PD testing in support of radical cure. The report also describes recommendations for evaluation of diagnostic tests for G6PD deficiency in support of radical cure.
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Pharmacokinetic properties of artemether, dihydroartemisinin, lumefantrine, and quinine in pregnant women with uncomplicated plasmodium falciparum malaria in Uganda.
Antimicrob. Agents Chemother.
PUBLISHED: 08-05-2013
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Pregnancy alters the pharmacokinetic properties of many drugs used in the treatment of malaria, usually resulting in lower drug exposures. This increases the risks of treatment failure, adverse outcomes for the fetus, and the development of resistance. The pharmacokinetic properties of artemether and its principal metabolite dihydroartemisinin (n = 21), quinine (n = 21), and lumefantrine (n = 26) in pregnant Ugandan women were studied. Lumefantrine pharmacokinetics in a nonpregnant control group (n = 17) were also studied. Frequently sampled patient data were evaluated with noncompartmental analysis. No significant correlation was observed between estimated gestational age and artemether, dihydroartemisinin, lumefantrine, or quinine exposures. Artemether/dihydroartemisinin and quinine exposures were generally low in these pregnant women compared to values reported previously for nonpregnant patients. Median day 7 lumefantrine concentrations were 488 (range, 30.7 to 3,550) ng/ml in pregnant women compared to 720 (339 to 2,150) ng/ml in nonpregnant women (P = 0.128). There was no statistical difference in total lumefantrine exposure or maximum concentration. More studies with appropriate control groups in larger series are needed to characterize the degree to which pregnant women are underdosed with current antimalarial dosing regimens.
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Targeting Plasmodium PI(4)K to eliminate malaria.
Nature
PUBLISHED: 07-23-2013
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Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.
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Optimal sampling designs for estimation of Plasmodium falciparum clearance rates in patients treated with artemisinin derivatives.
Malar. J.
PUBLISHED: 07-10-2013
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The emergence of Plasmodium falciparum resistance to artemisinins in Southeast Asia threatens the control of malaria worldwide. The pharmacodynamic hallmark of artemisinin derivatives is rapid parasite clearance (a short parasite half-life), therefore, the in vivo phenotype of slow clearance defines the reduced susceptibility to the drug. Measurement of parasite counts every six hours during the first three days after treatment have been recommended to measure the parasite clearance half-life, but it remains unclear whether simpler sampling intervals and frequencies might also be sufficient to reliably estimate this parameter.
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Ancillary Care: From Theory to Practice in International Clinical Research.
Public Health Ethics
PUBLISHED: 06-13-2013
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How international research might contribute to justice in global health has not been substantively addressed by bioethics. This article describes how the provision of ancillary care can link international clinical research to the reduction of global health disparities. It identifies the ancillary care obligations supported by a theory of global justice, showing that Jennifer Rugers health capability paradigm requires the delivery of ancillary care to trial participants for a limited subset of conditions that cause severe morbidity and mortality. Empirical research on the Shoklo Malaria Research Units (SMRU) vivax malaria treatment trial was then undertaken to demonstrate whether and how these obligations might be upheld in a resource-poor setting. Our findings show that fulfilment of the ancillary care obligations is feasible where there is commitment from chief investigators and funders and is strongly facilitated by SMRUs dual role as a research unit and medical non-governmental organization.
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Challenges and prospects for dengue and malaria control in Thailand, Southeast Asia.
Trends Parasitol.
PUBLISHED: 06-08-2013
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Despite significant advances in the search for potential dengue vaccines and new therapeutic schemes for malaria, the control of these diseases remains difficult. In Thailand, malaria incidence is falling whereas that of dengue is rising, with an increase in the proportion of reported severe cases. In the absence of antiviral therapeutic options for acute dengue, appropriate case management reduces mortality. However, the interruption of transmission still relies on vector control measures that are currently insufficient to curtail the cycle of epidemics. Drug resistance in malaria parasites is increasing, compromising malaria control and elimination. Deficiencies in our knowledge of vector biology and vectorial capacity also hinder public health efforts for vector control. Challenges to dengue and malaria control are discussed, and research priorities identified.
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Exploitation and community engagement: Can Community Advisory Boards successfully assume a role minimising exploitation in international research?
