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Genotyping of Orientia Tsutsugamushi from Humans with Scrub Typhus

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Genotyping of Orientia Tsutsugamushi from Humans with Scrub Typhus LETTERS Acknowledgments viron Microbiol. 2004;70:3733–5. DOI: scrub typhus (3). Two amplifi cation We thank the following persons and 10.1128/AEM.70.6.3733-3735.2004 reactions were performed, a real-time institutions for their assistance in obtain- quantitative PCR with a probe target- Address for correspondence: Julie R. Sinclair, ing and identifying specimens: Amy Han- ing the O. tsutsugamushi 47-kDa outer CDC Quarantine Station–Philadelphia, P.O. cock-Ronemus, Paul Mead, Ted Nuttall, membrane protein gene with appropri- Box 144, Essington, PA 19029, USA; email: Brigette Husband, and Kerry Pollard; the ate primers and probes (4) and a stan- [email protected] Department of Parasitology, College of dard PCR targeting a 372-nt fragment Veterinary Medicine, University of Penn- of the 56-kDa protein gene (3). sylvania; the Schuylkill Wildlife Rehabili- Buffy coat samples from 11 tation Center; and the Philadelphia Depart- (17.5%) patients were positive for O. ment of Public Health. tsutsugamushi in the real-time quanti- tative PCR and 56-kDa antigen gene Julie R. Sinclair, Alisa Newton, PCR (Table). All 11 patients were Keith Hinshaw, George Fraser, Genotyping from Vientiane or Vientiane Prov- Patrina Ross, Esther Chernak, of Orientia ince. PCR products for the 56-kDa Caroline Johnson, tsutsugamushi from gene fragments were purifi ed and se- quenced as described (3). Comparison and Nancy Warren Humans with Scrub Author affi liations: Centers for Disease (3,5) of amplicons for the 11 patients Control and Prevention, Atlanta, Geor- Typhus, Laos with each other and with GenBank gia, USA (J.R. Sinclair); Philadelphia Zoo, sequences identifi ed 6 genotypes. Per- To the Editor: Rickettsial dis- Philadelphia, Pennsylvania, USA (A. New- centages of nucleotide sequence simi- eases have been only recently identi- ton, K. Hinshaw); Pennsylvania Bureau of larity with other sequences available in fi ed as underrecognized but important Laboratories, Lionville, Pennsylvania, USA GenBank ranged from 95.9% to 100% causes of fever of unknown origin (G. Fraser, N. Warren); and Philadelphia (Table). Interpretation of our results in Laos. In 2006, 63 (14.8%) of 427 Department of Public Health, Philadelphia was also supported by recent phyloge- adults with negative blood cultures (P. Ross, E. Chernak, C. Johnson) netic studies that compared sequences admitted to Mahosot Hospital in Vien- of the entire 56-kDa type-specifi c anti- DOI: 10.3201/eid1409.071690 tiane had scrub typhus, an infection gen gene of isolates from Thailand (6). caused by Orientia tsutsugamushi and LaoUF238 and LaoUF220 genotypes References transmitted by the bite of larval trom- clustered with those of strains related biculid mites (1). O. tsutsugamushi to the Karp serotype, and LaoUF136 1. Farlow J, Wagner DM, Dukerich M, Stan- is characterized by a wide antigenic ley M, Chu M, Kubota K, et al. Francisella and LaoUF187 clustered with geno- tularensis in the United States. Emerg In- diversity, and isolates are convention- types of strains related to the Gilliam fect Dis. 2005;11:1835–41. ally classifi ed on the basis of reactiv- serotype (2). Other genotypes found in 2. Feldman KA. Tularemia. J Am Vet Med ity with hyperimmune serum against this study were grouped in 2 clusters Assoc. 2003;222:725–30. DOI: 10.2460/ prototype strains (e.g., Karp, Kato, javma.2003.222.725 that contained genotypes identifi ed in 3. Dennis DT, Inglesby TV, Henderson HA, Gilliam, Kawasaki, Kuroki, or Shimo- Thailand (5) and Taiwan (7) that have Bartlett JG, Ascher MS, Eitzen E, et al. goshi). The 4 hypervariable regions not been linked to a reference serotype Tularemia as a biological weapon. JAMA. within the 56-kDa type-specifi c anti- (Table). 2001;285:2763–73. DOI: 10.1001/jama. gen of O. tsutsugamushi, which is lo- 285.21.2763 Detection of O. tsutsugamushi in 4. Anda P, Segura del Pozo J, Diaz Garcia cated on the outer membrane surface, humans in Laos provides useful infor- JM, Escudero R, Garcia Pena FJ, Lopez are considered to play an essential role mation on genotypes prevalent in this Velasco MC, et al. Waterborne outbreak of in type strain assignment (2). country. Our results were confi rmed tularemia associated with crayfi sh fi shing. In the Lao study (1), in addition Emerg Infect Dis. 2001;7:575–82. by using 2 target genes in 2 PCRs. No 5. Dvorak P. Health offi cials vigilant for ill- to acute-phase serum samples, a 5-mL differences were found between the ness after sensors detect bacteria on mall. blood sample anticoagulated with number of days of fever in 11 PCR- Washington Post. 2005 Oct 2 [cited 2008 EDTA was collected at admission from positive patients and number of days Mar 26]. Available from http://www. all patients. After