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

Abstract

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.

Keywords

• Plasmodium falciparum erythrocyte membrane protein 1
• Parasitemia
• Real-time PCR
• Genomic DNA