Dev World Bioeth
PUBLISHED: 06-04-2013
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It has been suggested that community advisory boards (CABs) can play a role in minimising exploitation in international research. To get a better idea of what this requires and whether it might be achievable, the paper first describes core elements that we suggest must be in place for a CAB to reduce the potential for exploitation. The paper then examines a CAB established by the Shoklo Malaria Research Unit under conditions common in resource-poor settings - namely, where individuals join with a very limited understanding of disease and medical research and where an existing organisational structure is not relied upon to serve as the CAB. Using the Tak Province Border Community Ethics Advisory Board (T-CAB) as a case study, we assess the extent to which it might be able to take on a role minimising exploitation were it to decide to do so. We investigate whether, after two years in operation, T-CAB is capable of assessing clinical trials for exploitative features and addressing those found to have them. The findings show that, although T-CAB members have gained knowledge and developed capacities that are foundational for one-day taking on a role to reduce exploitation, their ability to critically evaluate studies for the presence of exploitative elements has not yet been strongly demonstrated. In light of this example, we argue that CABs may not be able to perform such a role for a number of years after initial formation, making it an unsuitable responsibility for many short-term CABs.
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A prospective evaluation of real-time PCR assays for the detection of Orientia tsutsugamushi and Rickettsia spp. for early diagnosis of rickettsial infections during the acute phase of undifferentiated febrile illness.
Am. J. Trop. Med. Hyg.
PUBLISHED: 06-03-2013
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One hundred and eighty febrile patients were analyzed in a prospective evaluation of Orientia tsutsugamushi and Rickettsia spp. real-time polymerase chain reaction (PCR) assays for early diagnosis of rickettsial infections. By paired serology, 3.9% (7 of 180) and 6.1% (11 of 180) of patients were confirmed to have acute scrub or murine typhus, respectively. The PCR assays for the detection of O. tsutsugamushi and Rickettsia spp. had high specificity (99.4% [95% confidence interval (CI): 96.8-100] and 100% [95% CI: 97.8-100], respectively). The PCR results were also compared with immunoglobulin M (IgM) immunofluorescence assay (IFA) on acute sera. For O. tsutsugamushi, PCR sensitivity was twice that of acute specimen IgM IFA (28.6% versus 14.3%; McNemars P = 0.3). For Rickettsia spp., PCR was four times as sensitive as acute specimen IgM IFA (36.4% versus 9.1%; P = 0.08), although this was not statistically significant. Whole blood and buffy coat, but not serum, were acceptable specimens for these PCRs. Further evaluation of these assays in a larger prospective study is warranted.
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A three year descriptive study of early onset neonatal sepsis in a refugee population on the Thailand Myanmar border.
BMC Infect. Dis.
PUBLISHED: 05-14-2013
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Each year an estimated four million neonates die, the majority in the first week of life. One of the major causes of death is sepsis. Proving the incidence and aetiology of neonatal sepsis is difficult, particularly in resource poor settings where the majority of the deaths occur.
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High-throughput analysis of antimalarial susceptibility data by the WorldWide Antimalarial Resistance Network (WWARN) in vitro analysis and reporting tool.
Antimicrob. Agents Chemother.
PUBLISHED: 04-22-2013
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Assessment of in vitro susceptibility is a fundamental component of antimalarial surveillance studies, but wide variations in the measurement of parasite growth and the calculation of inhibitory constants make comparisons of data from different laboratories difficult. Here we describe a Web-based, high-throughput in vitro analysis and reporting tool (IVART) generating inhibitory constants for large data sets. Fourteen primary data sets examining laboratory-determined susceptibility to artemisinin derivatives and artemisinin combination therapy partner drugs were collated from 11 laboratories. Drug concentrations associated with half-maximal inhibition of growth (IC50s) were determined by a modified sigmoid Emax model-fitting algorithm, allowing standardized analysis of 7,350 concentration-inhibition assays involving 1,592 isolates. Examination of concentration-inhibition data revealed evidence of apparent paradoxical growth at high concentrations of nonartemisinin drugs, supporting amendment of the method for calculating the maximal drug effect in each assay. Criteria for defining more-reliable IC50s based on estimated confidence intervals and growth ratios improved correlation coefficients for the drug pairs mefloquine-quinine and chloroquine-desethylamodiaquine in 9 of 11 and 8 of 8 data sets, respectively. Further analysis showed that maximal drug inhibition was higher for artemisinins than for other drugs, particularly in ELISA (enzyme-linked immunosorbent assay)-based assays, a finding consistent with the earlier onset of action of these drugs in the parasite life cycle. This is the first high-throughput analytical approach to apply consistent constraints and reliability criteria to large, diverse antimalarial susceptibility data sets. The data also illustrate the distinct biological properties of artemisinins and underline the need to apply more sensitive approaches to assessing in vitro susceptibility to these drugs.
