Rapid detection of carbapenemase producing Enterobacteriaceae direct from blood using real-time PCR

Abstract

The emergence and dissemination of carbapenemase-producing Enterobacteriaceae (CPE) is recognised as an urgent threat to human health. Due to limited treatment options, infections with CPE are associated with high mortality rates ranging from 22% to 72%.1Hirsch E.B. Tam V.H. Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause of multidrug-resistant infection.J Antimicrob Chemother. 2010; 65: 1119-1125Crossref PubMed Scopus (385) Google Scholar,2Borer A. Saidel-Odes L. Riesenberg K. et al.Attributable mortality rate for carbapenem-resistant Klebsiella pneumoniae bacteremia.Infect Control Hosp Epidemiol. 2009; 30: 972-976Crossref PubMed Scopus (301) Google Scholar Polymerase chain reaction (PCR)-based assays have been developed for the rapid detection of CPE from rectal surveillance swabs,3Tenover F.C. Canton R. Kop J. et al.Detection of colonization by carbapenemase-producing Gram-negative Bacilli in patients by use of the Xpert MDRO assay.J Clin Microbiol. 2013; 51: 3780-3787Crossref PubMed Scopus (67) Google Scholar however the use of such methods for direct sampling from blood cultures is infrequently described. Implementation of rapid CPE diagnostics from clinical samples, particularly blood cultures, may significantly alter antimicrobial therapy and improve patient outcomes. Our healthcare institution reported the first outbreak of KPC-producing Klebsiella pneumoniae in Australia.4Cronin K.M. Poy Lorenzo Y.S. Olenski M.E. et al.Risk factors for KPC acquisition and infection in a healthcare setting with possible local transmission: a case control study.J Hosp Infect. 2017; 96: 111-115Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar In order to facilitate rapid detection of CPEs, two multiplex real-time PCR assays were assessed for their suitability for use at our institution; the Xpert Carba-R assay, version 2 (Cepheid, USA) and BD MAX CRE RUO assay (BD, USA). The Xpert Carba-R assay is a qualitative on-demand system for the detection of blaKPC, blaNDM, blaVIM, blaIMP-1 and blaOXA-48 genes (including blaOXA-181 and blaOXA-232). The GeneXpert Instrument Systems utilise single-use, disposable cartridges allowing for automated sample preparation, amplification, and real-time detection of gene targets in approximately 50 minutes. The BD MAX CRE assay includes reagents for the detection of blaKPC, blaNDM, and blaOXA-48 genes and automates sample lysis, DNA extraction, amplification and real-time detection of the gene targets in approximately 120 minutes. Although the turnaround time is longer in comparison to the Xpert Carba-R assay, up to 24 samples can be tested at one time. Both assays are designed for the rapid detection of carbapenemase genes from rectal swab specimens. The aim of this study was to validate two commercial molecular assays for the rapid detection of CPE from spiked blood culture bottles. Non-duplicated isolates were obtained from St Vincent's Hospital (Melbourne) and the Microbiological Diagnostic Unit Public Health Laboratory (MDU PHL) (The Doherty Institute, Melbourne). The isolates were originally isolated from clinical samples or from rectal screening swabs. The resistance genes associated with these isolates were determined by in-house PCR followed by whole genome sequencing as described by Kwong et al.5Kwong J.C. Lane C.R. Romanes R. et al.Translating genomics into practice for real-time surveillance and response to carbapenemase-producing Enterobacteriaceae: evidence from a complex multi-institutional KPC outbreak.Peer J. 2018; 6e4210Crossref PubMed Scopus (36) Google Scholar Isolates were stored at –70oC in glycerol and subcultured to MacConkey agar plates for overnight incubation in air at 35°C prior to testing. Signal-negative aerobic blood culture bottles submitted for routine microbiological diagnostics were used to spike with known CPE isolates. Following overnight incubation, a single colony was removed from the MacConkey plate and diluted to a 0.5 McFarland standard equivalent suspension. Two mL of the 0.5 MacFarlane suspension was inoculated into signal negative aerobic BD Bactec blood culture bottles. These were incubated in the BD Bactec 9000 blood culture instrument until bacterial growth was detected. At this time the blood culture bottles were removed from the BD Bactec 9000 and 50 μL of the blood culture media was added to either the sample reagent vial (Xpert Carba-R assay) or the sample buffer tube (BD MAX CRE assay). The tubes were vortexed