title: Use of Multiplex Real-Time PCR To Diagnose Scrub Typhus.
abstract: Scrub typhus, caused byOrientia tsutsugamushi, is a common cause of acute undifferentiated febrile illness in the Asia-Pacific region. However, its nonspecific clinical manifestation often prevents early diagnosis. We propose the use of PCR and serologic tests as diagnostic tools. Here, we developed a multiplex real-time PCR assay using hydrolysis (TaqMan) probes targetingO. tsutsugamushi47-kDa,groEL, and human interferon beta (IFN-β gene) genes to improve early diagnosis of scrub typhus. The amplification efficiency was higher than 94%, and the lower detection limit was 10 copies per reaction. We used a human gene as an internal DNA quality and quantity control. To determine the sensitivity of this PCR assay, we selected patients with confirmed scrub typhus who exhibited a clear 4-fold increase in the level of IgG and/or IgM. The PCR assay result was positive in 45 of 52 patients, indicating a sensitivity of 86.5% (95% confidence interval [CI]: 74.2 to 94.4). The PCR assessment was negative for all 136 non-scrub typhus patients, indicating a specificity of 100% (95% CI: 97.3 to 100). In addition, this test helped diagnose patients with inconclusive immunofluorescence assay (IFA) results and using single blood samples. In conclusion, the real-time PCR assay proposed here is sensitive and specific in diagnosing scrub typhus. Combining PCR and serologic tests will improve the diagnosis of scrub typhus among patients presenting with acute febrile illness.
authors.affiliation: Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand wiwit.tan@mahidol.ac.th yupin.sup@mahidol.ac.th.
authors.forename: Wiwit
authors.initials: W
authors.lastname: Tantibhedhyangkul
authors.affiliation: Division of Infectious Diseases and Tropical Medicine, Department of Internal Medicine, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Ekkarat
authors.initials: E
authors.lastname: Wongsawat
authors.affiliation: Division of Infectious Diseases and Tropical Medicine, Department of Internal Medicine, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Saowaluk
authors.initials: S
authors.lastname: Silpasakorn
authors.affiliation: Division of Infectious Diseases and Tropical Medicine, Department of Internal Medicine, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Duangdao
authors.initials: D
authors.lastname: Waywa
authors.affiliation: Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Nuttawut
authors.initials: N
authors.lastname: Saenyasiri
authors.affiliation: Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Jintapa
authors.initials: J
authors.lastname: Suesuay
authors.affiliation: Maharat Nakhon Ratchasima Hospital, Nakhon Ratchasima Province, Thailand.
authors.forename: Wilawan
authors.initials: W
authors.lastname: Thipmontree
authors.affiliation: Division of Infectious Diseases and Tropical Medicine, Department of Internal Medicine, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand wiwit.tan@mahidol.ac.th yupin.sup@mahidol.ac.th.
authors.forename: Yupin
authors.initials: Y
authors.lastname: Suputtamongkol
mesh_terms: D000328:Adult; D000907:Antibodies, Bacterial / Q000097:blood; D058491:Bacterial Load / Q000379:methods; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics*; D018834:Chaperonin 60 / Q000032:analysis / Q000235:genetics*; D005334:Fever / Q000175:diagnosis / Q000382:microbiology; D005784:Gene Amplification; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D007075:Immunoglobulin M / Q000097:blood; D016899:Interferon-beta / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D012285:Orientia tsutsugamushi / Q000235:genetics* / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012612:Scrub Typhus / Q000175:diagnosis* / Q000382:microbiology; D012680:Sensitivity and Specificity; D058248:Serum Bactericidal Antibody Assay / Q000379:methods*
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D018834:Chaperonin 60; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D016899:Interferon-beta
keywords: Orientia tsutsugamushi; real-time PCR; scrub typhus