J Korean Med Sci.  2013 May;28(5):672-679. 10.3346/jkms.2013.28.5.672.

Improvement of the Diagnostic Sensitivity of Scrub Typhus Using a Mixture of Recombinant Antigens Derived from Orientia tsutsugamushi Serotypes

Affiliations
  • 1Immunemed, Chuncheon, Korea. ywkim@hallym.ac.kr
  • 2Medical Science Institute, Hallym University, Chuncheon, Korea.
  • 3Department of Microbiology, College of Medicine, Hallym University, Chuncheon, Korea.
  • 4Department of Life Science, Hallym University, Chuncheon, Korea.
  • 5Department of Microbiology, College of Medicine, Korea University, Seoul, Korea.
  • 6Division of Pulmonology, Department of Internal Medicine, Medical Center, Hallym University, Chuncheon, Korea.
  • 7Department of Microbiology, College of Medicine, Seoul National University, Seoul, Korea.

Abstract

Diagnosis of scrub typhus is difficult because its symptoms are very similar to other acute febrile illnesses, such as leptospirosis, murine typhus, and other viral hemorrhagic fevers. To differentiate scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. We have developed a chimeric recombinant antigen cr56 and two other recombinant antigens, r21 and kr56, from various serotypes of Orientia tsutsugamushi. They were tested for the detection of antibodies against O. tsutsugamushi in the patient's serum samples using enzyme-linked immunosorbent assay (ELISA) and dot-blot analyses. As of conventional immunofluorescence assay (IFA), when the mixture of these three recombinant antigens was used, both sensitivity and specificity of the recombinant antigens were increased up to 98% in IgM and IgG at ELISA and dot blotting. Additionally, both sensitivity and specificity by detection of IgM and IgG antibodies at rapid diagnostic test (RDT), using the mixture of three antigens and gold conjugated antibodies, were 99%. Our results suggest the use of mixture of these recombinant antigen proteins in ELISA or RDT is suitable as a diagnostic test for scrub typhus.

Keyword

Scrub Typhus; Orientia tsutsugamushi; Recombinant Antigen; IFA; ELISA; RDT

MeSH Terms

Antibodies, Bacterial/blood/chemistry/immunology
Antigens, Bacterial/diagnostic use/genetics/metabolism
Enzyme-Linked Immunosorbent Assay
Fluorescent Antibody Technique, Indirect
Gold/chemistry
Humans
Immunoassay
Immunoglobulin G/blood
Immunoglobulin M/blood
Orientia tsutsugamushi/immunology/*metabolism
Recombinant Proteins/biosynthesis/diagnostic use/genetics
Scrub Typhus/*diagnosis
Sensitivity and Specificity
Serotyping
Antibodies, Bacterial
Antigens, Bacterial
Immunoglobulin G
Immunoglobulin M
Recombinant Proteins
Gold

Figure

  • Fig. 1 PCR amplification of antigenic genes from O. tsutsugamushi strains, Gilliam (lane 1: 952 bp), Karp (lane 2: 873 bp), Kato (lane 3: 886 bp), Boryoung (lane 4: 549 bp) and Kangwon 87-61 (lane 5: 1,086 bp) strains. The molecular size marker, 1kb (lane M).

  • Fig. 2 SDS-PAGE of recombinant antigen protein r21(21 kDa), kr56 (41 kDa) and cr56 (101 kDa). Asterisks indicate the induced antigens. IPTG, Isoproyl-b-D-thiogalactopyranoside

  • Fig. 3 Western blot analysis of the scrub typhus patients' serum samples using recombinant antigens, cr56, kr56 and r2. Lane 1-4: serum samples from scrub typhus patients (OT1, OT2, OT3, OT4), Lane 5: serum of healthy control (N), Lane 6 and 7: serum samples of HFRS patients (H1 and H2), Lane 8 and 9: serum samples of leptospirosis patients (L1 and L2). Loading amount of each antigen onto membrane was confirmed by Western blot, using anti-His (Anti-His).


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