A comparative study of a flow-cytometry-based assessment of in vitro Plasmodium falciparum drug sensitivity.
BACKGROUND
Recently developed Sybr Green-based in vitro Plasmodium falciparum drug sensitivity assays provide an attractive alternative to current manual and automated methods. The present study evaluated flow cytometry measurement of DNA staining with Sybr Green in comparison with the P. falciparum lactate dehydrogenase assay, the tritiated hypoxanthine incorporation assay, a previously described Sybr Green based plate reader assay and light microscopy.


METHODS
All assays were set up in standardized format in 96-well plates. The 50% inhibitory concentrations (IC50) of chloroquine, mefloquine and dihydroartemisinin against the laboratory adapted P. falciparum strains 3D7, E8B, W2mef and Dd2 were determined using each method.


RESULTS
The resolution achieved by flow cytometry allowed quantification of the increase in individual cell DNA content after an incubation period of only 24 h. Regression, and Bland and Altman analyses showed that the IC50 values determined using the flow cytometry assay after 24 h agreed well with those obtained using the hypoxanthine incorporation assay, the P. falciparum lactate dehydrogenase assay, the Sybr Green plate reader assay and light microscopy. However the values obtained with the flow cytometry assay after 48 h of incubation differed significantly from those obtained with the hypoxanthine incorporation assay, and the P. falciparum lactate dehydrogenase assay at low IC50 values, but agreed well with the Sybr Green plate reader assay and light microscopy.


CONCLUSIONS
Although flow cytometric equipment is expensive, the necessary reagents are inexpensive, the procedure is simple and rapid, and the cell volume required is minimal. This should allow field studies using fingerprick sample volumes.
Karl
Stephan
S
School of Physics, M013, The University of Western Australia, 35 Stirling Highway, Crawley WA 6009, Australia.
Wong
Rina Pm
RP
St Pierre
Tim G
TG
Davis
Timothy Me
TM
D000962:Antimalarials / Q000494:pharmacology*; D052160:Benzothiazoles; D016054:DNA, Protozoan / Q000187:drug effects*; D003959:Diamines; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D020128:Inhibitory Concentration 50; D007770:L-Lactate Dehydrogenase / Q000097:blood; D016778:Malaria, Falciparum / Q000175:diagnosis / Q000469:parasitology; D008297:Male; D008853:Microscopy; D009930:Organic Chemicals; D021261:Parasitic Sensitivity Tests / Q000379:methods; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000254:growth & development / Q000302:isolation & purification; D011804:Quinolines
D000962:Antimalarials; D052160:Benzothiazoles; D016054:DNA, Protozoan; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D007770:L-Lactate Dehydrogenase