A 24-year-old woman visited the emergency room with fever and chills. The patient was an American without underlying diseases and had lived in Korea for about 6 months. She had traveled to Jeonju, Gyeongju, and Suwon, but did not visit the northern part of Gyeonggi-do or Gangwon-do, which are endemic areas of malaria in Korea. However, she stayed in Ghana, West Africa for about 5 months from August to December 2010. At that time, she did not have any symptoms related to malaria and did not receive any treatment for malaria. In addition, she did not travel to any other malaria endemic areas after that. The complete blood cell count showed slight leukopenia (WBC 3,240/µl), slight thrombocytopenia (platelet 138,000/µl) and hemoglobin 13.2g/dl. Biochemical tests (total protein, albumin, glucose, total bilirubin, aspartate aminotransferase, alanine aminotransferase, creatine phosphokinase, amylase, urea nitrogen, creatinine, sodium, potassium, and chloride) were all normal, but lactate dehydrogenase (LDH) increased slightly (322 U/L; reference range 106-230 U/L).

The rapid diagnostic test (RDT) for malarial antigen (SD Bioline Malaria antigen P.f/Pan rapid kit, Standard Diagnostics, Inc., Yongin, Korea) at the first visit was negative, and the fever had subsided, so the patient was discharged. However, on the next day, malarial parasites were found in the Wright-Giemsa stained peripheral blood smear. The patient was told to visit the hospital, but she did not return until she developed fever again 2 days later.

The Wright-Giemsa stained peripheral blood smear showed that the RBCs infected with malarial parasites were slightly enlarged and had fimbriated margins, which was suspicious of P. ovale (Fig. 1). After counting the number of malarial parasites per 200 WBCs using a microscope at ×1,000, the density of malarial parasites was calculated indirectly by counting the number of malarial parasites per µl using the number of WBCs measured by an automated hematology analyzer (XE-2100 hematology analyzer, Sysmex, Kobe, Japan). The densities of malarial parasites for the first and second visit were 583/µl and 2,478/µl, respectively.

The patient was treated with hydroxychloroquine sulfate (immediate dose of 800 mg, followed by 400 mg at 6, 12, 18, and 24 hr) and primaquine (15 mg/day for 2 weeks). Her symptoms began to subside after 2 days of treatment, and no malarial parasites were observed in the peripheral blood on day 5 of treatment. On the 10th day of treatment, the complete blood cell count and biochemical test returned to normal, and malarial parasites were not observed.

Plasmodium genus-specific real-time PCR was performed with TaqMan fluorogenic probes targeting 18S rRNA (small subunit ribosomal RNA) genes to evaluate the presence of Plasmodium. The EDTA-anticoagulated blood specimen from the second visit was used. The AccuPower Malaria Quantitative PCR kit (Bioneer Co., Daejeon, Korea) and Exicycler™ 96 Real-Time Quantitative Thermal Block (Bioneer) for the thermal cycler were used. The PCR reagent mixture contained the Plasmodium genus-specific primer set, dual-labeled fluorogenic probes, DNA polymerase, dNTPs, and stabilizers. DNA was extracted with a DNA extraction kit from the same manufacturer (AccuPrep genomic DNA extraction kit, Bioneer). Real-time PCR was performed according to the manufacturer's instructions. The experiment included an internal positive control (IPC), 5 different concentrations of standards, a negative control (reaction mixture without DNA), and the patient's specimen, which was run in duplicate. The cycle threshold (Ct) for a positive result was <40. ExiDiagnosis analysis software (Bioneer) was used for the data analysis. Amplification curves of the 5 standards and IPC were determined to be valid. The patient's specimens showed Ct values of 25.7 and 25.5, indicating positive for Plasmodium spp. (Fig. 2).

Nested PCR targeting 18S rRNA genes was performed with primers and amplification conditions described by Snounou et al. [7] to identify Plasmodium spp. DNA was extracted from EDTA-anticoagulated blood samples with the QIAamp DNA Mini kit (QIAGEN Inc., Chatsworth, California, USA). The Plasmodium genus-specific primer set (rPLU5/rPLU6) was used for the first-round PCR. A second-round PCR was performed to identify the parasitic species present in the patient's sample. Four species-specific primer sets (P. falciparum, rFAL1/rFAL2; P. vivax, rVIV1/rVIV2; P. malariae, rMAL1/rMAL2; P. ovale, rOVA1/rOVA2) were used in the second-round PCR. Nested PCR was performed using a DNA thermal cycler (iCycler Thermal Cycler, Bio-Rad Laboratories, Hercules, California, USA). Thermal cycling was carried out under the following conditions: initial denaturation at 95℃ for 5 min, annealing at 58℃ for 2 min, and extension at 72℃ for 2 min (25 cycles); denaturation at 94℃ for 1 min, final annealing at 58℃ for 2 min, and final extension at 72℃ for 5 min (30 cycles). The 4 previously diagnosed clinical samples (P. falciparum, P. vivax, P. malariae, and P. ovale) and sterile distilled water were used as the positive and negative controls, respectively. The amplified DNA products corresponded to Plasmodium, 1,100 bp; P. falciparum, 205 bp; P. vivax, 120 bp; P. malariae, 144 bp; and P. ovale, 800 bp (Fig. 3). The presence of P. ovale in the patient's specimen was confirmed by nested PCR.

The amplification products acquired from the second PCR (800 bp) were extracted from the agarose gel to perform sequencing analysis. The primer sets used in the nested PCR were also used, and the Big Dye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, California, USA) and ABI 3730 XL autoanalyzer (Applied Biosystems) were used. The sequence alignment was performed using the single-stranded rRNA sequences in GenBank (P. ovale A-type, accession no. L48987) [8]. The isolated strain showed 99% similarity with P. ovale.