- INTRODUCTION
- MATERIALS AND METHODS
- Human samples.
- Ethics statement.
- Real-time PCR.
- Indirect IFA.
- Type-specific antigen gene cloning.
- Expression and purification of recombinant TSA protein.
- Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis.
- Recombinant TSA-based ELISA.
- Determination of cutoff value of the recombinant TSA-based ELISA.
- Statistical and genetic analyses.
- RESULTS
- Expression of recombinant TSA proteins from various O. tsutsugamushi strains.
- Distribution of IFA titer of acute-phase serum samples from PCR-positive, scrub typhus patients and non-scrub typhus cases.
- Performance evaluation of ELISAs using cutoff values based on non-scrub typhus negative control specimens at the mean plus three SDs.
- Performance evaluation of ELISAs using cutoff values determined using ROC analysis.
- Correlation between IFA titers and the mixed-TSA ELISA OD values.
- DISCUSSION
- Supplementary Files
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Tamura A, Ohashi N, Urakami H, Miyamura S, 1995. Classification of Rickettsia tsutsugamushi in a new genus, Orientia gen. nov., as Orientia tsutsugamushi comb. nov. Int J Syst Bacteriol 45: 589–591.
-
2.
Walker DH, 2003. Rickettsial diseases in travelers. Trav Med Infect Dis 1: 35–40.
-
3.
Kelly DJ, Fuerst PA, Ching WM, Richards AL, 2009. Scrub typhus: the geographic distribution of phenotypic and genotypic variants of Orientia tsutsugamushi. Clin Infect Dis 48: S203–S230.
-
4.
Watt G, Strickman D, 1994. Life-threatening scrub typhus in a traveler returning from Thailand. Clin Infect Dis 18: 624–626.
-
5.
Paris DH, Shelite TR, Day NP, Walker DH, 2013. Unresolved problems related to scrub typhus: a seriously neglected life-threatening disease. Am J Trop Med Hyg 89: 301–307.
-
6.
Xu G, Walker DH, Jupiter D, Melby PC, Arcari CM, 2017. A review of the global epidemiology of scrub typhus. PLoS Negl Trop Dis 11: e0006062.
-
7.
Seong SY, Choi MS, Kim IS, 2001. Orientia tsutsugamushi infection: overview and immune responses. Microbes Infect 3: 11–21.
-
8.
Brown GW, Saunders JP, Singh S, Huxsoll DL, Shirai A, 1978. Single dose doxycycline therapy for scrub typhus. Trans R Soc Trop Med Hyg 72: 412–416.
-
9.
Chen CC, Juan CJ, Juan CW, Zeng XC, Huang M, 2007. Multi-organ dysfunction caused by scrub typhus initially misinterpreted as acute tonsillitis. J Emerg Crit Care Med 18: 161–166.
-
10.
Robinson DM, Brown G, Gan E, Huxsoll DL, 1976. Adaptation of a microimmunofluorescence test to the study of human Rickettsia tsutsugamushi antibody. Am J Trop Med Hyg 25: 900–905.
-
11.
Koh GCKW, Maude RJ, Paris DH, Newton PN, Blacksell SD, 2010. Diagnosis of scrub typhus. Am J Trop Med Hyg 82: 368–370.
-
12.
Hanson B, 1985. Identification and partial characterization of Rickettsia tsutsugamushi major protein immunogens. Infect Immun 50: 603–609.
-
13.
Tamura A, Ohashi N, Urakami H, Takahashi K, Oyanagi M, 1985. Analysis of polypeptide composition and antigenic components of Rickettsia tsutsugamushi by polyacrylamide gel electrophoresis and immunoblotting. Infect Immun 48: 671–675.
-
14.
Ohashi N, Tamura A, Ohta M, Hayashi K, 1989. Purification and partial characterization of a type-specific antigen of Rickettsia tsutsugamushi. Infect Immun 57: 1427–1431.
-
15.
Lu HY, Tsai KH, Yu SK, Cheng CH, Yang JS, Su CL, Hu HC, Wang HC, Huang JH, Shu PY, 2010. Phylogenetic analysis of 56-kDa type-specific antigen gene of Orientia tsutsugamushi isolates in Taiwan. Am J Trop Med Hyg 83: 658–663.
-
16.
Yang HH, Huang IT, Lin CH, Chen TY, Chen LK, 2012. New genotypes of Orientia tsutsugamushi isolated from humans in eastern Taiwan. PLoS One 7: e46997.
-
17.
Kim G et al. 2017. Diversification of Orientia tsutsugamushi genotypes by intragenic recombination and their potential expansion in endemic areas. PLOS Negl Trop Dis 11: e0005408.
-
18.
