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Plasmodium vivax Adherence to Placental Glycosaminoglycans
Kesinee Chotivanich, Rachanee Udomsangpetch, Rossarin Suwanarusk, Sasithon Pukrittayakamee, Polrat Wilairatana, James G. Beeson, Nicholas P. J. Day, Nicholas J. White
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0034509
Abstract: Background Plasmodium vivax infections seldom kill directly but do cause indirect mortality by reducing birth weight and causing abortion. Cytoadherence and sequestration in the microvasculature are central to the pathogenesis of severe Plasmodium falciparum malaria, but the contribution of cytoadherence to pathology in other human malarias is less clear. Methodology The adherence properties of P. vivax infected red blood cells (PvIRBC) were evaluated under static and flow conditions. Principal Findings P. vivax isolates from 33 patients were studied. None adhered to immobilized CD36, ICAM-1, or thrombospondin, putative ligands for P. falciparum vascular cytoadherence, or umbilical vein endothelial cells, but all adhered to immobilized chondroitin sulphate A (CSA) and hyaluronic acid (HA), the receptors for adhesion of P. falciparum in the placenta. PvIRBC also adhered to fresh placental cells (N = 5). Pre-incubation with chondroitinase prevented PvIRBC adherence to CSA, and reduced binding to HA, whereas preincubation with hyaluronidase prevented adherence to HA, but did not reduce binding to CSA significantly. Pre-incubation of PvIRBC with soluble CSA and HA reduced binding to the immobilized receptors and prevented placental binding. PvIRBC adhesion was prevented by pre-incubation with trypsin, inhibited by heparin, and reduced by EGTA. Under laminar flow conditions the mean (SD) shear stress reducing maximum attachment by 50% was 0.06 (0.02) Pa but, having adhered, the PvIRBC could then resist detachment by stresses up to 5 Pa. At 37°C adherence began approximately 16 hours after red cell invasion with maximal adherence at 30 hours. At 39°C adherence began earlier and peaked at 24 hours. Significance Adherence of P. vivax-infected erythrocytes to glycosaminoglycans may contribute to the pathogenesis of vivax malaria and lead to intrauterine growth retardation.
The effects of serum lipids on the in vitro activity of lumefantrine and atovaquone against Plasmodium falciparum
Kesinee Chotivanich, Mathirut Mungthin, Ronnatrai Ruengweerayuth, Rachanee Udomsangpetch, Arjen M Dondorp, Pratap Singhasivanon, Sasithon Pukrittayakamee, Nicholas J White
Malaria Journal , 2012, DOI: 10.1186/1475-2875-11-177
Abstract: Serum was obtained from non-immune volunteers under fasting conditions and after ingestion of a high fat meal and used in standard Plasmodium falciparum in-vitro susceptibility assays. Anti-malarial drugs, including lumefantrine, atovaquone and chloroquine in five-fold dilutions (range 0.05?ng/ml – 1?ug/mL) were diluted in culture medium supplemented with fasting or post-prandial 10% donor serum. The in-vitro drug susceptibility of parasite isolates was determined using the 3H-hypoxanthine uptake inhibition method and expressed as the concentration which gave 50% inhibition of hypoxanthine uptake (IC50).Doubling plasma triglyceride concentrations (from 160?mg/dL to 320?mg/dL), resulted in an approximate doubling of the IC50 for lumefantrine (191?ng/mL to 465?ng/mL, P?<?0.01) and a 20-fold increase in the IC50 for atovaquone (0.5?ng/mL to 12?ng/ml; P?<?0.01). In contrast, susceptibility to the hydrophilic anti-malarial chloroquine did not change in relation to triglyceride content of the medium.Lipidaemia reduces the anti-malarial activity of lipophilic anti-malarial drugs. This is an important confounder in laboratory in vitro testing and it could have therapeutic relevance.
