Using CF11 cellulose columns to inexpensively and effectively remove human DNA from Plasmodium falciparum-infected whole blood samples.
BACKGROUND
Genome and transcriptome studies of Plasmodium nucleic acids obtained from parasitized whole blood are greatly improved by depletion of human DNA or enrichment of parasite DNA prior to next-generation sequencing and microarray hybridization. The most effective method currently used is a two-step procedure to deplete leukocytes: centrifugation using density gradient media followed by filtration through expensive, commercially available columns. This method is not easily implemented in field studies that collect hundreds of samples and simultaneously process samples for multiple laboratory analyses. Inexpensive syringes, hand-packed with CF11 cellulose powder, were recently shown to improve ex vivo cultivation of Plasmodium vivax obtained from parasitized whole blood. This study was undertaken to determine whether CF11 columns could be adapted to isolate Plasmodium falciparum DNA from parasitized whole blood and achieve current quantity and purity requirements for Illumina sequencing.


METHODS
The CF11 procedure was compared with the current two-step standard of leukocyte depletion using parasitized red blood cells cultured in vitro and parasitized blood obtained ex vivo from Cambodian patients with malaria. Procedural variations in centrifugation and column size were tested, along with a range of blood volumes and parasite densities.


RESULTS
CF11 filtration reliably produces 500 nanograms of DNA with less than 50% human DNA contamination, which is comparable to that obtained by the two-step method and falls within the current quality control requirements for Illumina sequencing. In addition, a centrifuge-free version of the CF11 filtration method to isolate P. falciparum DNA at remote and minimally equipped field sites in malaria-endemic areas was validated.


CONCLUSIONS
CF11 filtration is a cost-effective, scalable, one-step approach to remove human DNA from P. falciparum-infected whole blood samples.
Howard Hughes Medical Institute, University of Maryland School of Medicine, Baltimore, MD, USA.
Meera
M
Venkatesan
Chanaki
C
Amaratunga
Susana
S
Campino
Sarah
S
Auburn
Oliver
O
Koch
Pharath
P
Lim
Sambunny
S
Uk
Duong
D
Socheat
Dominic P
DP
Kwiatkowski
Rick M
RM
Fairhurst
Christopher V
CV
Plowe
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001769:Blood / Q000469:parasitology*; D002160:Cambodia; D002648:Child; D002675:Child, Preschool; D002845:Chromatography / Q000379:methods*; D016054:DNA, Protozoan / Q000302:isolation & purification*; D006801:Humans; D007223:Infant; D008288:Malaria / Q000175:diagnosis*; D008875:Middle Aged; D010274:Parasitology / Q000379:methods*; D010963:Plasmodium falciparum / Q000235:genetics*; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D055815:Young Adult
D016054:DNA, Protozoan