title: Assessing anti-malarial drug effects ex vivo using the haemozoin detection assay.
abstract: BACKGROUND
  In vitro sensitivity assays are crucial to detect and monitor drug resistance. Plasmodium falciparum has developed resistance to almost all anti-malarial drugs. Although different in vitro drug assays are available, some of their inherent characteristics limit their application, especially in the field. A recently developed approach based on the flow cytometric detection of haemozoin (Hz) allowed reagent-free monitoring of parasite maturation and detection of drug effects in culture-adapted parasites. In this study, the set-up, performance and usefulness of this novel assay were investigated under field conditions in Gabon.
  
  
  METHODS
  An existing flow cytometer (Cyflow Blue) was modified on site to detect light depolarization caused by Hz. Blood from malaria patients was incubated for 72 hrs with increasing concentrations of chloroquine, artesunate and artemisinin. The percentage of depolarizing red blood cells (RBC) was used as maturation indicator and measured at 24, 48 and 72 hrs of incubation to determine parasite growth and drug effects.
  
  
  RESULTS
  The flow cytometer was easily adapted on site to detect light depolarization caused by Hz. Analysis of ex vivo cultures of parasites, obtained from blood samples of malaria patients, showed four different growth profiles. In 39/46 samples, 50% inhibitory concentrations (IC50) were successfully determined. IC50 values for chloroquine were higher than 200 nM in 70% of the samples, indicating the presence of chloroquine-resistant parasites. For artesunate and artemisinin, IC50 values ranged from 0.9 to 60 nM and from 2.2 nM to 124 nM, respectively, indicating fully sensitive parasites.
  
  
  CONCLUSIONS
  Flow cytometric detection of Hz allowed the detection of drug effects in blood samples from malaria patients, without using additional reagents or complex protocols. Adjustment of the initial parasitaemia was not required, which greatly simplifies the protocol, although it may lead to different IC50 values. Further investigation of set-up conditions of the Hz assay, as well as future studies in various settings should be performed to further determine the usefulness of this assay as a tool for rapid resistance testing in malaria-endemic countries.
authors: Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Av Prof Egas Moniz, Lisbon, P-1649-028, Portugal. mariarebelo@fm.ul.pt.|Centre de Recherches Médicales de Lambaréné - CERMEL, Albert Schweitzer Hospital, Lambaréné, Gabon. mariarebelo@fm.ul.pt.
authors: Maria
authors: M
authors: Rebelo
authors: Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Av Prof Egas Moniz, Lisbon, P-1649-028, Portugal. ctempera@fm.ul.pt.
authors: Carolina
authors: C
authors: Tempera
authors: Centre de Recherches Médicales de Lambaréné - CERMEL, Albert Schweitzer Hospital, Lambaréné, Gabon. jose.fernandes@lambarene.org.|Institut für Tropenmedizin, Universität Tübingen, Tübingen, Germany. jose.fernandes@lambarene.org.|Centre of Tropical Medicine and Travel Medicine, Amsterdam Medical Centre, University of Amsterdam, Amsterdam, The Netherlands. jose.fernandes@lambarene.org.
authors: José F
authors: JF
authors: Fernandes
authors: Centre de Recherches Médicales de Lambaréné - CERMEL, Albert Schweitzer Hospital, Lambaréné, Gabon. m.p.grobusch@amc.uva.nl.|Institut für Tropenmedizin, Universität Tübingen, Tübingen, Germany. m.p.grobusch@amc.uva.nl.|Centre of Tropical Medicine and Travel Medicine, Amsterdam Medical Centre, University of Amsterdam, Amsterdam, The Netherlands. m.p.grobusch@amc.uva.nl.
authors: Martin P
authors: MP
authors: Grobusch
authors: Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Av Prof Egas Moniz, Lisbon, P-1649-028, Portugal. t.hanscheid@fm.ul.pt.|Centre de Recherches Médicales de Lambaréné - CERMEL, Albert Schweitzer Hospital, Lambaréné, Gabon. t.hanscheid@fm.ul.pt.|Instituto de Microbiologia, Faculdade de Medicina, Lisbon, Portugal. t.hanscheid@fm.ul.pt.
authors: Thomas
authors: T
authors: Hänscheid
mesh_terms: D000962:Antimalarials / Q000494:pharmacology* / Q000627:therapeutic use; D037621:Artemisinins / Q000494:pharmacology / Q000627:therapeutic use; D001773:Blood Cells / Q000469:parasitology*; D002738:Chloroquine / Q000494:pharmacology / Q000627:therapeutic use; D005434:Flow Cytometry / Q000379:methods; D006420:Hemeproteins / Q000032:analysis*; D006801:Humans; D020128:Inhibitory Concentration 50; D016778:Malaria, Falciparum / Q000188:drug therapy* / Q000469:parasitology*; D010274:Parasitology / Q000379:methods; D010963:Plasmodium falciparum / Q000187:drug effects*
chemical_list: D000962:Antimalarials; D037621:Artemisinins; D006420:Hemeproteins; C020753:hemozoin; D002738:Chloroquine