Volume 100, Issue 5
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645



In this diagnostic accuracy study, we evaluated data from 135 febrile patients from Chiang Rai, to determine the optimal optical density (OD) cutoffs for an in-house scrub typhus IgM ELISA. Receiver operating characteristic curves were generated using a panel of reference assays, including an IgM immunofluorescence assay (IFA), PCR, in vitro isolation, presence of an eschar, or a combination of these. Altogether, 33 patients (24.4%) were diagnosed as having scrub typhus. Correlation between positivity by IFA and increasing OD values peaked at a cutoff of 2.0, whereas there was little association between positivity by culture or eschar with increasing ELISA cutoffscutoffs of 3.0 and 4.0 were demonstrated to be optimal for the total absorbance of the OD at dilutions 1:100, 1:400, 1:1,600, and 1:6,400, for admission and convalescent samples, respectively. The optimal cutoff at a 1:100 dilution was found to be between 1.85 and 2.22 for admission samples and convalescent-phase samples, respectively. Sensitivities for the cutoffs varied from 57.1% to 90.0% depending on the reference test and sample timing, whereas specificities ranged from 85.2% to 99.0%. We therefore recommend a cutoff of around 2.0, depending on the sensitivity and specificity desired in clinical or epidemiological settings. The results demonstrate the ELISA to be a valuable diagnostic tool, suitable for use in resource-limited endemic regions, especially when used in combination with other diagnostic modalities such as the presence of an eschar.

