JCM Accepted Manuscript Posted Online 27 May 2015 J. Clin. Microbiol. doi:10.1128/JCM.01237-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Rapid Detection of KPC, NDM, and OXA48-like Carbapenemases by Real-time PCR from

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Rectal Swab Surveillance Samples

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Tracy D. Lee1, Kathleen Adie1, Alan McNabb1, Dale Purych2,3, Kulvinder Mannan3, Robert

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Azana1, Corrinne Ng1, Patrick Tang1, 2, and Linda Hoang1,2*

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Control, PHSA Laboratories, Vancouver, British Columbia, Canada

Public Health and Reference Microbiology Laboratories, British Columbia Centre for Disease

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Columbia, Vancouver, British Columbia, Canada

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Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British

Department of Laboratory Medicine and Pathology, Fraser Health, British Columbia, Canada

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*Corresponding author. Mailing address: Public Health and Advanced Bacteriology & Mycology

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Program, Room 3072A, BC Centre for Disease Control, 655 W 12th Avenue, Vancouver, British

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Columbia, V5Z 4R4, Canada.

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ABSTRACT

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We describe a multiplex real-time PCR assay for use on the ABI 7500 FAST Taqman platform

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to detect all currently described Klebsiella pneumoniae carbapenemases (KPC), New Delhi

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metallo-β-lactamases (NDM), and the OXA48-like family of carbapenemases from bacterial

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culture lysates or sample enrichment broth lysates.

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The prevention of transmission of carbapenemase producing organisms (CPO) within healthcare

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facilities requires accurate and rapid laboratory detection methods so infection control

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precautions may be quickly implemented. In the midst of an outbreak, rapid case identification is

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required and conventional methods can take too long and be very labour intensive, resulting in

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delays in infection control and significant costs to the healthcare system.

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Molecular methods are fast and generally have higher sensitivity and specificity than phenotypic

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methods, and in-house developed methods are typically less expensive than commercially

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available kits. Published methods for the molecular detection of Klebsiella pneumoniae

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carbapenemases (KPC), New Delhi metallo-β-lactamases (NDM), and OXA48 are typically

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singleplex reactions targeting only one carbapenemase or are intended for cultured isolates rather

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than surveillance samples (1-14). We identified the need for a highly sensitive and specific

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molecular assay that can detect these CPO gene targets directly from surveillance sample

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matrices, such as rectal swabs.

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We developed a FAST multiplex real-time 5’ exonuclease-based real-time PCR to detect the

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common carbapenemases (KPC, NDM, and OXA48-like) from rectal swabs in enrichment

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broths. Although there have been several reports of real-time PCR assays in the literature (1-14),

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to our knowledge, this is the first FAST multiplex real-time 5’ exonuclease-based real-time PCR

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to detect and differentiate the 3 carbapenemases, KPC, NDM, and OXA48-like from rectal

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swabs in enrichment broths in a single reaction. A culture enrichment step was incorporated to

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increase sensitivity (15).

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To date, 23 KPC, 16 NDM, and 11 OXA48-like (http://www.lahey.org/studies/other.asp#table1)

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genotypes have been described. We used the IDT OligoAnalyzer 3.1 to define a set of primers

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and probes that is predicted, in silico, to detect all described genotypes of blaKPC, blaNDM, and

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blaOXA48-like genes (Table 1). To validate this, real-time PCR reactions were performed on an

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ABI 7500 with recommended FAST thermal-cycling conditions in a 20 μL final volume using

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TaqMan® Fast Advanced Master Mix (Life Technologies), PCR grade water (Life Technologies,

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Burlington, ON), and each primer and probe at a final concentration of 200nM and 150nM

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respectively. 2 μL of sample lysate was added to each reaction. Real-time PCR controls, ATCC

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strains and clinical isolates previously characterized by conventional PCR (17) are listed in Table

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2. Bacterial culture lysates were prepared using Instagene Matrix (BioRad, Mississauga, ON),

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according to the manufacturer’s protocol. Broth lysates were prepared by 2 methods: Instagene

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Matrix and a water boil. For each lysis method, approximately 30 μL of enriched culture broth

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was added to 1mL of PCR grade water. Instagene lysates were completed according to the

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manufacture’s protocol. The tube for the boil method was heated to 100⁰C in a dry bath for 8

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minutes to lyse the bacterial cells. A 500bp oligonucleotide containing 1 copy of the

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concatenated target sequences of each assay was purchased from Integrated DNA Technologies

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(IDT, Toronto, ON). Pooled negative Instagene lysates were spiked with serial dilutions of this

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oligo ranging from 101 to 108 copies/ml. The limit of detection was assessed in both multiplex

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and simplex format. All 3 targets have a lower detection limit of 5 copies/μL extracted DNA.

