New Genotypes of Orientia tsutsugamushi Isolated from Humans in Eastern Taiwan

Chen L-K (2012) New Genotypes of Orientia tsutsugamushi Isolated from Humans in Eastern Taiwan. PLoS ONE 7(10): e46997. doi:10.1371/journal.pone.0046997 New Genotypes of Orientia tsutsugamushi Isolated from Humans in Eastern Taiwan Hui-Hua Yang 0 I-Tsong Huang 0 Chin-Hui Lin 0 Tren-Yi Chen 0 Li-Kuang Chen 0 Ulrike Gertrud Munderloh, University of Minnesota, United States of America 0 1 Institute of Medical Sciences, Tzu Chi University , Hualien, Taiwan , 2 Contract Laboratory of Viral and Rickettsial Infection, Buddhist Tzu Chi General Hospital , Hualien, Taiwan , 3 Emerging Infectious Pathogen Research Laboratory, Buddhist Tzu Chi General Hospital , Hualien, Taiwan , 4 Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital , Hualien, Taiwan , 5 School of Medicine of Chung Shan Medical University , Taichung City, Taiwan , 6 Emergency Medicine Buddhist Tzu Chi General Hospital , Hualien , Taiwan Scrub typhus, an acute febrile illness, is caused by the obligate intracellular bacterium Orientia tsutsugamushi. In our study, O. tsutsugamushi was rapidly detected and typed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis of the 56-kDa type-specific antigen (TSA) gene. To investigate the genotypes of clinical variants of O. tsutsugamushi, we collected 3223 blood samples from eastern Taiwanese patients with suspected scrub typhus from 2002 to 2008. In total, 505 samples were found to be positive for scrub typhus infection by PCR, and bacteria were isolated from 282 of them. Four prototype genotype strains (Karp, Kato, Kawasaki and Gilliam) and eleven different Taiwanese genotype isolates (Taiwan-A, -B, -C, -D, -E, -G, -H, -J, -N, -O and -P) were identified by RPLF analysis. Taiwan-H, the major genotype in eastern Taiwan, exhibited prevalence and isolation rates of 47.3% (239/505) and 42.6% (120/282), respectively. We also assessed the genetic relatedness of the 56-kDa TSA gene among eight Taiwan-H isolates, thirteen other Taiwanese isolates and 104 DNA sequences deposited in the GenBank database using MEGA version 5.0 and PHYLIP version 3.66. We found that the Taiwan-H isolates formed into a new cluster, which was designated the Taiwan Gilliamvariant (TG-v) cluster to distinguish it from the Japanese Gilliam-variant (JG-v) cluster. According to Simplot analysis, TG-v is a new recombinant strain among Gilliam, Ikeda and Kato. Moreover, the Gilliam-Kawasaki cluster had the highest percentage of RFLP cases and was the most frequently isolated type in eastern Taiwan (50.1%, 253/505; 44.0%, 124/282). These findings shed light on the genetic evolution of O. tsutsugamushi into different strains and may be useful in vaccine development and epidemic disease control in the future. - Funding: This work was supported by grants from the Centers for Disease Control, R.O.C (Taiwan) (DOH 92-DC-1205, 94x007, DOH 91-DC-1085, 92-DC-1073, 93DC-1022, CDC 94-RM-101, 95-RD-014). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Scrub typhus (tsutsugamushi disease), an acute febrile illness, is caused by the obligate intracellular bacterium Orientia tsutsugamushi, which is transmitted by the bite of the trombiculid mite (or its larva, the chigger) [1]. The disease is a public health concern with approximately one billion people are at a risk of infection by the bacterium in the geographical triangle (Figure 1) extending from northern Japan in the east to Pakistan and Afghanistan in the west and northern Australia in the south [2]. Scrub typhus is a reportable infectious disease in Taiwan [3], and thousands of patients are suspected of scrub typhus infection annually. The related syndromes include meningitis, eschar, disseminated intravascular coagulation and multiorgan failure [3]. The fatality rate can reach to 50% in the absence of suitable treatment; however, scrub typhus can be treated effectively with antibiotics [4]. No effective vaccine is currently available for the prevention of scrub typhus [5,6,7], and vaccine development has been impeded by the lack of availability of a determinant sequence or a systematic study of O. tsutsugamushi serotypes and genotypes, particularly clinical variants. Six antigenic variants of O. tsutsugamushi have been identified previously by different groups. Shishido et al. reported three prototype strains for the Gilliam, Karp and Kato strains [8]. Another three prototype strains Shimokoshi, Kawasaki and Kurokiisolated from patients in Japan were demonstrated to belong to different antigenic types by Ohashi et al. [9,10,11]. Using the outer membrane 56-kDa type specific antigen (TSA) protein gene, several investigators in Japan, Korea, Taiwan, Thailand, Australia and China have characterized antigenic variation of O. tsutsugamushi, from patients, wild rodents and trombiculid mites in different endemic areas. The 56kDa antigen is usually strain specific, but shows cross-reactivity to some monoclonal and polyclonal antibodies [9,12,13,14]. Most clinical isolates are antigenically identical but some differ from their prototypes [12,14,15] and cannot be classified or associated antigenicly with any prototype strains. Genotyping of different clinical isolates had shown to overcome these problems [13,14,16,17]. The first reported 56-kDa TSA nucleotide sequence of O. tsutsugamushi was that of the Karp strain [18]. Later, Tamura et al. used the nucleotide variation in the 56-kDa TSA gene to classify O. tsutsugamushi strains into the following subtypes: Gilliam [19], Japanese Gilliam (JG) [13], Karp [19], Japanese Karp type1(JP-1) [14], Japanese Karp type-2 (JP-2) [13,14], Kato [19,20], Kawasaki [20], Kuroki [10,20], Shimokoshi [20] and TW46-1 [15]. In addition, the 56-kDa TSA gene, consisting of four variant domains, has been a target for molecular detection and phylogenetic analysis [6,12,13,15]. Immunofluorescent antibodies have been the traditional method to analyze the variation in the 56-kDa TSA gene of O. tsutsugamushi in Taiwan [12,15,16]. Some studies have used the polymerase chain reaction (PCR) followed by the restriction fragment length polymorphism (RFLP) analysis to further characterize nucleotide sequence variation in the gene [12,15,17,21]. Six different Taiwanese genotypes, Taiwan-A, -B, -C, -D, -E and -F, were characterized by PCR-RFLP analysis of the variant domain I (VD-I) of the 56-kDa TSA gene at the Taiwan Centers for Disease Control (CDC) in 1999 [21]. Eastern Taiwan has the highest prevalence of scrub typhus in Taiwan. From 2002 to 2008, 3223 blood samples from patients with suspected scrub typhus were collected in eastern Taiwan to investigate the genotypes of clinical variants of O. tsutsugamushi. Nested PCR, performed by the Taiwan CDC-contracted laboratory at Tzu Chi Ge (...truncated)