title: Attenuation of Plasmodium falciparum in vitro drug resistance phenotype following culture adaptation compared to fresh clinical isolates in Cambodia.
abstract: BACKGROUND
  There is currently no standardized approach for assessing in vitro anti-malarial drug susceptibility. Potential alterations in drug susceptibility results between fresh immediate ex vivo (IEV) and cryopreserved culture-adapted (CCA) Plasmodium falciparum isolates, as well as changes in parasite genotype during culture adaptation were investigated.
  
  
  METHODS
  The 50 % inhibitory concentration (IC50) of 12 P. falciparum isolates from Cambodia against a panel of commonly used drugs were compared using both IEV and CCA. Results were compared using both histidine-rich protein-2 ELISA (HRP-2) and SYBR-Green I fluorescence methods. Molecular genotyping and amplicon deep sequencing were also used to compare multiplicity of infection and genetic polymophisms in fresh versus culture-adapted isolates.
  
  
  RESULTS
  IC50 for culture-adapted specimens were significantly lower compared to the original fresh isolates for both HRP-2 and SYBR-Green I assays, with greater than a 50 % decline for the majority of drug-assay combinations. There were correlations between IC50s from IEV and CCA for most drugs assays. Infections were nearly all monoclonal, with little or no change in merozoite surface protein 1 (MSP1), MSP2, glutamate-rich protein (GLURP) or apical membrane antigen 1 (AMA1) polymorphisms, nor differences in P. falciparum multidrug resistance 1 gene (PfMDR1) copy number or single nucleotide polymorphisms following culture adaptation.
  
  
  CONCLUSIONS
  The overall IC50 reduction combined with the correlation between fresh isolates and culture-adapted drug susceptibility assays suggests the utility of both approaches, as long as there is consistency of method, and remaining mindful of possible attenuation of resistance phenotype occurring in culture. Further study should be done in higher transmission settings where polyclonal infections are prevalent.
authors.affiliation: Department of Immunology and Medicine, Armed Forces Research Institute of Medical Science, Bangkok, Thailand. SuwannaC2.fsn@afrims.org.
authors.forename: Suwanna
authors.initials: S
authors.lastname: Chaorattanakawee
authors.affiliation: Department of Immunology and Medicine, Armed Forces Research Institute of Medical Science, Bangkok, Thailand. charlotte.a.lanteri.mil@mail.mil.|Microbiology Section, Department of Pathology and Area Laboratory Services, Brooke Army Medical Center, San Antonio, TX, USA. charlotte.a.lanteri.mil@mail.mil.
authors.forename: Charlotte A
authors.initials: CA
authors.lastname: Lanteri
authors.affiliation: Department of Immunology and Medicine, Armed Forces Research Institute of Medical Science, Bangkok, Thailand. SiratchanaS.ca@afrims.org.
authors.forename: Siratchana
authors.initials: S
authors.lastname: Sundrakes
authors.affiliation: Department of Immunology and Medicine, Armed Forces Research Institute of Medical Science, Bangkok, Thailand. KritsanaiY.fsn@afrims.org.
authors.forename: Kritsanai
authors.initials: K
authors.lastname: Yingyuen
authors.affiliation: Department of Immunology and Medicine, Armed Forces Research Institute of Medical Science, Bangkok, Thailand. PanitaG.fsn@afrims.org.
authors.forename: Panita
authors.initials: P
authors.lastname: Gosi
authors.affiliation: Department of Immunology and Medicine, Armed Forces Research Institute of Medical Science, Bangkok, Thailand. NitimaC.ca@afrims.org.
authors.forename: Nitima
authors.initials: N
authors.lastname: Chanarat
authors.affiliation: Department of Immunology and Medicine, Armed Forces Research Institute of Medical Science, Bangkok, Thailand. SaowalukW.ca@afrims.org.
authors.forename: Saowaluk
authors.initials: S
authors.lastname: Wongarunkochakorn
authors.affiliation: Department of Immunology and Medicine, Armed Forces Research Institute of Medical Science, Bangkok, Thailand. NillawanB.fsn@afrims.org.
authors.forename: Nillawan
authors.initials: N
authors.lastname: Buathong
authors.affiliation: Department of Immunology and Medicine, Armed Forces Research Institute of Medical Science, Bangkok, Thailand. ChannS.ctr@afrims.org.
authors.forename: Soklyda
authors.initials: S
authors.lastname: Chann
authors.affiliation: Department of Immunology and Medicine, Armed Forces Research Institute of Medical Science, Bangkok, Thailand. WorachetK.fsn@afrims.org.
authors.forename: Worachet
authors.initials: W
authors.lastname: Kuntawunginn
authors.affiliation: Department of Immunology and Medicine, Armed Forces Research Institute of Medical Science, Bangkok, Thailand. MontriA.fsn@afrims.org.
authors.forename: Montri
authors.initials: M
authors.lastname: Arsanok
authors.affiliation: Division of Infectious Diseases, School of Medicine, University of North Carolina, Chapel Hill, NC, USA. jessica_lin@med.unc.edu.
authors.forename: Jessica T
authors.initials: JT
authors.lastname: Lin
authors.affiliation: Division of Infectious Diseases, School of Medicine, University of North Carolina, Chapel Hill, NC, USA. jonathan_juliano@med.unc.edu.
authors.forename: Jonathan J
authors.initials: JJ
authors.lastname: Juliano
authors.affiliation: Department of Immunology and Medicine, Armed Forces Research Institute of Medical Science, Bangkok, Thailand. stuart.d.tyner.mil@mail.mil.|US Army Institute of Surgical Research, Joint Base San Antonio-Fort Sam Houston, San Antonio, TX, USA. stuart.d.tyner.mil@mail.mil.
authors.forename: Stuart D
authors.initials: SD
authors.lastname: Tyner
authors.affiliation: National Centre for Parasitology, Entomology and Malaria Control, Phnom Penh, Cambodia. mengchuor@gmail.com.
authors.forename: Mengchuor
authors.initials: M
authors.lastname: Char
authors.affiliation: Department of Immunology and Medicine, Armed Forces Research Institute of Medical Science, Bangkok, Thailand. ChanthapL@afrims.org.
authors.forename: Chanthap
authors.initials: C
authors.lastname: Lon
authors.affiliation: Department of Immunology and Medicine, Armed Forces Research Institute of Medical Science, Bangkok, Thailand. david.saunders@afrims.org.
authors.forename: David L
authors.initials: DL
authors.lastname: Saunders
mesh_terms: D000293:Adolescent; D000328:Adult; D000962:Antimalarials / Q000494:pharmacology*; D002160:Cambodia; D016054:DNA, Protozoan / Q000235:genetics; D004351:Drug Resistance; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D020128:Inhibitory Concentration 50; D016778:Malaria, Falciparum / Q000469:parasitology; D021261:Parasitic Sensitivity Tests / Q000379:methods*; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D055815:Young Adult
chemical_list: D000962:Antimalarials; D016054:DNA, Protozoan