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An Improved Sample Preparation Method for the Detection of Plasmodium falciparum in Blood Samples

Thavamani Rajapandi

Microscopy remains the gold standard for the detection of malaria parasites in blood samples, while rapid diagnostic tests (RDTs) and polymerase chain reaction (PCR)-based methods are increasingly employed for field applications, each with limitations. Using in vitro cultured Plasmodium falciparum parasite isolates, we analyzed several sample preparation methods for PCR-based detection. Test samples subjected to hypotonic lysis, followed by a near complete depletion of hemozoin (Hz) and hemoglobin (Hb) and subsequent PCR amplification of the msp2 gene resulted in a detection limit that was improved over samples prepared in isotonic buffer conditions. In addition, we amplified sequences of several target genes, including the msp2-, msp1-, 18S rRNA- and eba175 from parasite extracts prepared by hypotonic lysis. We identified a primer set which amplified a region within eba175 that gave a greater detection limit of 1 parasite per 5 × 107 red blood cells (RBCs), as well as a primer set for msp2 that gave a detection limit of 1 to 5 parasites per 5 × 107 RBCs by standard PCR analysis. The remaining targets exhibited maximum detection limits of 20 to 30 parasites per 5 × 107 RBCs. This newly developed method is 100 to 500 times more sensitive than currently available PCR-based methods which detect 30 to 100 parasites per 5 × 106 RBCs in 1 μl of blood). In addition, this method could be used in field deployable PCR-based diagnostics, as well as for parasite genotyping and quality control of failed RDTs.