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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. Source


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. Source


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.,Center for Tropical Medicine and Vaccinology | 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. Source


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. Source


Pratt B.,Johns Hopkins University | Lwin K.M.,Shoklo Malaria Research Unit SMRU | Zion D.,Monash University | Nosten F.,Shoklo Malaria Research Unit SMRU | Loff B.,Monash University
Developing World Bioethics | Year: 2015

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. © 2013 John Wiley & Sons Ltd. Source

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