TY - JOUR
T1 - Optimizing the HRP-2 in vitro malaria drug susceptibility assay using a reference clone to improve comparisons of Plasmodium falciparum field isolates
AU - Rutvisuttinunt, Wiriya
AU - Chaorattanakawee, Suwanna
AU - Tyner, Stuart D.
AU - Teja-Isavadharm, Paktiya
AU - Se, Youry
AU - Yingyuen, Kritsanai
AU - Chaichana, Panjaporn
AU - Bethell, Delia
AU - Walsh, Douglas S.
AU - Lon, Chanthap
AU - Fukuda, Mark
AU - Socheat, Duong
AU - Noedl, Harald
AU - Schaecher, Kurt
AU - Saunders, David L.
N1 - Funding Information:
This work was supported by funding provided by the DoD Global Emerging Infectious Surveillance (GEIS) and the Medicines for Malaria Venture (MMV). We thank Dr.William Ellis at the Walter Reed Army Institute of Research (WRAIR) for providing us with drug compounds, and Ms Somporn Krasaesub for advice on statistical analysis. In addition, we thank Ms Piyaporn Saingam, Ms Siratchana Sundrakes, Ms Raveewan Siripokasubkul and Ms Roongnapha Apinan for technical assistance. Dr Sea Darapiseth, and Messrs Sittidech Surasri, Worachet Kuntawunginn, Theera Wimonwattrawatee, Montri Arsanok, and Ms Monticha Kongthaisong conducted the clinical study and cultured isolates in the field.
PY - 2012
Y1 - 2012
N2 - Background: Apparent emerging artemisinin-resistant Plasmodium falciparum malaria in Southeast Asia requires development of practical tools to monitor for resistant parasites. Although in vitro anti-malarial susceptibility tests are widely used, uncertainties remain regarding interpretation of P. falciparum field isolate values. Methods. Performance parameters of the W2 P. falciparum clone (considered artemisinin sensitive) were evaluated as a reference for the HRP-2 immediate ex vivo assay. Variability in W2 IC50s was assessed, including intra- and inter-assay variability among and between technicians in multiple experiments, over five freeze-thaw cycles, over five months of continuous culture, and before and after transport of drug-coated plates to remote field sites. Nominal drug plate concentrations of artesunate (AS) and dihydroartemisinin (DHA) were verified by LC-MS analysis. Plasmodium falciparum field isolate IC50s for DHA from subjects in an artemisinin-resistant area in Cambodia were compared with W2 susceptibility. Results: Plate drug concentrations and day-to-day technical assay performance among technicians were important sources of variability for W2 IC50s within and between assays. Freeze-thaw cycles, long-term continuous culture, and transport to and from remote sites had less influence. Despite variability in W2 susceptibility, the median IC50s for DHA for Cambodian field isolates were higher (p <0.0001) than the W2 clone (3.9 nM), both for subjects with expected (less than 72hours; 6.3 nM) and prolonged (greater or equal to 72hours; 9.6 nM) parasite clearance times during treatment with artesunate monotherapy. Conclusion: The W2 reference clone improved the interpretability of field isolate susceptibility from the immediate ex vivo HRP-2 assay from areas of artemisinin resistance. Methods to increase the reproducibility of plate coating may improve overall assay interpretability and utility.
AB - Background: Apparent emerging artemisinin-resistant Plasmodium falciparum malaria in Southeast Asia requires development of practical tools to monitor for resistant parasites. Although in vitro anti-malarial susceptibility tests are widely used, uncertainties remain regarding interpretation of P. falciparum field isolate values. Methods. Performance parameters of the W2 P. falciparum clone (considered artemisinin sensitive) were evaluated as a reference for the HRP-2 immediate ex vivo assay. Variability in W2 IC50s was assessed, including intra- and inter-assay variability among and between technicians in multiple experiments, over five freeze-thaw cycles, over five months of continuous culture, and before and after transport of drug-coated plates to remote field sites. Nominal drug plate concentrations of artesunate (AS) and dihydroartemisinin (DHA) were verified by LC-MS analysis. Plasmodium falciparum field isolate IC50s for DHA from subjects in an artemisinin-resistant area in Cambodia were compared with W2 susceptibility. Results: Plate drug concentrations and day-to-day technical assay performance among technicians were important sources of variability for W2 IC50s within and between assays. Freeze-thaw cycles, long-term continuous culture, and transport to and from remote sites had less influence. Despite variability in W2 susceptibility, the median IC50s for DHA for Cambodian field isolates were higher (p <0.0001) than the W2 clone (3.9 nM), both for subjects with expected (less than 72hours; 6.3 nM) and prolonged (greater or equal to 72hours; 9.6 nM) parasite clearance times during treatment with artesunate monotherapy. Conclusion: The W2 reference clone improved the interpretability of field isolate susceptibility from the immediate ex vivo HRP-2 assay from areas of artemisinin resistance. Methods to increase the reproducibility of plate coating may improve overall assay interpretability and utility.
KW - Anti-malarial drugs
KW - Drug resistance
KW - Drug susceptibility test
KW - ELISA
KW - HRP-2
KW - Malaria
KW - Plasmodium falciparum
UR - http://www.scopus.com/inward/record.url?scp=84866101366&partnerID=8YFLogxK
U2 - 10.1186/1475-2875-11-325
DO - 10.1186/1475-2875-11-325
M3 - Article
C2 - 22974086
AN - SCOPUS:84866101366
SN - 1475-2875
VL - 11
JO - Malaria Journal
JF - Malaria Journal
M1 - 325
ER -