• 1.

    Groves MG, Harrington KS, 1994. Scrub typhus. Beran GW, ed. Handbook of Zoonoses. Section A: Bacterial, Rickettsial, Chlamydial, and Mycotic Zoonoses. 2nd ed. Boca Raton, FL: CRC Press, 463474.

    • Search Google Scholar
    • Export Citation
  • 2.

    Silpapojakul K, Varachit B, Silpapojakul K, 2004. Paediatric scrub typhus in Thailand: a study of 73 confirmed cases. Trans R Soc Trop Med Hyg 98: 354359.

    • Search Google Scholar
    • Export Citation
  • 3.

    Kelly DJ, Wong PW, Gan E, Lewis GE, 1988. Comparative evaluation of the indirect immunoperoxidase test for the serodiagnosis of rickettsial disease. Am J Trop Med Hyg 38: 400406.

    • Search Google Scholar
    • Export Citation
  • 4.

    Blacksell SD, Bryant NJ, Paris DH, Doust JA, Sakoda Y, Day NPJ, 2007. Scrub typhus serologic testing with the indirect immunofluorescence method as a diagnostic gold standard: a lack of consensus leads to a lot of confusion. Clin Infect Dis 44: 391401.

    • Search Google Scholar
    • Export Citation
  • 5.

    Kelly DJ, Fuerst PA, Ching W, Richards AL, 2009. Scrub typhus: the geographic distribution of phenotypic and genotypic variants of Orientia tsutsugamushi. Clin Infect Dis 48: S203S230.

    • Search Google Scholar
    • Export Citation
  • 6.

    Miesse M, Diercks F, Danauskas J, 1950. Strain differences among Rickettsia tsutsugamushi. Bacteriological Proc M45: 9091.

  • 7.

    Ree HI, Kim TE, Lee IY, Jeon SH, Hwang UW, Chang WH, 2001. Determination and geographical distribution of Orientia tsutsugamushi serotypes in Korea by nested polymerase chain reaction. Am J Trop Med Hyg 65: 528534.

    • Search Google Scholar
    • Export Citation
  • 8.

    Anonymous, 2001. Scrub typhus (Tsutsugamushi disease) in Japan, 1996–2000. Byogen Biseibutsu Kenshutsu Joho Geppo 22: 211212.

  • 9.

    Luksameetanasan R, Blacksell SD, Kalambaheti T, Wuthiekanun V, Chierakul W, Chueasuwanchai S, Apiwattanaporn A, Stenos J, Graves S, Peacock SJ, Day NPJ, 2007. Patient and sample-related factors that effect the success of in vitro isolation of Orientia tsutsugamushi. Southeast Asian J Trop Med Public Health 38: 9196.

    • Search Google Scholar
    • Export Citation
  • 10.

    Casleton BG, Salata K, Dasch GA, Strickman D, Kelly DJ, 1998. Recovery and viability of Orientia tsutsugamushi from packed red cells and the danger of acquiring scrub typhus from blood transfusion. Transfusion 38: 680689.

    • Search Google Scholar
    • Export Citation
  • 11.

    Kim DM, Kim HL, Park CY, Yang TY, Lee JH, Yang JT, Shim SK, Lee SH, 2006. Clinical usefulness of eschar polymerase chain reaction for the diagnosis of scrub typhus: a prospective study. Clin Infect Dis 43: 12961300.

    • Search Google Scholar
    • Export Citation
  • 12.

    Saisongkorn W, Chenchittikul M, Silpapojakul K, 2004. Evaluation of nested PCR for the diagnosis of scrub typhus among patients with acute pyrexia of unknown origin. Trans R Soc Trop Med Hyg 98: 360366.

    • Search Google Scholar
    • Export Citation
  • 13.

    Paris DH, Blacksell SD, Newton PN, Day NPJ, 2008. Simple, rapid and sensitive detection of Orientia tsutsugamushi by loop-isothermal DNA amplification. Trans R Soc Trop Med Hyg 102: 12391246.

    • Search Google Scholar
    • Export Citation
  • 14.

    Furuya Y, Yoshida Y, Katayama T, Yamamoto S, Kawamura A, 1993. Scrub typhus. J Clin Microbiol 31: 16371640.

