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A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus.

ABSTRACT: CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus).

SUBMITTER: Das S

PROVIDER: S-EPMC4575071 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus.

Das Sanchita S Rundell Mark S MS Mirza Aashiq H AH Pingle Maneesh R MR Shigyo Kristi K Garrison Aura R AR Paragas Jason J Smith Scott K SK Olson Victoria A VA Larone Davise H DH Spitzer Eric D ED Barany Francis F Golightly Linnie M LM

PloS one 20150918 9

CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in ...[more]

PMID: 26381398

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Project description:Crimean-Congo hemorrhagic fever virus (CCHFV) is a widespread tick-borne zoonotic virus that causes Crimean-Congo hemorrhagic fever (CCHF). CCHF is asymptomatic in infected animals but can develop into severe illness in humans, with high case-fatality rates. Due to complex environmental and socio-economic factors, the distribution of CCHFV vectors is changing, leading to disease occurrence in previously unaffected countries. Neither an effective treatment nor a vaccine has been developed against CCHFV; thus, surveillance programs are essential to limit and control the spread of the virus. Furthermore, the WHO highlighted the need of assays that can cover a range of CCHFV antigenic targets, DIVA (differentiating infected from vaccinated animals) assays, or assays for future vaccine evaluation. Here, we developed a multiplex assay, based on a suspension microarray, able to detect specific antibodies in ruminants to three recombinantly produced CCHFV proteins: the nucleocapsid (N) protein and two glycoproteins, GN ectodomain (GNe), and GP38. This triplex assay was used to assess the antibody response in naturally infected animals. Out of the 29 positive field sera to the N protein, 40% showed antibodies against GNe or GP38, with 11 out of these 12 samples being positive to both glycoproteins. To determine the diagnostic specificity of the test, a total of 147 sera from Spanish farms free of CCHFV were included in the study. This multiplex assay could be useful to detect antibodies to different proteins of CCHFV as vaccine target candidates and to study the immune response to CCHFV in infected animals and for surveillance programs to prevent the further spread of the virus. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) causes Crimean-Congo hemorrhagic fever, which is one of the most important tick-borne viral diseases of humans and has recently been found in previously unaffected countries such as Spain. The disease is asymptomatic in infected animals but can develop into severe illness in humans. As neither an effective treatment nor a vaccine has been developed against CCHFV, surveillance programs are essential to limit and control the spread of the virus. In this study, a multiplex assay detecting antibodies against different CCHFV antigens in a single sample and independent of the ruminant species has been developed. This assay could be very useful in surveillance studies, to control the spread of CCHFV and prevent future outbreaks, and to better understand the immune response induced by CCHFV.

Project description:Purpose of reviewThis article presents a comprehensive narrative review of reactive arthritis (ReA) with focus on articles published between 2018 and 2020. We discuss the entire spectrum of microbial agents known to be the main causative agents of ReA, those reported to be rare infective agents, and those reported to be new candidates causing the disease. The discussion is set within the context of changing disease terminology, definition, and classification over time. Further, we include reports that present at least a hint of effective antimicrobial therapy for ReA as documented in case reports or in double-blind controlled studies. Additional information is included on microbial products detected in the joint, as well as on the positivity of HLA-B27.Recent findingsRecent reports of ReA cover several rare causative microorganism such as Neisseria meningitides, Clostridium difficile, Escherichia coli, Hafnia alvei, Blastocytosis, Giardia lamblia, Cryptosporidium, Cyclospora cayetanensis, Entamoeba histolytica/dispar, Strongyloides stercoralis, β-haemolytic Streptococci, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Mycobacterium bovis bacillus Calmette-Guerin, and Rickettsia rickettsii. The most prominent new infectious agents implicated as causative in ReA are Staphylococcus lugdunensis, placenta- and umbilical cord-derived Wharton's jelly, Rothia mucilaginosa, and most importantly the SARS-CoV-2 virus. In view of the increasingly large spectrum of causative agents, diagnostic consideration for the disease must include the entire panel of post-infectious arthritides termed ReA. Diagnostic procedures cannot be restricted to the well-known HLA-B27-associated group of ReA, but must also cover the large number of rare forms of arthritis following infections and vaccinations, as well as those elicited by the newly identified members of the ReA group summarized herein. Inclusion of these newly identified etiologic agents must necessitate increased research into the pathogenic mechanisms variously involved, which will engender important insights for treatment and management of ReA.

Dataset Information

A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus.

Publications

A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus.

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