Laboratory diagnosis of a suspected case of imported Plasmodium ovale infection

Blood smear of suspected imported malaria patient was prepared and observed by microscopy after Giemsa staining.Initial microscopy of the blood sample was negative for malaria.Nested PCR and real-time PCR methods of genus-specific Plasmodium and four species-specific Plasmodium were performed to detect and differentiate the parasites using in-house designed primers and probes.Nested PCR with the genus-specific primers of Plasmodium showed that the blood sample produced an expected band of about 240bp in size.Plasmodium classification demonstrated that the blood sample only produced an expected amplification band of about 450bp of P.ovale in size and no bands of P.falciparum,P.vivax and P.malarie.Real time PCR with both Plasmodium and specific P.ovale displayed typical S-shaped positive curves.Sequence analysis showed that the amplified fragment was composed of 434 bases and 393 bases removing PCR primers showed 100% homology to the corresponding part of P.ovale SSU rRNA gene of GenBank accession number DQ845247.Blood smear was reviewed by microscopy.The ring trophozoites of P.ovale were founded in thin blood film and the parasitized cell was oval in shape with fimbriated edges.The suspected case of imported malaria patient was confirmed to infect P.ovale by nested PCR,real-time PCR,sequence analysis and microscopic examination.