  • 15.

    Sonthayanon P, Chierakul W, Wuthiekanun V, Blacksell SD, Pimda K, Suputtamongkol Y, Pukrittayakamee S, White NJ, Day NPJ, Peacock SJ, 2006. Rapid diagnosis of scrub typhus in rural Thailand using polymerase chain reaction. Am J Trop Med Hyg 75: 10991102.

    • Search Google Scholar
    • Export Citation
  • 16.

    Sonthayanon P, Chierakul W, Wuthiekanun V, Phimda K, Pukrittayakamee S, Day NPJ, Peacock SJ, 2009. Association of high Orientia tsutsugamushi DNA loads with disease of greater severity in adults with scrub typhus. J Clin Microbiol 47: 430434.

    • Search Google Scholar
    • Export Citation
  • 17.

    Jiang J, Chan T-C, Temenak JJ, Dasch GA, Ching W-M, Richards AL, 2004. Development of a quantitative real-time polymerase chain reaction assay specific for Orientia tsutsugamushi. Am J Trop Med Hyg 70: 351356.

    • Search Google Scholar
    • Export Citation
  • 18.

    Paris DH, Aukkanit N, Jenjaroen K, Blacksell SD, Day NPJ, 2009. A highly sensitive quantitative real-time PCR assay based on the groEL gene of contemporary Thai strains of Orientia tsutsugamushi. Clin Microbiol Infect 15: 488495.

    • Search Google Scholar
    • Export Citation
  • 19.

    Bozeman FM, Elisberg BL, 1963. Serological diagnosis of scrub typhus by indirect immunofluorescence. Proc Soc Exp Biol Med 112: 568573.

  • 20.

    Pradutkanchana J, Silpapojakul K, Paxton H, Pradutkanchana S, Kelly DJ, Strickman D, 1997. Comparative evaluation of four serodiagnostic tests for scrub typhus in Thailand. Trans R Soc Trop Med Hyg 91: 425428.

    • Search Google Scholar
    • Export Citation

 

 

 

 

Diagnosis of Scrub Typhus

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  • 1 Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Department of Medicine, University of Cambridge, Cambridge, United Kingdom; Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Centre for Clinical Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom; Wellcome Trust–Mahosot Hospital–Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Vientiane, Lao People's Democratic Republic

Scrub typhus is transmitted by trombiculid mites and is endemic to East and Southeast Asia and Northern Australia. The clinical syndrome classically consists of a fever, rash, and eschar, but scrub typhus also commonly presents as an undifferentiated fever that requires laboratory confirmation of the diagnosis, usually by indirect fluorescent antibody (IFA) assay. We discuss the limitations of IFA, debate the value of other methods based on antigen detection and nucleic acid amplification, and outline recommendations for future study.

Scrub typhus (Orientia tsutsugamushi infection) is transmitted by the bite of larval trombiculid mites and is endemic to the land mass within the triangle bounded by Japan to the north, Northern Australia to the south, and the Arabian Peninsula to the west. Mortality in the pre-antibiotic era was variable and in some series, approached 60%,1 but specific and effective antimicrobial chemotherapy is now available.

Scrub typhus often presents as fever with little to distinguish it clinically from co-endemic diseases such as typhoid, leptospirosis, and dengue. The presence of an eschar supports the diagnosis but is variably present.2 Diagnosis, therefore, depends on clinical suspicion, prompting the clinician to request an appropriate laboratory investigation, and failure to diagnose the disease will likely result in treatment with ineffective β-lactam–based regimens.

The mainstay in scrub-typhus diagnostics remains serology. The oldest test in current use is the Weil–Felix OX-K agglutination reaction, which is inexpensive, easy to perform, and results are available overnight; however, it lacks specificity and sensitivity3 (Table 1). The indirect fluorescent antibody (IFA) test is more sensitive, and results are available in a couple of hours; however, the test is more expensive and requires considerable training (Table 1). IFA uses fluorescent anti-human antibody to detect specific antibody from patient serum bound to a smear of scrub-typhus antigen and is currently the reference standard.4 Indirect immunoperoxidase (IIP) eliminates the expense of a fluorescent microscope by substituting peroxidase for fluorescein (Table 1).3

All currently available serological tests for scrub typhus have limitations in which clinicians need to be aware, despite their widespread use. Although agreement exists that a ≥ 4-fold increase in antibody titer between two consecutive samples is diagnostic,4 such a diagnosis is retrospective and cannot guide initial treatment.