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Genetic evaluation of the performance of malaria parasite clearance rate metrics.
J. Infect. Dis.
PUBLISHED: 04-16-2013
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Accurate measurement of malaria parasite clearance rates (CRs) following artemisinin (ART) treatment is critical for resistance surveillance and research, and various CR metrics are currently used. We measured 13 CR metrics in 1472 ART-treated hyperparasitemia infections for which 6-hour parasite counts and parasite genotypes (93 single nucleotide polymorphisms [SNPs]) were available. We used heritability to evaluate the performance of each metric. Heritability ranged from 0.06 ± 0.06 (SD) for 50% parasite clearance times to 0.67 ± 0.04 (SD) for clearance half-lives estimated from 6-hour parasite counts. These results identify the measures that should be avoided and show that reliable clearance measures can be obtained with abbreviated monitoring protocols.
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Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia.
Nat. Genet.
PUBLISHED: 04-04-2013
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We describe an analysis of genome variation in 825 P. falciparum samples from Asia and Africa that identifies an unusual pattern of parasite population structure at the epicenter of artemisinin resistance in western Cambodia. Within this relatively small geographic area, we have discovered several distinct but apparently sympatric parasite subpopulations with extremely high levels of genetic differentiation. Of particular interest are three subpopulations, all associated with clinical resistance to artemisinin, which have skewed allele frequency spectra and high levels of haplotype homozygosity, indicative of founder effects and recent population expansion. We provide a catalog of SNPs that show high levels of differentiation in the artemisinin-resistant subpopulations, including codon variants in transporter proteins and DNA mismatch repair proteins. These data provide a population-level genetic framework for investigating the biological origins of artemisinin resistance and for defining molecular markers to assist in its elimination.
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Microsatellite genotyping of Plasmodium vivax infections and their relapses in pregnant and non-pregnant patients on the Thai-Myanmar border.
Malar. J.
PUBLISHED: 04-03-2013
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Plasmodium vivax infections in pregnancy are associated with low birth weight and anaemia. This parasites species is also characterised by relapses, erythrocytic infections initiated by the activation of the dormant liver stages, the hypnozoites, to mature. Genotyping of P. vivax using microsatellite markers has opened the way to comparative investigations of parasite populations. The aim of the study was to assess whether there were any differences between the parasites found in pregnant and non-pregnant patients, and/or between the admission infections and recurrent episodes during follow-up.
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Serum antibody responses to pneumococcal colonisation in the first two years of life: results from a SE Asian longitudinal cohort study.
Clin. Microbiol. Infect.
PUBLISHED: 03-25-2013
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Assessment of antibody responses to pneumococcal colonisation in early childhood may aid our understanding of protection and inform vaccine antigen selection. Serum samples were collected from mother-infant pairs during a longitudinal pneumococcal colonisation study in Burmese refugees. Maternal and cord sera were collected at birth and infants were bled monthly (1-24 months of age). Nasopharyngeal swabs were taken monthly to detect colonisation. Serum IgG titres to 27 pneumococcal protein antigens were measured in 2,624 sera and IgG to dominant serotypes (6B,14,19F,19A,23F) were quantified in 864 infant sera. Antibodies to all protein antigens were detectable in maternal sera. Titres to four proteins (LytB,PcpA,PhtD,PhtE) were significantly higher in mothers colonised by pneumococci at delivery. Maternally-derived antibodies to PiuA and Spr0096 were associated with delayed pneumococcal acquisition in infants in univariate, but not multivariate models. Controlling for infant age and previous homologous serotype exposure, nasopharyngeal acquisition of serotypes 19A, 23F, 14, or 19F were associated significantly with a ?2-fold antibody response to the homologous capsule (OR 12.84, 7.52, 6.52, 5.33; p<0.05). Acquisition of pneumococcal serotypes in the nasopharynx of infants was not significantly associated with a ?2-fold rise in antibodies to any of the protein antigens studied. In conclusion, nasopharyngeal colonisation in young children resulted in demonstrable serum IgG responses to pneumococcal capsules and surface/virulence proteins. However, the relationship between serum IgG and the prevention of, or response to, pneumococcal nasopharyngeal colonisation remains complex. Mechanisms other than serum IgG are likely to have a role but are currently poorly understood. This article is protected by copyright. All rights reserved.
