Developing a multiplex loop-mediated isothermal amplification assay (LAMP) to determine severe fever with thrombocytopenia syndrome (SFTS) and scrub typhus

Severe fever with thrombocytopenia syndrome (SFTS) and scrub typhus are endemic zoonotic diseases that pose significant public health threats in East Asia. As these two diseases share common clinical features, as well as overlapping disease regions, it is difficult to differentiate between SFTS and scrub typhus. A multiplex reverse-transcription loop‑mediated isothermal amplification (RT-LAMP) assay was developed to detect large segments and GroES genes for SFTS virus (SFTSV) and Orientia tsutsugamushi (OT). The performance of the RT-LAMP assay was compared and evaluated with those of commercial PowerChek™ SFTSV real-time PCR and LiliF™ TSUTSU nested PCR for 23 SFTS and 12 scrub typhus clinical samples, respectively. The multiplex SFTSV/OT/Internal control (IC) RT-LAMP assay showed comparable sensitivity (91.3%) with that of commercial PowerChek™ SFTSV Real-time PCR (95.6%) and higher sensitivity (91.6%) than that of LiliF™ TSUTSU nested PCR (75%). In addition, the multiplex SFTSV/OT RT-LAMP assay showed 100% specificity and no cross-reactivity for blood from uninfected healthy patients and samples from patients infected with other fever viruses. Thus, the multiplex SFTSV/OT/IC RT-LAMP assay could serve as a useful point-of-care molecular diagnostic test for SFTS and scrub typhus.


Introduction
Severe fever with thrombocytopenia syndrome (SFTS) and scrub typhus are endemic zoonotic diseases that are becoming significant public health threats in East Asia [1,2]. The causative pathogen of SFTS is severe fever thrombocytopenia syndrome virus (SFTSV), which is a newly  (23) and scrub typhus (12) clinical samples were confirmed using the SFTSV qRT-PCR and O. tsutsugamushi qPCR, which were previously reported by Yoshikawa et. al. [35] and Tantibhedhyangkul et. al. [26], respectively. To assess the specificity of the multiplex SFTSV/OT/IC RT-LAMP assay, 100 clinical sample specimens from individuals with (38) and without (62) other viral infections were tested. Viral infection samples, as confirmed via PCR using qRT-PCR [36][37][38]

Primer design
The RT-LAMP primer sets for SFTSV and O. tsutsugamushi were designed from the conserved regions of the L segment and groES genes ( Table 1). The actin beta gene in humans was used as an internal control (IC), as previously reported [39]. All LAMP primers including two outer primers (forward primer F3 and backward primer B3), two inner primers (forward inner primer FIP and backward inner primer BIP), and two loop primers (forward loop primer LF and backward loop primer LB) were designed using the Primer Explorer v4 software (Eiken Chemical Co., Tokyo, Japan). For multiple LAMPs, we used a slightly modified DARQ probe method. Briefly, a dye-labeled artificial nucleic acid + FLP sequence probe and a quencherlabeled displacement probe complementary to the artificial nucleic acid sequence were used. In this study, two types of artificial nucleic acids (32mers and 35mers) were used for multiplexing different fluorescence (FAM/Hex and Cy5) quenched by BHQ1 and BHQ2, respectively. A FAM (or Hex)-labeled 32-artificial oligomer-SFTS (or IC) FLP was designed for SFTS (or IC) FLP probe 1 and a Cy5-labeled 35-artificial oligomer-OT FLP was designed for tsu FLP probe 2. The quencher-labeled 30-oligonucleotide (BHQ1) or 35-oligonucleotide (BHQ2) was complementary to artificial nucleic acids sequences of SFTS (or IC) FLP probe 1 and tsu FLP probe 2, respectively. Before use in LAMP, all primers were assessed for specificity by performing a BLAST search. All LAMP primers and probes were synthesized by Macrogen (Seoul, South Korea).

Real-time RT-PCR
To confirm SFTS and scrub typhus positive clinical samples, the SFTSV L, M, and S gene qRT-PCR primer sets [35] and O. tsutsugamushi groEL gene qPCR primer set [26] were used, and PCR conditions were set according to the protocol described by Yoshikawa

Multiplex RT-LAMP
For multiplex SFTSV/OT/IC RT-LAMP assay, the reaction mixture was prepared with 12.