Diagnosis based on a single acute-serum sample requires using a cut-off antibody titer. Cut offs ranging from 1:10 to 1:400 are quoted, often with little corroborating evidence4 and without establishing titers in the healthy local population (necessary to distinguish background immunity from acute infection); that cut off is then used for all patients, irrespective of whether or not they come from a scrub-typhus–endemic environment. Although IFA may be modified to report separate IgG and IgM titers, there is no consensus on when this is useful or how to interpret the results. Currently available rapid bedside tests are based on serological methods and share the same inherent problems as IFA.

Most frequently, IFA uses antigen from just three serotypes: Karp, Kato, and Gilliam. Yet, enormous antigenic variation has been found everywhere where it has been sought.5 Eight different serotypes were found in mites from a single field in Malaysia.6 In South Korea, > 75% of isolates are of the Boryong serotype.7 On the Japanese island of Kyushu, > 90% of the disease reported is of the Kawasaki or Kuroki serotypes.8 The Infectious Disease Surveillance Center in Japan, therefore, recommends a two-pronged approach to diagnosis. First, local strains are included in the IFA for each prefecture; second, PCR of the blood clot is performed on all specimens,8 although buffy coat might be preferable. This recommendation is not widely implemented outside of Japan.4

Isolating O. tsutsugamushi requires biosafety level-3 facilities and culture on cell monolayers; median time to positivity is 27 days.9 Mouse inoculation is even more laborious and intensive on resources.10 Current methods of isolation are, therefore, not appropriate for the routine diagnosis of scrub typhus. There is an urgent need for alternative diagnostic methods; however, evaluation is hampered, because the current gold standard (IFA) is imperfect. In a Korean polymerase chain reaction (PCR) study of eschars, O. tsutsugamushi DNA was detected in six of seven patients who tested negative for scrub typhus by IFA but had eschars typical of scrub typhus.11 In a study from Thailand, 3 of 20 (15%) patients with fever had positive O. tsutsugamushi PCR, despite negative serology.12 One recent study attempted to surmount this problem by evaluating the proposed test against a panel of serological and PCR-based methods,13 but PCR is itself beset with problems. The high resource costs and training required make it impractical for many areas where scrub typhus is endemic (Table 1). That aside, the most appropriate specimen to use remains unclear. PCR of eschar material is more sensitive than blood and remains positive even after the initiation of treatment.11 Unfortunately, in a setting where eschars are present in 7% of patients,2 for example, eschar-based tests can have a maximum sensitivity of only 7%. Using buffy coat could improve sensitivity compared with whole blood,13 but blood-based assays are positive only during the time window of rickettsemia. It the optimal PCR target remains unclear; nested PCR targeting the 56-kDa antigen has been shown to be highly specific,2,14 but sequence variability may affect primer annealing and therefore, test sensitivity.15 A whole-blood–based assay targeting the 16S rRNA gene showed a sensitivity of only 37.5–52.3% (95% confidence interval) in real-world conditions15,16 (probably because median copy number was only 13 copies/mL of blood16), but it performed better than the 56-kDa gene target in the same study (sensitivity = 22.5–36.1%); however, this may have been caused by differences in the assay rather than differences in the target genes.15 The 47-kDa outer-membrane protein is highly specific for O. tsutsugamushi,17 and species-specific primers also exist for the highly conserved molecular chaperone gene, groEL.13,18 But, it remains to be seen if either target will allay concerns about detecting infection caused by previously undescribed serotypes.

Loop isothermal amplification (LAMP) is a technique for amplifying DNA that makes use of three specially designed primer pairs and the Bst DNA polymerase. There is no complicated DNA extraction procedure, and unlike PCR, the entire reaction takes place at the same temperature. This means that only a water bath or heating block is required, whereas PCR requires a thermocycler. The reaction is read visually (a positive reaction produces a white pellet) and does not require special equipment (Table 1).13 A small proof-of-principle study (nine patients) showed that LAMP could detect DNA levels as low as 14 copies/µL compared with 3 copies/µL for real-time PCR).13 However, the technique has yet to be validated in a prospective clinical trial.

