Optimizing the HRP-2 in vitro malaria drug susceptibility assay using a reference clone to improve comparisons of Plasmodium falciparum field isolates

Malaria Journal

Research

Mark Fukuda¹  Kritsanai Yingyuen²  Stuart D Tyner²  Kurt Schaecher²  David L Saunders²  Paktiya Teja-isavadharm²  Panjaporn Chaichana²  Delia Bethell²  Suwanna Chaorattanakawee²  Douglas S Walsh²  Youry Se²  Chanthap Lon²  Wiriya Rutvisuttinunt²  Harald Noedl³  Duong Socheat⁴ 

[1] Armed Forces Health Surveillance Center, Silver Spring, MD, USA;Department of Immunology and Medicine, US Army Medical Corps, Armed Forces Research Institute of Medical Sciences (USAMC-AFRIMS), Bangkok, Thailand;Medical University of Vienna, Vienna, Austria;The National Center for Parasitology, Entomology and Malaria Control (CNM), Ministry of Health, Phnom Penh, Cambodia;

关键词: Malaria;    Plasmodium falciparum;    HRP-2;    ELISA;    Anti-malarial drugs;    Drug resistance;    Drug susceptibility test;   

DOI  :  10.1186/1475-2875-11-325

 received in 2012-06-21, accepted in 2012-08-28,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundApparent 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.MethodsPerformance 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.ResultsPlate 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 72 hours; 6.3 nM) and prolonged (greater or equal to 72 hours; 9.6 nM) parasite clearance times during treatment with artesunate monotherapy.ConclusionThe 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.

【 授权许可】

Unknown   
© Rutvisuttinunt et al.; licensee BioMed Central Ltd. 2012. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.