Cellulose filtration of blood from malaria patients for improving ex vivo growth of Plasmodium falciparum parasites.
Establishing in vitro Plasmodium falciparum culture lines from patient parasite isolates can offer deeper understanding of geographic variations of drug sensitivity and mechanisms of malaria pathogenesis and immunity. Cellulose column filtration of blood is an inexpensive, rapid and effective method for the removal of host factors, such as leucocytes and platelets, significantly improving the purification of parasite DNA in a blood sample.



In this study, the effect of cellulose column filtration of venous blood on the initial in vitro growth of P. falciparum parasite isolates from Tanzanian children admitted to hospital was tested. The parasites were allowed to expand in culture without subcultivation until 5 days after admission or the appearance of dead parasites and parasitaemia was determined daily. To investigate whether the filtration had an effect on clonality, P. falciparum merozoite surface protein 2 genotyping was performed using nested PCR on extracted genomic DNA, and the var gene transcript levels were investigated, using quantitative PCR on extracted RNA, at admission and 4 days of culture.



The cellulose-filtered parasites grew to higher parasitaemia faster than non-filtered parasites seemingly due to a higher development ratio of ring stage parasites progressing into the late stages. Cellulose filtration had no apparent effect on clonality or var gene expression; however, evident differences were observed after only 4 days of culture in both the number of clones and transcript levels of var genes compared to the time of admission.



Cellulose column filtration of parasitized blood is a cheap, applicable method for improving cultivation of P. falciparum field isolates for ex vivo based assays; however, when assessing phenotype and genotype of cultured parasites, in general, assumed to represent the in vivo infection, caution is advised.
Kilimanjaro Clinical Research Institute, Kilimanjaro Christian Medical University College, Moshi, Tanzania.
Sixbert I
SI
Mkumbaye
Korogwe Research Station, Tanga Centre, National Institute for Medical Research, Tanga, Tanzania.
Daniel T R
DT
Minja
Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, Centre for Medical Parasitology, University of Copenhagen, CSS Building 22-23, Øster Farimagsgade 5, PO Box 2099, 1014, Copenhagen K, Denmark.|Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark.
Jakob S
JS
Jespersen
Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, Centre for Medical Parasitology, University of Copenhagen, CSS Building 22-23, Øster Farimagsgade 5, PO Box 2099, 1014, Copenhagen K, Denmark.|Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark.
Michael
M
Alifrangis
Kilimanjaro Clinical Research Institute, Kilimanjaro Christian Medical University College, Moshi, Tanzania.
Reginald A
RA
Kavishe
Kilimanjaro Clinical Research Institute, Kilimanjaro Christian Medical University College, Moshi, Tanzania.
Steven B
SB
Mwakalinga
Korogwe Research Station, Tanga Centre, National Institute for Medical Research, Tanga, Tanzania.
John P
JP
Lusingu
Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, Centre for Medical Parasitology, University of Copenhagen, CSS Building 22-23, Øster Farimagsgade 5, PO Box 2099, 1014, Copenhagen K, Denmark.|Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark.
Thor G
TG
Theander
Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, Centre for Medical Parasitology, University of Copenhagen, CSS Building 22-23, Øster Farimagsgade 5, PO Box 2099, 1014, Copenhagen K, Denmark.|Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark.
Thomas
T
Lavstsen
Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, Centre for Medical Parasitology, University of Copenhagen, CSS Building 22-23, Øster Farimagsgade 5, PO Box 2099, 1014, Copenhagen K, Denmark. cwang@sund.ku.dk.|Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark. cwang@sund.ku.dk.
Christian W
0000-0003-3140-6989
CW
Wang
D000818:Animals; D001769:Blood; D002482:Cellulose; D002675:Child, Preschool; D003470:Culture Media / Q000737:chemistry*; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D005374:Filtration; D005838:Genotype; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D016778:Malaria, Falciparum / Q000469:parasitology*; D008297:Male; D059208:Parasite Load; D010963:Plasmodium falciparum / Q000145:classification / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D016053:RNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D013636:Tanzania
D003470:Culture Media; D016054:DNA, Protozoan; D016053:RNA, Protozoan; D002482:Cellulose
Cellulose column filtration; Ex vivo growth; Plasmodium falciparum