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One hundred and fifty-five blind coded Instagene lysates from archived clinical isolates

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previously tested by conventional PCR were tested by real-time PCR and results of each method

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were compared to determine the sensitivity and specificity. The multiplex assay correctly

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identified all 45 positive isolates as having at least one of the 3 carbapenemases tested (Table 3).

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The remaining 110 isolates tested were negative for KPC, NDM, and OXA48 by both

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conventional PCR and real-time PCR assays. There was no evidence of cross reaction among

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these three targets. The sensitivity and specificity is 100%.

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Two hundred and sixty-eight surveillance rectal swabs collected by the participating hospital, as

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part of their infection control surveillance program, were enriched in 5 mL TSB with a 10μg

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ertapenem disc for 18-24 hours incubation at 37°C, stationary, in an ambient air incubator and

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then lysed and tested with the multiplex real-time PCR. Results were compared to isolates

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recovered from the same broth (Table 4) and investigated as follows: isolates recovered from

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broths were obtained by sub-culturing the broths to 2 MacConkey agar plates (Oxoid) with

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imipenem and meropenem discs (BioRad, Mississauga, ON). Colonies growing within the zone

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of inhibition were speciated and antibiotic susceptibility testing was performed according to

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2014 CLSI guidelines. Isolates intermediate or resistant to imipenem or meropenem were

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submitted to the reference lab for conventional PCR testing. As there is no gold standard for the

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detection of carbapenemases direct from stool or rectal swab samples (18), we considered our

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current phenotypic screening followed by conventional PCR method as the reference method for

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comparison.

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One hundred percent of conventionally identified KPC/NDM/OXA48 positive screen isolates

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were identified by the real-time PCR assay. None of the screen isolates harbored VIM or IMP

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genes. Three enrichment broths had discordant results between phenotypic testing and real-time

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PCR. Enrichment broths 102 and 220 both tested as phenotypically ertapenem and meropenem

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susceptible, but both tested positive for the presence of NDM by real-time PCR. Broth 220 had a

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second enrichment broth tested which had an NDM-positive K. pneumoniae isolate recovered.

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Broth 102 had no carbapenem resistant organisms isolated and repeat testing could not be

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performed. Broth 239 tested as NDM-positive and OXA48-positive by real-time PCR, but only

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an NDM-positive E. coli was isolated initially. A second screen sample 1 week later from the

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same patient recovered an OXA48-positive K. pneumoniae.

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This real-time PCR assay is fast, simple to perform and the results are easier to interpret

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compared to the conventional PCR. Replacing the current method of conventional PCR with the

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real-time PCR will significantly reduce turn-around times for CPO detection, especially during

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an outbreak. For clinical laboratories, a real-time PCR application will reduce the turn-around

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time for implementing infection control measures and reduce the costs associated with CPO

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detection.

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ACKNOWLEDGEMENTS

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We thank Dr. Michael Mulvey and David Boyd form the National Microbiology Laboratory for

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confirming discrepant results, and Tony Wong from the BC Public Health Microbiology and

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Reference Laboratory for assisting in conventional PCR isolate testing.

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REFERENCES 1. CDC. Multiplex Real-Time PCR Detection of Klebsiella pneumoniae Carbapenemase

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(KPC) and New Delhi metallo-β-lactamase (NDM-1).

