Malaria is a parasitic disease caused by protozoa of the genus Plasmodium. It is spread by the female Anopheles mosquito and in 2012 resulted in over 200 million cases worldwide, according to the World Health Organization. Plasmodium falciparum causes the majority of deaths associated with malaria, but Plasmodium vivax is the main cause of recurring malaria. This is when dormant forms of the parasite reside in the liver and ‘activate’ to cause disease years after infection. Detection of the malarial parasite, in particular P. vivax, has proven to be a challenge due to limitations innate to current detection methods that are reliant on light microscopy and nucleic acid staining. In a recent study in Malaria Journal, John Adams from the University of South Florida, USA, and colleagues reveal a new technique for the detection of P. vivax that may overcome these limitations.
P. vivax is predominant in the Americas and Asia, whilst less common in Africa where the general human population lack the Duffy antigen through which P. vivax enters and infects red blood cells. Traditionally, light microscopy has been used in the field to detect malaria in blood smears. This method of detection can take a long time and also requires a skilled microscopist to identify the parasite which, in the case of P. vivax, is an extremely difficult task due to low levels of parasite-infected blood cells in the peripheral circulation. Consequently, the presence and numbers of parasites can be inaccurately read.
In more recent years, to address these issues, flow cytometry based detection methods using nucleic acid staining have been developed. These methods work on the premise that the only nucleic acid containing entities in the blood sample would be the malaria parasite, since white blood cells would have been removed and red blood cells have little or no nucleic acid content. A flow cytometer can then quantify the number of stained cells that pass through the detector.
However, nucleic acid based detection methods also have limitations as blood samples can still contain residual white blood cells. Moreover, anaemia caused by malaria increases the number of immature red blood cells called reticulocytes, which do contain nucleic acid. This is compounded by the fact that P. vivax has an affinity for reticulocytes, ultimately contributing to inaccurate detection and quantification of P. vivax in patient blood samples.
Adams and colleagues present a new method for detection, using flow cytometry, based around antibody staining against the Plasmodium falciparum BiP protein. This protein is highly expressed and found abundantly on endoplasmic reticulum of infected cells. Furthermore, it is well conserved and can therefore also be used as a marker for P. vivax. The antibody serum developed by the authors, called anti-PfBiP, was successfully used to accurately quantify the P. vivax parasite during its blood stage in both clinical isolates and in vitro.
The staining method presented by the authors in this study provides a robust and time sensitive detection process for P.vivax, removing the main obstacle found with existing nucleic acid staining techniques – namely the difficulty in distinguishing the parasite in nucleic acid filled reticulocytes. Although light microscopy is still the gold standard for detection of parasites, flow cytometry is becoming a readily available and affordable detection method for laboratories.
A rapid sensitive, flow cytometry-based method for the detection of
Plasmodium vivax-infected blood cells
Malaria Journal 2014, 13:55
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