Evaluating the efficacy of a Plasmodium species-specific Multiplex-Nest-PCR in malaria diagnosis using different DNA isolation methods

Malaria diagnosis and speciation still rely on microscopic identification in many settings. But, microscopy is tedious and lack sensitivity, particularly in areas under advanced eradication programs where low-density infections are increasingly reported. Species-specific molecular techniques are highly sensitive and reliable alternatives for Plasmodium parasites detection and speciation. Nevertheless, the efficacy of molecular techniques is directly affected by the purity and quality of isolated DNA templates. A Plasmodium species-specific multiplex-nested-PCR was assessed using DNA templates prepared separately by phenol-chloroform method, a DNA-precipitation commercial kit, and a chromatographic commercial kit from 126 EDTA-preserved whole blood samples. 115 samples were collected from malaria suspicious febrile patients in endemic southern region and 11 malaria positive samples from foreign and national visitors of non-endemic western region of Saudi Arabia. In total, 89 specimens were found malaria positive by at least one diagnostic method, out of which 82 (92%) were detected by microscopy. P. multiplex-N-PCR revealed 89 (100%), 77 (86.5%), and 85 (95.5%) positive samples using DNA templates extracted by the chromatographic kit, the DNA-precipitation kit, and phenol-chloroform standard method, respectively. P. falciparum parasites were detected in 86 samples and P. vivax in three samples from foreign visitors. Thus, P. multiplex-N-PCR applied to DNA templates isolated by chromatographic method achieved the highest sensitivity and was particularly useful for submicroscopic malaria cases in the endemic area where intensive elimination efforts are being deployed.