title: Multicentre evaluation of a real-time PCR assay to detect genes encoding clinically relevant carbapenemases in cultured bacteria.
abstract: The performance and portability of a multiplex real-time PCR assay to detect KPC, NDM, OXA-48-like and VIM carbapenemase gene families from bacterial isolates was assessed using Rotor-Gene Q and ABI 7500 instruments. Gram-negative bacterial isolates (n=502) were comprised of 100 isolates each with KPC, NDM, VIM or OXA-48-like carbapenemases (including 17 with OXA-181) and 2 isolates with NDM+OXA-48-like enzymes (including 1 with OXA-181) as well as 100 assay-negative isolates comprised of 24 IMP-producers, 24 carbapenem-resistant isolates with no known carbapenemase gene and 52 extended-spectrum β-lactamase-producing carbapenem-susceptible isolates. A multicentre evaluation was carried out in five laboratories using a subset of 100 isolates comprised of 22 isolates each with KPC, NDM, OXA-48-like or VIM alleles and 12 isolates that were negative for the assay targets. Initial validation of the assay on both the Rotor-Gene Q and ABI 7500 instruments demonstrated 100% sensitivity amongst the 402 isolates that were positive for KPC, NDM, OXA-48-like (including OXA-181) and VIM carbapenemase genes, whilst the 100 assay-negative samples were correctly identified indicating 100% specificity. During the multicentre evaluation the same 100% level of sensitivity and specificity was observed in each of the five centres that participated. Neither invalid nor false-positive results were observed. In conclusion, the assay offers a portable and reliable option for the detection of bacteria carrying clinically significant carbapenemases encoded by KPC, NDM, VIM and OXA-48-like carbapenemase genes using either of the two most common real-time PCR instrument platforms.
authors.affiliation: Public Health England, Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, Colindale, London NW9 5EQ, UK; Public Health England, Clinical and Public Health Microbiology Laboratory, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QW, UK. Electronic address: matthew.ellington@phe.gov.uk.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Ellington
authors.affiliation: Public Health England, Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, Colindale, London NW9 5EQ, UK.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Findlay
authors.affiliation: Public Health England, Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, Colindale, London NW9 5EQ, UK.
authors.forename: Katie L
authors.initials: KL
authors.lastname: Hopkins
authors.affiliation: Public Health England, Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, Colindale, London NW9 5EQ, UK.
authors.forename: Danièle
authors.initials: D
authors.lastname: Meunier
authors.affiliation: Public Health England, West Midlands Public Health Laboratory, Heart of England NHS Foundation Trust, Birmingham B9 5SS, UK.
authors.forename: Adela
authors.initials: A
authors.lastname: Alvarez-Buylla
authors.affiliation: Public Health England, Department of Microbiology, Leeds Teaching Hospitals NHS Trust, Leeds LS1 3EX, UK.
authors.forename: Carolyne
authors.initials: C
authors.lastname: Horner
authors.affiliation: Public Health England, Public Health Laboratory, Manchester, Manchester Royal Infirmary, Manchester M13 9WL, UK.
authors.forename: Ashley
authors.initials: A
authors.lastname: McEwan
authors.affiliation: Public Health England, Public Health Laboratory, Manchester, Manchester Royal Infirmary, Manchester M13 9WL, UK.
authors.forename: Malcolm
authors.initials: M
authors.lastname: Guiver
authors.affiliation: Public Health England, West Midlands Public Health Laboratory, Heart of England NHS Foundation Trust, Birmingham B9 5SS, UK.
authors.forename: Li-Xu
authors.initials: LX
authors.lastname: McCrae
authors.affiliation: Public Health England, Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, Colindale, London NW9 5EQ, UK.
authors.forename: Neil
authors.initials: N
authors.lastname: Woodford
authors.affiliation: Public Health England, West Midlands Public Health Laboratory, Heart of England NHS Foundation Trust, Birmingham B9 5SS, UK.
authors.forename: Peter
authors.initials: P
authors.lastname: Hawkey
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D006090:Gram-Negative Bacteria / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D001618:beta-Lactamases; C063912:carbapenemase
keywords: Enterobacteriaceae; KPC; Metallo-β-lactamase; NDM; OXA-48; VIM