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Journal of Clinical Microbiology, June 2007, p. 2084-2087, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.01334-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

CASE REPORT

First Case of Human Babesiosis in Korea: Detection and Characterization of a Novel Type of Babesia sp. (KO1) Similar to Ovine Babesia

Jung-Yeon Kim,^1, Shin-Hyeong Cho,^1, Hyun-Na Joo,¹ Masayoshi Tsuji,² Sung-Ran Cho,³ Il-Joong Park,³ Gyung-Tae Chung,¹ Jung-Won Ju,¹ Hyeng-Il Cheun,¹ Hyeong-Woo Lee,¹ Young-Hee Lee,¹ and Tong-Soo Kim^1*

Division of Malaria and Parasitic Diseases, National Institute of Health, KCDC, Seoul 122-701, Korea,¹ School of Veterinary Medicine, Rakuno-Gakuen University, Ebetsu 069-8501, Japan,² Department of Laboratory Medicine, Ajou University School of Medicine, Suwon 443-749, Korea³

Received 29 June 2006/ Returned for modification 11 September 2006/ Accepted 14 March 2007

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We report on the first case of human babesiosis in Korea. The intraerythrocytic parasite (KO1) in the patient's blood mainly appeared as paired pyriforms and ring forms; but Maltese cross forms were not seen, and the parasite showed morphological features consistent with those of the genus Babesia sensu stricto. The sequence of the 18S rRNA gene of KO1 was closely related to that of Babesia spp. isolated from sheep in China (similarity, 98%). The present study provides the first evidence of the presence of a hitherto unidentified, new type of Babesia parasite capable of infecting humans.

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A 75-year-old female residing in Gurae, Jeon-nam Province, Korea, was hospitalized at the Ajou University Medical Center, Suwon, Korea, on 5 June 2005 with symptoms of severe anemia, an irregular high fever, rigor, muscle pain, vomiting, diarrhea, and jaundice. Her medical history included gastric cancer, diagnosed in 1998, for which she had received treatments including gastric resection and splenectomy. Because of those operations, she had received blood transfusions twice in 1999. Initially, her disease was diagnosed as malaria because the clinical symptoms and morphologies of the parasites found in her blood smear were quite similar to those of the malaria parasite and because tertian malaria has often been found often in Korea in recent years. She was treated with quinine, an antimalarial drug, but it was apparently ineffective. Therefore, the patient's blood sample was transferred to the Division of Malaria and Parasitic Diseases, Korean Centers for Disease Control and Prevention, for systematic diagnosis on 20 June 2005.

On 22 June, her disease was diagnosed as babesiosis by microscopic examination and molecular biological methods. The patient was then treated with clindamycin on 23 June, which successfully reduced the parasitemia. The patient was discharged from the hospital on 1 July. When the second examination was done on 6 July, no parasite was detected by microscopic examination or PCR analysis.

Giemsa-stained blood smears from the patient revealed that intraerythrocytic parasites were most frequently observed as paired pyriforms and ring forms, but Maltese cross forms were not seen in most smears (Fig. 1). In general, the genus Babesia has been divided into two groups, namely "large" and "small" Babesia; the former is larger than 3 µm and frequently appears as unique paired pyriforms, while the latter is smaller than 3 µm and mainly appears as ring forms and, occasionally, Maltese cross forms (10). Hence, the parasite found in this study (KO1) can be classified as large Babesia on the basis of its size and shape.

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FIG. 1. Giemsa-stained smears of blood obtained on 2 July 2005 from a patient in Gurae, Korea, who had acquired babesiosis. (A) Paired pyriform parasites; (B) ring-form parasites. Bars, 5 µm.