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Triangular test design to evaluate tinidazole in the prevention of Plasmodium vivax relapse.
Malar. J.
PUBLISHED: 03-04-2013
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There are very few drugs that prevent the relapse of Plasmodium vivax malaria in man. Tinidazole is a 5-nitroimidazole approved in the USA for the treatment of indications including amoebiasis and giardiasis. In the non-human primate relapsing Plasmodium cynomolgi/macaque malaria model, tinidazole cured one of six macaques studied with an apparent mild delay to relapse in the other five of 14-28 days compared to 11-12 days in controls. One study has demonstrated activity against P. vivax in man. Presented here are the results of a pilot phase II, randomized, open-label study conducted along the Thai-Myanmar border designed to evaluate the efficacy of tinidazole to prevent relapse of P. vivax when administered with chloroquine.
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Pharmacokinetic predictors for recurrent malaria after dihydroartemisinin-piperaquine treatment of uncomplicated malaria in Ugandan infants.
J. Infect. Dis.
PUBLISHED: 02-27-2013
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Although dihydroartemisinin-piperaquine (DP) is used primarily in children, pharmacokinetic/pharmacodynamic (PK/PD) data on DP use in young children are lacking.
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A whole cell pathway screen reveals seven novel chemosensitizers to combat chloroquine resistant malaria.
Sci Rep
PUBLISHED: 02-15-2013
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Due to the widespread prevalence of resistant parasites, chloroquine (CQ) was removed from front-line antimalarial chemotherapy in the 1990s despite its initial promise of disease eradication. Since then, resistance-conferring mutations have been identified in transporters such as the PfCRT, that allow for the efflux of CQ from its primary site of action, the parasite digestive vacuole. Chemosensitizing/chemoreversing compounds interfere with the function of these transporters thereby sensitizing parasites to CQ once again. However, compounds identified thus far have disappointing in vivo efficacy and screening for alternative candidates is required to revive this strategy. In this study, we propose a simple and direct means to rapidly screen for such compounds using a fluorescent-tagged CQ molecule. When this screen was applied to a small library, seven novel chemosensitizers (octoclothepin, methiothepin, metergoline, loperamide, chlorprothixene, L-703,606 and mibefradil) were quickly elucidated, including two which showed greater potency than the classical chemosensitizers verapamil and desipramine.
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Malaria in the post-partum period; a prospective cohort study.
PLoS ONE
PUBLISHED: 01-28-2013
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Several studies have shown a prolonged or increased susceptibility to malaria in the post-partum period. A matched cohort study was conducted to evaluate prospectively the susceptibility to malaria of post-partum women in an area where P.falciparum and P.vivax are prevalent.
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Malaria burden and artemisinin resistance in the mobile and migrant population on the Thai-Myanmar border, 1999-2011: an observational study.
PLoS Med.
PUBLISHED: 01-24-2013
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The Shoklo Malaria Research Unit has been working on the Thai-Myanmar border for 25 y providing early diagnosis and treatment (EDT) of malaria. Transmission of Plasmodium falciparum has declined, but resistance to artesunate has emerged. We expanded malaria activities through EDT and evaluated the impact over a 12-y period.
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Changes in the body weight of term infants, born in the tropics, during the first seven days of life.
BMC Pediatr
PUBLISHED: 01-22-2013
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BACKGROUND: Identifying unwell neonates, particularly in the first week of life, is often subjective. If normal values are known, calculating the weight lost or gained from birth weight can be a useful adjunct in the evaluation of the health of a neonate. METHODS: Serial body weights of well, term, breast fed infants who were attending for routine follow up, were recorded at the Shoklo Malaria Research Unit clinic in Maela Camp for displaced persons on the Thailand Myanmar border. Newborn examination was routine. Weight loss, expressed as percent weight lost from birth weight, and weight gain, expressed as a velocity (g/kg/day), was calculated for the first seven days of life. The results from normal birth weight infants, low birth weight infants (<2.5 kg) and small for gestational age infants (SGA) were examined. RESULTS: In the first week of life there were no significant differences in weight gained or lost across the three study groups. The maximum weight lost was 4.4% (95% CI 4.1 -- 4.6 %), which occurred on day three. Weight gain ranged from 13 g/kg/day [95% CI 10 -- 16] on day four to 18 g/kg/day [95% CI 15 -- 20] on days six and seven. CONCLUSIONS: Use of these normal values for weight gain and loss, allows infants falling outside of the expected range (95% CI) to be easily identified and subsequently highlighted as needing further medical review.
