Species-specific PCR techniques are highly sensitive and reliable alternatives to classical methods for malaria diagnosis and speciation, especially in endemic regions under advanced control or elimination programs where asymptomatic and low-density infections are increasingly reported. Nevertheless, the performance of these techniques is directly affected by the quality of isolated DNA templates. A Plasmodium falciparum/vivax-specific diagnostic Nested-PCR (Pf/Pv N-PCR) was used to assess three DNA preparation methods, Qiagen® Mini-Chromatographic kit (QIAmp) and Jena-Biosciences DNA isolation kit (JB) for genomic DNA extraction from EDTA-preserved whole blood samples, and Whatman-FTA® purification reagent (FTA®) for DNA preparation from dry blood spots (DBS) collected onto FTA®- cards. A total of 84 out of 137 blood specimens collected from malaria suspicious febrile patients who visited five health care centres in south-western endemic localities of Saudi Arabia were found P. falciparum positive by at least one method. Among these, only 76 (90%) were reported P. falciparum malaria positive by two expert microscopists. No other species of Plasmodium
were detected. Pf/Pv N-PCR revealed 84/84 (100%), 75/84 (89%), and 81 (96%) P. falciparum positive samples using DNA templates prepared by QIAmp®, JB®, and FTA® purification methods, respectively. Therefore, Pf/Pv N-PCR, when applied to QIAmp® DNA templates showed to be a highly sensitive diagnostic method, particularly useful for submicroscopic specimens from clinically malaria suspicious patients in endemic areas. On the other hand, Pf/Pv N-PCR of FTA®-DBS DNA templates revealed 5 positive cases missed by microscopy, encouraging its use as an affordable field semi-adapted protocol for malaria active screening, especially in remote rural regions with limited laboratory infrastructure.