Practical PCR genotyping protocols for Plasmodium vivax using Pvcs and Pvmsp1
Mallika Imwong, Sasithon Pukrittayakamee, Anne Grüner, Laurent Rénia, Frank Letourneur, Sornchai Looareesuwan, Nicholas J White, Georges Snounou
Malaria Journal , 2005, DOI: 10.1186/1475-2875-4-20
Abstract: Practical PCR genotyping protocols based on polymorphic loci present in two P. vivax genetic markers, Pvcs and Pvmsp1, were developed. The methodology was evaluated using 100 P. vivax isolates collected in Thailand.Analysis revealed that P. vivax populations in Thailand are highly diverse genetically, with mixed genotype infections found in 26 % of the samples (average multiplicity of infection = 1.29). A large number of distinguishable alleles were found for the two markers, 23 for Pvcs and 36 for Pvmsp1. These were generally randomly distributed amongst the isolates. A total of 68 distinct genotypes could be enumerated in the 74 isolates with a multiplicity of infection of 1.These results indicate that the genotyping protocols presented can be useful in the assessment of in vivo drug efficacy clinical trials conducted in endemic areas and for epidemiological studies of P. vivax infections.Plasmodium vivax is globally distributed and is the dominant species in many countries. Infection by this species is generally regarded as benign and mortality is a rare outcome. 1.2 billion inhabitants of the South East Asian and Pacific countries are at risk from malaria transmission, representing about a third of world population exposed to Plasmodium parasites [1,2]. Most of the populations at risk in these countries (corresponding to the WHO regional groupings SEARO and WPRO) reside in hypoendemic and mesoendemic areas, where half of the recorded infections are due to P. vivax, suggesting that the global burden of vivax malaria morbidity must be close to that of falciparum malaria. It has recently been established that P. vivax infections during pregnancy are associated with reduced birthweight [3], and thereby, increase the risk of neonatal deaths. Thus, the urgency to develop and implement measures to control P. vivax should equal that attending P. falciparum.The emergence and global spread of P. falciparum parasites resistant to chloroquine and pyrimethamine – sulfadoxine
The Diversity and Geographical Structure of Orientia tsutsugamushi Strains from Scrub Typhus Patients in Laos
Rattanaphone Phetsouvanh?,Piengchan Sonthayanon?,Sasithon Pukrittayakamee,Daniel H. Paris?,Paul N. Newton?,Edward J. Feil?,Nicholas P. J. Day
PLOS Neglected Tropical Diseases , 2015, DOI: 10.1371/journal.pntd.0004024
Abstract: Orientia tsutsugamushi is the causative agent of scrub typhus, a disease transmitted by Leptotrombidium mites which is responsible for a severe and under-reported public health burden throughout Southeast Asia. Here we use multilocus sequence typing (MLST) to characterize 74 clinical isolates from three geographic locations in the Lao PDR (Laos), and compare them with isolates described from Udon Thani, northeast Thailand. The data confirm high levels of diversity and recombination within the natural O. tsutsugamushi population, and a rate of mixed infection of ~8%. We compared the relationships and geographical structuring of the strains and populations using allele based approaches (eBURST), phylogenetic approaches, and by calculating F-statistics (FST). These analyses all point towards low levels of population differentiation between isolates from Vientiane and Udon Thani, cities which straddle the Mekong River which defines the Lao/Thai border, but with a very distinct population in Salavan, southern Laos. These data highlight how land use, as well as the movement of hosts and vectors, may impact on the epidemiology of zoonotic infections.
