Abstract
Scrub typhus is caused by an obligated intracellular organism, Orientia tsutsugamushi (Orientia). The disease was traditionally thought to be limited in the tsutsugamushi triangle. Recently, scrub typhus has been confirmed in areas outside the triangle. Serological diagnosis of scrub typhus relies on indirect immunofluorescence assay (IFA). Molecular assays such as PCR, qPCR, loop-mediated isothermal amplification, and recombinase polymerase amplification are often targeting a single copy gene. These assays are sensitive and specific, yet they are not broadly used in clinical settings possibly due to low circulating Orientia in blood. In this study, we compared qPCR results using a multiple copy (traD) gene with those using a single copy (47 kDa) gene to assess the improvement of sensitivity and limit of detection. Our results demonstrate that the qPCR using the traD gene provides superior sensitivity in 15 Orientia strains. The limit of detection is below single Orientia genome equivalent and the assay retains specificity with excessive DNA from mouse, chiggers and human. The clinical utility was evaluated using confirmed scrub typhus positive and negative samples. The results show 100% sensitivity and specificity in these samples suggesting that the traD gene qPCR may be useful for clinical diagnosis of Orientia infection.
Highlights
Scrub typhus (ST) is an acute febrile illness caused by an obligate intracellular bacterium, Orientia tsutsugamushi (Orientia)
We demonstrated that the assay is broadly reactive to detect Orientia DNA from 15 strains previously isolated in the tsutsugamushi triangle
It was clear that the traD gene qPCR consistently showed lower cycle threshold (Ct) in comparison to the 47 kDa gene qPCR
Summary
Scrub typhus (ST) is an acute febrile illness caused by an obligate intracellular bacterium, Orientia tsutsugamushi (Orientia). The disease is prevalent in the tsutsugamushi triangle encompassing a broad Asia–Pacific region where over 1 billion people are at risk with estimated 1 million cases annually. The disease can contribute up to 20% of all acute undifferentiated febrile illnesses. Among the blood culture-negative fever patients, as high as 27% of these patients are scrub typhus positive [3,4,5]. The mortality of this high prevalence disease ranges from as low as 1.4% to as high as 70%. The disease has recently been found outside the traditional triangle in places like Middle East, South America and Africa [9,10,11,12], highlighting the global need for a sensitive diagnostic assay
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