Evaluation of an Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay for Diagnosis of Orientia tsutsugamushi Infection

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Abstract
To differentiate scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. We developed an immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) for diagnosis of recent Orientia tsutsugamushi infections in humans. The 56-kDa major outer membrane protein of O. tsutsugamushi is well known as the most immunodominant antigen in scrub typhus. The test is based on the use of the biotinylated recombinant 56-kDa protein of O. tsutsugamushi Boryong, Bor56, which was expressed as a fusion protein with a maltose-binding protein in Escherichia coli . In the test, the serum IgM antibodies were captured by anti-human IgM antibodies coated onto a microtiter plate. The captured IgM antibodies were revealed through sequential addition of biotinylated Bor56 antigen and peroxidase-conjugated streptavidin to the plate. The IgM capture ELISA was compared with the immunofluorescence antibody assay (IFA) by testing 176 serum samples from patients with diagnosed cases of rickettsial disease and patients with other acute febrile diseases. Of the 81 IgG IFA-positive samples, 78 tested positive (sensitivity, 96.3%) and all 31 IgM IFA-positive samples tested positive (sensitivity, 100%) by the IgM capture ELISA. The specificity of the IgM capture ELISA was 99%, and 1 of the 95 IFA-negative samples was positive in the assay. These results strongly suggest that IgM capture ELISA using the recombinant Bor56 antigen is a reliable and detailed method for the detection of early O. tsutsugamushi infection.