Limits of detection
pTOP Blunt V2 plasmids, including partial L segment gene sequences of SFTSV and groES gene sequences of O. tsutsugamushi, were used to test the limit of detection (LOD) of the RT-LAMP assay. All plasmids were constructed by Macrogen. To determine the LOD of the monoplex SFTSV RT-LAMP, monoplex OT RT-LAMP, and multiplex SFTSV/OT/IC RT-LAMP assays, the plasmids were serially diluted 10-fold from 1 × 10 6 to 1 × 10 0 copies/μL. This test was repeated 3 times. In addition, the LODs of the monoplex SFTSV RT-LAMP, monoplex OT RT-LAMP, and multiplex SFTSV/OT/IC RT-LAMP assays were compared with those of the commercial PowerChek™ SFTSV Real-time PCR kit and LiliF™ TSUTSU nested PCR kit for 2-fold serial dilutions of clinical samples from patients infected with SFTSV or O. tsutsugamushi. According to FDA EUA guidelines for COVID-19 diagnostic tests, the LODs of the monoplex SFTSV RT-LAMP, monoplex OT RT-LAMP, and multiplex SFTSV/OT/IC RT-LAMP assays were determined as the minimum concentration in a 2-fold dilution series at which 19 of 20 replicates amplify [40,41]. the LODs of commercial PowerChek™ SFTSV Realtime PCR kit and LiliF™ TSUTSU nested PCR kit were repeated five times and determined as the minimum concentration in a 2-fold dilution series at which 5 of 5 replicates amplify.

Comparison of detection limits of the multiplex SFTSV/OT/IC RT-LAMP assay with those of commercial kits for serial diluted SFTS and scrub typhus clinical samples
The LODs of the monoplex and multiplex SFTSV/OC/IC LAMP primer sets were compared to those of two commercial kits, the PowerChek™ SFTSV Real-time PCR kit (Kogene Biotech, Seoul, Korea) and LiliF™ TSUTSU nested PCR kit (iNtRON, Seongnam-Si, South Korea), for 2-fold serial diluted SFTSV serum samples and scrub typhus blood samples (range 2 8 -2 15 and 2 1 −2 8 , respectively) ( Table 3). For serial two-fold diluted SFTS clinical samples, monoplex and PowerChekTM SFTSV Real-time PCR kit showed the same LODs of 2 10 , whereas the LOD of  multiplex SFTSV/OC/IC LAMP assay was 2 12 , which is one step lower than results of others. However, for scrub typhus clinical samples, both of the monoplex and multiplex SFTSV/OC/ IC LAMP assay showed the superior LOD (2 5 ) to that (2 3 ) of the LiliFTM TSUTSU nested PCR kit.

Comparison of the clinical performance of the multiplex SFTSV/OT/IC RT-LAMP assay with those of the commercial PowerChek™ SFTSV Realtime PCR kit and LiliF™ TSUTSU nested PCR kit using clinical samples
To confirm the clinical performance of the multiplex SFTSV/OT/IC RT-LAMP assay, the sensitivities of the assays were compared to those of the commercial PowerChek™ SFTSV Realtime PCR kit and LiliF™ TSUTSU nested PCR kit for 35 clinical samples from patients infected with SFTSV (23) and O. tsutsugamushi (12). For the specificities of these three assays, 100 clinical sample specimens from individuals with (38) and without (62) other viral infections were used (Table 4). For the SFTS clinical samples (n = 23), the sensitivity of the PowerChek™ SFTSV Real-time PCR kit was 95.6% (S gene: 95.6%, and M genes: 86.9%). The sensitivity of the multiplex SFTSV/OT/IC RT-LAMP assay was 91.3%. For scrub typhus clinical samples (n = 12), the sensitivity of the LiliF™ TSUTSU nested PCR kit was 75% and that of the multiplex SFTSV/OT/IC RT-LAMP assay was 91.6%. Overall, the multiplex SFTSV/OT/IC RT-LAMP assay showed similar sensitivities to that of the commercial PowerChek™ SFTSV Real-time PCR kit and superior sensitivity to the LiliF™ TSUTSU nested PCR kit. For 100 Table 3. negative clinical samples (non-infection/other viral infections; n = 64/38), the specificity of the two assays was 100%, except for the LiliF™ TSUTSU nested PCR kit (98%) ( Table 4).

Cross-reactivity tests of SFTSV/OT/IC RT-LAMP assay with other fever viruses
To confirm the absence of cross-reactivity with other common fever viruses, a total of 38 viral RNA samples, including Hantaan virus, Dengue virus, chikungunya virus, influenza virus A, influenza virus B, RSV A, RSV B, coronavirus 229E, NL63, and OC43, were tested using the multiplex SFTSV/OT/IC RT-LAMP assay ( Table 5). As a result, the multiplex SFTSV/OT/IC RT-LAMP assay did not show cross-reactivity for all the tested samples.