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Made in Europe: will artemisinin resistance emerge in French Guiana?
Malar. J.
PUBLISHED: 01-15-2013
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Resistance to artemisinin casts a shadow on the fight against malaria. The importance of illegal gold miners and of malaria in isolated regions of French Guiana constitutes a threat that endangers the fight against malaria in the Amazon. The hurdles of French laws and the remoteness of the territory from France make it impossible for the system to adapt to the problem of total inaccessibility of an important part of the malaria problem. Transmission is high in these areas and gold miners self-medicate with erratic regimens of artemisinin combinations, thus creating perfect conditions for the emergence of resistance. What needs to be done is being done, but within the limits of national law, with some results. However, facing the same difficult problem, Suriname shows more flexibility and is doing much better than French Guiana despite having lower resources. Local authorities in French Guiana cannot overrule the laws that block appropriate malaria care from reaching a third of malaria-exposed persons. Thus the health authorities in France should take immediate calibrated legislative and financial measures to avoid a predictable disaster.
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Giemsa-stained wet mount based method for reticulocyte quantification: a viable alternative in resource limited or malaria endemic settings.
PLoS ONE
PUBLISHED: 01-11-2013
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The quantity of circulating reticulocytes is an important indicator of erythropoietic activity in response to a wide range of haematological pathologies. While most modern laboratories use flow cytometry to quantify reticulocytes, most field laboratories still rely on subvital staining. The specialist subvital stains, New Methylene Blue (NMB) and Brilliant Crésyl Blue are often difficult to procure, toxic, and show inconsistencies between batches. Here we demonstrate the utility of Giemsas stain (commonly used microbiology and parasitology) in a subvital manner to provide an accurate method to visualize and count reticulocytes in blood samples from normal and malaria-infected individuals.
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High rates of pneumonia in children under two years of age in a South East Asian refugee population.
PLoS ONE
PUBLISHED: 01-08-2013
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There are an estimated 150 million episodes of childhood pneumonia per year, with 11-20 million hospital admissions and 1.575 million deaths. Refugee children are particularly vulnerable, with poorly defined pneumonia epidemiology.
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Antigenicity and immunogenicity of Plasmodium vivax merozoite surface protein-3.
PLoS ONE
PUBLISHED: 01-04-2013
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A recent clinical trial in African children demonstrated the potential utility of merozoite surface protein (MSP)-3 as a vaccine against Plasmodium falciparum malaria. The present study evaluated the use of Plasmodium vivax MSP-3 (PvMSP-3) as a target antigen in vaccine formulations against malaria caused by P. vivax. Recombinant proteins representing MSP-3? and MSP-3? of P. vivax were expressed as soluble histidine-tagged bacterial fusions. Antigenicity during natural infection was evaluated by detecting specific antibodies using sera from individuals living in endemic areas of Brazil. A large proportion of infected individuals presented IgG antibodies to PvMSP-3? (68.2%) and at least 1 recombinant protein representing PvMSP-3? (79.1%). In spite of the large responder frequency, reactivity to both antigens was significantly lower than was observed for the immunodominant epitope present on the 19-kDa C-terminal region of PvMSP-1. Immunogenicity of the recombinant proteins was studied in mice in the absence or presence of different adjuvant formulations. PvMSP-3?, but not PvMSP-3?, induced a TLR4-independent humoral immune response in the absence of any adjuvant formulation. The immunogenicity of the recombinant antigens were also tested in formulations containing different adjuvants (Alum, Salmonella enterica flagellin, CpG, Quil A,TiterMax® and incomplete Freunds adjuvant) and combinations of two adjuvants (Alum plus flagellin, and CpG plus flagellin). Recombinant PvMSP-3? and PvMSP-3? elicited higher antibody titers capable of recognizing P. vivax-infected erythrocytes harvested from malaria patients. Our results confirm that P. vivax MSP-3 antigens are immunogenic during natural infection, and the corresponding recombinant proteins may be useful in elucidating their vaccine potential.
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Effective preparation of Plasmodium vivax field isolates for high-throughput whole genome sequencing.
