Ex vivo drug sensitivity profiles of Plasmodium falciparum field isolates from Cambodia and Thailand, 2005 to 2010, determined by a histidine-rich protein-2 assay

Abstract

BackgroundIn vitro drug susceptibility assay of Plasmodium falciparum field isolates processed “immediate ex vivo” (IEV), without culture adaption, and tested using histidine-rich protein-2 (HRP-2) detection as an assay, is an expedient way to track drug resistance.MethodsFrom 2005 to 2010, a HRP-2 in vitro assay assessed 451 P. falciparum field isolates obtained from subjects with malaria in western and northern Cambodia, and eastern Thailand, processed IEV, for 50% inhibitory concentrations (IC50) against seven anti-malarial drugs, including artesunate (AS), dihydroartemisinin (DHA), and piperaquine.ResultsIn western Cambodia, from 2006 to 2010, geometric mean (GM) IC50 values for chloroquine, mefloquine, quinine, AS, DHA, and lumefantrine increased. In northern Cambodia, from 2009–2010, GM IC50 values for most drugs approximated the highest western Cambodia GM IC50 values in 2009 or 2010.ConclusionsWestern Cambodia is associated with sustained reductions in anti-malarial drug susceptibility, including the artemisinins, with possible emergence, or spread, to northern Cambodia. This potential public health crisis supports continued in vitro drug IC50 monitoring of P. falciparum isolates at key locations in the region.

Ex Vivo Activity of Endoperoxide Antimalarials, Including Artemisone and Arterolane, against Multidrug-Resistant Plasmodium falciparum Isolates from Cambodia

“…Isolates from this trial were collected at the Tasanh Health Center, located in Battambang Province in western Cambodia, near the Thailand border and directly south of Pailin. A total of 166 isolates were also collected in 2009 and 2010 through the AFRIMS malaria drug resistance surveillance protocol in Cambodia under protocol number WRAIR 1576 (21), with a study population of individuals Ն13 years old living in areas with relatively high incidence rates of malaria in northern and western Cambodia. Individuals who gave a history of antimalarial drug use within the previous 7 days or who showed symptoms of severe or complicated malaria were excluded from participation.…”

“…Dried drug-coated plates were prepared at the Armed Forces Research Institute of Medical Sciences (AFRIMS) laboratory in Bangkok as previously described (21). Standard malaria drugs included in the routine test panel were provided by the Walter Reed Army Institute of Research (WRAIR) (Silver Spring, MD, USA).…”

“…After obtaining informed consent from clinical study volunteers, venous blood was collected in sodium heparin tubes from malaria patients who were confirmed by microscopy to have P. falciparum monoinfection for ex vivo drug sensitivity testing. Within 6 h after phlebotomy, fresh isolates (without culture adaptation) were added to 96-well dried drug-coated plates and evaluated using the HRP-2 ELISA, as previously described (21,24,25). Prior to transfer to dried drug-coated plates, samples with a parasitemia of Յ0.5% were adjusted to 1.5% hematocrit in 0.5% Albumax RPMI, whereas those with Ͼ0.5% parasitemia were diluted to the plate parasitemia range of 0.2 to 0.5% and adjusted to a 1.5% hematocrit by adding 50% hematocrit human Oϩ red blood cells in 10% serum-RPMI 1640.…”

Polymorphisms in Pf mdr1 , Pf crt , and Pf nhe1 Genes Are Associated with Reduced In Vitro Activities of Quinine in Plasmodium falciparum Isolates from Western Kenya

“…QN was prepared in 5 ml of 70% ethanol to attain 5 mg/ml, which was lowered to 2,554 nM in tissue culture medium. QN was then diluted across 10 concentrations ranging from 2,554 nM to 5 nM and predosed on 96-well microtiter plates as previously described (36). The assay was initiated by addition of 100 l reconstituted parasite components to each well of the drug plates and incubation at 37°C as previously described (35).…”

Atovaquone-Proguanil Remains a Potential Stopgap Therapy for Multidrug-Resistant Plasmodium falciparum in Areas along the Thai-Cambodian Border

“…ATQ was dissolved in dimethyl sulfoxide (DMSO) and diluted in 70% ethanol and then sterile water for a final concentration range of 0.14 to 100 ng/ml, while the conditions used for other drugs were as previously described (13). Susceptibility was measured by histidine-rich protein-2 (HRP-2) enzyme-linked immunosorbent assay (ELISA) testing on fresh isolates within 4 h of phlebotomy after being incubated for 72 h in 0.5% AlbuMAX with RPMI medium on drug-coated plates, according to previously published methods (13,14). Figure 1 shows the ex vivo susceptibility results for all isolates, and the values were comparable to our other recent ex vivo observations for all drugs except piperaquine, which was more resistant (15).…”