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      Plasmodium berghei bio-burden correlates with parasite lactate dehydrogenase: application to murine Plasmodium diagnostics

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          Abstract

          Background

          The spectrum of techniques to detect malaria parasites in whole blood is limited to measuring parasites in circulation. One approach that is currently used to enumerate total parasite bio-burden involves the use of bio-luminescent parasites. As an alternative approach, this study describes the use of a commercial ELISA human parasite lactate dehydrogenase (pLDH) detection kit to estimate total parasite bio-burden in murine malaria models.

          Methods

          The cross reactivity of pLDH in a commercial human malaria pLDH diagnostic kit was established in different components of blood for different murine malaria models. The use of pLDH as a measure of parasite bio-burden was evaluated by examining pLDH in relation to peripheral blood parasitaemia as determined by microscopy and calculating total parasite bio-burden using a bio-luminescent Plasmodium berghei ANKA luciferase parasite.

          Results

          The pLDH antigen was detected in all four murine Plasmodium species and in all components of Plasmodium-infected blood. A significant correlation ( r = 0.6922, P value <0.0001) was observed between total parasite bio-burden, measured as log average radiance, and concentration of pLDH units.

          Conclusions

          This high throughput assay is a suitable measure of total parasite bio-burden in murine malaria infections. Unlike existing methods, it permits the estimation of both circulating and sequestered parasites, allowing a more accurate assessment of parasite bio-burden.

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          Most cited references42

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          Rapid diagnostic tests for malaria parasites.

          Malaria presents a diagnostic challenge to laboratories in most countries. Endemic malaria, population movements, and travelers all contribute to presenting the laboratory with diagnostic problems for which it may have little expertise available. Drug resistance and genetic variation has altered many accepted morphological appearances of malaria species, and new technology has given an opportunity to review available procedures. Concurrently the World Health Organization has opened a dialogue with scientists, clinicians, and manufacturers on the realistic possibilities for developing accurate, sensitive, and cost-effective rapid diagnostic tests for malaria, capable of detecting 100 parasites/microl from all species and with a semiquantitative measurement for monitoring successful drug treatment. New technology has to be compared with an accepted "gold standard" that makes comparisons of sensitivity and specificity between different methods. The majority of malaria is found in countries where cost-effectiveness is an important factor and ease of performance and training is a major consideration. Most new technology for malaria diagnosis incorporates immunochromatographic capture procedures, with conjugated monoclonal antibodies providing the indicator of infection. Preferred targeted antigens are those which are abundant in all asexual and sexual stages of the parasite and are currently centered on detection of HRP-2 from Plasmodium falciparum and parasite-specific lactate dehydrogenase or Plasmodium aldolase from the parasite glycolytic pathway found in all species. Clinical studies allow effective comparisons between different formats, and the reality of nonmicroscopic diagnoses of malaria is considered.
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            High efficiency transfection of Plasmodium berghei facilitates novel selection procedures.

            The use of transfection in the study of the biology of malaria parasites has been limited due to poor transfection efficiencies (frequency of 10(-6) to 10(-9)) and a paucity of selection markers. Here, a new method of transfection, using non-viral Nucleofector technology, is described for the rodent parasite Plasmodium berghei. The transfection efficiency obtained (episomal and targeted integration into the genome) is in the range of 10(-2) to 10(-3). Such high transfection efficiency strongly reduces the time, number of laboratory animals and amount of materials required to generate transfected parasites. Moreover, it allows different experimental strategies for reverse genetics to be developed and we demonstrate direct selection of stably and non-reversibly transformed, fluorescent protein (FP)-expressing parasites using FACS. Since there is no need to use a drug-selectable marker, this method increases the (low) number of selectable markers available for transformation of P. berghei and can in principle be extended to utilise additional FP. Furthermore the FACS-selected, FP-expressing parasites may serve as easily visualized reference lines that may still be genetically manipulated with the existing drug-selectable markers. The combination of enhanced transfection efficiency and a versatile rodent model provides a basis for the further development of novel tools for high throughput genome manipulation.
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              Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration.

              Sequestration of malaria-parasite-infected erythrocytes in the microvasculature of organs is thought to be a significant cause of pathology. Cerebral malaria (CM) is a major complication of Plasmodium falciparum infections, and PfEMP1-mediated sequestration of infected red blood cells has been considered to be the major feature leading to CM-related pathology. We report a system for the real-time in vivo imaging of sequestration using transgenic luciferase-expressing parasites of the rodent malaria parasite Plasmodium berghei. These studies revealed that: (i) as expected, lung tissue is a major site, but, unexpectedly, adipose tissue contributes significantly to sequestration, and (ii) the class II scavenger-receptor CD36 to which PfEMP1 can bind is also the major receptor for P. berghei sequestration, indicating a role for alternative parasite ligands, because orthologues of PfEMP1 are absent from rodent malaria parasites, and, importantly, (iii) cerebral complications still develop in the absence of CD36-mediated sequestration, dissociating parasite sequestration from CM-associated pathology. Real-time in vivo imaging of parasitic processes may be used to evaluate the molecular basis of pathology and develop strategies to prevent pathology.
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                Author and article information

                Contributors
                s.de@griffith.edu.au
                d.stanisic@griffith.edu.au
                fabian.rivera@qimrberghofer.edu.au
                m.batzloff@griffith.edu.au
                Christian.Engwerda@qimrberghofer.edu.au
                michael.good@griffith.edu.au
                Journal
                Malar J
                Malar. J
                Malaria Journal
                BioMed Central (London )
                1475-2875
                4 January 2016
                4 January 2016
                2016
                : 15
                : 3
                Affiliations
                [ ]Institute for Glycomics, Griffith University, Gold Coast, QLD Australia
                [ ]QIMR Berghofer Medical Research Institute, Brisbane, QLD Australia
                Article
                1027
                10.1186/s12936-015-1027-2
                4700574
                26729268
                1d2bf79d-fefe-4ebf-8956-21e3ef2ca42c
                © De et al. 2015

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 4 September 2015
                : 2 December 2015
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100000925, National Health and Medical Research Council (AU);
                Award ID: 1037304
                Award Recipient :
                Categories
                Methodology
                Custom metadata
                © The Author(s) 2016

                Infectious disease & Microbiology
                malaria,pldh,murine plasmodium,parasite bio-burden
                Infectious disease & Microbiology
                malaria, pldh, murine plasmodium, parasite bio-burden

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