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Antimicrobial Agents and Chemotherapy, April 2007, p. 1172-1178, Vol. 51, No. 4
0066-4804/07/$08.00+0     doi:10.1128/AAC.01313-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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{triangledown}

David J. Bacon,1* Christine Latour,2 Carmen Lucas,1 Olga Colina,1 Pascal Ringwald,3 and Stéphane Picot2

Parasitology Program, Naval Medical Research Center Detachment, Lima, Peru,1 EA3732, Parasitology and Tropical Medicine, University Lyon 1, Lyon, France,2 Global Malaria Programme, World Health Organization, 20 Av. Appia, 1211 Geneva 27, Switzerland3

Received 20 October 2006/ Returned for modification 20 November 2006/ Accepted 30 December 2006

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.

* Corresponding author. Present address: Parasitology Program, Naval Medical Research Center Detachment, American Embassy, APO AA 34031. Phone: 511 562 3848, ext. 183. Fax: 511 561 3042. E-mail: bacon{at}nmrcd.med.navy.mil

{triangledown} Published ahead of print on 12 January 2007.

Antimicrobial Agents and Chemotherapy, April 2007, p. 1172-1178, Vol. 51, No. 4
0066-4804/07/$08.00+0     doi:10.1128/AAC.01313-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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