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http://www.cdc.gov/HAI/settings/lab/kpc-ndm1-lab-protocol.html

115 116

2. Cunningham, S. A., Noorie, T., Meunier, D., Woodford, N., & Patel, R. (2013). Rapid

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and simultaneous detection of genes encoding Klebsiella pneumoniae carbapenemase

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(blaKPC) and New Delhi metallo-β-lactamase (blaNDM) in Gram-negative bacilli. J Clin

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Microbiol, 51(4), 1269-1271. doi: 10.1128/JCM.03062-12

120 121

3. Hindiyeh, M., Smollen, G., Grossman, Z., Ram, D., Davidson, Y., Mileguir, F., . . .

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Keller, N. (2008). Rapid detection of blaKPC carbapenemase genes by real-time PCR. J

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Clin Microbiol, 46(9), 2879-2883. doi: 10.1128/JCM.00661-08

124 125

4. Hofko, M., Mischnik, A., Kaase, M., Zimmermann, S., & Dalpke, A. H. (2014).

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Detection of carbapenemases by real-time PCR and melt curve analysis on the BD Max

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system. J Clin Microbiol, 52(5), 1701-1704. doi: 10.1128/JCM.00373-14

128 129

5. Mangold, K. A., Santiano, K., Broekman, R., Krafft, C. A., Voss, B., Wang, V., . . .

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Peterson, L. R. (2011). Real-time detection of blaKPC in clinical samples and

131

surveillance specimens. J Clin Microbiol, 49(9), 3338-3339. doi: 10.1128/JCM.00268-11

132

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133

6. Monteiro, J., Widen, R. H., Pignatari, A. C., Kubasek, C., & Silbert, S. (2012). Rapid

134

detection of carbapenemase genes by multiplex real-time PCR. J Antimicrob Chemother,

135

67(4), 906-909. doi: 10.1093/jac/dkr563

136 137 138 139 140 141

7. Naas, T., Ergani, A., Carrër, A., & Nordmann, P. (2011). Real-time PCR for detection of NDM-1 carbapenemase genes from spiked stool samples. Antimicrob Agents Chemother, 55(9), 4038-4043. doi: 10.1128/AAC.01734-10 8. Naas, T., Cotellon, G., Ergani, A., & Nordmann, P. (2013). Real-time PCR for

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detection of blaOXA-48 genes from stools. J Antimicrob Chemother, 68(1), 101-104. doi:

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10.1093/jac/dks340

144 145

9. Schechner, V., Straus-Robinson, K., Schwartz, D., Pfeffer, I., Tarabeia, J.,

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Moskovich, R., . . . Navon-Venezia, S. (2009). Evaluation of PCR-based testing for

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surveillance of KPC-producing carbapenem-resistant members of the Enterobacteriaceae

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family. J Clin Microbiol, 47(10), 3261-3265. doi: 10.1128/JCM.02368-08

149 150

10. Singh, K., Mangold, K. A., Wyant, K., Schora, D. M., Voss, B., Kaul, K. L., . . .

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Peterson, L. R. (2012). Rectal screening for Klebsiella pneumoniae carbapenemases:

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comparison of real-time PCR and culture using two selective screening agar plates. J Clin

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Microbiol, 50(8), 2596-2600. doi: 10.1128/JCM.00654-12

154 155

11. Swayne, R. L., Ludlam, H. A., Shet, V. G., Woodford, N., & Curran, M. D. (2011).

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Real-time TaqMan PCR for rapid detection of genes encoding five types of non-metallo-

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(class A and D) carbapenemases in Enterobacteriaceae. Int J Antimicrob Agents, 38(1),

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35-38. doi: 10.1016/j.ijantimicag.2011.03.010

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159 160

12. van der Zee, A., Roorda, L., Bosman, G., Fluit, A. C., Hermans, M., Smits, P. H., . . .

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Ossewaarde, J. M. (2014). Multi-centre evaluation of real-time multiplex PCR for

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detection of carbapenemase genes OXA-48, VIM, IMP, NDM and KPC. BMC Infect Dis,

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14, 27. doi: 10.1186/1471-2334-14-27

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13. Vasoo, S., Cunningham, S. A., Kohner, P. C., Mandrekar, J. N., Lolans, K., Hayden,

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M. K., & Patel, R. (2013). Rapid and direct real-time detection of blaKPC and blaNDM

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from surveillance samples. J Clin Microbiol, 51(11), 3609-3615. doi:

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10.1128/JCM.01731-13

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14. Zheng, F., Sun, J., Cheng, C., & Rui, Y. (2013). The establishment of a duplex real-

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time PCR assay for rapid and simultaneous detection of blaNDM and blaKPC genes in

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bacteria. Ann Clin Microbiol Antimicrob, 12, 30. doi: 10.1186/1476-0711-12-30

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15. Hedman, J., Lövenklev, M., Wolffs, P., Löfström, C., Knutsson, R., Rådström, P.

