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.

Conclusion: 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.

Figure 1

Flow cytometry modifications to detect light depolarization. The optical set-up of the Cyflow (Partec, Münster, Germany) (A) was easily modified to allow the detection of light depolarization. Four optical components from the original set-up (blue boxes in A) had to be replaced by the optical components shown in B, as described in the Methods section. Filters were simply replaced by unscrewing the original ones from the filter holder, or by removing the metallic ring (second row in B). This was accomplished using simple tools (bottom row in B). Note that in this case, polarization filters were glued inside the holder (arrows in B) so that they would not move, since to attain the best depolarization signal polarizers should be perpendicular to each other. The final optical layout to detect light depolarization is presented in C.

Figure 2

Representative analysis of drug effects assessed after ex vivo culture of blood samples from malaria patients. Representative plots of flow cytometric analysis of an uninfected blood sample (A) and a sample from a malaria patient with 1.5% parasitaemia (B). At time point 0 of incubation, no difference in depolarizing events is observed between the infected samples and the uninfected control (A and B). However, Hz is produced as the parasite matures, and after 24 hrs of incubation an increase in the percentage of depolarizing cells is detected (B) from 0.02 to 0.36. In this example, drug effects could be determined after only 24 hrs of incubation (C). Contrary to chloroquine, where resistance was observed, artesunate and artemisinin are still effective drugs (C).

Figure 3

Inhibitory 50% concentrations obtained for artesunate and artemisinin. Using the Hz detection assay, IC50 values for artesunate and artemisinin differed between samples ranging from 0.9 to 60 nM and from 2.2 nM to 124 nM, respectively. IC50 values obtained by the HRP-2 ELISA ranged from 0.6 to 31 nM for artesunate and from 0.6 to 94.8 nM for artemisinin.