title: Direct comparison of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR green I fluorescence (MSF) drug sensitivity tests in Plasmodium falciparum reference clones and fresh ex vivo field isolates from Cambodia.
abstract: BACKGROUND
  Performance of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR Green I fluorescence (MSF) drug sensitivity tests were directly compared using Plasmodium falciparum reference strains and fresh ex vivo isolates from Cambodia against a panel of standard anti-malarials. The objective was to determine which of these two common assays is more appropriate for studying drug susceptibility of "immediate ex vivo" (IEV) isolates, analysed without culture adaption, in a region of relatively low malaria transmission.
  
  
  METHODS
  Using the HRP-2 and MSF methods, the 50% inhibitory concentration (IC50) values against a panel of malaria drugs were determined for P. falciparum reference clones (W2, D6, 3D7 and K1) and 41 IEV clinical isolates from an area of multidrug resistance in Cambodia. Comparison of the IC50 values from the two methods was made using Wilcoxon matched pair tests and Pearson's correlation. The lower limit of parasitaemia detection for both methods was determined for reference clones and IEV isolates. Since human white blood cell (WBC) DNA in clinical samples is known to reduce MSF assay sensitivity, SYBR Green I fluorescence linearity of P. falciparum samples spiked with WBCs was evaluated to assess the relative degree to which MSF sensitivity is reduced in clinical samples.
  
  
  RESULTS
  IC50 values correlated well between the HRP-2 and MSF methods when testing either P. falciparum reference clones or IEV isolates against 4-aminoquinolines (chloroquine, piperaquine and quinine) and the quinoline methanol mefloquine (Pearson r = 0.85-0.99 for reference clones and 0.56-0.84 for IEV isolates), whereas a weaker IC50 value correlation between methods was noted when testing artemisinins against reference clones and lack of correlation when testing IEV isolates. The HRP-2 ELISA produced a higher overall success rate (90% for producing IC50 best-fit sigmoidal curves), relative to only a 40% success rate for the MSF assay, when evaluating ex vivo Cambodian isolates. Reduced sensitivity of the MSF assay is likely due to an interference of WBCs in clinical samples.
  
  
  CONCLUSIONS
  For clinical samples not depleted of WBCs, HRP-2 ELISA is superior to the MSF assay at evaluating fresh P. falciparum field isolates with low parasitaemia (<0.2%) generally observed in Southeast Asia.
authors.affiliation: Department of Immunology and Medicine, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.
authors.forename: Suwanna
authors.initials: S
authors.lastname: Chaorattanakawee
authors.forename: Stuart D
authors.initials: SD
authors.lastname: Tyner
authors.forename: Chanthap
authors.initials: C
authors.lastname: Lon
authors.forename: Kritsanai
authors.initials: K
authors.lastname: Yingyuen
authors.forename: Wiriya
authors.initials: W
authors.lastname: Ruttvisutinunt
authors.forename: Siratchana
authors.initials: S
authors.lastname: Sundrakes
authors.forename: Piyaporn
authors.initials: P
authors.lastname: Sai-gnam
authors.forename: Jacob D
authors.initials: JD
authors.lastname: Johnson
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Walsh
authors.forename: David L
authors.initials: DL
authors.lastname: Saunders
authors.forename: Charlotte A
authors.initials: CA
authors.lastname: Lanteri
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000953:Antigens, Protozoan / Q000032:analysis; D000962:Antimalarials / Q000494:pharmacology*; D052160:Benzothiazoles; D002160:Cambodia; D003959:Diamines; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D005453:Fluorescence; D006801:Humans; D020128:Inhibitory Concentration 50; D016778:Malaria, Falciparum / Q000469:parasitology*; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009930:Organic Chemicals / Q000378:metabolism; D021261:Parasitic Sensitivity Tests / Q000379:methods; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000302:isolation & purification; D011506:Proteins; D015800:Protozoan Proteins / Q000032:analysis; D011804:Quinolines; D013194:Staining and Labeling / Q000379:methods; D055815:Young Adult
chemical_list: D000953:Antigens, Protozoan; D000962:Antimalarials; D052160:Benzothiazoles; D003959:Diamines; C514457:HRP-2 antigen, Plasmodium falciparum; D009930:Organic Chemicals; D011506:Proteins; D015800:Protozoan Proteins; D011804:Quinolines; C098022:SYBR Green I