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Vicentin E.C.,University of Sao Paulo | Francoso K.S.,University of Sao Paulo | Rocha M.V.,University of Sao Paulo | Iourtov D.,Instituto Butantan | And 12 more authors.
Infection and Immunity | Year: 2014

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 Plasmodium 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 primeboost 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 enterica serovar Typhimurium, saponin Quil A, or incomplete Freund's 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, and no microbial contaminants and reproduced the experimental immunizations. Most relevant for vaccine development was the fact that immunization with PvAMA-1 elicited invasioninhibitory 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 preclinical and clinical studies. © 2014, American Society for Microbiology.


Lee W.-C.,University of Malaya | Russell B.,University of Malaya | Russell B.,National University of Singapore | Lau Y.-L.,University of Malaya | And 8 more authors.
PLoS ONE | Year: 2013

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 Giemsa's 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. © 2013 Lee et al.


Nair S.,Southwest Foundation for Biomedical Research SFBR | Nkhoma S.,Southwest Foundation for Biomedical Research SFBR | Nosten F.,Shoklo Malaria Research Unit SMRU | Nosten F.,Churchill Hospital | And 5 more authors.
Molecular and Biochemical Parasitology | Year: 2010

Comparative genomic hybridization studies have revealed elevated copy number (CN) at the reticulocyte-binding protein 1 gene (PfRh1) in fast growing lab-adapted parasites, while genetic manipulation demonstrates a causal link between cell invasion and PfRh1 CN. We therefore examined PfRh1 copy number variation (CNV) in 202 single clone parasite isolates from four countries to quantify the extent of CNV within natural populations. Surprisingly, we found that no natural parasite infections showed elevated CN. In contrast, 4/28 independent laboratory reference strains show elevated CN. One possibility is that amplification of PfRh1 (or neighboring loci) is selected during laboratory culture. In the case of FCR3 group of parasites, clone trees show that PfRh1 amplification arose in laboratory lines following establishment in culture. These data show that CNV at PfRh1 is rare or non-existent in natural populations, but can arise during laboratory propagation. We conclude that PfRh1 CNV is not an important determinant of gene expression, cell invasion or growth rate in natural parasite populations. © 2010 Elsevier B.V.


Anderson T.J.C.,Southwest Foundation for Biomedical Research SFBR | Williams J.T.,Southwest Foundation for Biomedical Research SFBR | Nair S.,Southwest Foundation for Biomedical Research SFBR | Sudimack D.,Southwest Foundation for Biomedical Research SFBR | And 6 more authors.
Proceedings of the Royal Society B: Biological Sciences | Year: 2010

Malaria parasites vary in phenotypic traits of biomedical or biological interest such as growth rate, virulence, sex ratio and drug resistance, and there is considerable interest in identifying the genes that underlie this variation. An important first step is to determine trait heritability (H 2). We evaluate two approaches to measuring H2in natural parasite populations using relatedness inferred from genetic marker data. We collected single-clone Plasmodium falciparum infections from 185 patients from the Thailand-Burma border, monitored parasite clearance following treatment with artemisinin combination therapy (ACT), measured resistance to six antimalarial drugs and genotyped parasites using 335 microsatellites. We found strong relatedness structure. There were 27 groups of two to eight clonally identical (CI) parasites, and 74 per cent of parasites showed significant relatedness to one or more other parasites. Initially, we used matrices of allele sharing and variance components (VC) methods to estimate H2. Inhibitory concentrations (IC2) for six drugs showed significant H 2(0.24 to 0.79, p = 0.06 to 2.85 × 10-9), demonstrating that this study design has adequate power. However, a phenotype of current interest-parasite clearance following ACT-showed no detectable heritability (H2= 0-0.09, ns) in this population. The existence of CI parasites allows the use of a simple ANOVA approach for quantifying H 2, analogous to that used in human twin studies. This gave similar results to the VC method and requires considerably less genotyping information. We conclude (i) that H2can be effectively measured in malaria parasite populations using minimal genotype data, allowing rational design of genome-wide association studies; and (ii) while drug response (IC50) shows significant H2, parasite clearance following ACT was not heritable in the population studied. © 2010 The Royal Society.


