title: Comparison of a SYBR green I-based assay with a histidine-rich protein II enzyme-linked immunosorbent assay for in vitro antimalarial drug efficacy testing and application to clinical isolates.
abstract: In vitro drug susceptibility testing with the malaria parasite has been used to assess the antimalarial activities of new compounds and to monitor drug resistance in field isolates. We investigated the validity of a SYBR green I fluorescent-based assay under various culture conditions and compared the assay results to those of previously published histidine-rich protein II (HRPII) enzyme-linked immunosorbent assay (ELISA) methods. Reference strains of Plasmodium falciparum were cultured in vitro by using standard conditions in complete medium with and without phenol red before they were dispensed into 96-well plates predosed with chloroquine, mefloquine, or quinine. Following incubation, the culture supernatants were divided and the 50% inhibitory concentrations (IC50s) were determined by using a SYBR green I-based method and the HRPII capture ELISA method. There were no significant differences in IC50 values when phenol red was included in the medium. The IC50s and the IC90s of the antimalarials tested by both methods were similar or identical for each of the reference strains. Fresh clinical isolates of P. falciparum collected from imported cases of malaria in Lyon, France, were tested for in vitro resistance to chloroquine and mefloquine by using the validated SYBR green I and HRPII ELISA methods. The SYBR green I-based method was able to calculate IC50 and IC90 values similar or identical to those calculated by the HRPII assay with fresh clinical samples without removal of white blood cells. The SYBR green I-based method for determination of drug sensitivity levels produced results comparable to those produced by other methods, showing that this method can be used routinely to conduct surveillance for drug resistance in P. falciparum with fresh or cultured parasites.
authors.affiliation: Parasitology Program, Naval Medical Research Center Detachment, American Embassy, APO AA 34031, Lima, Peru. bacon@nmrcd.med.navy.mil
authors.forename: David J
authors.initials: DJ
authors.lastname: Bacon
authors.forename: Christine
authors.initials: C
authors.lastname: Latour
authors.forename: Carmen
authors.initials: C
authors.lastname: Lucas
authors.forename: Olga
authors.initials: O
authors.lastname: Colina
authors.forename: Pascal
authors.initials: P
authors.lastname: Ringwald
authors.forename: Stéphane
authors.initials: S
authors.lastname: Picot
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D052160:Benzothiazoles; D001681:Biological Assay / Q000191:economics / Q000379:methods; D003959:Diamines; D004351:Drug Resistance; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D009930:Organic Chemicals; D021261:Parasitic Sensitivity Tests / Q000379:methods; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000201:enzymology / Q000302:isolation & purification; D011506:Proteins; D015800:Protozoan Proteins / Q000032:analysis* / Q000096:biosynthesis; D011804:Quinolines; D011933:Reagent Kits, Diagnostic; D000099053:Histidine-Rich Glycoprotein
chemical_list: D000962:Antimalarials; D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011506:Proteins; D015800:Protozoan Proteins; D011804:Quinolines; D011933:Reagent Kits, Diagnostic; D000099053:Histidine-Rich Glycoprotein; C098022:SYBR Green I