Clinicians will remain dependent on serological methods until these issues are resolved, but work can be done to optimize their performance. Cut offs must be validated locally, and previously undiscovered serotypes must be assiduously searched for by examining rodents and chiggers, not merely patient isolates. We propose that new diagnostic assays not be validated against IFA alone but instead, be compared against a panel of both serological and antigen-detection assays (e.g., IFA with 47-kDA and/or 56-kDa PCR).

Acknowledgment:

The authors thank Professors Sharon J. Peacock and Nicholas P. J. Day for their advice and support.

  • 1.

    Groves MG, Harrington KS, 1994. Scrub typhus. Beran GW, ed. Handbook of Zoonoses. Section A: Bacterial, Rickettsial, Chlamydial, and Mycotic Zoonoses. 2nd ed. Boca Raton, FL: CRC Press, 463474.

    • Search Google Scholar
    • Export Citation
  • 2.

    Silpapojakul K, Varachit B, Silpapojakul K, 2004. Paediatric scrub typhus in Thailand: a study of 73 confirmed cases. Trans R Soc Trop Med Hyg 98: 354359.

    • Search Google Scholar
    • Export Citation
  • 3.

    Kelly DJ, Wong PW, Gan E, Lewis GE, 1988. Comparative evaluation of the indirect immunoperoxidase test for the serodiagnosis of rickettsial disease. Am J Trop Med Hyg 38: 400406.

    • Search Google Scholar
    • Export Citation
  • 4.

    Blacksell SD, Bryant NJ, Paris DH, Doust JA, Sakoda Y, Day NPJ, 2007. Scrub typhus serologic testing with the indirect immunofluorescence method as a diagnostic gold standard: a lack of consensus leads to a lot of confusion. Clin Infect Dis 44: 391401.

    • Search Google Scholar
    • Export Citation
  • 5.

    Kelly DJ, Fuerst PA, Ching W, Richards AL, 2009. Scrub typhus: the geographic distribution of phenotypic and genotypic variants of Orientia tsutsugamushi. Clin Infect Dis 48: S203S230.

    • Search Google Scholar
    • Export Citation
  • 6.

    Miesse M, Diercks F, Danauskas J, 1950. Strain differences among Rickettsia tsutsugamushi. Bacteriological Proc M45: 9091.

  • 7.

    Ree HI, Kim TE, Lee IY, Jeon SH, Hwang UW, Chang WH, 2001. Determination and geographical distribution of Orientia tsutsugamushi serotypes in Korea by nested polymerase chain reaction. Am J Trop Med Hyg 65: 528534.

    • Search Google Scholar
    • Export Citation
  • 8.

    Anonymous, 2001. Scrub typhus (Tsutsugamushi disease) in Japan, 1996–2000. Byogen Biseibutsu Kenshutsu Joho Geppo 22: 211212.

  • 9.

    Luksameetanasan R, Blacksell SD, Kalambaheti T, Wuthiekanun V, Chierakul W, Chueasuwanchai S, Apiwattanaporn A, Stenos J, Graves S, Peacock SJ, Day NPJ, 2007. Patient and sample-related factors that effect the success of in vitro isolation of Orientia tsutsugamushi. Southeast Asian J Trop Med Public Health 38: 9196.

    • Search Google Scholar
    • Export Citation
  • 10.

    Casleton BG, Salata K, Dasch GA, Strickman D, Kelly DJ, 1998. Recovery and viability of Orientia tsutsugamushi from packed red cells and the danger of acquiring scrub typhus from blood transfusion. Transfusion 38: 680689.

    • Search Google Scholar
    • Export Citation
  • 11.

    Kim DM, Kim HL, Park CY, Yang TY, Lee JH, Yang JT, Shim SK, Lee SH, 2006. Clinical usefulness of eschar polymerase chain reaction for the diagnosis of scrub typhus: a prospective study. Clin Infect Dis 43: 12961300.

