Next, 100L of 10% NaCl solution is usually added to each tube, and the mixtures are left undisturbed at room temperature for 2h. SJ infected cell strains by building a relationship between cell number and copy number of the pathogen using a quantitative PCR-based standard curve. The assay also exhibited outstanding specificity, with no false positives observed against other bacterial species, includingEscherichia coli,Salmonella,Staphylococcus aureus, andListeria monocytogenes. In summary, an ICA which is sensitive, specific, and easy to operate was successfully established for the detection ofO. tsutsugamushiin scrub typhus, potentially enabling early quick point-of-care diagnosis of scrub typhus. Glycerol phenylbutyrate Keywords:O. tsutsugamushi, monoclonal antibody, polyclonal antibody, immunochromatography, colloidal platinum == 1. Glycerol phenylbutyrate Introduction == Scrub typhus is an acute zoonotic febrile illness caused byOrientia tsutsugamushi, an obligate intracellular bacterium (Xu et al., 2017). This disease threatens approximately one billion people worldwide and causes nearly one million cases annually (Bhandari et al., 2022;John and Varghese, 2020). The clinical manifestations of scrub typhus closely resemble other acute febrile illnesses, such as murine typhus, malaria, dengue fever, and viral hemorrhagic fever. Moreover, it is not responsive to standard antibiotics, leading to a high risk of misdiagnosis, delayed treatment, and potentially fatal outcomes (Ching et al., 2001). Therefore, accurate and timely diagnostic methods are crucial for the effective management of scrub typhus. Current diagnostic methods for scrub typhus include pathogen isolation and culture, nucleic acid detection, and immunological assays. Blood samples from scrub typhus patients and organs or blood from infected animals Nrp2 (mainly rodents) can be used for pathogen isolation and culture. Various experimental animals, such as mice and chicken embryo yolk sacs, and established cell lines, including L929, HeLa, BHK21, and Vero, can be employed for the cultivation ofO. tsutsugamushi(La Scola and Raoult, 1997;Tamura et al., 1995). Further identification is usually primarily performed through PCR amplification and gene sequencing ofO. tsutsugamushi-specific genes. However, the culture conditions forO. tsutsugamushiare stringent, and its reproduction is slow, requiring an environment of 35C and 5% CO2for cell contamination and growth (Luksameetanasan et al., 2007). Additionally, the isolation and culture ofO. tsutsugamushipose security Glycerol phenylbutyrate concerns and must be conducted in biosafety facilities (Paris and Dumler, 2016;Kim et al., 2006). Consequently, pathogen isolation is usually primarily used for laboratory research and is not suitable for routine diagnosis of scrub typhus due to its complex and time-consuming procedures (Luce-Fedrow et al., 2015). Nucleic acid-based detection methods offer high specificity and sensitivity, enabling early detection of bacterial infections. Genes such as the 56 kDa type-specific antigen gene (Kumar et al., 2019), GroEL gene (Paris et al., 2009), 16S rRNA gene (Sonthayanon et al., 2009), TraD conjugative transfer protein gene (Prakash et al., 2022), and 47 kDa membrane protease gene (Jiang et al., 2004) are commonly used as target genes for specific nucleic acid detection ofO. tsutsugamushi. Nucleic acid detection methods include polymerase chain reaction (PCR), multiplex PCR (mPCR), real-time fluorescence quantitative PCR (qPCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification, and electrochemical DNA sensors. Although these nucleic acid-based detection methods offer numerous advantages, their application is limited by the need for expensive gear, specialized personnel, and the potential for contamination. Immunological detection methods, such as the WeilFelix reaction (Dasch et al., 1979), indirect fluorescent immunoassay (IFA) (Blacksell et al., 2015), enzyme-linked immunosorbent assay (ELISA) (Xue et al., 2019), and immunochromatographic assay (ICA) (Tang et al., 2022), are also widely used for scrub typhus diagnosis. The Weil-Felix reaction, despite its low specificity, can be employed for preliminary screening of scrub typhus in rural and grassroots areas with inadequate diagnostic facilities. Both ELISA and IFA have the advantages of sensitivity and specificity, but they lack standardized background controls for antibody detection. Therefore, research in different regions is required to determine their detection thresholds and distinguish patients from healthy individuals based on antibody background levels (Saraswati et al., 2019). IFA is recognized as the gold standard for the diagnosis of scrub typhus, but it requires expensive gear and trained professionals, limiting its application in remote areas and epidemic settings. In recent years, the colloidal platinum immunochromatography method has gained attention as a research hotspot for the diagnosis of scrub typhus due to its accuracy, convenience, and quick results. However, most relevant studies focus on the detection of specific antibodies againstO. tsutsugamushi(Kim et al., 2013;Cao et al., 2007;Kingston et al.,.
