Early Diagnosis of Scrub Typhus with a Rapid Flow Assay Using Recombinant Major Outer Membrane Protein Antigen (r56) of Orientia tsutsugamushi

Abstract

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. We developed a rapid immunochromatographic flow assay (RFA) for the detection of immunoglobulin M (IgM) and IgG antibodies to O. tsutsugamushi. The RFA employs a truncated recombinant 56-kDa protein from the Karp strain as the antigen. The performance of the RFA was evaluated with a panel of 321 sera (serial bleedings of 85 individuals suspected of scrub typhus) which were collected in the Pescadore Islands, Taiwan, from 1976 to 1977. Among these 85 individuals, IgM tests were negative for 7 cases by both RFA and indirect fluorescence assay (IFA) using Karp whole-cell antigen. In 29 cases specific responses were detected by the RFA earlier than by IFA, 44 cases had the same detection time, and 5 cases were detected earlier by IFA than by RFA. For IgG responses, 4 individuals were negative with both methods, 37 cases exhibited earlier detection by RFA than IFA, 42 cases were detected at the same time, and 2 cases were detected earlier by IFA than by RFA. The sensitivities of RFA detection of antibody in sera from confirmed cases were 74 and 86% for IgM and IgG, respectively. When IgM and IgG results were combined, the sensitivity was 89%. A panel of 78 individual sera collected from patients with no evidence of scrub typhus was used to evaluate the specificity of the RFA. The specificities of the RFA were 99% for IgM and 97% for IgG. The sensitivities of IFA were 53 and 73% for IgM and IgG, respectively, and were 78% when the results of IgM and IgG were combined. The RFA test was significantly better than the IFA test for the early detection of antibody to scrub typhus in primary infections, while both tests were equally sensitive with reinfected individuals.

Differentiation of Rickettsiae by groEL Gene Analysis

“…Diagnosis of scrub typhus is generally based on clinical presentation, the history of the patient, and serological assays. However, the differentiation of scrub typhus from other acute febrile illnesses is difficult because the symptoms are very similar (2,15). In particular, the differentiation of scrub typhus from other rickettsial diseases, such as spotted fever diseases, murine typhus, and epidemic typhus, is very difficult.…”

Comparative evaluation of selected diagnostic assays for the detection of IgG and IgM antibody to Orientia tsutsugamushi in Thailand.

“…18 Selection of this protein was based on the fact that sera from 95-99% of acutely ill and convalescent patients with scrub typhus recognize the native 56-kd protein of O. tsutsugamushi. Because of its strong immunoreactivity, the r56 recombinant protein has been incorporated into ELISAs 18,19 and dipstick and immunochromatographic 24 assays to diagnose scrub typhus. We evaluate and compare 5 assays (IFA, a pooled-antigen ELISA, a r56 recombinant-antigen ELISA, a Pan-Bio [Brisbane, Australia] dipstick assay, and an Integrated Diagnostics [Baltimore, MD] dipstick assay) for the diagnosis of scrub typhus, using the IIP test as the reference standard.…”

“…1 The IIP test has been evaluated in a series of studies 1,11,12,14,15 and seems to perform similarly to the IFA. A variety of studies have evaluated the performance of enzyme-linked immunosorbent assays (ELISAs), [16][17][18][19] dot-blot immunoassays, 1,[20][21][22][23] a rapid flow assay, 24 and a passive hemagglutination assay. 25 Although the performance of some of these assays was as good or better than the IFA or IIP, none of these assays is used routinely on a widespread basis for the diagnosis of scrub typhus.…”

“…25 Although the performance of some of these assays was as good or better than the IFA or IIP, none of these assays is used routinely on a widespread basis for the diagnosis of scrub typhus. The introduction of recombinant proteins into the development of diagnostic assays for scrub typhus [17][18][19]24,25 should allow for their increased use, however, because of an enhanced stability and consistency of the antigen preparation and a reduction in cost, transport, and reproducibility problems presently associated with whole-cell antigen preparations. A truncated nonfused recombinant protein of 56 kd antigen from Karp strain (r56) was expressed, purified, and refolded into a structure similar to its native form at the Naval Medical Research Institute.…”

“…The INDX assay evaluated by Pradutkanchana et al 1 used native O. tsutsugamushi antigen as described by Weddle et al, 20 whereas the INDX assay we evaluated used the r56 recombinant antigen. 18 As we have already described, however, the r56 recombinant antigen used in this study has been incorporated into a variety of assays, 18,19,24 in which it has sensitivity and specificity comparable or better than that obtained using native O. tsutsugamushi antigen. We can offer no logical explanation for the poor performance of the INDX assay in this study.…”