[open-access] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


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  1. Blacksell SD, Lim C, Tanganuchitcharnchai A, Jintaworn S, Kantipong P, Richards AL, Paris DH, Limmathurotsakul D, Day NP, , 2016. Optimal cutoff and accuracy of an IgM enzyme-linked immunosorbent assay for diagnosis of acute scrub typhus in northern Thailand: an alternative reference method to the IgM immunofluorescence assay. J Clin Microbiol 54: 14721478. [Google Scholar]
  2. Blacksell SD, Paris DH, Chierakul W, Wuthiekanun V, Teeratakul A, Kantipong P, Day NP, , 2012. Prospective evaluation of commercial antibody-based rapid tests in combination with a loop-mediated isothermal amplification PCR assay for detection of Orientia tsutsugamushi during the acute phase of scrub typhus infection. Clin Vaccine Immunol 19: 391395. [Google Scholar]
  3. Isaac R, Varghese GM, Mathai E, Manjula J, Joseph I, , 2004. Scrub typhus: prevalence and diagnostic issues in rural southern India. Clin Infect Dis 39: 13951396. [Google Scholar]
  4. Tamura A, Ohashi N, Urakami H, Miyamura S, , 1995. Classification of Rickettsia tsutsugamushi in a Orientia gen. nov., as Orientia tsutsugamushi New Genus, comb. nov. Int J Syst Bacteriol 45: 589591. [Google Scholar]
  5. Lerdthusnee K, 2003. Scrub typhus. Ann New York Acad Sci 990: 2535. [Google Scholar]
  6. Rajapakse S, Rodrigo C, Fernando D, , 2012. Scrub typhus: pathophysiology, clinical manifestations and prognosis. Asian Pac J Trop Med 5: 261264. [Google Scholar]
  7. Koh GC, Maude RJ, Paris DH, Newton PN, Blacksell SD, , 2010. Diagnosis of scrub typhus. Am J Trop Med Hyg 82: 368370. [Google Scholar]
  8. Blacksell SD, Bryant NJ, Paris DH, Doust JA, Sakoda Y, Day NP, , 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. [Google Scholar]
  9. Bonell A, Lubell Y, Newton PN, Crump JA, Paris DH, , 2017. Estimating the burden of scrub typhus: a systematic review. PLoS Negl Trop Dis 11: e0005838. [Google Scholar]
  10. Koraluru M, Bairy I, Varma M, Vidyasagar S, , 2015. Diagnostic validation of selected serological tests for detecting scrub typhus. Microbiol Immunol 59: 371374. [Google Scholar]
  11. Rahi M, Gupte MD, Bhargava A, Varghese GM, Arora R, , 2015. DHR-ICMR guidelines for diagnosis & management of rickettsial diseases in India. Indian J Med Res 141: 417422. [Google Scholar]
  12. Blacksell SD, Lim C, Tanganuchitcharnchai A, Jintaworn S, Kantipong P, Richards AL, Paris DH, Limmathurotsakul D, Day NPJ, , 2016. Optimal cutoff and accuracy of an IgM enzyme-linked immunosorbent assay for diagnosis of acute scrub typhus in northern Thailand: an alternative reference method to the IgM immunofluorescence assay. J Clin Microbiol 54: 14721478. [Google Scholar]
  13. Luce-Fedrow A, Mullins K, Kostik AP, St John HK, Jiang J, Richards AL, , 2015. Strategies for detecting rickettsiae and diagnosing rickettsial diseases. Future Microbiol 10: 537564. [Google Scholar]
  14. Blacksell SD, Tanganuchitcharnchai A, Nawtaisong P, Kantipong P, Laongnualpanich A, Day NP, Paris DH, , 2015. Diagnostic accuracy of the InBios scrub typhus detect enzyme-linked immunoassay for the detection of IgM antibodies in northern Thailand. Clin Vaccine Immunol 23: 148154. [Google Scholar]
  15. Land MV, Ching WM, Dasch GA, Zhang Z, Kelly DJ, Graves SR, Devine PL, , 2000. Evaluation of a commercially available recombinant-protein enzyme-linked immunosorbent assay for detection of antibodies produced in scrub typhus rickettsial infections. J Clin Microbiol 38: 27012705. [Google Scholar]
  16. Luksameetanasan R, Blacksell SD, Kalambaheti T, Wuthiekanun V, Chierakul W, Chueasuwanchai S, Apiwattanaporn A, Stenos J, Graves S, Peacock SJ, , 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. [Google Scholar]
  17. Wangrangsimakul T, Althaus T, Mukaka M, Kantipong P, Wuthiekanun V, Chierakul W, Blacksell SD, Day NP, Laongnualpanich A, Paris DH, , 2018. Causes of acute undifferentiated fever and the utility of biomarkers in Chiangrai, northern Thailand. PLoS Negl Trop Dis 12: e0006477. [Google Scholar]
  18. Chao CC, Zhang Z, Belinskaya T, Thipmontree W, Tantibhedyangkul W, Silpasakorn S, Wongsawat E, Suputtamongkol Y, Ching WM, , 2017. An ELISA assay using a combination of recombinant proteins from multiple strains of Orientia tsutsugamushi offers an accurate diagnosis for scrub typhus. BMC Infect Dis 17: 413. [Google Scholar]
  19. Suwanabun N, Chouriyagune C, Eamsila C, Watcharapichat P, Dasch GA, Howard RS, Kelly DJ, , 1997. Evaluation of an enzyme-linked immunosorbent assay in Thai scrub typhus patients. Am J Trop Med Hyg 56: 3843. [Google Scholar]
  20. Lim C, Blacksell SD, Laongnualpanich A, Kantipong P, Day NP, Paris DH, Limmathurotsakul D, , 2015. Optimal cutoff titers for indirect immunofluorescence assay for diagnosis of scrub typhus. J Clin Microbiol 53: 36633666. [Google Scholar]
