Scrub typhus is an acute febrile human disease caused by mite vectors, which are infected by a gram negative bacterium O. tsutsugamushi. Previously endemic throughout the Pacific Rim of Asia, including China, Japan, Korea, Pakistan, India, Thailand, Malaysia and Australia(Kelly et al., 2009; Tamura et al., 2001; Chang et al., 1990; Richards and Jiang, 2020; Akhunji et al., 2019; Fournier et al., 2008; Mohamed Zan et al., 2016; Odorico et al., 1998). Recent studies have shown that scrub typhus is no longer limited to this “tsutsugamushi triangle” (Xu et al.,2017). Scrub typhus has been reported in Africa, the Middle East and South America (Jiang and Richards, 2018; Weitzel et al., 2016). it is estimated that 1 billion people are at risk for infection and at least 1 million cases annually are reported in Southeast Asia alone (Xu et al., 2017; Kala et al., 2020). In China, scrub typhus was traditionally prevalent in the tropical and subtropical regions of the south, but in recent years, the epidemic areas of scrub typhus have expanded north and south-west (Latif et al., 2017). Cases have been reported in all provinces of mainland China and small-scale outbreaks regularly occur in many areas. Scrub typhus is becoming a serious public health disease in China and other epidemic countries (Peng et al., 2022).

Infection by O. tsutsugamushi causes clinical symptoms similar to other febrile diseases such as leptospirosis, murine typhus, dengue fever, influenza, and other viral hemorrhagic fevers, which prevents timely and accurate diagnosis. (Furuya et al., 1991). In addition, many epidemic areas are relatively remote and suffer from inadequate medical facilities (Janardhanan et al., 2014). Therefore, it is necessary to establish a simple and portable diagnostic method for fast and early diagnosis of scrub typhus.

Molecular methods and serological tests are currently the most common methods used to diagnose scrub typhus. Molecular methods, although sensitive and specific, is time-consuming, expensive, and requires specialized instrumentation and personnel, as such it is not practical for most areas where scrub typhus is endemic. The simplest and quickest serological test is the Weil-Felix test, although it is not sensitive or specific. Other serological tests, such as complement fixation, indirect immunofluorescence (IFA), and enzyme-linked immunosorbent assay (ELISA) suffer from many of the same shortcomings as the nucleic acid tests in that they require specialized equipment and personnel (Dasch et al., 1979; Blacksell et al., 2016; Xue et al., 2019; Tang et al., 2022).

Immunochromatographic test (ICT) technologies however, provide a mechanism for efficient point-of-care and field-use serological testing. Colloidal gold immunochromatography has been successfully used to test for scrub typhus. However false positives are not uncommon because of the background color from the test serum. It can also difficult to distinguish low level positives if reading the test strip in daylight, which is usual under field conditions. In these respects, fluorescent microspheres (FMs) perform better than colloidal gold nanoparticles, neither serum color nor bright ambient light interferes with signal intensity, thus the test is more sensitive and less prone to false positives (Xue et al., 2019).

The 56 kDa outer membrane protein of O. tsutsugamushi (which contains the group-specific and serotype-specific epitopes) is the diagnostic antigen for colloidal gold immunochromatography (Tang et al., 2022). Antigenic variation among O. tsutsugamushi strains has been attributed to the 56 kDa antigen, so it is necessary to use a mixture of 56 kDa proteins from common strain types in order to provide a reliable scrub typhus diagnosis (Elisberg et al., 1968; Jang et al., 2003; Kim et al., 2013a). In this work, we used a mixture of recombinant 56 kDa proteins of SJ, Ptan, and Gilliam O. tsutsugamushi strains, which are representative of epidemic strains in China, as diagnostic antigens in developing a fluorescent microsphere based immunochromatographic assay for the rapid detection of O. tsutsugamushi antibodies in human serum samples.