Ching WM, Wang H, Eamsila C, Kelly D, Dasch G, 1998. Expression and refolding of truncated recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi and its use in enzyme-linked immunosorbent assays. Clin Diagn Lab Immunol 5: 519–526.
-
19.
Blacksell SD, Jenjaroen K, Phetsouvanh R, Tanganuchitcharnchai A, Phouminh P, Phongmany S, Day NP, Newton PN, 2010. Accuracy of rapid IgM-based immunochromatographic and immunoblot assays for diagnosis of acute scrub typhus and murine typhus infections in Laos. Am J Trop Med Hyg 83: 365–369.
-
20.
Ching WM, Rowland D, Zhang Z, Bourgeois A, Kelly D, Dasch G, Devine P, 2001. Early diagnosis of scrub typhus with a rapid flow assay using recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi. Clin Diagn Lab Immunol 8: 409–414.
-
21.
Jiang J, Marienau KJ, May LA, Beecham HJ, Wilkinson R, Ching W-M, 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: 60–66.
-
22.
Jang WJ, Huh MS, Park KH, Choi MS, Kim IS, 2003. Evaluation of an immunoglobulin M capture enzyme-linked immunosorbent assay for diagnosis of Orientia tsutsugamushi infection. Clin Diagn Lab Immunol 10: 394–398.
-
23.
Blacksell SD, Jenjaroen K, Phetsouvanh R, Wuthiekanun V, Day NP, Newton PN, Ching WM, 2010. Accuracy of AccessBio immunoglobulin M and total antibody rapid immunochromatographic assays for the diagnosis of acute scrub typhus infection. Clin Vaccine Immunol 17: 263–266.
-
24.
Chao CC, Huber ES, Porter TB, Zhang Z, Ching WM, 2011. Analysis of the cross-reactivity of various 56 kdD recombinant protein antigens with serum samples collected after Orientia tsutsugamushi infection by ELISA. Am J Trop Med Hyg 84: 967–972.
-
25.
Tsai KH, Lu HY, Tsai JJ, Yu SK, Huang JH, Shu PY, 2008. Human case of Rickettsia felis infection, Taiwan. Emerg Infect Dis 14: 1970–1972.
-
26.
Frey A, Di Canzio J, Zurakowski D, 1998. A statistically defined endpoint titer determination method for immunoassays. J Immunol Methods 221: 35–41.
-
27.
Kumar R, Indrayan A, 2011. Receiver operating characteristic (ROC) curve for medical researchers. Indian Pediatr 48: 277–287.
-
28.
Thompson JD, Higgins DG, Gibson TJ, 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673–4680.
-
29.
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.
-
30.
Kim DM, Lee YM, Back JH, Yang TY, Lee JH, Song HJ, Shim SK, Hwang KJ, Park MY, 2010. A serosurvey of Orientia tsutsugamushi from patients with scrub typhus. Clin Microbiol Infect 16: 447–451.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 2626 | 1905 | 360 |
| Full Text Views | 846 | 17 | 0 |
| PDF Downloads | 285 | 17 | 0 |
Evaluation of Enzyme-Linked Immunosorbent Assay Using Recombinant 56-kDa Type-Specific Antigens Derived from Multiple Orientia tsutsugamushi Strains for Detection of Scrub Typhus Infection
Su-Lin Yang
Su-Lin Yang
Center for Diagnostics and Vaccine Development, Centers for Disease Control, Ministry of Health and Welfare, Taipei, Republic of China;
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Kun-Hsien Tsai
Kun-Hsien Tsai
Department of Public Health and Institute of Environmental Health, College of Public Health, National Taiwan University, Taipei, Republic of China
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Hsiang-Fei Chen
Hsiang-Fei Chen
Center for Diagnostics and Vaccine Development, Centers for Disease Control, Ministry of Health and Welfare, Taipei, Republic of China;
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Jun-Yu Luo
Jun-Yu Luo
Center for Diagnostics and Vaccine Development, Centers for Disease Control, Ministry of Health and Welfare, Taipei, Republic of China;
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Pei-Yun Shu
Pei-Yun Shu
Center for Diagnostics and Vaccine Development, Centers for Disease Control, Ministry of Health and Welfare, Taipei, Republic of China;
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Scrub typhus is caused by the intracellular bacterium Orientia tsutsugamushi. The 56-kDa type-specific antigen (TSA) displays a significant antigenic variation across different O. tsutsugamushi strains. To minimize the influence of the antigenic diversity of TSA on assay sensitivity, we developed a mixed-TSA enzyme-linked immunosorbent assay (mixed-TSA ELISA) using a mixture of recombinant TSAs of prototype (Karp, Gilliam, and Kato) and local (TW-1, TW-10, TW-19, and TW-22) O. tsutsugamushi strains as antigens to detect immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against O. tsutsugamushi. These four local strains covered a major part of the total genetic diversity of TSA gene of O. tsutsugamushi in Taiwan. A total of 109 acute-phase serum samples from O. tsutsugamushi polymerase chain reaction–positive, scrub typhus patients, and 82 negative control serum samples from non-scrub typhus cases were used for evaluation of the recombinant TSA-based ELISA. We compared the performance of the mixed-TSA ELISA with immunofluorescence assay (IFA), which is considered the gold standard method for the serological diagnosis of scrub typhus. The results indicated that the sensitivity of IgM mixed-TSA ELISA (80.7%) was significantly higher than that of IgM IFA (68.8%). We demonstrated that the mixed-TSA ELISA had a high sensitivity and specificity and can be used for screening of scrub typhus patient in the early phase of the disease.
