A 44-yr-old male was admitted to hospital with fever of three days duration in October 2004. He had worked as a woodcutter in Côte d'Ivoire in tropical West Africa from May 1995 to January 2004. He had suffered from malaria in October 2002 and was treated with antimalarial drugs, but he did not remember the type of malaria or treatment regimen applied. He returned to Korea in January 2004 and has never since traveled abroad. His body temperature at presentation was 40.4℃. Physical examination findings were unremarkable. A routine complete blood count with differential counts revealed anemia (hemoglobin, 12.3 g/dL) and thrombocytopenia (44,000/µL). Lactate dehydrogenase (660 IU/L), alanine aminotransferase (63 IU/L), and γ-glutamyl transpeptidase (124 IU/L) were increased. Other hematological and biochemical profiles were within normal limits. Plasmodium lactate dehydrogenase (OptiMal Rapid Malaria Test, DiaMed, Morat, Switzerland) was negative. However, Wright-Giemsa-stained peripheral blood smear showed various stages (ring forms, schizonts, and gametocytes) of a Plasmodium species. Plasmodium-infected RBCs were enlarged and showed fimbriated margins (Fig. 1). Parasite density was 3,000 parasites/µL. The patient was treated with hydroxychloroquine for 3 days and was discharged with clinical improvement and prescribed primaquine for 14 days to prevent relapse. A subsequent visit to the outpatient clinic after one month showed normal hematological and biochemical profiles. No Plasmodium species was detected on the follow-up peripheral blood smear.

To differentiate the species of Plasmodium and evaluate mixed species infection, a nested PCR targeting the SSU rRNA gene was performed and P. ovale single infection was confirmed.

DNA was isolated from blood samples of the patient using QIAamp DNA Blood Mini kit (QIAGEN GmbH, Hilden, Germany). A nested PCR targeting the SSU rRNA gene was performed according to the protocols described by Snounou et al. (9). A set of genus specific primers (rPLU5/rPLU6) was used for the first round of amplification and four sets of species-specific primers (rFAL1/rFAL3 for P. falciparum, rVIV1/rVIV2 for P. vivax, rMAL1/rMAL2 for P. malariae, and rOVA1/rOVA2 for P. ovale) were used for the second round. Amplification was performed in a 20 µL reaction mixture containing the following: 0.2 µM of each primer; 200 µM (each) dATP, dCTP, dTTP, and dGTP; 10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl₂, 40 mM KCl; 1 unit of Taq polymerase (Perkin-Elmer, Cetus, CT, U.S.A.); and 1 µL of DNA sample (or 1 µL of first-round PCR product for the second-round amplification). First- and second-round PCRs were performed using the same amplification condition. Forty cycles of amplification were preformed in a DNA thermal cycler (iCycler Thermal Cycler, Bio-Rad Laboratories, Hercules, CA, U.S.A.). Each cycle consisted of the followings: predenaturation at 95℃ for 5 min, 5 amplification cycles of (denaturation at 95℃ for 1 min, primer annealing at 58℃ for 2 min, and extension at 72℃ for 2 min), 35 amplification cycles of (denaturation at 95℃ for 1 min, primer annealing at 55℃ for 2 min, and extension at 72℃ for 2 min), and 72℃ for 10 min. The sizes of the amplified DNA fragments were -1,000 bp, 205 bp, 144 bp, 120 bp, and 788 bp for genus Plasmodium, P. falciparum, P. vivax, P. malariae, and P. ovale, respectively. Only P. ovale DNA was detected in the patient's peripheral blood (Fig. 2).

The PCR product was sequenced at Solgent (Daejeon, Korea). For the SSU rRNA gene, rPLU5 and RPLU6 were used. It was sequenced in both directions using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, U.S.A.). Sequencing products were resolved with an ABI 3730 XL autoanalyzer (Applied Biosystems). To compare gene sequences for the SSU rRNA genes, Nigerian I/CDC strain (L48997), NAG/Cameroon (AJ00157), MAL/MAI (X99790), and Papua New Guinea (AF145337) were used. We chose P. falciparum (AF145334), P. vivax (AF145335), and P. malariae (AF145336) as an outgroup. Nucleotide sequences were aligned using BioEdit (North Carolina State University, Raleigh, NC, U.S.A.). The aligned sequences were analyzed by the maximum likelihood method using fastDNAmL and distance methods using the neighboring distance method included in PHYLIP (Version 3.63, University of Washington, Seattle, WA, U.S.A.). The alignment of the P. ovale partial sequence of the SSU rRNA gene showed that our isolate (SM04-119, DQ104413) was more similar to isolates of Nigerian I/CDC and Papua New Guinea (identity 98.3% and 97.9%, respectively) than to those of NGA/Cameroon and MAL/MAI (95.8% and 95.4% identity, respectively) (Fig. 3).