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Author affiliation: Author affiliation: University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
Scrub typhus, caused by Orientia tsutsugamushi bacteria and transmitted by Trombiculid mites, affects >1 million persons each year (1,2). Clinical disease is characterized by fever, headache, and eschars. Untreated, case-fatality rates for the disease can exceed 30% (2,3).
Most cases of scrub typhus occur in Asia and the Pacific. Although the vectors, commonly known as chiggers, are widely distributed across North America, no autochthonous (i.e., locally acquired) cases have been reported in the United States. In 2023, however, researchers identified O. tsutsugamushi in chiggers collected in North Carolina (4). To investigate potential transmission to humans in North Carolina, we sought to estimate the seroprevalence of O. tsutsuguamushi antibodies and assess the clinical characteristics of persons with evidence of exposure. We hypothesized that cases of eschar are more likely to be caused by endemic tickborne Rickettsiaceae, such as Rickettsia parkeri bacteria (5).
The protocols of the parent study have been published (6). In brief, we collected remanent serum specimens from adult patients undergoing testing for spotted fever group Rickettsia or Ehrlichia spp. bacteria as part of routine care for acute febrile illness. For the purposes of this study, we selected serum specimens from patients with documented eschar.
We performed serologic testing in parallel by using 2 commercially available kits. We performed all tests according to manufacturers’ instructions and ran the tests with the included positive and negative controls. We first tested samples by using IgM-specific indirect immunofluorescence antibody (IFA) assays against O. tsutsugamushi (Fuller Laboratories, https://fullerlaboratories.com) (7). We diluted samples to a reciprocal titer of 1:64. In parallel, specimens underwent testing with the Scrub Typhus Detect ELISA-based assays that detect IgM and IgG (InBios International Inc., https://inbios.com). We diluted samples to a reciprocal titer of 1:100. The IgG ELISA kit instructions suggested an optical density (OD) cutoff of 0.37, but the IgM kit did not suggest a cutoff. We applied the 0.37 OD cutoff to the IgG results but also applied several IgM and IgG OD cutoffs from existing literature (8,9) (Table 1). We estimated seropositivity and used the Clopper-Pearson exact method to calculate 95% CIs.
A total of 138 (5.3%) of 2,593 persons had an eschar documented. Of those, 101 had an adequate sample volume. On the basis of the number of slides and kits available, we selected 87 (86.1%) samples for IFA and 92 (91.1%) samples for ELISA from 83 unique person; some persons had both acute and convalescent samples tested. Of the 83 persons, 35 (42.1%) had illnesses that had been classified as confirmed, probable, or suspected cases of spotted fever group Rickettsia (10) (Appendix Table 1).
By IFA result, we classified 18 (20.6%) of 87 samples as indeterminate (i.e., weakly positive). By ELISA result, 4 (4.3%) of 92 were positive for O. tsutsugamushi IgM and 8 (8.9%) of 90 for IgG; no samples were positive for both. None of the 18 samples that were indeterminate by IFA were positive by ELISA. The 8 samples IgG-positive by ELISA results underwent confirmatory IgG IFA testing at Fuller Laboratories (Appendix Table 2). We observed 50% (4 of 8) agreement in IgG reactivity between ELISA and IFA results at an endpoint titer of 1:128.
Of the 11 persons with either a reactive IgG or IgM, 7 (63.6%) reported a tick bite. None of the clinical encounters were associated with travel. Fever and myalgia were the most common clinical syndrome (Table 2). All patients were seen in outpatient settings, and none were hospitalized.
Our study identified North Carolina residents with antibodies reactive against O. tsutsugamushi bacteria. However, those results must be interpreted cautiously. The presence of antibodies does not indicate active infection but can be a marker of prior exposure. In addition, in a setting where transmission has not been previously reported, positive results are more likely to be falsely positive because of imperfect test performance. However, the presence of antibodies, especially when indicated by both assays, was unexpected and merits investigation.
Including only persons who had reactive antibodies by both assays (n = 4), our estimate of IgG seroprevalence is 4.3% (95% CI 1.2%–10.8%), but depending on the assay and cutoffs applied might be as high as 8.9. However, if O. tsutsugamushi bacteria were being locally transmitted, we would expect cases of severe illness, which we did not observe.
One limitation of this study is its reliance on serologic results and absence of contextual information, including travel histories and clinical outcomes. In addition, most patients did not have convalescent titers drawn, so we were unable to confirm cases.
We believe the current evidence does not support transmission of virulent O. tsutsugamushi strains in North Carolina. Further investigation, ideally by using molecular approaches from clinical samples (e.g., eschar swab samples), is needed. Clinicians should maintain a high level of suspicion for R. parkeri rickettsiosis, the seroprevalence of which was nearly 10 times greater than O. tsutsugamushi, when evaluating patients with arthropod-associated eschars.
Ms. Abernathy and Ms. Ursery are doctoral students in epidemiology at the University of North Carolina at Chapel Hill Gillings School of Global Public Health. Ms. Abernathy’s research interests include the laboratory diagnosis of infectious diseases. Ms. Ursery’s research interests include infections in pregnancy and early childhood.
We thank InBios for providing the ELISA kits at no cost to the study and Fuller Laboratories for performing confirmatory testing of samples. We acknowledge Christopher Paddock for reviewing drafts of the manuscript.
Study activities for the parent study were reviewed by the UNC Institutional Review Board (21-0356). Some activities, including the collection and testing of remnant samples, were granted a waiver of informed consent.
Deidentified individual data that supports the results will be shared after publication provided the investigator who proposes to use the data has approval from an institutional review board, independent ethics committee, or research ethics board, as applicable, and executes a data use and sharing agreement with University of North Carolina.
Funding for creation of the biorepository was provided by the Centers for Disease Control and Prevention Epidemiology and Laboratory Capacity program through the North Carolina Division of Public Health (award no. 5 NU50CK000530-05-00). Research components were funded by a Creativity Hub Award from the UNC Office of the Vice Chancellor for Research to R.M.B. H.A.A is supported by the Southeastern Center of Excellence in Vector Borne Diseases (U01CK000662). Database support (REDCap) was provided by the National Institutes of Health National Center for Advancing Translational Sciences (grant no. UM1TR004406).
Author contributions: study conception and design, R.M.B. and H.A.A; funding, R.M.B.; study implementation, all authors; data analysis, R.M.B., H.A.A., and L.U.; first draft of manuscript, R.M.B., H.A.A., and L.U.; and revisions, all authors.
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