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(2013). Pre-PCR Processing Strategies, p 5-6. In Weissensteiner, Thomas, Tania Nolan,

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Stephen A. Bustin, Hugh G. Griffin, and Annette Griffin, eds. PCR technology: current

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innovations. CRC press, London, UK.

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16. Swayne, R. L., Ludlam, H. A., Shet, V. G., Woodford, N., & Curran, M. D. (2011). Real-time TaqMan PCR for rapid detection of genes encoding five types of non-metallo-

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(class A and D) carbapenemases in Enterobacteriaceae. Int J Antimicrob Agents, 38(1),

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35-38. doi: 10.1016/j.ijantimicag.2011.03.010

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17. Mataseje LF, Bryce E, Roscoe D, Boyd DA, Embree J, Gravel D, Katz K, Kibsey P,

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Kuhn M, Mounchili A, Simor A, Taylor G, Thomas E, Turgeon N, Mulvey MR;

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Canadian Nosocomial Infection Surveillance Program. 2012.

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Gram-negative bacilli in Canada 2009-10: results from the Canadian Nosocomial

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Infection Surveillance Program (CNISP). Journal of Antimicrobial Chemotherapy. June;

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67(6): 1359-67.

Carbapenem-resistant

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18. Public Health England, S. I., Microbiology Services. (2014). Laboratory Detection and

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Reporting of Bacteria with Carbapenem-Hydrolysing β-lactamases (Carbapenemases)

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(Vol. P8): UK Protocols. link

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Table 1. Primers and Probes Sequence

Name

Probe Dye

Final Concentration Reference (nM) 13 200

GGCCGCCGTGCAATAC

KPC-F

GCCGCCCAACTCCTTCA

KPC-R

TGATAACGCCGCCGCCAATTTGT

KPC-P

ATTAGCCGCTGCATTGATGC

NDM-F

200

CCGGCATGTCGAGATAGGAA

NDM-R

200

CTG +CCA +GAC +ATT +CGGTGCa

NDM-P

CAATGGYGACTATATTATTCGGGC

OXA48F-F

200

CCACACATTATCATCMAGTTCAACC

OXA48F-R

200

CTAAGATTGGCTGGTGGGT

OXA48F-P

201

a

202

b

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assays

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c

200 MAX/ZEN

LNA 6FAM

MGB NED

150 19b

150 this studyc

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LNA base pairs preceded by (+) with modifications to the published primers to improve annealing temperatures in Taqman

based on the sequence described by Swayne et al. (16)

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Table 2. Summary of positive controls used in real-time and conventional PCR’s Resistance gene Strain Organism Method Real-time and conventional K. pneumoniae KPC ATCC-1705 NDM Real-time E. coli ATCC 2452 Conventional E. coli NML-1049 OXA-48 Real-time K. pneumoniae NCTC13443 F189043 Conventional E. coli

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Table 3: Summary of clinical isolate results, real-time PCR, and conventional PCR

KPC

Total # of positives by real time PCR a 9

Total # of positives by conventional PCR a 9

NDM

30

30

ΟXΑ48

8

8

Carbapenemase

Species isolated K. oxytoca (1) K. pneumoniae (7) Raoultella planticola (1) Enterobacter cloacae (6) E. coli (7) K. oxytoca (1) K. pneumoniae (16)

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E. coli (2) K. pneumoniae (6) a 2 K. pneumoniae isolates tested positive for multiple targets. Isolate 45 tested as KPC+ and

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NDM+. Isolate 54 tested as NDM+ and OXA48+. All other isolates tested positive for only 1

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target.

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Table 4: Summary of surveillance sample results, real-time PCR, and phenotypic screen isolates

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Phenotypic Screen Isolates Total # of positives Carbapenemase by conventional Species isolated PCR KPC 0 NDM 20 Citrobacter freundii (1) Klebsiella pneumoniae (10) Mixed C. freundii/K. pneumoniae (5) Escherichia coli (4) 0 1b ΟXΑ48 a broth 102 and broth 220 both tested as phenotypically ertapenem and meropenem susceptible

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however both tested positive for NDM.

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b

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broth. A repeat screen sample 1 week later had an OXA48+ K. pneumoniae isolated.

Total # of positives by real time PCR 0 22a

broth 239 tested as NDM+ and OXA48+, but only an NDM+ E. coli was recovered from the

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