title: Improved detection of gastrointestinal pathogens using generalised sample processing and amplification panels.
abstract: We aimed to streamline the diagnosis of gastrointestinal disease by producing multiplexed real time polymerase chain reaction (PCR) panels employing universal sample processing for DNA and RNA containing pathogens. A total of 487 stored, previously characterised stool samples comprising bacterial, viral, protozoan and Clostridium difficile positive samples were tested using four multiplexed real time PCR panels. A further 81 pre-selected clinical samples from a teaching hospital were included to provide an independent validation of assay performance. Improved sensitivity was achieved using the protozoan panels and 16 more mixed infections were observed compared to tests with conventional methods. Using the C. difficile panels, 100% sensitivity was achieved when compared to the gold standard of toxigenic culture. In addition, hypervirulent strains including ribotype 027 could be identified directly from primary sample without the need for ribotyping methods. Bacterial and viral panels detecting Salmonella, Shigella, Campylobacter, Yersinia enterocolitica, Listeria monocytogenes, norovirus groups I and II, rotavirus A, astrovirus, sapovirus, rotavirus B, adenovirus and adenovirus 40/41 performed as well as conventional methods, whilst allowing detection in 3 hours from processing to result. Multiplex real time PCR panels with universal sample preparation allow streamlined, rapid diagnosis of gastrointestinal pathogens whilst extending the characterisation of pathogens present in stool samples from affected patients.
authors.affiliation: 1Genetic Signatures, c/o Virology Research Laboratories, Prince of Wales Hospital, Randwick 2Microbiology Division, SEALS, Prince of Wales Hospital, Randwick 3Virology Division, SEALS Microbiology, Prince of Wales, Randwick 4Department of Microbiology & Infectious Diseases, Pacific Laboratory Medicine Services, Royal North Shore Hospital, St Leonards 5Microbiology Department, St Vincent's Hospital, Darlinghurst 6General Microbiology, CIDMLS, Institute of Clinical Pathology and Medical Research, Westmead, NSW, Australia.
authors.forename: Shoo Peng
authors.initials: SP
authors.lastname: Siah
authors.forename: Juan
authors.initials: J
authors.lastname: Merif
authors.forename: Kiran
authors.initials: K
authors.lastname: Kaur
authors.forename: Jiny
authors.initials: J
authors.lastname: Nair
authors.forename: Peter G
authors.initials: PG
authors.lastname: Huntington
authors.forename: Thomas
authors.initials: T
authors.lastname: Karagiannis
authors.forename: Damien
authors.initials: D
authors.lastname: Stark
authors.forename: William
authors.initials: W
authors.lastname: Rawlinson
authors.forename: Tom
authors.initials: T
authors.lastname: Olma
authors.forename: Lee
authors.initials: L
authors.lastname: Thomas
authors.forename: John R
authors.initials: JR
authors.lastname: Melki
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Millar
mesh_terms: D000818:Animals; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D016360:Clostridioides difficile / Q000235:genetics / Q000302:isolation & purification; D005243:Feces / Q000382:microbiology / Q000469:parasitology; D005767:Gastrointestinal Diseases / Q000175:diagnosis* / Q000382:microbiology / Q000469:parasitology; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010271:Parasites / Q000235:genetics / Q000302:isolation & purification*; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013997:Time Factors; D014780:Viruses / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D011933:Reagent Kits, Diagnostic