A Novel Flow Cytometric Hemozoin Detection Assay for Real-Time Sensitivity Testing of Plasmodium Falciparum

PLoS One. 2013 Apr 24;8(4):e61606. doi: 10.1371/journal.pone.0061606. Print 2013.

Abstract

Resistance of Plasmodium falciparum to almost all antimalarial drugs, including the first-line treatment with artemisinins, has been described, representing an obvious threat to malaria control. In vitro antimalarial sensitivity testing is crucial to detect and monitor drug resistance. Current assays have been successfully used to detect drug effects on parasites. However, they have some limitations, such as the use of radioactive or expensive reagents or long incubation times. Here we describe a novel assay to detect antimalarial drug effects, based on flow cytometric detection of hemozoin (Hz), which is rapid and does not require any additional reagents. Hz is an optimal parasite maturation indicator since its amount increases as the parasite matures. Due to its physical property of birefringence, Hz depolarizes light, hence it can be detected using optical methods such as flow cytometry. A common flow cytometer was adapted to detect light depolarization caused by Hz. Synchronized in vitro cultures of P. falciparum were incubated for 48 hours with several antimalarial drugs. Analysis of depolarizing events, corresponding to parasitized red blood cells containing Hz, allowed the detection of parasite maturation. Moreover, chloroquine resistance and the inhibitory effect of all antimalarial drugs tested, except for pyrimethamine, could be determined as early as 18 to 24 hours of incubation. At 24 hours incubation, 50% inhibitory concentrations (IC50) were comparable to previously reported values. These results indicate that the reagent-free, real-time Hz detection assay could become a novel assay for the detection of drug effects on Plasmodium falciparum.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antimalarials / pharmacology*
  • Chloroquine / pharmacology
  • Drug Resistance
  • Erythrocytes / parasitology
  • Flow Cytometry
  • Hemeproteins / metabolism*
  • Humans
  • Inhibitory Concentration 50
  • Plasmodium falciparum / drug effects*
  • Plasmodium falciparum / growth & development
  • Plasmodium falciparum / metabolism*

Substances

  • Antimalarials
  • Hemeproteins
  • hemozoin
  • Chloroquine

Grant support

This work was supported by the Luso-American Foundation (FLAD-LACR grant: B-A.V-109-09/07). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.