Highly Sensitive Molecular Diagnostic Platform for Scrub Typhus Diagnosis Using O. tsutsugamushi Enrichment and Nucleic Acid Extraction

Abstract

Scrub typhus is caused by the Gram-negative obligate intracellular bacterium Orientia tsutsugamushi, and this tick-borne disease is difficult to distinguish from other acute febrile illnesses as it typically presents with symptoms such as rash, crusting at the bite site, headache, myalgia, lymphadenopathy, and elevated liver transaminases. It can often be diagnosed clinically, but not all patients present with characteristic symptoms, so serological diagnosis and molecular techniques may be required. However, existing diagnostic tests often have low sensitivity and specificity, making early detection difficult. This study presents a nucleic acid extraction method using large volumes of plasma and buffy coat to increase sensitivity, as well as an improved detection method using two target genes. Using the I-PULL device, nucleic acids can be extracted from up to 4 mL of sample in 30 min, avoiding contamination. The extracted DNA detects two genes of O. tsutsugamushi, increasing sensitivity compared to single-gene detection. Clinical validation in 38 patient samples showed 100% specificity and 95.24% sensitivity for the single target gene, with specificity and sensitivity rising to 100% when both genes are analyzed. This molecular diagnostic platform can be useful for distinguishing scrub typhus from similar diseases.

Keywords: DNA detection kit; molecular diagnostics; sample preparation; scrub typhus.

Figure 1

Schematic diagram of rapid pathogen diagnostic systems using I-PULL device. In situ sample processing involves pathogen enrichment and DNA extraction from plasma and buffy coats using amine-functionalized diatomaceous earth within 40 min. Detection for scrub typhus DNA used another gene. This novel system can be used for the rapid, simple and sensitive diagnosis of scrub typhus. The nucleic acid extraction step using the I-PULL instrument is divided into two main steps. The first step is the concentration of the pathogen and the binding of the nucleic acid to the surface of the ADE. The whole process takes place inside a sealed instrument, safe from external contamination. The solution in the container can be passed through the PTFE membrane by pulling down on the handle. Next, the filter membrane is removed, and the elution solution is injected to separate the nucleic acids.

Figure 2

Optimization and characterization of amine-functionalized diatomaceous earth (ADE). (A) Comparison of nucleic acid extraction efficiency and ADE size. (B,C) SEM images of 20 um size and sieved ADE.

Figure 3

Characterization of the I-PULL device. (A) I-PULL device 3D illustration. The dimensions of the device are 12.5 cm high by 4.5 cm wide. The device consists of a container and a body. (B) Schematic of the I-PULL device. The I-PULL is divided into three main parts. (C,D) Selection of filter pore size and material used in the device. (E) Comparison of pathogen enrichment efficiency of the same concentration in different PBS volumes.

Figure 4

Optimization of nucleic acid extraction and PCR kit detection efficiency using the I-PULL system. The DNA extraction process was optimized and evaluated based on the cycle threshold (C_t) values after performing real-time quantitative PCR (qPCR). (A–D) Optimization of DNA extraction conditions. Results as a function of (A) DMS concentration and (B) ADE volume for DNA extraction. (C) Optimization of incubation time and (D) elution time for binding of ADE to DNA. (E) Limits of detection for DNA extraction based on optimizations results. (F) Comparison experiment of DNA extraction using patient samples. Plasma and buffy coat from five scrub typhus patients were used to extract DNA using two different methods (I-PULL and a commercial kit). Each patient was represented by a different color. (G) A serial dilution of synthetic DNA was prepared. The amplification plot is represented.

Figure 5

Clinical application of the scrub typhus diagnostic system with plasma and buffy coat. Plasma and buffy coat isolated from 38 different febrile patients were analyzed by nucleic acid extraction using I-PULL device followed by qPCR. (A) Comparison of Ct values of 21 scrub-typhus-positive patients and 17 negative patients for target gene A and target gene B (B). (C) The ROC graph shows the results of using target genes A and B individually and the results of using both target genes together. The diagnostic result using the two target genes is 100% specificity and sensitivity.