title: Rapid and direct real-time detection of blaKPC and blaNDM from surveillance samples.
abstract: We assessed the performance of a duplex real-time PCR assay for bla(KPC) and bla(NDM) performed directly (D-PCR) on perianal and perirectal swabs and stool. Spiked specimens and 126 clinical surveillance swabs (comprising a sensitivity panel of 46 perirectal double swabs previously determined to be culture positive for bla(KPC)-PCR-positive Enterobacteriaceae and a specificity panel of 80 perianal swabs from patients at risk of carbapenemase-producing Enterobacteriaceae [CPE] colonization) were studied. For the surveillance swabs, D-PCR was compared to PCR after broth enrichment (BE-PCR) and two culture-based methods: the HardyCHROM ESBL agar (HC-A) and the CDC screening (CDC-A) methods. PCR was performed on morphologically distinct colonies that were isolated by culture. All of the initial PCR testing was done without extraction using a simple lysis procedure. The analytical sensitivities of D-PCR for bla(KPC) were 9 CFU/μl (for swabs) and 90 CFU/μl (for stool), and for bla(NDM), it was 1.9 CFU/μl (for both swabs and stool). In the clinical sensitivity panel, D-PCR and BE-PCR were initially positive for bla(KPC) in 41/46 (89.1%) and 43/46 (93.5%) swabs, respectively. The swabs that were initially negative by D-PCR (n = 5) and BE-PCR (n = 3) were visibly stool soiled; all swabs were bla(KPC) positive upon repeat testing after lysate extraction. The CDC-A and HC-A yielded bla(KPC)-positive Enterobacteriaceae from 36/46 (78.3%) and 35/46 (76.1%) swabs, respectively (sensitivities of D-PCR/BE-PCR postextraction of soiled specimens versus HC-A, P = 0.0009, and versus CDC-A, P = 0.0016). All swabs in the specificity panel were negative for CPE by all four methods. D-PCR allows for the timely detection of bla(KPC) and bla(NDM) carriage with excellent sensitivity when specimens visibly soiled with stool undergo preparatory extraction.
authors: Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology.
authors: Shawn
authors: S
authors: Vasoo
authors: Scott A
authors: SA
authors: Cunningham
authors: Peggy C
authors: PC
authors: Kohner
authors: Jayawant N
authors: JN
authors: Mandrekar
authors: Karen
authors: K
authors: Lolans
authors: Mary K
authors: MK
authors: Hayden
authors: Robin
authors: R
authors: Patel
mesh_terms: D004755:Enterobacteriaceae / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D004756:Enterobacteriaceae Infections / Q000382:microbiology*; D062665:Epidemiological Monitoring; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D001618:beta-Lactamases
references: Antimicrob Agents Chemother. 2006 Oct;50(10):3396-406
references: 17005822
references: J Clin Microbiol. 2013 Apr;51(4):1269-71
references: 23345290
references: J Antimicrob Chemother. 2013 Jan;68(1):101-4
references: 22969079
references: Infect Control Hosp Epidemiol. 2012 Oct;33(10):984-92
references: 22961017
references: J Clin Microbiol. 2011 Jun;49(6):2239-42
references: 21471338
references: Int J Antimicrob Agents. 2011 Feb;37(2):124-8
references: 21177079
references: Antimicrob Agents Chemother. 2000 Mar;44(3):622-32
references: 10681329
references: Infect Control Hosp Epidemiol. 2010 Apr;31(4):341-7
references: 20175685
references: J Clin Microbiol. 2011 Sep;49(9):3338-9
references: 21734030
references: Infect Control Hosp Epidemiol. 2013 Jan;34(1):56-61
references: 23221193
references: Ann Lab Med. 2012 Sep;32(5):359-61
references: 22950072
references: Diagn Microbiol Infect Dis. 2013 Feb;75(2):214-7
references: 23142168
references: MMWR Morb Mortal Wkly Rep. 2013 Feb 15;62(6):108
references: 23407128
references: J Clin Microbiol. 2012 Feb;50(2):477-9
references: 22116154
references: J Clin Microbiol. 2008 Sep;46(9):3110-1
references: 18632915
references: J Clin Microbiol. 2002 Jun;40(6):2153-62
references: 12037080
references: J Clin Microbiol. 2010 Mar;48(3):836-41
references: 20071553
references: J Clin Microbiol. 2009 Oct;47(10):3261-5
references: 19675211
references: J Clin Microbiol. 2009 Feb;47(2):322-6
references: 19036932
references: J Clin Microbiol. 2012 May;50(5):1718-20
references: 22378915
references: J Clin Microbiol. 1996 Dec;34(12):2909-13
references: 8940421
references: J Microbiol Methods. 2010 Nov;83(2):185-7
references: 20807554
references: Infect Control Hosp Epidemiol. 2009 May;30(5):447-52
references: 19301985
references: Antimicrob Agents Chemother. 2011 Sep;55(9):4038-43
references: 21690281
references: Antimicrob Agents Chemother. 2000 Apr;44(4):891-7
references: 10722487
references: Antimicrob Agents Chemother. 2004 Jan;48(1):15-22
references: 14693513
references: J Clin Microbiol. 2012 Sep;50(9):3115-8
references: 22785190
references: J Clin Microbiol. 2013 Jun;51(6):1948-50
references: 23554195
references: J Clin Microbiol. 2012 Aug;50(8):2596-600
references: 22622443
references: J Clin Microbiol. 2008 Sep;46(9):2879-83
references: 18614657
references: Sci Transl Med. 2012 Aug 22;4(148):148ra116
references: 22914622