Molecular epidemiology of Orientia tsutsugamushi in Cambodia and Central Vietnam reveals a broad region-wide genetic diversity Veasna Duong a , Trinh Thi Xuan Mai b , Kim Blasdell a , Le Viet Lo b , Claire Morvan a , Sreyrath Lay a , Wichittra Anukool c , Patimaporn Wongprompitak d , Yupin Suputtamongkol e , Denis Laurent f , Beat Richner f , Chheang Ra g , Bui Trong Chien b , Roger Frutos h , Philippe Buchy a,⇑ a Institut Pasteur in Cambodia, Virology Unit, 5 Monivong Blvd., Phnom Penh, Cambodia b Institut Pasteur in Nha Trang, 8 Tran Phu street, Nha Trang, Viet Nam c Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand d Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand e Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand f Kantha Bopha Hospital, Street 61, Phnom Penh, Cambodia g Calmette Hospital, 3 Monivong Boulevard, Phnom Penh, Cambodia h CIRAD, UMR 17, TA A17/G, Campus International de Baillarguet, 34398 Montpellier Cedex 5, France article info Article history: Available online 15 January 2011 Keywords: Orientia tsutsugamushi 56-kDa type specific antigen gene Phylogenetic analysis Genetic diversity Cambodia Vietnam abstract Scrub typhus is an acute infectious disease caused by an obligate intracellular bacterium Orientia tsutsu- gamushi following the bite of infected trombiculid mites of the genus Leptotrombidium. This zoonotic dis- ease is a major cause of febrile illness in the Asia-Pacific region, with a large spectrum of clinical manifestations from unapparent or mild disease to fatal disease. O. tsutsugamushi is characterized by a very high genomic plasticity and a large number of antigenic variants amongst strains. The 56-kDa type specific antigen (TSA) gene, encoding the major antigenic protein, was used as reference to investigate the genetic relationships between the strains and to genotype O. tsutsugamushi isolates. The open reading frame of the 56-kDa TSA gene of 41 sequences (28 Cambodian and 13 Vietnamese strains) from patient samples were sequenced and used for genotyping. The 28 Cambodian isolates clustered into 5 major groups, including Karp (43.5%), JG-v (25%), Kato/TA716 (21.5%), TA763 (3.5%) and Gilliam (3.5%). Karp (77%), TA763 (15.5%) and JG-v (7.5%) strains were identified amongst the 13 Vietnamese isolates. This is the first countrywide genotyping description in Cambodia and in Central Vietnam. These results dem- onstrate the considerable diversity of genotypes in co-circulation in both countries. The genotyping result might raise awareness amongst Cambodian and Vietnamese clinicians of the high genetic diversity of cir- culating O. tsutsugamushi strains and provides unique and beneficial data for serological and molecular diagnosis of scrub typhus infections as well as raw materials for future studies and vaccine development. Ó 2011 Elsevier B.V. All rights reserved. 1. Introduction Scrub typhus is an acute infectious disease caused by the bacte- rium Orientia tsutsugamushi following the bite of infected trombic- ulid mites of the genus Leptotrombidium (Traub and Wisseman, 1974). This zoonotic disease is transmitted to rodents and eventu- ally to humans who are dead-end accidental hosts (Duong et al., 2010). Clinical presentation in humans can vary from mild or unap- parent disease, to fatal disease in the absence of appropriate antibi- otic treatment, and the severity of the disease appears to be strain dependant. O. tsutsugamushi is an obligate intracellular Gram- negative bacterium from the family of Rickettsiaceae. As antigenic variation between Orientia strains is very high, information on strain prevalence is important for development of serodiagnostic tests and vaccines. Characterization of Orientia strains was initially performed using various serological methods such as complement fixation (CF), direct fluorescent antibody assay and indirect fluores- cent antibody assay (Bozeman and Elisberg, 1963; Shirai et al., 1979; Shirai and Wisseman, 1975; Shishido, 1964). In 1964, Shishido identified 3 unique prototype strains, namely Karp, Gilliam and Kato, using CF (Shishido, 1964) and later Elisberg et al. (1967) described 5 new serologically unique strains in Thailand: TA678, TA686, TA716, TA763 and TH1817. More recently, O. tsutsugamushi strains were classified into 5 major serotypes: Boryong, Gilliam, Karp, Kato and Kawazaki (Tamura et al., 1995). However, these serological techniques require comparison with well-characterized prototypes bacteria available but the Orientia species are difficult to cultivate and require biosafety-level 3 laboratory facilities. 1567-1348/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2011.01.004 ⇑ Corresponding author. Tel.: +855 12802982. E-mail address: pbuchy@pasteur-kh.org (P. Buchy). Infection, Genetics and Evolution 15 (2013) 35–42 Contents lists available at SciVerse ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid