title: Drying anti-malarial drugs in vitro tests to outsource SYBR green assays.
abstract: BACKGROUND
  Measurement of anti-malarial drug efficacy and resistance relies mainly on in vivo clinical trials, in vitro/ex vivo assays and molecular markers detection. The existing in vitro/ex vivo assays, in particular those that are using non-radioactive devices, need to be standardized and adapted to field conditions. SYBR Green assay offers a rapid and cheap alternative to other in vitro assays, but it requires tools not commonly available in field laboratories. Here is described a modified SYBR green I protocol to perform the parasite growth test with blood samples in endemic areas, followed later by the SYBR green fluorescence assay performed at a specialized laboratory level.
  
  
  METHODS
  In vitro susceptibility of Plasmodium falciparum clones HB3, 3D7, W2 and 7G8 to chloroquine (CQ), dihydroartemisinin (DHA), pyronaridine (PYD) and piperaquine (PPQ) was tested. Fresh isolates of P. falciparum from imported malaria cases were collected for ex vivo assays. The parasite suspension was added in 96-well plates predosed with anti-malarial drugs and incubated for 72 hours at 37°C, 5% CO2. SYBR green I protocol was modified to dry the plates after freeze-thawed process to mimic storage and shipping conditions. The plates were rehydrated with 200 μl of complete RPMI medium for fluorescence assay.
  
  
  RESULTS
  There were no significant differences in IC₅₀ values of CQ, DHA, PYD and PPQ, determined by the modified protocol, compared to standard protocol. Longer storage did not affect the IC₅₀ values.
  
  
  CONCLUSIONS
  The SYBR green I modified protocol produced reliable results and could be a suitable method for in vitro/ex vivo assays in field.
authors.affiliation: Malaria Research and Training Center, DEAP/FMPOS, UMI3189, Université des Sciences, des Techniques et des Technologies de Bamako, BP 1805, Bamako, Mali. karim@icermali.org.|Malaria Research Unit, SMITH, ICBMS, UMR 5246 CNRS-INSA-CPE-University Claude Bernard Lyon1, 8 Avenue Rockefeller, 69373, Lyon, Cedex 08, France. karim@icermali.org.
authors.forename: Karim
authors.initials: K
authors.lastname: Traore
authors.affiliation: Malaria Research Unit, SMITH, ICBMS, UMR 5246 CNRS-INSA-CPE-University Claude Bernard Lyon1, 8 Avenue Rockefeller, 69373, Lyon, Cedex 08, France. adeline.lavoignat@univ-lyon1.fr.
authors.forename: Adeline
authors.initials: A
authors.lastname: Lavoignat
authors.affiliation: Malaria Research Unit, SMITH, ICBMS, UMR 5246 CNRS-INSA-CPE-University Claude Bernard Lyon1, 8 Avenue Rockefeller, 69373, Lyon, Cedex 08, France. guillaume.bonnot@univ-lyon1.fr.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Bonnot
authors.affiliation: Malaria Research Unit, SMITH, ICBMS, UMR 5246 CNRS-INSA-CPE-University Claude Bernard Lyon1, 8 Avenue Rockefeller, 69373, Lyon, Cedex 08, France. fatimata.sow@etu.univ-lyon1.fr.
authors.forename: Fatimata
authors.initials: F
authors.lastname: Sow
authors.affiliation: Institut de Parasitologie et Mycologie Médicale, Hospices Civils de Lyon, Lyon, France. giuliana.constantino-bess@etu.univ-lyon1.fr.
authors.forename: Giuliana C
authors.initials: GC
authors.lastname: Bess
authors.affiliation: Institut de Parasitologie et Mycologie Médicale, Hospices Civils de Lyon, Lyon, France. chavant.marjorie@gmail.com.
authors.forename: Marjorie
authors.initials: M
authors.lastname: Chavant
authors.affiliation: AP-HP, Service de Parasitologie-Mycologie, Université Pierre et Marie Curie Paris 6, Paris, France. frederick.gay@upmc.fr.
authors.forename: Frederick
authors.initials: F
authors.lastname: Gay
authors.affiliation: Malaria Research and Training Center, DEAP/FMPOS, UMI3189, Université des Sciences, des Techniques et des Technologies de Bamako, BP 1805, Bamako, Mali. okd@icermali.org.
authors.forename: Ogobara
authors.initials: O
authors.lastname: Doumbo
authors.affiliation: Malaria Research Unit, SMITH, ICBMS, UMR 5246 CNRS-INSA-CPE-University Claude Bernard Lyon1, 8 Avenue Rockefeller, 69373, Lyon, Cedex 08, France. stephane.picot@univ-lyon1.fr.|Institut de Parasitologie et Mycologie Médicale, Hospices Civils de Lyon, Lyon, France. stephane.picot@univ-lyon1.fr.
authors.forename: Stephane
authors.initials: S
authors.lastname: Picot
mesh_terms: D000962:Antimalarials / Q000494:pharmacology*; D052160:Benzothiazoles; D003959:Diamines; D004351:Drug Resistance; D005456:Fluorescent Dyes / Q000737:chemistry*; D020128:Inhibitory Concentration 50; D009930:Organic Chemicals / Q000737:chemistry*; D010274:Parasitology / Q000379:methods*; D010963:Plasmodium falciparum / Q000187:drug effects*; D011804:Quinolines
chemical_list: D000962:Antimalarials; D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I