title: Improvement of culture conditions for long-term in vitro culture of Plasmodium vivax.
abstract: BACKGROUND
  The study of the biology, transmission and pathogenesis of Plasmodium vivax is hindered due to the lack of a robustly propagating, continuous culture of this parasite. The current culture system for P. vivax parasites still suffered from consistency and difficulties in long-term maintenance of parasites in culture and for providing sufficient biological materials for studying parasite biology. Therefore, further improvement of culture conditions for P. vivax is needed.
  
  
  METHODS
  Clinical samples were collected from patients diagnosed with P. vivax in western Thailand. Leukocyte-depleted P. vivax infected blood samples were cultured in a modified McCoy's 5A medium at 5% haematocrit under hypoxic condition (5% O2, 5% CO2, and 90% N2). Reticulocytes purified from adult peripheral blood were added daily to maintain 4% reticulocytes. Parasites were detected by microscopic examination of Giemsa-stained smears and molecular methods.
  
  
  RESULTS
  The effects of culture variables were first analysed in order to improve the culture conditions for P. vivax. Through analysis of the sources of host reticulocytes and nutrients of culture medium, the culture conditions better supporting in vitro growth and maturation of the parasites were identified. Using this system, three of 30 isolates could be maintained in vitro for over 26 months albeit parasite density is low.
  
  
  CONCLUSIONS
  Based on the analysis of different culture variables, an improved and feasible protocol for continuous culture of P. vivax was developed.
authors.affiliation: Mahidol Vivax Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. wanlapa.ros@mahidol.edu.
authors.forename: Wanlapa
authors.initials: W
authors.lastname: Roobsoong
authors.affiliation: Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand. chayada.si@cmu.ac.th.
authors.forename: Chayada S
authors.initials: CS
authors.lastname: Tharinjaroen
authors.affiliation: Mahidol Vivax Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. nattawan.rac@mahidol.ac.th.
authors.forename: Nattawan
authors.initials: N
authors.lastname: Rachaphaew
authors.affiliation: Mahidol Vivax Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. ppchobson@gmail.com.
authors.forename: Porpimon
authors.initials: P
authors.lastname: Chobson
authors.affiliation: Department of Infectious Diseases, Walter and Elisa Hall Institute of Medical Research, Melbourne, Australia. schofield@wehi.edu.au.
authors.forename: Louis
authors.initials: L
authors.lastname: Schofield
authors.affiliation: Department of Entomology, The Pennsylvania State University, University park, PA, USA. luc2@psu.edu.
authors.forename: Liwang
authors.initials: L
authors.lastname: Cui
authors.affiliation: Department of Global Health, University of South Florida, Tampa, USA. usfmalaria@gmail.com.
authors.forename: John H
authors.initials: JH
authors.lastname: Adams
authors.affiliation: Mahidol Vivax Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. jetsumon.pra@mahidol.edu.
authors.forename: Jetsumon
authors.initials: J
authors.lastname: Sattabongkot
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D003470:Culture Media / Q000737:chemistry; D004912:Erythrocytes / Q000469:parasitology; D006801:Humans; D016780:Malaria, Vivax / Q000469:parasitology*; D059208:Parasite Load; D010966:Plasmodium vivax / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D012156:Reticulocytes
chemical_list: D003470:Culture Media