Quantification of Plasmodium ex vivo drug susceptibility by flow cytometry

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Abstract

© 2015 Wirjanata et al. 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 IC 50 values varied from -5.4 to 6.1 nM, associated with 0.83-1.23 fold change in IC 50 values between LM and FC. In 15 assays (5.1 %), the derived difference of IC 50 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 IC 50 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.

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Wirjanata, G., Handayuni, I., Prayoga, P., Apriyanti, D., Chalfein, F., Sebayang, B. F., … Marfurt, J. (2015). Quantification of Plasmodium ex vivo drug susceptibility by flow cytometry. Malaria Journal, 14(1). https://doi.org/10.1186/s12936-015-0940-8

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