Abstract
BackgroundMalaria 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.MethodsThe 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.ResultsPfEMP1 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.ConclusionsThese 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.
Highlights
Malaria remains a global public health problem responsible for 445,000 deaths in 2016
Given the lack of knowledge on the minimum number of parasites that could be present in asymptomatic individuals and limitations in volume of blood used in detection assays, molecular methods of improved sensitivity are still needed to identify all Plasmodium infections in endemic areas
Human blood samples Plasmodium falciparum-infected blood smear positive samples from patients in Ghana used for this study were collected on Whatman® FTA Elute cards (GE Lifesciences, MA, USA) at Kpone Katamanso District Hospital in the Greater Accra Region of Ghana, a malaria endemic country which recorded about 10 million malaria cases in the year 2015
Summary
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. The gold standard of malaria parasite detection is direct visual confirmation of intraerythrocytic parasites in a blood film. This method is time and labourintensive and can only reach typical detection limits of 5000–150,000 parasites/mL of blood [8]. Given the lack of knowledge on the minimum number of parasites that could be present in asymptomatic individuals and limitations in volume of blood used in detection assays, molecular methods of improved sensitivity are still needed to identify all Plasmodium infections in endemic areas
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