PLoS ONE
PUBLISHED: 01-04-2013
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Whole genome sequencing (WGS) of Plasmodium vivax is problematic due to the reliance on clinical isolates which are generally low in parasitaemia and sample volume. Furthermore, clinical isolates contain a significant contaminating background of host DNA which confounds efforts to map short read sequence of the target P. vivax DNA. Here, we discuss a methodology to significantly improve the success of P. vivax WGS on natural (non-adapted) patient isolates. Using 37 patient isolates from Indonesia, Thailand, and travellers, we assessed the application of CF11-based white blood cell filtration alone and in combination with short term ex vivo schizont maturation. Although CF11 filtration reduced human DNA contamination in 8 Indonesian isolates tested, additional short-term culture increased the P. vivax DNA yield from a median of 0.15 to 6.2 ng µl(-1) packed red blood cells (pRBCs) (p = 0.001) and reduced the human DNA percentage from a median of 33.9% to 6.22% (p = 0.008). Furthermore, post-CF11 and culture samples from Thailand gave a median P. vivax DNA yield of 2.34 ng µl(-1) pRBCs, and 2.65% human DNA. In 22 P. vivax patient isolates prepared with the 2-step method, we demonstrate high depth (median 654X coverage) and breadth (?89%) of coverage on the Illumina GAII and HiSeq platforms. In contrast to the A+T-rich P. falciparum genome, negligible bias was observed in coverage depth between coding and non-coding regions of the P. vivax genome. This uniform coverage will greatly facilitate the detection of SNPs and copy number variants across the genome, enabling unbiased exploration of the natural diversity in P. vivax populations.
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Significant biochemical, biophysical and metabolic diversity in circulating human cord blood reticulocytes.
PLoS ONE
PUBLISHED: 01-01-2013
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The transition from enucleated reticulocytes to mature normocytes is marked by substantial remodeling of the erythrocytic cytoplasm and membrane. Despite conspicuous changes, most studies describe the maturing reticulocyte as a homogenous erythropoietic cell type. While reticulocyte staging based on fluorescent RNA stains such as thiazole orange have been useful in a clinical setting; these sub-vital stains may confound delicate studies on reticulocyte biology and may preclude their use in heamoparasite invasion studies.
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Neonatal intensive care in a karen refugee cAMP: a 4 year descriptive study.
PLoS ONE
PUBLISHED: 01-01-2013
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A third of all deaths in children aged <5 years occur in the neonatal period. Neonatal intensive care is often considered too complex and expensive to be implemented in resource poor settings. Consequently the reductions that have been made in infant mortality in the poorest countries have not been made in the neonatal period. This manuscript describes the activities surrounding the introduction of special care baby unit (SCBU) in a refugee setting and the resulting population impact.
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Nonlinear mixed-effects modelling of in vitro drug susceptibility and molecular correlates of multidrug resistant Plasmodium falciparum.
PLoS ONE
PUBLISHED: 01-01-2013
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The analysis of in vitro anti-malarial drug susceptibility testing is vulnerable to the effects of different statistical approaches and selection biases. These confounding factors were assessed with respect to pfmdr1 gene mutation and amplification in 490 clinical isolates. Two statistical approaches for estimating the drug concentration associated with 50% effect (EC50 ) were compared: the commonly used standard two-stage (STS) method, and nonlinear mixed-effects modelling. The in vitro concentration-effect relationships for, chloroquine, mefloquine, lumefantrine and artesunate, were derived from clinical isolates obtained from patients on the western border of Thailand. All isolates were genotyped for polymorphisms in the pfmdr1 gene. The EC50 estimates were similar for the two statistical approaches but 15-28% of isolates in the STS method had a high coefficient of variation (>15%) for individual estimates of EC50 and these isolates had EC50 values that were 32 to 66% higher than isolates derived with more precision. In total 41% (202/490) of isolates had amplification of pfmdr1 and single nucleotide polymorphisms were found in 50 (10%). Pfmdr1 amplification was associated with an increase in EC50 for mefloquine (139% relative increase in EC50 for 2 copies, 188% for 3+ copies), lumefantrine (82% and 75% for 2 and 3+ copies respectively) and artesunate (63% and 127% for 2 and 3+ copies respectively). In contrast pfmdr1 mutation at codons 86 or 1042 were associated with an increase in chloroquine EC50 (44-48%). Sample size calculations showed that to demonstrate an EC50 shift of 50% or more with 80% power if the prevalence was 10% would require 430 isolates and 245 isolates if the prevalence was 20%. In conclusion, although nonlinear mixed-effects modelling did not demonstrate any major advantage for determining estimates of anti-malarial drug susceptibility, the method includes all isolates, thereby, potentially improving confirmation of candidate molecular markers of anti-malarial drug susceptibility.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.