Abstract
BackgroundPlasmodium vivax preferentially infects Duffy-positive reticulocytes and infections typically have few parasite-infected cells in the peripheral circulation. These features complicate detection and quantification by flow cytometry (FC) using standard nucleic acid-based staining methods. A simple antibody-based FC method was developed for rapid parasite detection along with simultaneous detection of other parasite and erythrocyte markers.MethodsClinical samples were collected from patients diagnosed with P. vivax at a district Malaria Clinic in Kanchanaburi, Thailand. One μL of infected blood was washed, fixed, stained with a Plasmodium pan-specific anti-PfBiP antibody conjugated with Alexa Fluor 660, and analysed by FC. Additional primary conjugated antibodies for stage-specific markers of P. vivax for late trophozoite-early schizonts (MSP1-Alexa Fluor 660), late-stage schizonts (DBP-Alexa Fluor 555), and sexual stages (Pvs16) were used to differentiate intra-erythrocytic developmental stages.ResultsThe percentages of P. vivax-infected cells determined by the FC method and manually by microscopic examination of Giemsa-stained thick blood smears were positively correlated by Spearman’s rank correlation coefficient (R 2 = 0.93843) from 0.001 to 1.00% P. vivax-infected reticulocytes.ConclusionsThe FC-based method is a simple, robust, and efficient method for detecting P. vivax-infected reticulocytes.
Highlights
Plasmodium vivax preferentially infects Duffy-positive reticulocytes and infections typically have few parasite-infected cells in the peripheral circulation
Plasmodium Binding immunoglobulin protein of endoplasmic reticulum (BiP) is conserved Plasmodium falciparum BiP is an abundantly expressed protein of the endoplasmic reticulum (ER) involved in retrograde transport
The immunofluorescence assay (IFA) results in this study reconfirmed these observations demonstrating this anti-Plasmodium falciparum BiP (PfBiP) reacted with all intra-erythrocytic developmental stages of P. falciparum (Figure 1A)
Summary
Plasmodium vivax preferentially infects Duffy-positive reticulocytes and infections typically have few parasite-infected cells in the peripheral circulation. In recent years numerous flow cytometric-based methods have been developed to detect and quantify Plasmodium falciparum in the laboratory [4,5,6,7,8,9] Many of these methods use nucleic acid staining to detect parasite-infected cells, since nucleated white blood cells (WBCs) are removed for culture and mature erythrocytes retain virtually no nucleic acid. Constitutive expression of BiP through asexual blood-stage development coinciding with continued ER development has led to the common use of anti-PfBiP as reference antigen in P. falciparum studies [13,14] The antibodies to this conserved ER-resident protein were reactive with P. vivax and quantification of parasite-infected reticulocytes correlated accurately with light microscopic calculations
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