Sensitive real-time PCR detection of Plasmodium falciparum parasites in whole blood by erythrocyte membrane protein 1 gene amplification

Abstract

Background: Malaria remains a global public health problem responsible for 445,000 deaths in 2016. While microscopy remains the mainstay of malaria diagnosis, highly sensitive molecular methods for detection of low-grade sub-microscopic infections are needed for surveillance studies and identifying asymptomatic reservoirs of malaria transmission. Methods: The Plasmodium falciparum genome sequence was analysed to identify high copy number genes that improve P. falciparum parasite detection in blood by RT-PCR. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)-specific primers were evaluated for P. falciparum detection in hospital-based microscopically positive dried blood spots and field-acquired whole blood from asymptomatic individuals from Ghana. Results: PfEMP1 outperformed the Pf18S sequence for amplification-based P. falciparum detection. PfEMP1 primers exhibited sevenfold higher sensitivity compared to Pf18S primers for parasite genomic DNA. Probit analysis established a 95% detection threshold of 9.3 parasites/mL for PfEMP1 compared to 98.2 parasites/mL for Pf18S primers. The PfEMP1 primers also demonstrated superior clinical sensitivity, identifying 100% (20/20) of dried blood spot samples and 70% (69/98) of asymptomatic individuals as positive versus 55% (11/20) and 54% (53/98), respectively, for Pf18S amplification. Conclusions: These results establish PfEMP1 as a novel amplification target for highly sensitive detection of both acute infections from filter paper samples and submicroscopic asymptomatic low-grade infections.