Dataset Information

Clinical Usefulness of Nested Reverse-Transcription Polymerase Chain Reaction for the Diagnosis of Severe Fever with Thrombocytopenia Syndrome.

ABSTRACT: Severe fever with thrombocytopenia syndrome (SFTS), caused by SFTS virus (SFTSV) is an emerging tick-borne infectious disease. Few studies have assessed the clinical usefulness of nested reverse-transcription polymerase chain reaction (RT-PCR) for diagnosing SFTS. We performed conventional RT-PCR targeting the M segment, nested RT-PCR targeting M and S segments, and real-time RT-PCR targeting the S segment of SFTSV for four patients with suspected SFTS. Although conventional RT-PCR results for the first two patients were negative at admission, nested RT-PCR using the S or M targets was positive for the same samples. Likewise, in the other two patients, initial samples were confirmed positive in all three tests, but follow-up testing demonstrated negative conventional RT-PCR and positive nested RT-PCR results. Thus, delayed testing using conventional RT-PCR or real-time RT-PCR in symptomatic patients with SFTS may result in missed diagnoses, and compared with these methods, nested RT-PCR may increase the window for obtaining positive SFTSV PCR results. Meanwhile, the indirect immunofluorescence assay showed seroconversion to SFTSV antibodies in all four patients. Nested RT-PCR for SFTSV M and S segments could help diagnose SFTS in patients testing negative by conventional RT-PCR.

SUBMITTER: Kim CM

PROVIDER: S-EPMC8592136 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Clinical Usefulness of Nested Reverse-Transcription Polymerase Chain Reaction for the Diagnosis of Severe Fever with Thrombocytopenia Syndrome.

Kim Choon-Mee CM Kim Dong-Min DM Yun Na-Ra NR

The American journal of tropical medicine and hygiene 20210802 4

Severe fever with thrombocytopenia syndrome (SFTS), caused by SFTS virus (SFTSV) is an emerging tick-borne infectious disease. Few studies have assessed the clinical usefulness of nested reverse-transcription polymerase chain reaction (RT-PCR) for diagnosing SFTS. We performed conventional RT-PCR targeting the M segment, nested RT-PCR targeting M and S segments, and real-time RT-PCR targeting the S segment of SFTSV for four patients with suspected SFTS. Although conventional RT-PCR results for t ...[more]

PMID: 34339382

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Project description:UnlabelledSevere fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen that was first reported in China in 2009. Phylogenetic analysis of the viral genome showed that SFTS virus represents a new lineage within the Phlebovirus genus, distinct from the existing sandfly fever and Uukuniemi virus groups, in the family Bunyaviridae. SFTS disease is characterized by gastrointestinal symptoms, chills, joint pain, myalgia, thrombocytopenia, leukocytopenia, and some hemorrhagic manifestations with a case fatality rate of about 2 to 15%. Here we report the development of reverse genetics systems to study STFSV replication and pathogenesis. We developed and optimized functional T7 polymerase-based M- and S-segment minigenome assays, which revealed errors in the published terminal sequences of the S segment of the Hubei 29 strain of SFTSV. We then generated recombinant viruses from cloned cDNAs prepared to the antigenomic RNAs both of the minimally passaged virus (HB29) and of a cell culture-adapted strain designated HB29pp. The growth properties, pattern of viral protein synthesis, and subcellular localization of viral N and NSs proteins of wild-type HB29pp (wtHB29pp) and recombinant HB29pp viruses were indistinguishable. We also show that the viruses fail to shut off host cell polypeptide production. The robust reverse genetics system described will be a valuable tool for the design of therapeutics and the development of killed and attenuated vaccines against this important emerging pathogen.ImportanceSFTSV and related tick-borne phleboviruses such as Heartland virus are emerging viruses shown to cause severe disease in humans in the Far East and the United States, respectively. Study of these novel pathogens would be facilitated by technology to manipulate these viruses in a laboratory setting using reverse genetics. Here, we report the generation of infectious SFTSV from cDNA clones and demonstrate that the behavior of recombinant viruses is similar to that of the wild type. This advance will allow for further dissection of the roles of each of the viral proteins in the context of virus infection, as well as help in the development of antiviral drugs and protective vaccines.

Project description:The definitive diagnosis of lyssavirus infection (including rabies) in animals and humans is based on laboratory confirmation. The reference techniques for post-mortem rabies diagnosis are still based on direct immunofluorescence and virus isolation, but molecular techniques, such as polymerase chain reaction (PCR) based methods, are increasingly being used and now constitute the principal tools for diagnosing rabies in humans and for epidemiological analyses. However, it remains a key challenge to obtain relevant specificity and sensitivity with these techniques while ensuring that the genetic diversity of lyssaviruses does not compromise detection. We developed a dual combined real-time reverse transcription polymerase chain reaction (combo RT-qPCR) method for pan-lyssavirus detection. This method is based on two complementary technologies: a probe-based (TaqMan) RT-qPCR for detecting the RABV species (pan-RABV RT-qPCR) and a second reaction using an intercalating dye (SYBR Green) to detect other lyssavirus species (pan-lyssa RT-qPCR). The performance parameters of this combined assay were evaluated with a large panel of primary animal samples covering almost all the genetic variability encountered at the viral species level, and they extended to almost all lyssavirus species characterized to date. This method was also evaluated for the diagnosis of human rabies on 211 biological samples (positive n = 76 and negative n = 135) including saliva, skin and brain biopsies. It detected all 41 human cases of rabies tested and confirmed the sensitivity and the interest of skin biopsy (91.5%) and saliva (54%) samples for intra-vitam diagnosis of human rabies. Finally, this method was successfully implemented in two rabies reference laboratories in enzootic countries (Cambodia and Morocco). This combined RT-qPCR method constitutes a relevant, useful, validated tool for the diagnosis of rabies in both humans and animals, and represents a promising tool for lyssavirus surveillance.

Dataset Information

Clinical Usefulness of Nested Reverse-Transcription Polymerase Chain Reaction for the Diagnosis of Severe Fever with Thrombocytopenia Syndrome.

Publications

Clinical Usefulness of Nested Reverse-Transcription Polymerase Chain Reaction for the Diagnosis of Severe Fever with Thrombocytopenia Syndrome.

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