Quantification of Plasmodium ex vivo drug susceptibility by flow cytometry

Malaria Journal

Methodology

Boni F. Sebayang¹  Rintis Noviyanti¹  Dwi Apriyanti¹  Steven Kho²  Irene Handayuni²  Jutta Marfurt²  Ric N. Price³  Grennady Wirjanata⁴  Brice Campo⁵  Pak Prayoga⁶  Ferryanto Chalfein⁶  Enny Kenangalem⁷  Jeanne Rini Poespoprodjo⁸ 

[1] Eijkman Institute for Molecular Biology, Jl. Diponegoro 69, 10430, Jakarta, Indonesia;Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, PO Box 41096, Casuarina, 0811, Darwin, Australia;Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, PO Box 41096, Casuarina, 0811, Darwin, Australia;Nuffield Department of Clinical Medicine, Centre for Tropical Medicine and Global Health, University of Oxford, Oxford, UK;Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, PO Box 41096, Casuarina, 0811, Darwin, Australia;Papuan Health and Community Development Foundation (PHCDF), Timika, Papua, Indonesia;Medicines for Malaria Venture (MMV), PO Box 1826, 20 rte de Pré-Bois, 1215, Geneva 15, Switzerland;Papuan Health and Community Development Foundation (PHCDF), Timika, Papua, Indonesia;Papuan Health and Community Development Foundation (PHCDF), Timika, Papua, Indonesia;District Health Authority, Timika, Papua, Indonesia;Papuan Health and Community Development Foundation (PHCDF), Timika, Papua, Indonesia;District Health Authority, Timika, Papua, Indonesia;Department of Paediatrics, Faculty of Medicine, Gadjah Mada University, Yogyakarta, Indonesia;

关键词: Malaria;    Plasmodium falciparum;    Plasmodium vivax;    Ex vivo drug susceptibility;    Drug resistance;    Flow cytometry;   

DOI  :  10.1186/s12936-015-0940-8

 received in 2015-08-17, accepted in 2015-10-11,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundThe 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.MethodsField 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.ResultsIn 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.ConclusionsA 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.

【 授权许可】

CC BY   
© Wirjanata et al. 2015