To investigate the parasite's molecular characteristics, we performed nested PCR to detect the 18S rRNA gene of Babesia microti (12, 17), because it was regarded as the most probable cause of human babesiosis and it has been observed in wild mice in Korea. The parasite DNAs, prepared from the patient's blood sample by using a Nucleospin blood DNA extraction kit (Macherey-Nagel, Germany), were used for the first round of PCR with the primer set of Bab1A (5'-GTCTTAGTATAAGCTTTTATACAGCG-3') and Bab4A (5'-GATAGGTCAGAAACTTGAATGATACATCG-3'), followed by a second round of PCR with 1 µl of the first-round PCR product with the primer set Bab2A (5'-CAGTTATAGTTTATTTGATGTTCGTTTTAC-3') and Bab3A (5'-CGGCAAAGCCATGCGATTCGCTAAT-3') (17). The results were negative, indicating that B. microti was not present. At the same time, another nested PCR was conducted independently with primer sets that universally detect the 18S rRNA genes of most Babesia spp., namely, primers Bab5 (5'-AATTACCCAATCCTGACACAGG-3') and Bab8 (5'-TTTGGCAGTAGT TCGTCTTTAACA-3') for the first round of amplification and primers Bab6 (5'-GACACAGGG AGGTAGTGACAAGA-3') and Bab7 (5'-CCCAACTGCTCCTATTAACCATTAC-3') for the second round of PCR (17). As a result, a PCR product of about 400 bp was obtained. The PCR product was purified and cloned into a plasmid vector by using a Zero Blunt TOPO PCR cloning kit (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. The nucleotide sequences were determined by using a BigDye chain termination kit (Applied Biosystems, Foster City, CA) with an ABI 3100 automated sequencer (Applied Biosystems). The sequence was used to search the database of the National Center for Biotechnology Information (NCBI) with the Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov/BLAST/) for similarity analysis. Interestingly, the result revealed that the sequence from the patient was virtually identical to that of a Babesia sp. isolated from a sheep in Hebei, China (GenBank accession number DQ159074). Thus, for a more accurate analysis we again amplified the full-sized sequence (1.7 kb) encoding the 18S rRNA gene of the Babesia parasite from the patient with the primer set A (5'-ACCTGGTTGATCCTGCCAGT-3') and B (5'-TGATCCTTCTGCAGGTTCACCTAC-3') described by Medlin et al. (9), cloned the PCR product, and sequenced it as described above. The sequence obtained was highly similar (98%) to that registered in GenBank under accession number DQ159074. Phylogenetic analysis was carried out with the MacVector software package (version 8.1.1; Genetics Computer Group, Inc., Madison, WI). A total of 1,684 bp of the 18S rRNA sequence obtained in this study and the 18S rRNA gene sequences available from NCBI's database were aligned with the program ClustalW, followed by alignment with the phylogenetic analysis program available in the same package. The phylogenetic tree constructed by the neighbor-joining method is depicted in Fig. 2, which clearly demonstrates their close relationship. The Babesia sp. from the Korean patient formed a clade that included several Babesia spp. previously reported from sheep in China (18). Meanwhile, the Babesia spp. previously known to be the agents of human babesiosis, such as B. divergens, B. microti, and B. duncani, were placed into the phylogenetic tree very distant from the Babesia sp. found in this study. It was also shown that the new Babesia sp. was apparently included in the genus Babesia sensu stricto (i.e., large Babesia), but morphologically it was clearly distinguishable from B. divergens parasites and some other B. divergens-like parasites, such as MO-1 (5) and EU-1 (6), which are the sole species in the genus Babesia sensu stricto that have been confirmed to infect humans in Europe (4, 6) and, very recently, in the United States (7). The great resemblance of the morphologies of the intraerythrocytic parasites found in the Korean patient and those reported for the Babesia sp. from sheep in Gansu, China (1), is also noteworthy. Whether or not the patient was infected with parasites naturally maintained in sheep in Korea, however, must await further epizootiologic investigations, because our preliminary surveys have yet to provide any evidence of the natural vertebrate hosts or the ticks involved in transmission. Until enough knowledge is accumulated to give certain specific nomenclature, we temporarily designate this parasite strain KO1.