Genotyping of Plasmodium vivax Reveals Both Short and Long Latency Relapse Patterns in Kolkata
Jung-Ryong Kim, Amitabha Nandy, Ardhendu Kumar Maji, Manjulika Addy, Arjen M. Dondorp, Nicholas P. J. Day, Sasithon Pukrittayakamee, Nicholas J. White, Mallika Imwong
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0039645
Abstract: Background The Plasmodium vivax that was once prevalent in temperate climatic zones typically had an interval between primary infection and first relapse of 7–10 months, whereas in tropical areas P.vivax infections relapse frequently at intervals of 3–6 weeks. Defining the epidemiology of these two phenotypes from temporal patterns of illness in endemic areas is difficult or impossible, particularly if they overlap. Methods A prospective open label comparison of chloroquine (CQ) alone versus CQ plus unobserved primaquine for either 5 days or 14 days was conducted in patients presenting with acute vivax malaria in Kolkata. Patients were followed for 15 months and primary and recurrent infections were genotyped using three polymorphic antigen and up to 8 microsatellite markers. Results 151 patients were enrolled of whom 47 (31%) had subsequent recurrent infections. Recurrence proportions were similar in the three treatment groups. Parasite genotyping revealed discrete temporal patterns of recurrence allowing differentiation of probable relapse from newly acquired infections. This suggested that 32 of the 47 recurrences were probable relapses of which 22 (69%) were genetically homologous. The majority (81%) of probable relapses occurred within three months (16 homologous, 10 heterologous) and six genetically homologous relapses (19%) were of the long latency (8–10 month interval) phenotype. Conclusions With long follow-up to assess temporal patterns of vivax malaria recurrence, genotyping of P.vivax can be used to assess relapse rates. A 14 day unobserved course of primaquine did not prevent relapse. Genotyping indicates that long latency P.vivax is prevalent in West Bengal, and that the first relapses after long latent periods are genetically homologous. Trial Registration Controlled-Trials.com ISRCTN14027467
Directly-observed therapy (DOT) for the radical 14-day primaquine treatment of Plasmodium vivax malaria on the Thai-Myanmar border
Rie Takeuchi, Saranath Lawpoolsri, Mallika Imwong, Jun Kobayashi, Jaranit Kaewkungwal, Sasithon Pukrittayakamee, Supalap Puangsa-art, Nipon Thanyavanich, Wanchai Maneeboonyang, Nicholas PJ Day, Pratap Singhasivanon
Malaria Journal , 2010, DOI: 10.1186/1475-2875-9-308
Abstract: A randomized trial was conducted from May 2007 to January 2009 in a low malaria transmission area along the Thai-Myanmar border. Patients aged ≥ 3 years diagnosed with P. vivax by microscopy, were recruited. All patients were treated with the national standard regimen of chloroquine for three days followed by primaquine for 14 days. Patients were randomized to receive DOT or self-administered therapy (SAT). All patients were followed for three months to check for any reappearance of P. vivax.Of the 216 patients enrolled, 109 were randomized to DOT and 107 to SAT. All patients recovered without serious adverse effects. The vivax reappearance rate was significantly lower in the DOT group than the SAT group (3.4/10,000 person-days vs. 13.5/10,000 person-days, p = 0.021). Factors related to the reappearance of vivax malaria included inadequate total primaquine dosage received (< 2.75 mg/kg), duration of fever ≤ 2 days before initiation of treatment, parasite count on admission ≥ 10,000/μl, multiple P. vivax-genotype infection, and presence of P. falciparum infection during the follow-up period.Adherence to the 14-day primaquine regimen is important for the radical cure of P. vivax malaria infection. Implementation of DOT reduces the reappearance rate of the parasite, and may subsequently decrease P. vivax transmission in the area.Globally, over 3 billion people live in areas at risk of malaria infection; about one billion of these live in countries outside Africa, where malaria transmission is low and Plasmodium vivax is most prevalent [1,2]. Unlike Plasmodium falciparum, P. vivax infection rarely develops into complicated malaria and death is unusual. However, P. vivax has a dormant stage (the hypnozoite) that persists in the human liver and may cause relapse weeks, months, or even years later. Therefore, P. vivax infection is considered to have greater impact on morbidity than mortality, resulting in significant social and economic burdens [1]. Moreover, it is very di
