Biophysical characterisation of P. falciparum infected-red blood cells: consequences for diagnosis

Diagnosis of malaria in asymptomatic individuals is hampered by the lack of tools able to detect very low parasite densities. Microfluidics is emerging as a possible approach to address this issue. In particular, label-free sorting (i.e. exploiting the cell biophysical properties) of P. falciparum-infected red blood cells (iRBCs) from uninfected cells, and subsequent enrichment of parasite-infected RBCs within a blood sample may be an efficient and cost-effective way to increase the sensitivity of available methods used for malaria diagnosis in the field. However, the development of any sorting device based on cell biophysical properties requires a priori knowledge of when the difference in these properties between infected and uninfected RBCs (uRBCs) occurs. By the employment of leading-edge techniques in the field (real-time deformability cytometry (RT-DC) and microfluidic impedance cytometry (MIC)), the mechanical and dielectric properties of infected and uRBCs were studied over the P. falciparum-intraerythrocytic life cycle in vitro. Significant differences in biophysical properties between iRBCs and uRBCs occurred at around 12 hours post-invasion (hpi). Furthermore, the parasite age (defined as hpi) of P. falciparum parasites in vivo was also studied using a newly-developed method. This method applied real-time quantitative PCR (RT-qPCR) of three time-specific genes which are differentially expressed over the ring stage of the parasite, the only stage present in the peripheral blood of an infected person. Results obtained from the RT-qPCR analysis of highly synchronised in vitro grown ring-stage parasites at different times post-invasion were used to generate a predictive model which allowed the estimation of parasite ages (hpi). Moreover, discrimination between synchronous (with a single parasite age) or asynchronous (with multiple parasite ages) infections was achieved in naturally infected individuals. This novel method was further utilised to estimate parasite age and synchronicity status in samples from patients with uncomplicated malaria collected in Accra, Ghana. By coupling the results obtained from the study of the biophysical properties in vitro and parasite ages in vivo it was possible to determine the potential of mechanical and dielectric properties for label-free sorting of P. falciparum-infected and uninfected RBCs