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FIG. 2. Phylogenetic analysis of the 18S rRNA gene sequences of Babesia sp. strain KO1 (newly found in the Korean index case patient) and closely related Babesia spp. The phylogenetic tree was built by using the neighbor-joining method. Multiple-sequence alignment was carried out by use of the ClustalW alignment program in the MacVector software package, and the aligned sequence was used to construct phylogenetic trees by using the Phylogenetic Analysis program in the same software package. The numbers on the nodes indicate the percentage of replicates of 1,000 samplings in which the given branching pattern was obtained. The scale bar indicates 0.02 nucleotide substitutions per site. The designations in brackets are GenBank accession numbers.

We conducted further epidemiological surveys with blood samples from the residents in the patient's village, Gurae, in southwestern South Korea. For this survey we were able to collect blood samples from 68 residents (30 males and 38 females; age range, 37 to 100 years), and we extracted DNA for parasite detection by PCR. PCR amplification was done with primers Bab5 and Bab8 for the first round of PCR and primers Bab6 and Bab7 for the second round of PCR. Three cases were confirmed to be positive (Fig. 3). Sequencing analysis of the three PCR products showed sequences identical to those detected in the patient (KO1) (data not shown). These results indicate that there may be some asymptomatic carriers in this village. We have also attempted to detect the Babesia parasite in 17 goats raised in the village, but unfortunately, no positive signal was detected.

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FIG. 3. PCR amplification for detection of Babesia spp. from the inhabitants in the village where the patient lives (lanes 1 to 7). DNA from the patient (lane P) was used as a positive control. The products of the second PCR with primers Bab6 and Bab7 are shown. Lanes M, molecular size marker.

Babesiosis, caused by infection with parasites of the genus Babesia, is one of the most ubiquitous infections in wild and domestic animals worldwide. The disease is transmitted by ixodid ticks to vertebrate hosts. Recently, it has increasingly gained attention as an emerging zoonosis in humans (4, 8). Two main species of Babesia parasites, namely, B. microti (so-called small Babesia) and B. divergens (large Babesia, or the genus Babesia sensu stricto) have been known to be involved in human infections in the United States and Europe, respectively (4, 6, 7). In addition, a newly emerging Babesia species, referred to as WA1 (13, 16) and CA1 (11), that causes human babesiosis has been reported and was recently identified as B. duncani sp. nov. in the United States (3). In East Asia, cases of human babesiosis have been reported in Taiwan (15) and Japan (14). Both of these were caused by B. microti-like parasites, which often appear asymptomatically. Although field surveys of wild rodents and cattle have suggested the presence of Babesia parasites in Korea (2, 19), human babesiosis has not yet been reported. In this study we report the first case of human babesiosis in Korea, which was found to be a novel large Babesia parasite infecting a human and which was nearly fatal.

In conclusion, this is the first report of human babesiosis in Korea which was apparently caused by a hitherto unidentified, new type of Babesia sp. capable of infecting humans. The sequence of this new parasite is similar (98%) to that of Babesia spp. detected from sheep in China. Research collaboration will apparently be needed to further investigate the interrelationship between the parasites found in the Korean index case patient and those reported from sheep in China.

Nucleotide sequence accession number. The 18S rRNA gene sequence of strain KO1 can be found in the GenBank database under accession number DQ346955.

 
This work was supported by an intramural grant from the National Institute of Health, Korea (NIH-347-6111-158).

 
* Corresponding author. Mailing address: Division of Malaria and Parasitic Diseases, National Institute of Health, Korean Centers for Disease Control and Prevention, Seoul 122-701, Korea. Phone: 82-2-380-1505. Fax: 82-2-380-1560. E-mail: nihkim{at}nih.go.kr

Published ahead of print on 28 March 2007.

J.-Y. Kim and S.-H. Cho have contributed equally to the work.

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Journal of Clinical Microbiology, June 2007, p. 2084-2087, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.01334-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, J.-Y. Articles by Kim, T.-S. Search for Related Content PubMed PubMed Citation Articles by Kim, J.-Y. Articles by Kim, T.-S.