Abstract
BackgroundIn 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.MethodsAn 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.ResultsThe 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.ConclusionFlow 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.
Highlights
In vitro sensitivity assays are crucial to detect and monitor drug resistance
Ex vivo cultures of infected red blood cells (RBC) showed that parasites had four different growth profiles (Table): (i) seven samples showed no maturation, as defined by an increase in depolarization; (ii) another eight samples showed maturation at 24 hrs and replication at 48 hrs; (iii) 17 samples showed maturation at 24 hrs but no reinvasion occurred; and, (iv) 14 samples had a delay in parasite growth, with maturation being observed at 48 hrs and replication at 72 hrs
Opening new avenues for anti-malarial drug testing in the field This study showed that drug effects of clinically relevant anti-malarial drugs as well as resistance to chloroquine could be assessed by detecting Hz
Summary
In vitro sensitivity assays are crucial to detect and monitor drug resistance. Plasmodium falciparum has developed resistance to almost all anti-malarial drugs. The number of malaria deaths has decreased in large part due to the availability of effective treatments, in particular artemisinin combination therapy (ACT) [1]. These achievements are in danger and might even be reversed because parasites with prolonged parasite clearance times (PCT), observed in patients treated with ACT, have emerged in Southeast. It is not unlikely that it will emerge in sub-Saharan Africa, and drug sensitivities should be monitored pro-actively In this scenario, in vitro sensitivity assays may play a crucial role in the Rebelo et al Malaria Journal (2015) 14:140 future. In vitro assays allow reducing host-related factors and provide an objective insight into the intrinsic sensitivity of malaria parasites
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