Development and validation of a pan-Plasmodium molecular assay as a confirmatory method for blood donor screening.

BACKGROUND

Transfusion-transmitted malaria remains a concern in non-endemic regions due to asymptomatic parasitemia in donors with prior residence in or travel to malaria-endemic areas. Nucleic acid testing (NAT)-based screening has been proposed to mitigate this risk, and supplemental assays are needed to confirm reactive donations. We developed a pan-Plasmodium reverse transcription quantitative PCR (RT-qPCR) assay with species identification capability as a supplemental tool for malaria donor screening.

STUDY DESIGN AND

METHODS

The assay targets a conserved region of the 18S rRNA shared by all five human-infecting Plasmodium species and is compatible with whole blood lysed using Grifols' parasite transport medium (PTM). Analytical performance was evaluated using in vitro transcripts and infected red blood cells (iRBCs), with the limit of detection (LoD) determined by probit analysis. Specificity was assessed against Babesia microti and 300 non-exposed US donor samples. Clinical sensitivity was evaluated using infected specimens; species identification was performed by sequencing of RT-qPCR amplicons.

RESULTS

The assay demonstrated high amplification efficiency (97.2%) and linearity (R2 = 0.99). The 95% LoD was 5.3 iRBCs/mL (95% CI: 3.2-8.6), comparable to the Procleix Plasmodium Assay. Clinical sensitivity was 100% across all five Plasmodium species, and clinical specificity was 100% (95% CI: 99-100%), with no cross-reactivity with Babesia. Amplicon sequencing enabled accurate species-level identification of all sequenced specimens.

DISCUSSION: This assay provides a sensitive confirmatory tool for malaria NAT-based donor screening. Its compatibility with PTM lysates and species identification capabilities supports regulatory applications and research into asymptomatic parasitemia in semi-immune donors.