Improving detection of Plasmodium falciparum from low density parasitemia using ultrasensitive PCR Assay

Understanding the epidemiology of malaria in low transmission settings is essential to design meaningful strategies to detect a large reservoir of individuals infected with sub-microscopic and often asymptomatic infections and this helps to control and eliminate the disease. To achieve this goal molecular assays have been increasingly used. However, one major limitation continues to be the volume of the blood sample collected and analysed (5-50 μL). This study aimed to increase sensitivity to detect P. falciparum infections by increasing blood volume for DNA extraction, detection, and quantification of P. falciparum using qPCR. This study compared the feasibility to extract 1000 μL vs 500 μL of whole blood at 0.1% parasite density and the result revealed no significant difference between the mean DNA concentrations in the 1000 μL whole blood sample compared to 500 μL (1000μL=31.50ng/ μL vs 500 μL= 30.32ng/ μL, P > 0.05). Further, qPCR sensitivity was analysed using 500 μL blood volumes and 7.8 μL of blood from a dried blood spot following a series of dilutions of 3D7 P. falciparum infected RBC with non-infected human whole blood ranging from 1x10-3 to 1x10-6 parasite densities. For all parasite densities, 500 μL aliquot and 50 μL dried blood spot was prepared, DNA extracted with Qiagen DNA mini kit and suspended in 150 μL and 200 μL respectively. 5 μL of the sample were used for detection and quantification by qPCR. Detection limit using 500 μL blood for DNA extraction was 0.05 parasites/ μL whereas for dried blood spot was 5 parasites/ μL. Therefore, 500 μL whole blood for extraction increases sensitivity by 100-fold compared to the currently used collection method with dried blood spots. In conclusion, a high-throughput ultra-sensitive PCR assay from high blood volume using 96-well plates Qiagen DNA extraction mini kit allows detection and quantification of P. falciparum parasites densities of 0.000001%. This finding should be translated into field epidemiological study to see whether the collection of higher blood volumes increases the prevalence of asymptomatic carriers in areas of low transmission intensity.