Flow Cytometric Sorting of Infected Erythrocytes Demonstrates Reliable Detection of Individual Ring-Stage Plasmodium falciparum Parasites by Plasmodium 18S rRNA Reverse Transcription Polymerase Chain Reaction

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  • 1 Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington;
  • | 2 Center for Emerging and Re-emerging Infectious Diseases, University of Washington, Seattle, Washington;
  • | 3 Department of Microbiology, University of Washington, Seattle, Washington

Molecular diagnostic tests for Plasmodium falciparum parasites are increasingly used to enable ultrasensitive detection of infection in clinical trials and field surveillance studies. Ribonucleic acid (RNA)-based assays targeting 18S rRNA are particularly sensitive with limits of detection reported to comprise a single infected red blood cell (RBC) in a relatively large volume of blood. However, the validation testing at such limiting concentrations is hampered by the so-called Poisson distribution of such rare events, which can lead laboratorians to inaccurately set the limit of detection higher (i.e., less sensitive) than the assay can actually detect. Here we set out to formally demonstrate the analytical sensitivity of the Plasmodium 18S rRNA quantitative reverse transcription PCR (qRT-PCR). Fluorescence-activated cell sorting (FACS) was used on synchronous P. falciparum cultures doubly stained for DNA and RNA and was followed by qRT-PCR on the individual sorted cells spiked with negative whole blood. Over 95% of individual single-ring infected RBCs were detected by qRT-PCR. The formally measured median 18S rRNA content per individual ring-stage P. falciparum parasite was 9,550 copies (interquartile range 8,130–12,300). Thus, one can confidently rely on Plasmodium 18S rRNA qRT-PCR to detect one parasite per 50-µL blood sample.

Author Notes

Address correspondence to Sean C. Murphy, University of Washington, 750 Republican St., F873, Seattle, WA 98109. E-mail: murphysc@uw.edu

Financial support: This study was supported by the Department of Laboratory Medicine and Pathology, the University of Washington, and the Bill & Melinda Gates Foundation (OPP1212966).

Author’s addresses: Jokichi Matsubara, Ming Chang, and Annette M. Seilie, Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, and Center for Emerging and Re-emerging Infectious Diseases, University of Washington, Seattle, WA, E-mails: jmm189@students.jefferson.edu, minchang@uw.edu, and amseilie@uw.edu. Sean C. Murphy, Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, Center for Emerging and Re-emerging Infectious Diseases, University of Washington, Seattle, WA, and Department of Microbiology, University of Washington, Seattle, WA, E-mail: murphysc@uw.edu.

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