    • Search Google Scholar
    • Export Citation
  • 12.

    Saisongkorn W, Chenchittikul M, Silpapojakul K, 2004. Evaluation of nested PCR for the diagnosis of scrub typhus among patients with acute pyrexia of unknown origin. Trans R Soc Trop Med Hyg 98: 360366.

    • Search Google Scholar
    • Export Citation
  • 13.

    Paris DH, Blacksell SD, Newton PN, Day NPJ, 2008. Simple, rapid and sensitive detection of Orientia tsutsugamushi by loop-isothermal DNA amplification. Trans R Soc Trop Med Hyg 102: 12391246.

    • Search Google Scholar
    • Export Citation
  • 14.

    Furuya Y, Yoshida Y, Katayama T, Yamamoto S, Kawamura A, 1993. Scrub typhus. J Clin Microbiol 31: 16371640.

  • 15.

    Sonthayanon P, Chierakul W, Wuthiekanun V, Blacksell SD, Pimda K, Suputtamongkol Y, Pukrittayakamee S, White NJ, Day NPJ, Peacock SJ, 2006. Rapid diagnosis of scrub typhus in rural Thailand using polymerase chain reaction. Am J Trop Med Hyg 75: 10991102.

    • Search Google Scholar
    • Export Citation
  • 16.

    Sonthayanon P, Chierakul W, Wuthiekanun V, Phimda K, Pukrittayakamee S, Day NPJ, Peacock SJ, 2009. Association of high Orientia tsutsugamushi DNA loads with disease of greater severity in adults with scrub typhus. J Clin Microbiol 47: 430434.

    • Search Google Scholar
    • Export Citation
  • 17.

    Jiang J, Chan T-C, Temenak JJ, Dasch GA, Ching W-M, Richards AL, 2004. Development of a quantitative real-time polymerase chain reaction assay specific for Orientia tsutsugamushi. Am J Trop Med Hyg 70: 351356.

    • Search Google Scholar
    • Export Citation
  • 18.

    Paris DH, Aukkanit N, Jenjaroen K, Blacksell SD, Day NPJ, 2009. A highly sensitive quantitative real-time PCR assay based on the groEL gene of contemporary Thai strains of Orientia tsutsugamushi. Clin Microbiol Infect 15: 488495.

    • Search Google Scholar
    • Export Citation
  • 19.

    Bozeman FM, Elisberg BL, 1963. Serological diagnosis of scrub typhus by indirect immunofluorescence. Proc Soc Exp Biol Med 112: 568573.

  • 20.

    Pradutkanchana J, Silpapojakul K, Paxton H, Pradutkanchana S, Kelly DJ, Strickman D, 1997. Comparative evaluation of four serodiagnostic tests for scrub typhus in Thailand. Trans R Soc Trop Med Hyg 91: 425428.

    • Search Google Scholar
    • Export Citation

Author Notes

*Address correspondence to Gavin C. K. W. Koh, Mahidol–Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand. E-mail: gavin.koh@gmail.com

Financial support: Gavin C. K. W. Koh, Daniel H. Paris, Paul N. Newton, and Stuart D. Blacksell are funded by the Wellcome Trust of Great Britain. Richard J. Maude is funded by the Wellcome Trust of Great Britain, The Bill and Melinda Gates Foundation, and a British Infection Society Fellowship. Gavin C. K. W. Koh and Daniel H. Paris both hold Wellcome Trust Clinical Training Fellowships (Grants 086532/Z/08/Z and 078990/Z/06/Z, respectively).

Authors' addresses: Gavin C. K. W. Koh, Richard J. Maude, Daniel H. Paris, and Stuart D. Blacksell, Mahidol–Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, E-mails: gavin.koh@gmail.com, richardmaude@gmail.com, parigi@tropmedres.ac, and stuart@tropmedres.ac. Gavin C. K. W. Koh, Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. Gavin C. K. W. Koh, Department of Medicine, Addenbrooke's Hospital, University of Cambridge, Cambridge, United Kingdom. Paul N. Newton, Wellcome Trust–Mahosot Hospital–Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Vientiane, Lao People's Democratic Republic, E-mail: paul@tropmedres.ac.

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