Dihydroartemisinin-piperaquine versus chloroquine to treat vivax malaria in Afghanistan: an open randomized, non-inferiority, trial
Ghulam Awab, Sasithon Pukrittayakamee, Mallika Imwong, Arjen M Dondorp, Charles J Woodrow, Sue Lee, Nicholas PJ Day, Pratap Singhasivanon, Nicholas J White, Faizullah Kaker
Malaria Journal , 2010, DOI: 10.1186/1475-2875-9-105
Abstract: Between July 2007 and February 2009, an open-label, randomized controlled trial of chloroquine and dihydroartemisinin-piperaquine in patients aged three months and over with slide-confirmed P. vivax mono-infections was conducted. Consistent with current national guidelines, primaquine was not administered. Subjects were followed up daily during the acute phase of illness (days 0-3) and weekly until day 56. The primary endpoint was the overall cumulative parasitological failure rate at day 56 after the start of treatment, with the hypothesis being that dihydroartemisinin-piperaquine was non-inferior compared to chloroquine (Δ = 5% difference in proportion of failures).Of 2,182 individuals with positive blood films for P. vivax, 536 were enrolled in the trial. The day 28 cure rate was 100% in both treatment groups. Parasite clearance was more rapid with dihydroartemisinin-piperaquine than chloroquine. At day 56, there were more recurrent infections in the chloroquine arm (8.9%, 95% CI 6.0-13.1%) than the dihydroartemisinin-piperaquine arm (2.8%, 95% CI 1.4-5.8%), a difference in cumulative recurrence rate of 6.1% (2-sided 90%CI +2.6 to +9.7%). The log-rank test comparing the survival curves confirmed the superiority of dihydroartemisinin-piperaquine over chloroquine (p = 0.003). Multivariate analysis showed that a lower initial haemoglobin concentration was also independently associated with recurrence. Both regimens were well tolerated and no serious adverse events were reported.Chloroquine remains an efficacious treatment for the treatment of vivax malaria in Afghanistan. In a setting where radical therapy cannot be administered, dihydroartemisinin-piperaquine provides additional benefit in terms of post-treatment prophylaxis, reducing the incidence of recurrence from 4-8 weeks after treatment.The trial was registered at ClinicalTrials.gov under identifier NCT00682578.Plasmodium vivax is associated with less mortality than Plasmodium falciparum, but still exerts a c
Genetic diversity of Plasmodium vivax in Kolkata, India
Jung-Ryong Kim, Mallika Imwong, Amitabha Nandy, Kesinee Chotivanich, Apichart Nontprasert, Naowarat Tonomsing, Ardhendu Maji, Manjulika Addy, Nick PJ Day, Nicholas J White, Sasithon Pukrittayakamee
Malaria Journal , 2006, DOI: 10.1186/1475-2875-5-71
Abstract: Blood from 151 patients with P. vivax infection diagnosed in Kolkata between April 2003 and September 2004 was genotyped at three polymorphic loci: the P. vivax circumsporozoite protein (pvcs), the merozoite surface protein 1 (pvmsp1) and the merozoite surface protein 3-alpha (pvmsp3-alpha).Analysis of these three genetic markers revealed that P. vivax populations in Kolkata are highly diverse. A large number of distinguishable alleles were found from three genetic markers: 11 for pvcs, 35 for pvmsp1 and 37 for pvmsp3-alpha. These were, in general, randomly distributed amongst the isolates. Among the 151 isolates, 142 unique genotypes were detected the commonest genotype at a frequency of less than 2% (3/151). The overall rate of mixed genotype infections was 10.6%.These results indicate that the P. vivax parasite population is highly diverse in Kolkata, despite the low level of transmission. The genotyping protocols used in this study may be useful for differentiating re-infection from relapse and recrudescence in studies assessing of malarial drug efficacy in vivax malaria.Malaria remains one of the most important communicable diseases in the world. Despite enormous control efforts over many decades malaria is still a significant health problem. It is estimated that around 300–500 million cases occur each year with one to three million deaths. The problem is compounded by multiple drug resistance in Plasmodium falciparum and chloroquine resistance in Plasmodium vivax [1]. The global burden of malaria due to P. vivax is 70–80 million cases annually. Vivax malaria is usually a non-lethal infection but its prolonged and recurrent infection can have major deleterious effects on personal well-being, growth and on the economic performance at the individual, family, community and national levels [2]. The recent emergence of chloroquine-resistant strains is of great concern [3-5].P. vivax causes about 60–65% of all malaria infections in India [6,7,42]. The frequency of re