Author Notes
Conflicts of Interest: PYS and SLY have a patent detection kit for diagnosis of scrub typhus and detection method thereof pending.
Financial support: This work was supported in part by grants MOHW104-CDC-C-315-000115 and MOHW105-CDC-C-315-123504 from Centers for Disease Control, Ministry of Health and Welfare, Taiwan, Republic of China.
Authors’ addresses: Su-Lin Yang, Hsiang-Fei Chen, Jun-Yu Luo, and Pei-Yun Shu, Center for Diagnostics and Vaccine Development, Centers for Disease Control, Ministry of Health and Welfare, Taipei, Republic of China, E-mails: cerline@cdc.gov.tw, xiangfei@cdc.gov.tw, lions8022@cdc.gov.tw, and pyshu@cdc.gov.tw. Kun-Hsien Tsai, Department of Public Health and Institute of Environmental Health, College of Public Health, National Taiwan University, Taipei, Republic of China, E-mail: kunhtsai@ntu.edu.tw.
- INTRODUCTION
- MATERIALS AND METHODS
- Human samples.
- Ethics statement.
- Real-time PCR.
- Indirect IFA.
- Type-specific antigen gene cloning.
- Expression and purification of recombinant TSA protein.
- Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis.
- Recombinant TSA-based ELISA.
- Determination of cutoff value of the recombinant TSA-based ELISA.
- Statistical and genetic analyses.
- RESULTS
- Expression of recombinant TSA proteins from various O. tsutsugamushi strains.
- Distribution of IFA titer of acute-phase serum samples from PCR-positive, scrub typhus patients and non-scrub typhus cases.
- Performance evaluation of ELISAs using cutoff values based on non-scrub typhus negative control specimens at the mean plus three SDs.
- Performance evaluation of ELISAs using cutoff values determined using ROC analysis.
- Correlation between IFA titers and the mixed-TSA ELISA OD values.
- DISCUSSION
- Supplementary Files
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Tamura A, Ohashi N, Urakami H, Miyamura S, 1995. Classification of Rickettsia tsutsugamushi in a new genus, Orientia gen. nov., as Orientia tsutsugamushi comb. nov. Int J Syst Bacteriol 45: 589–591.
-
2.
Walker DH, 2003. Rickettsial diseases in travelers. Trav Med Infect Dis 1: 35–40.
-
3.
Kelly DJ, Fuerst PA, Ching WM, Richards AL, 2009. Scrub typhus: the geographic distribution of phenotypic and genotypic variants of Orientia tsutsugamushi. Clin Infect Dis 48: S203–S230.
-
4.
Watt G, Strickman D, 1994. Life-threatening scrub typhus in a traveler returning from Thailand. Clin Infect Dis 18: 624–626.
-
5.
Paris DH, Shelite TR, Day NP, Walker DH, 2013. Unresolved problems related to scrub typhus: a seriously neglected life-threatening disease. Am J Trop Med Hyg 89: 301–307.
-
6.
Xu G, Walker DH, Jupiter D, Melby PC, Arcari CM, 2017. A review of the global epidemiology of scrub typhus. PLoS Negl Trop Dis 11: e0006062.
-
7.
Seong SY, Choi MS, Kim IS, 2001. Orientia tsutsugamushi infection: overview and immune responses. Microbes Infect 3: 11–21.
-
8.
Brown GW, Saunders JP, Singh S, Huxsoll DL, Shirai A, 1978. Single dose doxycycline therapy for scrub typhus. Trans R Soc Trop Med Hyg 72: 412–416.
-
9.
Chen CC, Juan CJ, Juan CW, Zeng XC, Huang M, 2007. Multi-organ dysfunction caused by scrub typhus initially misinterpreted as acute tonsillitis. J Emerg Crit Care Med 18: 161–166.
-
10.
Robinson DM, Brown G, Gan E, Huxsoll DL, 1976. Adaptation of a microimmunofluorescence test to the study of human Rickettsia tsutsugamushi antibody. Am J Trop Med Hyg 25: 900–905.
