title: Nonlinear mixed-effects modelling of in vitro drug susceptibility and molecular correlates of multidrug resistant Plasmodium falciparum.
abstract: The analysis of in vitro anti-malarial drug susceptibility testing is vulnerable to the effects of different statistical approaches and selection biases. These confounding factors were assessed with respect to pfmdr1 gene mutation and amplification in 490 clinical isolates. Two statistical approaches for estimating the drug concentration associated with 50% effect (EC50 ) were compared: the commonly used standard two-stage (STS) method, and nonlinear mixed-effects modelling. The in vitro concentration-effect relationships for, chloroquine, mefloquine, lumefantrine and artesunate, were derived from clinical isolates obtained from patients on the western border of Thailand. All isolates were genotyped for polymorphisms in the pfmdr1 gene. The EC50 estimates were similar for the two statistical approaches but 15-28% of isolates in the STS method had a high coefficient of variation (>15%) for individual estimates of EC50 and these isolates had EC50 values that were 32 to 66% higher than isolates derived with more precision. In total 41% (202/490) of isolates had amplification of pfmdr1 and single nucleotide polymorphisms were found in 50 (10%). Pfmdr1 amplification was associated with an increase in EC50 for mefloquine (139% relative increase in EC50 for 2 copies, 188% for 3+ copies), lumefantrine (82% and 75% for 2 and 3+ copies respectively) and artesunate (63% and 127% for 2 and 3+ copies respectively). In contrast pfmdr1 mutation at codons 86 or 1042 were associated with an increase in chloroquine EC50 (44-48%). Sample size calculations showed that to demonstrate an EC50 shift of 50% or more with 80% power if the prevalence was 10% would require 430 isolates and 245 isolates if the prevalence was 20%. In conclusion, although nonlinear mixed-effects modelling did not demonstrate any major advantage for determining estimates of anti-malarial drug susceptibility, the method includes all isolates, thereby, potentially improving confirmation of candidate molecular markers of anti-malarial drug susceptibility.
authors.affiliation: Centre for Molecular, Environmental, Genetic & Analytic Epidemiology, Melbourne School of Population and Global Health, University of Melbourne, Melbourne, Australia. julieas@unimelb.edu.au
authors.forename: Julie A
authors.initials: JA
authors.lastname: Simpson
authors.forename: Kris M
authors.initials: KM
authors.lastname: Jamsen
authors.forename: Tim J C
authors.initials: TJ
authors.lastname: Anderson
authors.forename: Sophie
authors.initials: S
authors.lastname: Zaloumis
authors.forename: Shalini
authors.initials: S
authors.lastname: Nair
authors.forename: Charles
authors.initials: C
authors.lastname: Woodrow
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: White
authors.forename: Francois
authors.initials: F
authors.lastname: Nosten
authors.forename: Ric N
authors.initials: RN
authors.lastname: Price
mesh_terms: D000962:Antimalarials / Q000494:pharmacology*; D037621:Artemisinins / Q000494:pharmacology; D000077332:Artesunate; D002738:Chloroquine / Q000494:pharmacology; D004983:Ethanolamines / Q000494:pharmacology; D005449:Fluorenes / Q000494:pharmacology; D000078102:Lumefantrine; D015767:Mefloquine / Q000494:pharmacology; D015233:Models, Statistical; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics; D011110:Polymorphism, Genetic / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D015800:Protozoan Proteins / Q000235:genetics
chemical_list: D000962:Antimalarials; D037621:Artemisinins; D000077332:Artesunate; D002738:Chloroquine; D004983:Ethanolamines; D005449:Fluorenes; D000078102:Lumefantrine; D015767:Mefloquine; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D015800:Protozoan Proteins; C513498:Mdr1 protein, Plasmodium falciparum
references.citation: Br J Clin Pharmacol. 2006 Mar;61(3):289-300
references.pmid: 16487222
references.citation: Antimicrob Agents Chemother. 2008 Mar;52(3):1040-5
references.pmid: 18180357
references.citation: Antimicrob Agents Chemother. 2003 Aug;47(8):2418-23
references.pmid: 12878499
references.citation: Clin Infect Dis. 2006 Jun 1;42(11):1570-7
references.pmid: 16652314
references.citation: Antimicrob Agents Chemother. 1999 Dec;43(12):2943-9
references.pmid: 10582887
references.citation: Malar J. 2007 Jan 17;6:4
references.pmid: 17229314
references.citation: Lancet. 2004 Jul 31-Aug 6;364(9432):438-447
references.pmid: 15288742
references.citation: PLoS One. 2009;4(2):e4551
references.pmid: 19234601
references.citation: Exp Parasitol. 2006 Sep;114(1):34-9
references.pmid: 16545375
references.citation: J Immunol Methods. 2011 Mar 31;367(1-2):1-16
references.pmid: 21296083
references.citation: Antimicrob Agents Chemother. 2006 Jul;50(7):2281-5
references.pmid: 16801402
references.citation: Comput Methods Programs Biomed. 2004 Aug;75(2):85-94
references.pmid: 15212851
references.citation: Environ Toxicol Pharmacol. 1996 Oct 15;2(2-3):197-9
references.pmid: 21781727
references.citation: Malar J. 2012 Aug 30;11:303
references.pmid: 22931058
references.citation: Trans R Soc Trop Med Hyg. 2000 Sep-Oct;94(5):537-44
references.pmid: 11132385
references.citation: Antimicrob Agents Chemother. 2010 Jun;54(6):2611-7
references.pmid: 20368402
references.citation: Trans R Soc Trop Med Hyg. 2001 Jul-Aug;95(4):347-51
references.pmid: 11579871
references.citation: Evol Appl. 2009 Feb;2(1):52-61
references.pmid: 20526409
references.citation: Comput Methods Programs Biomed. 2005 Sep;79(3):241-57
references.pmid: 16023764
references.citation: Drug Metab Rev. 1984;15(1-2):265-92
references.pmid: 6745083
references.citation: Trends Parasitol. 2003 Apr;19(4):175-81
references.pmid: 12689648
references.citation: Nature. 2000 Feb 24;403(6772):906-9
references.pmid: 10706290
references.citation: Antimicrob Agents Chemother. 2005 Jun;49(6):2180-8
references.pmid: 15917511