Optimal Cutoff and Accuracy of an IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Scrub Typhus in Northern Thailand: an Alternative Reference Method to the IgM Immunofluorescence Assay

Abstract

The enzyme-linked immunosorbent assay (ELISA) has been proposed as an alternative serologic diagnostic test to the indirect immunofluorescence assay (IFA) for scrub typhus. Here, we systematically determine the optimal sample dilution and cutoff optical density (OD) and estimate the accuracy of IgM ELISA using Bayesian latent class models (LCMs). Data from 135 patients with undifferentiated fever were reevaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar and tested with a blood culture for Orientia tsutsugamushi, three different PCR assays, and an IgM IFA. The IgM ELISA was performed for every sample at sample dilutions from 1:100 to 1:102,400 using crude whole-cell antigens of the Karp, Kato, and Gilliam strains of O. tsutsugamushi developed by the Naval Medical Research Center. We used Bayesian LCMs to generate unbiased receiver operating characteristic curves and found that the sample dilution of 1:400 was optimal for the IgM ELISA. With the optimal cutoff OD of 1.474 at a sample dilution of 1:400, the IgM ELISA had a sensitivity of 85.7% (95% credible interval [CrI], 77.4% to 86.7%) and a specificity of 98.1% (95% CrI, 97.2% to 100%) using paired samples. For the ELISA, the OD could be determined objectively and quickly, in contrast to the reading of IFA slides, which was both subjective and labor-intensive. The IgM ELISA for scrub typhus has high diagnostic accuracy and is less subjective than the IgM IFA. We suggest that the IgM ELISA may be used as an alternative reference test to the IgM IFA for the serological diagnosis of scrub typhus.

Evaluation of a broad-ranging and convenient enzyme-linked immunosorbent assay using the lysate of infected cells with five serotypes of Orientia tsutsugamushi, a causative agent of scrub typhus

“…In the case of scrub typhus, our previous report showed that the serum antibody level of some patients was only elevated against limited serotypes, whereas that of most patients was elevated against all serotypes [7]. In some previous studies conducted outside of Japan, serological diagnostic methods such as ELISA were evaluated using only certain reference serotypes that did not include some of the most prevalent serotypes in Japan [8, 9], which can show wider variation than those of other countries. Accordingly, the sera from patients infected with a different serotype in Japan might not react to the serotypes used in these methods tested in previous reports.…”

“…That is, the serum antibody level of the patient will only be elevated against a specific serotype and not in response to the other serotypes. These facts clearly suggest that five or more strains should be used in serological tests of scrub typhus in Japan, although some previous reports from other countries used only a few standard strains in serological tests [8, 9]. …”

“…In addition to the objectivity advantage, this method has additional convenience, since previous methods for ELISA for rickettsia have involved a purification step of the sample by ultracentrifugation as the antigen [8, 9, 14]. We evaluated the accuracy of this novel broad-ranging and simplified ELISA in comparison with the micro-IFA for the serological diagnosis of scrub typhus in Japan.…”

State of the art of diagnosis of rickettsial diseases: the use of blood specimens for diagnosis of scrub typhus, spotted fever group rickettsiosis, and murine typhus

“…Anti- O. tsutsugamushi IgG can persist leading to high RDT false-positive rates in endemic areas, for which assay adjustments might be required. Currently available RDTs are immunochromatographic or semiquantitative dot-blot assays, increasingly incorporating recombinant antigens, allowing greater standardization, and simple readout for point-of-care testing in resource-constrained settings [ 16 , 35 – 38 ].…”

“…The dissemination dynamics of Rickettsia and Orientia with their early limited bacteremic phase and subsequent appearance of antibodies have hindered the development of effective diagnostic tools for targeted early antirickettsial therapy. Especially in disease endemic areas, the occurrence of high background antibody titers poses an additional challenge to the already difficult serodiagnosis [ 16 , 17 ▪▪ , 18 , 19 ]. More hurdles involve translating promising technologies with high analytical sensitivity and specificity into clinically useful tests [ 2 , 20 ▪ ].…”