-
11.
Koh GCKW, Maude RJ, Paris DH, Newton PN, Blacksell SD, 2010. Diagnosis of scrub typhus. Am J Trop Med Hyg 82: 368–370.
-
12.
Hanson B, 1985. Identification and partial characterization of Rickettsia tsutsugamushi major protein immunogens. Infect Immun 50: 603–609.
-
13.
Tamura A, Ohashi N, Urakami H, Takahashi K, Oyanagi M, 1985. Analysis of polypeptide composition and antigenic components of Rickettsia tsutsugamushi by polyacrylamide gel electrophoresis and immunoblotting. Infect Immun 48: 671–675.
-
14.
Ohashi N, Tamura A, Ohta M, Hayashi K, 1989. Purification and partial characterization of a type-specific antigen of Rickettsia tsutsugamushi. Infect Immun 57: 1427–1431.
-
15.
Lu HY, Tsai KH, Yu SK, Cheng CH, Yang JS, Su CL, Hu HC, Wang HC, Huang JH, Shu PY, 2010. Phylogenetic analysis of 56-kDa type-specific antigen gene of Orientia tsutsugamushi isolates in Taiwan. Am J Trop Med Hyg 83: 658–663.
-
16.
Yang HH, Huang IT, Lin CH, Chen TY, Chen LK, 2012. New genotypes of Orientia tsutsugamushi isolated from humans in eastern Taiwan. PLoS One 7: e46997.
-
17.
Kim G et al. 2017. Diversification of Orientia tsutsugamushi genotypes by intragenic recombination and their potential expansion in endemic areas. PLOS Negl Trop Dis 11: e0005408.
-
18.
Ching WM, Wang H, Eamsila C, Kelly D, Dasch G, 1998. Expression and refolding of truncated recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi and its use in enzyme-linked immunosorbent assays. Clin Diagn Lab Immunol 5: 519–526.
-
19.
Blacksell SD, Jenjaroen K, Phetsouvanh R, Tanganuchitcharnchai A, Phouminh P, Phongmany S, Day NP, Newton PN, 2010. Accuracy of rapid IgM-based immunochromatographic and immunoblot assays for diagnosis of acute scrub typhus and murine typhus infections in Laos. Am J Trop Med Hyg 83: 365–369.
-
20.
Ching WM, Rowland D, Zhang Z, Bourgeois A, Kelly D, Dasch G, Devine P, 2001. Early diagnosis of scrub typhus with a rapid flow assay using recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi. Clin Diagn Lab Immunol 8: 409–414.
-
21.
Jiang J, Marienau KJ, May LA, Beecham HJ, Wilkinson R, Ching W-M, 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: 60–66.
-
22.
Jang WJ, Huh MS, Park KH, Choi MS, Kim IS, 2003. Evaluation of an immunoglobulin M capture enzyme-linked immunosorbent assay for diagnosis of Orientia tsutsugamushi infection. Clin Diagn Lab Immunol 10: 394–398.
-
23.
Blacksell SD, Jenjaroen K, Phetsouvanh R, Wuthiekanun V, Day NP, Newton PN, Ching WM, 2010. Accuracy of AccessBio immunoglobulin M and total antibody rapid immunochromatographic assays for the diagnosis of acute scrub typhus infection. Clin Vaccine Immunol 17: 263–266.
-
24.
Chao CC, Huber ES, Porter TB, Zhang Z, Ching WM, 2011. Analysis of the cross-reactivity of various 56 kdD recombinant protein antigens with serum samples collected after Orientia tsutsugamushi infection by ELISA. Am J Trop Med Hyg 84: 967–972.
-
25.
Tsai KH, Lu HY, Tsai JJ, Yu SK, Huang JH, Shu PY, 2008. Human case of Rickettsia felis infection, Taiwan. Emerg Infect Dis 14: 1970–1972.
-
26.
Frey A, Di Canzio J, Zurakowski D, 1998. A statistically defined endpoint titer determination method for immunoassays. J Immunol Methods 221: 35–41.
-
27.
Kumar R, Indrayan A, 2011. Receiver operating characteristic (ROC) curve for medical researchers. Indian Pediatr 48: 277–287.
-
28.
Thompson JD, Higgins DG, Gibson TJ, 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673–4680.
-
29.
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.
-
30.
Kim DM, Lee YM, Back JH, Yang TY, Lee JH, Song HJ, Shim SK, Hwang KJ, Park MY, 2010. A serosurvey of Orientia tsutsugamushi from patients with scrub typhus. Clin Microbiol Infect 16: 447–451.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 2626 | 1905 | 360 |
| Full Text Views | 846 | 17 | 0 |
| PDF Downloads | 285 | 17 | 0 |