  21. Paris DH, Blacksell SD, Nawtaisong P, Jenjaroen K, Teeraratkul A, Chierakul W, Wuthiekanun V, Kantipong P, Day NP, , 2011. Diagnostic accuracy of a loop-mediated isothermal PCR assay for detection of Orientia tsutsugamushi during acute scrub typhus infection. PLoS Negl Trop Dis 5: e1307. [Google Scholar]
  22. Sonthayanon P, Chierakul W, Wuthiekanun V, Blacksell SD, Pimda K, Suputtamongkol Y, Pukrittayakamee S, White NJ, Day NP, Peacock SJ, , 2006. Rapid diagnosis of scrub typhus in rural Thailand using polymerase chain reaction. Am J Trop Med Hyg 75: 10991102. [Google Scholar]
  23. Paris DH, Blacksell SD, Stenos J, Graves SR, Unsworth NB, Phetsouvanh R, Newton PN, Day NP, , 2008. Real-time multiplex PCR assay for detection and differentiation of rickettsiae and orientiae. Trans R Soc Trop Med Hyg 102: 186193. [Google Scholar]
  24. Paris DH, Aukkanit N, Jenjaroen K, Blacksell SD, Day NP, , 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. [Google Scholar]
  25. Dasch GA, Halle S, Bourgeois AL, , 1979. Sensitive microplate enzyme-linked immunosorbent assay for detection of antibodies against the scrub typhus rickettsia, Rickettsia tsutsugamushi. J Clin Microbiol 9: 3848. [Google Scholar]
  26. Kocher C, 2017. Serologic evidence of scrub typhus in the Peruvian Amazon. Emerg Infect Dis 23: 13891391. [Google Scholar]
  27. Jiang J, Marienau KJ, May LA, Beecham HJ, III Wilkinson R, Ching WM, Richards AL, , 2003. Laboratory diagnosis of two scrub typhus outbreaks at Camp Fuji, Japan in 2000 and 2001 by enzyme-linked immunosorbent assay, rapid flow assay, and western blot assay using outer membrane 56-kD recombinant proteins. Am J Trop Med Hyg 69: 6066. [Google Scholar]
  28. Paris DH, 2015. A nonhuman primate scrub typhus model: protective immune responses induced by pKarp47 DNA vaccination in cynomolgus macaques. J Immunol 194: 17021716. [Google Scholar]
  29. Khan SA, Bora T, Chattopadhyay S, Jiang J, Richards AL, Dutta P, , 2016. Seroepidemiology of rickettsial infections in northeast India. Trans R Soc Trop Med Hyg 110: 487494. [Google Scholar]
  30. Coleman RE, 2002. Comparative evaluation of selected diagnostic assays for the detection of IgG and IgM antibody to Orientia tsutsugamushi in Thailand. Am J Trop Med Hyg 67: 497503. [Google Scholar]
  31. Varghese GM, 2013. Scrub typhus in south India: clinical and laboratory manifestations, genetic variability, and outcome. Int J Infect Dis 17: e981e987. [Google Scholar]
  32. Sood AK, Chauhan L, Gupta H, , 2016. CNS manifestations in Orientia tsutsugamushi disease (scrub typhus) in north India. Indian J Pediatr 83: 634639. [Google Scholar]
  33. Bhargava A, Kaushik R, Kaushik RM, Sharma A, Ahmad S, Dhar M, Mittal G, Khanduri S, Pant P, Kakkar R, , 2016. Scrub typhus in Uttarakhand & adjoining Uttar Pradesh: seasonality, clinical presentations & predictors of mortality. Indian J Med Res 144: 901909. [Google Scholar]
  34. Sengupta M, Benjamin S, Prakash JA, , 2014. Scrub typhus continues to be a threat in pregnancy. Int J Gynaecol Obstet 127: 212. [Google Scholar]
  35. Kim DM, Byun JN, , 2008. Effects of antibiotic treatment on the results of nested PCRs for scrub typhus. J Clin Microbiol 46: 34653466. [Google Scholar]
  36. Lim C, Paris DH, Blacksell SD, Laongnualpanich A, Kantipong P, Chierakul W, Wuthiekanun V, Day NP, Cooper BS, Limmathurotsakul D, , 2015. How to determine the accuracy of an alternative diagnostic test when it is actually better than the reference tests: a re-evaluation of diagnostic tests for scrub typhus using bayesian LCMs. PLoS One 10: e0114930. [Google Scholar]
  37. Parola P, Miller RS, McDaniel P, Telford SR, 3rd Rolain JM, Wongsrichanalai C, Raoult D, , 2003. Emerging rickettsioses of the Thai-Myanmar border. Emerg Infect Dis 9: 592595. [Google Scholar]
  38. Gupta N, Chaudhry R, Thakur CK, , 2016. Determination of cutoff of ELISA and immunofluorescence assay for scrub typhus. J Glob Infect Dis 8: 9799. [Google Scholar]
  39. Blacksell SD, Luksameetanasan R, Kalambaheti T, Aukkanit N, Paris DH, McGready R, Nosten F, Peacock SJ, Day NP, , 2008. Genetic typing of the 56-kDa type-specific antigen gene of contemporary Orientia tsutsugamushi isolates causing human scrub typhus at two sites in north-eastern and western Thailand. FEMS Immunol Med Microbiol 52: 335342. [Google Scholar]
  40. Wongprompitak P, Anukool W, Wongsawat E, Silpasakorn S, Duong V, Buchy P, Morand S, Frutos R, Ekpo P, Suputtamongkol Y, , 2013. Broad-coverage molecular epidemiology of Orientia tsutsugamushi in Thailand. Infect Genet Evol 15: 5358. [Google Scholar]

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  • Received : 17 Aug 2018
  • Accepted : 27 Jan 2019
  • Published online : 11 Mar 2019

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