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

Abstract

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.

Conflict of interest statement

Conflicts of Interest: PYS and SLY have a patent detection kit for diagnosis of scrub typhus and detection method thereof pending.

Figures

Figure 1.

Analysis of Escherichia coli expressing recombinant type-specific antigen (TSA) proteins. (A) Polymerase chain reaction–amplified products of the 56-kDa TSA from genomic DNA of Orientia tsutsugamushi strains, including TW-1, TW-10, TW-19, TW-22, Kato, Gilliam, and Karp (lane 1–7). Lane M, 1-Kb DNA ladder marker. (B) Purified TSA proteins were resolved by 12% Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and stained with Coomassie Blue. Lane 1–7, TSA of TW-1, TW-10, TW-19, TW-22, Kato, Gilliam, and Karp. Lane M, protein molecular weight marker. (C) Western blot analysis of recombinant TSA proteins from different strains. An anti-His tag antibody and anti-TSA monoclonal antibody were used to detect recombinant TSA proteins on membrane. This figure appears in color at www.ajtmh.org.

Figure 2.

The distribution of immunofluorescence assay (IFA) IgM and IgG titers. (A) One hundred and nine serum samples from qPCR-positive, scrub typhus patients. (B) Eighty-two serum samples from non-scrub typhus cases. The numbers shown on the top of each bar indicate the number of samples with their respective IFA titer.

Figure 3.

The distribution of optical density (OD) values obtained by the KGK enzyme-linked immunosorbent assay (ELISA) on non-scrub typhus and scrub typhus specimens. (A) For IgM ELISA, the cutoff values derived from mean plus three SDs (dotted line) was 0.34 and the cutoff value derived from receiver operating characteristic (ROC) analysis (solid line) was 0.30. (B) For IgG ELISA, the cutoff values derived from mean plus three SDs (dotted line) was 0.35 and the cutoff value derived from ROC analysis (solid line) was 0.37. This figure appears in color at www.ajtmh.org.

Figure 4.

The distribution of optical density (OD) values obtained by the mixed-TSA enzyme-linked immunosorbent assay (ELISA) on non-scrub typhus and scrub typhus specimens. (A) For IgM ELISA, the cutoff values derived from mean plus three SDs (dotted line) was 0.28 and the cutoff value derived from receiver operating characteristic (ROC) analysis (solid line) was 0.27. (B) For IgG ELISA, the cutoff values derived from mean plus three SDs (dotted line) was 0.32 and the cutoff value derived from ROC analysis (solid line) was 0.36. This figure appears in color at www.ajtmh.org.

Figure 5.

Correlation of the mixed-TSA enzyme-linked immunosorbent assay (ELISA) optical density (OD) values with immunofluorescence assay (IFA) titers. (A) The correlation of IgM ELISA OD values and IgM IFA titers. The Pearson correlation coefficient was 0.59 and the Spearman correlation coefficient was 0.61 (P < 0.0001). (B) The correlation of IgG ELISA OD values and IgG IFA titers. The Pearson correlation coefficient was 0.47 and the Spearman correlation coefficient was 0.48 (P < 0.0001). This figure appears in color at www.ajtmh.org.

Figure 6.

A phylogenetic tree of Orientia tsutsugamushi. The phylogenetic tree is based on the complete 56-kDa type-specific antigen gene (TSA) sequences of O. tsutsugamushi strains. The tree was constructed by the neighbor-joining method and the maximum composite likelihood model. Bootstrap support values greater than 75 are shown. The Taiwanese TSA sequence types are designated in solid circles (●) and prototype TSA sequences are designated in solid triangles (▲). Type-specific antigen sequences were identified by using the nomenclature of OT (Orientia tsutsugamushi)/country/strain/year of isolation/GenBank accession number/sequence type. The scale bar on the left indicates substitutions per site.