A Population Survey of the Glucose-6-Phosphate Dehydrogenase (G6PD) 563C>T (Mediterranean) Mutation in Afghanistan
Natsuda Jamornthanyawat, Ghulam R. Awab, Naowarat Tanomsing, Sasithon Pukrittayakamee, Fazel Yamin, Arjen M. Dondorp, Nicholas P. J. Day, Nicholas J. White, Charles J. Woodrow, Mallika Imwong
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0088605
Abstract: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzyme defect and an important problem in areas with Plasmodium vivax infection because of the risk of haemolysis following administration of primaquine to treat the liver forms of the parasite. We undertook a genotypic survey of 713 male individuals across nine provinces of Afghanistan in which malaria is found, four in the north and five in the east. RFLP typing at nucleotide position 563 detected 40 individuals with the Mediterranean mutation 563C>T, an overall prevalence of 5.6%. This varied according to self-reported ethnicity, with prevalence in the Pashtun/Pashai group of 33/369 (8.9%) compared to 7/344 individuals in the rest of the population (2.0%; p<0.001, Chi-squared test). Multivariate analysis of ethnicity and geographical location indicated an adjusted odds ratio of 3.50 (95% CI 1.36–9.02) for the Pashtun/Pashai group, while location showed only a trend towards higher prevalence in eastern provinces (adjusted odds ratio = 1.73, 0.73–4.13). Testing of known polymorphic markers (1311C>T in exon 11, and C93T in intron XI) in a subset of 82 individuals wild-type at C563 revealed a mixture of 3 haplotypes in the background population and was consistent with data from the 1000 Genomes Project and published studies. By comparison individuals with G6PD deficiency showed a highly skewed haplotype distribution, with 95% showing the CT haplotype, a finding consistent with relatively recent appearance and positive selection of the Mediterranean variant in Afghanistan. Overall, the data confirm that the Mediterranean variant of G6PD is common in many ethnic groups in Afghanistan, indicating that screening for G6PD deficiency is required in all individuals before radical treatment of P. vivax with primaquine.
Estimating the True Accuracy of Diagnostic Tests for Dengue Infection Using Bayesian Latent Class Models
Wirichada Pan-ngum, Stuart D. Blacksell, Yoel Lubell, Sasithon Pukrittayakamee, Mark S. Bailey, H. Janaka de Silva, David G. Lalloo, Nicholas P. J. Day, Lisa J. White, Direk Limmathurotsakul
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0050765
Abstract: Background Accuracy of rapid diagnostic tests for dengue infection has been repeatedly estimated by comparing those tests with reference assays. We hypothesized that those estimates might be inaccurate if the accuracy of the reference assays is not perfect. Here, we investigated this using statistical modeling. Methods/Principal Findings Data from a cohort study of 549 patients suspected of dengue infection presenting at Colombo North Teaching Hospital, Ragama, Sri Lanka, that described the application of our reference assay (a combination of Dengue IgM antibody capture ELISA and IgG antibody capture ELISA) and of three rapid diagnostic tests (Panbio NS1 antigen, IgM antibody and IgG antibody rapid immunochromatographic cassette tests) were re-evaluated using Bayesian latent class models (LCMs). The estimated sensitivity and specificity of the reference assay were 62.0% and 99.6%, respectively. Prevalence of dengue infection (24.3%), and sensitivities and specificities of the Panbio NS1 (45.9% and 97.9%), IgM (54.5% and 95.5%) and IgG (62.1% and 84.5%) estimated by Bayesian LCMs were significantly different from those estimated by assuming that the reference assay was perfect. Sensitivity, specificity, PPV and NPV for a combination of NS1, IgM and IgG cassette tests on admission samples were 87.0%, 82.8%, 62.0% and 95.2%, respectively. Conclusions Our reference assay is an imperfect gold standard. In our setting, the combination of NS1, IgM and IgG rapid diagnostic tests could be used on admission to rule out dengue infection with a high level of accuracy (NPV 95.2%). Further evaluation of rapid diagnostic tests for dengue infection should include the use of appropriate statistical models.
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