centrifugation, buffy washingtonpost.com/wp-dyn/content/ of fever in 52 PCR-negative patients. article/2005/10/01/AR2005100101209. coat of the serum sample was removed However, the PCR-negative patients html and stored at –80°C (1). DNA was ex- may not have had bacteremia at the 6. Petersen JM, Schriefer ME, Gage KL, tracted from buffy coat samples of 63 time of sample collection. Montenieri JA, Carter LG, Stanley M, et patients whose conditions were diag- al. Methods for enhanced culture recov- Diversity of O. tsutsugamushi ery of Francisella tularensis. Appl En- nosed by imunofl uorescence assay as genotypes found in Laos includes Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 9, September 2008 1483 LETTERS genotypes closely related to genotypes gene in isolates from Laos might limit 56-kDa protein–encoding gene and from Thailand and Taiwan. This diver- sensitivity and specifi city of serologic of other genes of O. tsutsugamushi sity raises doubt about usual concepts methods. A recent study showed that would help to better characterize the because it has been thought that O. addition of a serotype to the panel of new genotypes identifi ed in our study tsutsugamushi genotypes are restrict- O. tsutsugamushi antigens used for and their relationship with known se- ed to specifi c geographic areas and to testing improved sensitivity of anti- rotypes. Expanding the panel of anti- specifi c mite vectors (8). Furthermore, body detection in patients in Thailand gens used to test patients suspected of these results might have clinical reper- (10). We demonstrated that, in analy- having scrub typhus to take into ac- cussions because sequence variations sis of sera in the diagnosis of scrub count local antigenic diversity would within the 56-kDa protein gene corre- typhus contracted in Laos, antigen improve sensitivity of serologic as- late with antigenic diversity of geno- pools should contain at least Karp and says for this disease. types of O. tsutsugamushi. This fi nd- Gilliam strain antigens. Furthermore, ing is supported by data for sequences new genotypes identifi ed in patients Acknowledgments of the entire 56-kDa gene of different in Laos might be related to previously We thank Khalid El Karkouri for help isolates (6) and for monoclonal and unrecognized type strains. However, with phylogenetic studies; the patients, human and animal polyclonal antibod- cross-reactivity with Gilliam, Kato, Vimone Soukkhaseum, Khamphong Phi- ies used to map antigenic differences and Kawasaki serotypes enabled se- asakha, Surn Soukkhaseum, Khamthavi among isolates with known sequence rologic diagnosis in the initial study, Frichithavong, Vang Chu, Valy Keol- variations (9). including 1 patient infected with a ouangkhot, Bertrand Martinez-Aussel, Although our data are prelimi- Karp-related bacteria (1). Ko Chang, Chirapha Darasavath, Oud- nary, diversity of nucleotide sequenc- Phylogenetic studies based on ayvone Rattanavong, Siho Sisouphone, es of the 56-kDa protein–encoding larger fragments of sequences of the Mayfong Mayxay, Sisouphane Vidamaly, Table. Serologic results for 11 patients from Laos positive by real-time quantitative PCR for the 47-kDa outer membrane protein and a PCR for a fragment of the 56-kDa protein–encoding gene of Orientia tsutsugamushi* GenBank accession no. Highest % identity with IgG/IgM titers, acute phase–late phase of 56-kDa–amplified GenBank sequences Patient Gilliam Kato Kawasaki gene fragment (reference) LaoUF74 64/1,048– 128/256–128/256 128/256–128/256 EU168798 These 4 genotypes showed 64/1,048 100% identity with each other LaoUF317 2,048/1,024–ND 128/1,024–ND 2,048/1,024–ND EU168799 and 96.6% with strain BB23 LaoUF366 2,048/512– 128/32–128/32 2,048/512– EU168797 from Thailand (5) 2,048/512 2,048/512 LaoUF396 256/1,024– 128/64–128/64 256/1,024– EU168796 256/1,024 256/1,024 LaoUF220 2,048/512– 2,048/512– 1,024/256– EU168801 These 2 genotypes showed 2,048/1,024 2,048/512 2,048/512 100% identity with UT150 and LaoUF244 1,024/128–ND 256/64–ND 128/64–ND EU168800 UT332, which are related to Karp serotype isolates (6), BB29 from patients in Thailand (5), and TWyU81 from chiggers in Taiwan (7) LaoUF83 128/1,024–ND 0/1,024–ND 0/128–ND EU168795 95.9% with BB23 from Thailand (5) LaoUF136 2,048/1,024– 2,048/1,024– 2,048/1,024– EU168804 99.5% with UT144, UT125, and 2,048/1,024 2,048/1,024 2,048/1,024 UT196 from Thailand, which are related to Gilliam serotype isolates (6) LaoUF186 128/1,024– 128/1,024– 128/1,024– EU168805 100% with BB23 from 2,048/1,024 2,048/1,024 2,048/1,024 Thailand (5) LaoUF187 256/1,024– 256/0–256/0 0/0–256/1,024 EU168803 100% with UT144, UT125, and 256/1,024 UT196 from Thailand (6) LaoUF238 2,048/1,024– 2,048/1,024– 0/512–256/512 EU168802 100% with UT219, UT395, 2,048/1,024 2,048/1,024 UT221, and UT213, which are related to Karp serotype–related isolates (7), and FPW2031 from Thailand (6) *Ig, immunoglobulin; ND, not done.
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