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Improved In Vitro Culture of Plasmodium falciparum Permits Establishment of Clinical Isolates with Preserved Multiplication, Invasion and Rosetting Phenotypes

Ribacke, U.; Moll, K.; Albrecht, L.; Ismail, H. A.; Normark, J.; Flaberg, E.; Szekely, L.; Hultenby, K.; Persson, Kristina LU and Egwang, T. G., et al. (2013) In PLoS ONE 8(7).
Abstract
To be able to robustly propagate P. falciparum at optimal conditions in vitro is of fundamental importance for genotypic and phenotypic studies of both established and fresh clinical isolates. Cryo-preserved P. falciparum isolates from Ugandan children with severe or uncomplicated malaria were investigated for parasite phenotypes under different in vitro growth conditions or studied directly from the peripheral blood. The parasite cultures showed a minimal loss of parasite-mass and preserved percentage of multiple infected pRBCs to that in peripheral blood, maintained adhesive phenotypes and good outgrowth and multiplication rates when grown in suspension and supplemented with gas. In contrast, abnormal and greatly fluctuating levels of... (More)
To be able to robustly propagate P. falciparum at optimal conditions in vitro is of fundamental importance for genotypic and phenotypic studies of both established and fresh clinical isolates. Cryo-preserved P. falciparum isolates from Ugandan children with severe or uncomplicated malaria were investigated for parasite phenotypes under different in vitro growth conditions or studied directly from the peripheral blood. The parasite cultures showed a minimal loss of parasite-mass and preserved percentage of multiple infected pRBCs to that in peripheral blood, maintained adhesive phenotypes and good outgrowth and multiplication rates when grown in suspension and supplemented with gas. In contrast, abnormal and greatly fluctuating levels of multiple infections were observed during static growth conditions and outgrowth and multiplication rates were inferior. Serum, as compared to Albumax, was found necessary for optimal presentation of PfEMP1 at the pRBC surface and/or for binding of serum proteins (immunoglobulins). Optimal in vitro growth conditions of P. falciparum therefore include orbital shaking (50 rev/min), human serum (10%) and a fixed gas composition (5% O2, 5% CO2, 90% N2). We subsequently established 100% of 76 frozen patient isolates and found rosetting with schizont pRBCs in every isolate (>26% schizont rosetting rate). Rosetting during schizogony was often followed by invasion of the bound RBC as seen by regular and time-lapse microscopy as well as transmission electron microscopy. The peripheral parasitemia, the level of rosetting and the rate of multiplication correlated positively to one another for individual isolates. Rosetting was also more frequent with trophozoite and schizont pRBCs of children with severe versus uncomplicated malaria (p<0.002; p<0.004). The associations suggest that rosetting enhances the ability of the parasite to multiply within the human host (Less)
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keywords
red-blood-cells, malaria-infected erythrocytes, surface-antigen, expression, virulence protein pfemp1, human cerebral malaria, uncomplicated malaria, alpha-thalassemia, serum, cytoadherence, association
in
PLoS ONE
volume
8
issue
7
publisher
Public Library of Science
external identifiers
  • scopus:84880690027
ISSN
1932-6203
DOI
10.1371/journal.pone.0069781
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English
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87227a03-504e-4fd9-952f-3c65ca98613a (old id 8726669)
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2016-02-19 13:09:33
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@article{87227a03-504e-4fd9-952f-3c65ca98613a,
  abstract     = {To be able to robustly propagate P. falciparum at optimal conditions in vitro is of fundamental importance for genotypic and phenotypic studies of both established and fresh clinical isolates. Cryo-preserved P. falciparum isolates from Ugandan children with severe or uncomplicated malaria were investigated for parasite phenotypes under different in vitro growth conditions or studied directly from the peripheral blood. The parasite cultures showed a minimal loss of parasite-mass and preserved percentage of multiple infected pRBCs to that in peripheral blood, maintained adhesive phenotypes and good outgrowth and multiplication rates when grown in suspension and supplemented with gas. In contrast, abnormal and greatly fluctuating levels of multiple infections were observed during static growth conditions and outgrowth and multiplication rates were inferior. Serum, as compared to Albumax, was found necessary for optimal presentation of PfEMP1 at the pRBC surface and/or for binding of serum proteins (immunoglobulins). Optimal in vitro growth conditions of P. falciparum therefore include orbital shaking (50 rev/min), human serum (10%) and a fixed gas composition (5% O2, 5% CO2, 90% N2). We subsequently established 100% of 76 frozen patient isolates and found rosetting with schizont pRBCs in every isolate (&gt;26% schizont rosetting rate). Rosetting during schizogony was often followed by invasion of the bound RBC as seen by regular and time-lapse microscopy as well as transmission electron microscopy. The peripheral parasitemia, the level of rosetting and the rate of multiplication correlated positively to one another for individual isolates. Rosetting was also more frequent with trophozoite and schizont pRBCs of children with severe versus uncomplicated malaria (p&lt;0.002; p&lt;0.004). The associations suggest that rosetting enhances the ability of the parasite to multiply within the human host},
  articleno    = {e69781},
  author       = {Ribacke, U. and Moll, K. and Albrecht, L. and Ismail, H. A. and Normark, J. and Flaberg, E. and Szekely, L. and Hultenby, K. and Persson, Kristina and Egwang, T. G. and Wahlgren, M.},
  issn         = {1932-6203},
  keyword      = {red-blood-cells,malaria-infected erythrocytes,surface-antigen,expression,virulence protein pfemp1,human cerebral malaria,uncomplicated malaria,alpha-thalassemia,serum,cytoadherence,association},
  language     = {eng},
  number       = {7},
  publisher    = {Public Library of Science},
  series       = {PLoS ONE},
  title        = {Improved In Vitro Culture of Plasmodium falciparum Permits Establishment of Clinical Isolates with Preserved Multiplication, Invasion and Rosetting Phenotypes},
  url          = {http://dx.doi.org/10.1371/journal.pone.0069781},
  volume       = {8},
  year         = {2013},
}