Discussion
Severe fever with thrombocytopenia syndrome virus (SFTSV) and O. tsutsugamushi are not transmitted by the same vectors; however, these two diseases share a common point as they are transmitted by arthropod bites mostly during outdoor activities [18,42]. In addition, these two diseases share common clinical features, such as fever, nausea, vomiting, diarrhea, headache, and muscle pain, as well as overlapping disease regions. Furthermore, many cases of coinfection with SFTSV and O. tsutsugamushi have been reported [43][44][45].
Here, we developed a multiplex SFTSV/OT/IC RT-LAMP assay to detect SFTSV L segment, groES, and actin beta genes. In sensitivity tests for SFTS and scrub typhus clinical samples, the multiplex SFTSV/OT/IC RT-LAMP assay showed 91.3% and 91.6% for SFTS (n = 23) and scrub typhus (n = 12) clinical samples, respectively. These results are comparable to those (M gene: 86.9% and S gene: 95.6%) of the commercial PowerChek™ SFTSV Real-time PCR, and superior to 75% of LiliF™ TSUTSU nested PCR kit. The sensitivity and specificity results of PowerChek™ SFTSV Real-time PCR were within the scope of previous results (sensitivity: 100% (CI: 73.2%-100%) and sensitivity: 98.1% (CI: 92.5%-99.7%)) reported by Yoo. et al [46]. Therefore, the results of this test are considered reliable, although they were tested with a small number of samples. In addition, the LODs for the multiplex SFTSV/OT/IC RT-LAMP for To simultaneously detect SFTSV and O. tsutsugamushi, nucleic acid (DNA/RNA) extraction from whole blood is an important step in multiplex SFTSV/OT/IC RT-LAMP assay. Here, Nextractor 1 NX-48S, which is an automated nucleic acid extraction instrument, was used for fast (within 20 min) and contamination-free extraction of nucleic acids from serum or whole blood. Unfortunately, the multiplex SFTSV/OT/IC RT-LAMP assay could not detect OT signals with nucleic acids (DNA/RNA) extracted from the serum of patients infected with O. tsutsugamushi. This may be because O. tsutsugamushi is present at low concentrations in the serum. In fact, most of the O. tsutsugamushi qPCR and LAMP assays developed to date were performed using DNA extracted from whole blood, buffy coat, and eschars of patients infected with O. tsutsugamushi [30,47,48]. In the sensitivity test, we used 24 SFTS serum clinical samples to confirm the performance of the multiplex SFTSV/OT/IC RT-LAMP assay because of the small number of SFTS whole blood samples. Thus, to confirm whether the multiplex SFTSV/OT/IC RT-LAMP assay can detect SFTSV in whole blood samples, an additional 12 whole blood samples, which were matched with the positive SFTS serum sample, were tested and confirmed to be positive in the multiplex SFTSV/OT/IC RT-LAMP assay (S1 Table).
Our study has some limitations. First, the multiplex SFTSV/OT/IC RT-LAMP assay was performed with a relatively small sample size of positive SFTS (23) and scrub typhus (12) clinical samples, which resulted in widened confidence intervals for our estimates of diagnostic accuracy. However, considering that the multiplex SFTSV/OT/IC RT-LAMP assay showed the same or higher sensitivity compared to the two commercially available diagnostic kits, and 100% specificity to the negative samples, the multiplex SFTSV/OT/IC RT-LAMP assay is sufficiently competitive in commercial development. In addition, it can be applied quickly and cost-effectively, particularly in the early stages of fever in patients in endemic areas. Second, in SFTS LOD test, we used SFTS DNA plasmid as a standard material for LOD test of the multiplex SFTSV/OT/IC RT-LAMP assay. However, there is the deficiency of characterizing an RT-LAMP assay with a DNA standard because it cannot confirm the function of reverse transcriptase. Thus, using SFTS and scrub typhus clinical samples, we reconfirmed the limit of detection of monoplex and multiplex SFTSV/OT/IC RT-LAMP assay compared to those of commercial LiliF™ TSUTSU nested PCR and PowerChekTM SFTSV Real-time PCR kit.
In this study, we developed a multiplex SFTSV/OT/IC RT-LAMP assay capable of simultaneous diagnosis of SFTS and scrub typhus within 40 min. For sensitivity and specificity tests with SFTS and scrub typhus clinical samples, the multiplex SFTSV/OT/IC RT-LAMP assay showed similar or superior performance compared with commercial PowerChek™ SFTSV Real-time PCR and LiliF™ TSUTSU nested PCR. Thus, the multiplex SFTSV/OT/IC RT-LAMP assay could serve as a useful point-of-care molecular diagnostic test for SFTS and scrub typhus.
Supporting information S1 Table. Sensitivities the multiplex SFTSV/OT/IC RT-LAMP assay for whole blood SFTS clinical samples.