Quantification of Plasmodium ex vivo drug susceptibility by flow cytometry.
BACKGROUND
The emergence and spread of multidrug-resistant Plasmodium falciparum and Plasmodium vivax highlights the need for objective measures of ex vivo drug susceptibility. Flow cytometry (FC) has potential to provide a robust and rapid quantification of ex vivo parasite growth.


METHODS
Field isolates from Papua, Indonesia, underwent ex vivo drug susceptibility testing against chloroquine, amodiaquine, piperaquine, mefloquine, and artesunate. A single nucleic acid stain (i.e., hydroethidine (HE) for P. falciparum and SYBR Green I (SG) for P. vivax) was used to quantify infected red blood cells by FC-based signal detection. Data derived by FC were compared to standard quantification by light microscopy (LM). A subset of isolates was used to compare single and double staining techniques.


RESULTS
In total, 57 P. falciparum and 23 P. vivax field isolates were collected for ex vivo drug susceptibility testing. Reliable paired data between LM and FC was obtained for 88 % (295/334) of these assays. The median difference of derived IC50 values varied from -5.4 to 6.1 nM, associated with 0.83-1.23 fold change in IC50 values between LM and FC. In 15 assays (5.1 %), the derived difference of IC50 estimates was beyond the 95 % limits of agreement; in eleven assays (3.7 %), this was attributable to low parasite growth (final schizont count < 40 %), and in four assays (1.4 %) due to low initial parasitaemia at the start of assay (<2000 µl(-1)). In a subset of seven samples, LM, single and double staining FC techniques generated similar IC50 values.


CONCLUSIONS
A single staining FC-based assay using a portable cytometer provides a simple, fast and versatile platform for field surveillance of ex vivo drug susceptibility in clinical P. falciparum and P. vivax isolates.
Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, PO Box 41096, Casuarina, 0811, Darwin, Australia. Grennady.Wirjanata@menzies.edu.au.|Papuan Health and Community Development Foundation (PHCDF), Timika, Papua, Indonesia. Grennady.Wirjanata@menzies.edu.au.
Grennady
G
Wirjanata
Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, PO Box 41096, Casuarina, 0811, Darwin, Australia. Irene.Handayuni@menzies.edu.au.
Irene
I
Handayuni
Papuan Health and Community Development Foundation (PHCDF), Timika, Papua, Indonesia. prayogatimika@gmail.com.
Pak
P
Prayoga
Eijkman Institute for Molecular Biology, Jl. Diponegoro 69, Jakarta, 10430, Indonesia. dwiapriyanti@eijkman.go.id.
Dwi
D
Apriyanti
Papuan Health and Community Development Foundation (PHCDF), Timika, Papua, Indonesia. ferryanto_cs@yahoo.com.
Ferryanto
F
Chalfein
Eijkman Institute for Molecular Biology, Jl. Diponegoro 69, Jakarta, 10430, Indonesia. boni_sebayang@fmi.com.
Boni F
BF
Sebayang
Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, PO Box 41096, Casuarina, 0811, Darwin, Australia. Steven.Kho@menzies.edu.au.
Steven
S
Kho
Eijkman Institute for Molecular Biology, Jl. Diponegoro 69, Jakarta, 10430, Indonesia. rintis@eijkman.go.id.
Rintis
R
Noviyanti
Papuan Health and Community Development Foundation (PHCDF), Timika, Papua, Indonesia. ennykenangalem@yahoo.com.|District Health Authority, Timika, Papua, Indonesia. ennykenangalem@yahoo.com.
Enny
E
Kenangalem
Medicines for Malaria Venture (MMV), 20 rte de Pré-Bois, PO Box 1826, 1215, Geneva 15, Switzerland. campob@mmv.org.
Brice
B
Campo
Papuan Health and Community Development Foundation (PHCDF), Timika, Papua, Indonesia. didot2266@yahoo.com.|District Health Authority, Timika, Papua, Indonesia. didot2266@yahoo.com.|Department of Paediatrics, Faculty of Medicine, Gadjah Mada University, Yogyakarta, Indonesia. didot2266@yahoo.com.
Jeanne Rini
JR
Poespoprodjo
Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, PO Box 41096, Casuarina, 0811, Darwin, Australia. ricprice99@googlemail.com.|Nuffield Department of Clinical Medicine, Centre for Tropical Medicine and Global Health, University of Oxford, Oxford, UK. ricprice99@googlemail.com.
Ric N
RN
Price
Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, PO Box 41096, Casuarina, 0811, Darwin, Australia. jutta.marfurt@menzies.edu.au.
Jutta
J
Marfurt
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000962:Antimalarials / Q000494:pharmacology*; D002648:Child; D004912:Erythrocytes / Q000469:parasitology; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007214:Indonesia; D008297:Male; D008875:Middle Aged; D021261:Parasitic Sensitivity Tests / Q000379:methods*; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000254:growth & development; D010966:Plasmodium vivax / Q000187:drug effects* / Q000254:growth & development; D013194:Staining and Labeling / Q000379:methods; D055815:Young Adult
D000962:Antimalarials