PubMed | Shoklo Malaria Research Unit SMRU, Mahidol University, University of Oxford and Christian Medical College
Type: Journal Article | Journal: PLoS neglected tropical diseases | Year: 2014

There is a paucity of published reports on pregnancy outcome following scrub and murine typhus despite these infections being leading causes of undifferentiated fever in Asia. This study aimed to relate pregnancy outcome with treatment of typhus.Data were analyzed from: i) pregnant women with a diagnosis of scrub and/or murine typhus from a fever cohort studies; ii) case series of published studies in PubMed using the search terms scrub typhus (ST), murine typhus (MT), Orientia tsutsugamushi, Rickettsia tsutsugamushi, Rickettsia typhi, rickettsiae, typhus, or rickettsiosis; and pregnancy, until February 2014 and iii) an unpublished case series. Fever clearance time (FCT) and pregnancy outcome (miscarriage and delivery) were compared to treatment. Poor neonatal outcome was a composite measure for pregnancies sustained to 28 weeks or more of gestation ending in stillbirth, preterm birth, or delivery of a growth restricted or low birth weight newborn.There were 26 women in the fever cohort. MT and ST were clinically indistinguishable apart from two ST patients with eschars. FCTs (median [range] hours) were 25 [16-42] for azithromycin (n=5), 34 [20-53] for antimalarials (n=5) and 92 [6-260] for other antibiotics/supportive therapy (n=16). There were 36.4% (8/22) with a poor neonatal outcome. In 18 years, 97 pregnancies were collated, 82 with known outcomes, including two maternal deaths. Proportions of miscarriage 17.3% (14/81) and poor neonatal outcomes 41.8% (28/67) were high, increasing with longer FCTs (p=0.050, linear trend). Use of azithromycin was not significantly associated with improved neonatal outcomes (p=0.610).The published ST and MT world literature amounts to less than 100 pregnancies due to under recognition and under diagnosis. Evidence supporting the most commonly used treatment, azithromycin, is weak. Collaborative, prospective clinical trials in pregnant women are urgently required to reduce the burden of adverse maternal and newborn outcomes and to determine the safety and efficacy of antimicrobial treatment.


McGready R.,Shoklo Malaria Research Unit SMRU | McGready R.,Mahidol University | White N.J.,Mahidol University | White N.J.,Churchill Hospital | And 2 more authors.
BJOG: An International Journal of Obstetrics and Gynaecology | Year: 2011

Background: Pregnant women are at increased risk from malaria. Resistance to all classes of antimalarials has affected the treatment and prevention of malaria in pregnancy. Objectives To review the therapeutic efficacy of antimalarials used for treatment and intermittent preventive treatment (IPT) in pregnancy. Search strategy We searched MEDLINE and the Cochrane Library between January 1998 and December 2009 for publications using the medical subject headings: efficacy, antimalarials, malaria, pregnancy, pharmacokinetics, treatment, IPT and placenta positive. In May 2010 we searched the register of clinical trials (http://clinicaltrials.gov/) and of WHO (http://apps.who.int/ trialsearch/) using 'malaria', and 'pregnancy' and 'treatment'. Selection criteria We identified 233 abstracts, reviewed 83 full text articles and included 60 studies. Data collection and analysis Two authors entered extracted data to an excel spreadsheet. Main results Parasitological failure rates, placenta positivity rates (assessed by microscopy) or both were reported in 44% (21/48), 46% (22/48) and 10% (5/48) of articles, respectively. Most pharmacokinetic studies (9/12) suggested dose optimisation. In 23 treatment studies 17 different antimalarial drugs were delivered in 53 study arms; 43.4% (23/53) reported a failure rate of < 5%; 83.3% of sulphadoxine-pyrimethamine (SP) arms and 9% of artemisinin combination therapy (ACT) arms had failure rates ≥ 10%. Placenta-positive rates (mostly reported in the context of IPT in pregnancy) were > 10% in 68% (23/34) of SP trial arms and > 15% in all seven chloroquine arms. The ACT provided lower parasitological failure and gametocyte carriage rates. Author's conclusions Drugs used in pregnancy should aim for 95% efficacy but many currently deployed regimens are associated with much lower cure rates. © RCOG 2010 BJOG An International Journal of Obstetrics and Gynaecology.


