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

Abstract

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.

Accuracy of reverse‐transcription polymerase chain reaction and loop‐mediated isothermal amplification in diagnosing severe fever with thrombocytopenia syndrome: A systematic review and meta‐analysis

“…After reading the full text of the articles, 103 were discarded, including 48 that did not use clinical patient samples or lacked data, 35 that were not on nucleic acid‐related diagnostic techniques, 14 that lacked reference standards, and 6 that were not on or related to SFTS. A final total of 16 articles were included, seven on RT‐PCR 19 , 20 , 24 , 25 , 26 , 27 , 28 and seven on RT‐LAMP, 16 , 21 , 29 , 30 , 31 , 32 , 33 and two that were on both RT‐PCR and RT‐LAMP. 15 , 34 A flowchart of the research process is shown in Figure 1 .…”

“… 40 , 41 , 42 Even if the conditions are limited, primary clinical decisions can still be implemented. However, for use in scientific research, more sensitive multiplex detection experiments still need be developed for the more rapid detection of multiple viral diseases, as demonstrated in the study by Jang et al, 16 in which the simultaneous detection of SFTS and scrub typhus was attempted.…”

“…Diagnostic techniques used to identify SFTS include traditional pathogen detection, such as virus isolation and serum antigen testing, as well as antibody detection techniques, such as enzyme‐linked immunosorbent assay (ELISA), and neutralization test 2 . With the progress of technology, significant developments have been likewise made in nucleic acid molecular diagnosis testing, expanding its applications to various scenarios and detection targets 15–17 and turning it into the gold standard of many virological diseases, such as coronavirus disease 2019, an acute infectious disease that is spreading globally 18 . Meanwhile, ELISA and other traditional virus detection techniques have some disadvantages; they are time‐consuming, expensive, and complicated to perform 4,19 .…”

Advancements in the Worldwide Detection of Severe Fever with Thrombocytopenia Syndrome Virus Infection from 2009 to 2023

“…Detection of SFTSV genome could be achieved by different nucleic acid detection techniques such as RT-PCR (13,(40)(41)(42)(43), real-time RT-PCR (23,28,41,(44)(45)(46)(47)(48), LAMP (24,(49)(50), as well as RPA (25,(51)(52).…”

“…The ability to differentiate between SFTSV strains can be facilitated by the M segment. A one-step RT-PCR assay targeting this M segment was developed by Sun et al (23), which exhibited high specificity and sensitivity and was capable of detecting as few as 10 copies of the viral RNA per reaction.…”