A rapid sensitive, flow cytometry-based method for the detection of Plasmodium vivax-infected blood cells.

BACKGROUND

Plasmodium 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.

METHODS

Clinical 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.

RESULTS

The 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 (R2=0.93843) from 0.001 to 1.00% P. vivax-infected reticulocytes.

CONCLUSIONS

The FC-based method is a simple, robust, and efficient method for detecting P. vivax-infected reticulocytes.