Watthanaworawit W.,Shoklo Malaria Research Unit SMRU | Watthanaworawit W.,Mahidol University | Turner P.,Shoklo Malaria Research Unit SMRU | Turner P.,Mahidol University | And 11 more authors.
Transactions of the Royal Society of Tropical Medicine and Hygiene | Year: 2011

Clinically useful diagnostic tests of dengue virus infection are lacking. We prospectively evaluated the performance of real-time reverse transcriptase (rRT)-PCR, NS-1 antigen and IgM antibody tests to confirm dengue virus infection in acute blood specimens from 162 patients presenting with undifferentiated febrile illness compatible with dengue infection. rRT-PCR was the most sensitive test (89%) and potentially could be used as a single test for confirmation of dengue infection. NS-1 antigen and IgM antibody were not sufficiently sensitive to be used as a single confirmatory test with sensitivities of 54% and 17% respectively. The specificities of rRT-PCR, NS-1 antigen and IgM antibody tests were 96%, 100% and 88% respectively. Combining NS-1 and rRT-PCR or the combination of all three tests resulted in the highest sensitivity (93%) but specificities dropped to 96% and 83% respectively. We conclude that at least the combination of two tests, either agent detection (rRT-PCR) or antigen detection (NS-1) plus IgM antibody detection should be used for laboratory confirmation of dengue infection. © 2010 Royal Society of Tropical Medicine and Hygiene.


Bergstrand M.,Uppsala University | Nosten F.,Mahidol University | Nosten F.,University of Oxford | Nosten F.,Shoklo Malaria Research Unit SMRU | And 6 more authors.
Science Translational Medicine | Year: 2014

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.


Rijken M.,Shoklo Malaria Research Unit SMRU | Moroski W.,Shoklo Malaria Research Unit SMRU | Kiricharoen S.,Shoklo Malaria Research Unit SMRU | Karunkonkowit N.,Shoklo Malaria Research Unit SMRU | And 11 more authors.
Malaria Journal | Year: 2012

Background. The presence of malaria parasites and histopathological changes in the placenta are associated with a reduction in birth weight, principally due to intrauterine growth restriction. The aim of this study was to examine the feasibility of studying early pregnancy placental volumes using three-dimensional (3D) ultrasound in a malaria endemic area, as a small volume in the second trimester may be an indicator of intra-uterine growth restriction and placental insufficiency. Methods. Placenta volumes were acquired using a portable ultrasound machine and a 3D ultrasound transducer and estimated using the Virtual Organ Computer-aided AnaLysis (VOCAL) image analysis software package. Intra-observer reliability and limits of agreement of the placenta volume measurements were calculated. Polynomial regression models for the mean and standard deviation as a function of gestational age for the placental volumes of uninfected women were created and tested. Based on these equations each measurement was converted into a z -score. The z-scores of the placental volumes of malaria infected and uninfected women were then compared. Results. Eighty-four women (uninfected = 65; infected = 19) with a posterior placenta delivered congenitally normal, live born, single babies. The mean placental volumes in the uninfected women were modeled to fit 5th, 10th, 50th, 90th and 95th centiles for 14-24 weeks' gestation. Most placenta volumes in the infected women were below the 50th centile for gestational age; most of those with Plasmodium falciparum were below the 10th centile. The 95% intra-observer limits of agreement for first and second measurements were 37.0 mL and 25.4 mL at 30 degrees and 15 degrees rotation respectively. Conclusion. The new technique of 3D ultrasound volumetry of the placenta may be useful to improve our understanding of the pathophysiological constraints on foetal growth caused by malaria infection in early pregnancy. © 2011 Rijken et al; licensee BioMed Central Ltd.


PubMed | Shoklo Malaria Research Unit SMRU, Uppsala University and Mahidol University
Type: Journal Article | Journal: Science translational medicine | Year: 2014

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 manufacturers 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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