for 10–20 s. The specific assay protocols were then followed as if the sample was a rectal swab. The manual handling time required is less than 5 min with an overall processing time of less than 60 min for the Xpert Carba-R assay and less than 130 min for the BD MAX CRE assay. All discrepant results were evaluated. The performance characteristics (sensitivity, specificity) for detecting CPE genes from blood were calculated for each assay, using whole genome sequencing as the reference standard. The Xpert Carba-R assay was used to test a total of 49 isolates including K. pneumoniae (n=37), Escherichia coli (n=4), and other Gram-negative bacilli (n=8) (Table 1). Seven isolates contained multiple resistance genes, and two isolates of carbapenem resistant K. pneumoniae which did not harbour resistance genes on sequencing, were used as negative controls. The sensitivity and specificity of the assay was 100% for the detection of blaKPC (n=20), blaNDM (n=15) and blaOXA-48 genes (n=10). The Xpert Carba-R assay detected all blaOXA-232 (n=7), blaOXA-181 (n=2) and blaOXA-48 (n=1) genes, which are part of the OXA-48-like enzyme group. The assay did not detect blaOXA-23, blaOXA-24/40 and blaOXA-51 genes, which are not part of the OXA-48-like carbapenemases. The assay demonstrated low sensitivity (25%) for the detection of blaVIM genes with one of four isolates detected. The assay detected the only isolate containing a blaIMP-1 gene. Although the Xpert Carba-R assay is designed to detect blaIMP-1, the assay also detected a blaIMP-4, however the blaIMP-14 remained undetected. There was no assay inhibition detected in any of the tests performed.Table 1Spiked blood culture samplesIsolateOrganismSequencingBD Max CREXpert Carba-RDiscordant results1P. aeruginosablaIMP-1Not testedIMP-12K. pneumoniaeblaIMP-14Not testedNil detected3S. marcescensblaIMP-4Not testedIMP-14K. pneumoniaeblaKPC-2KPCKPC5K. pneumoniaeblaKPC-2KPCKPC6K. pneumoniaeblaKPC-2KPCKPC7K. pneumoniaeblaKPC-2KPCKPC8K. pneumoniaeblaKPC-2KPCKPC9K. pneumoniaeblaKPC-2KPCKPC10K. pneumoniaeblaKPC-2KPCKPC11K. pneumoniaeblaKPC-2KPCKPC12K. pneumoniaeblaKPC-2KPCKPC13K. pneumoniaeblaKPC-2KPCKPC14K. pneumoniaeblaKPC-2KPCKPC15C. farmeriiblaKPC-2Not testedKPC16C. farmeriiblaKPC-2Not testedKPC17K. pneumoniaeblaKPC-2Not testedKPC18K. pneumoniaeblaKPC-2Not testedKPC19K. pneumoniaeblaKPC-2Not testedKPC20K. pneumoniaeblaKPC-2Not testedKPC21K. pneumoniaeblaKPC-2Not testedKPC22K. pneumoniaeblaKPC-2Not testedKPC23K. pneumoniaeblaKPC-3KPCKPC24K. pneumoniaeblaNDM-1NDMNDM25C. braakiiblaNDM-4NDMNDM26K. pneumoniaeblaNDM-5Not testedNDM27K. pneumoniaeblaNDM-5NDMNDM28K. pneumoniaeblaNDM-5NDM, OXA-48NDMBD Max false positive29E. coliblaNDM-5Not testedNDM30K. pneumoniaeblaNDM-5NDMNDM31K. pneumoniaeblaNDM-5 blaOXA-232Not testedNDMOXA-4832K. pneumoniaeblaNDM-5 blaOXA-232NDMOXA-48NDMOXA-4833K. pneumoniaeblaNDM-5 blaOXA-232NDMOXA-48NDMOXA-4834K. pneumoniaeblaNDM-5 blaOXA-232NDMOXA-48NDMOXA-4835K. pneumoniaeblaNDM-5 blaOXA-232Not testedNDMOXA-4836K. pneumoniaeblaNDM-5 blaOXA-232 blaVIM-2NDMOXA-48NDMOXA-48 Nil detectedXpert false negative37K. pneumoniaeblaNDM-5OXA-232Not testedNDM OXA-4838E. coliblaNDM-7NDMNDM39K. pneumoniaeblaOXA-181OXA-48OXA-4840E. coliblaOXA-181Not testedOXA-4841E. coliblaOXA-23-likeNil detectedNil detected42A. baumanniiblaOXA-24/40-likeNil detectedNil detected43K. pneumoniaeblaOXA-48OXA-48OXA-4844E. cloacaeblaOXA-51-likeNil detectedNil detected45K. pneumoniaeblaVIM-1Not testedNil detectedXpert false negative46P. aeruginosablaVIM-2Not testedVIM47K. pneumoniaeblaVIM-5Nil detectedNil detectedXpert false negative48K. pneumoniaeNil detectedNil detectedNil detected49K. pneumoniaeNil detectedNil detectedNil detectedOXA-23-like and OXA-24/40-like beta-lactamases have been described in Acinetobacter species and species belonging to Enterobacterales. These genes have not been described in Pseudomonas species. Bold text indicates discrepant results. Open table in a new tab OXA-23-like and OXA-24/40-like beta-lactamases have been described in Acinetobacter species and species belonging to Enterobacterales. These genes have not been described in Pseudomonas species. Bold text indicates discrepant results. Of the 49 spiked blood culture samples tested using the Xpert Carba-R assay, a subset of 30 samples were tested using the BD MAX CRE assay due to kit availability. In addition, blaIMP and blaVIM genes are not detected by the BD MAX CRE assay, therefore these samples were largely excluded. The sensitivity and specificity of the assay was 100% for the detection blaKPC (n=12) and blaNDM genes (n=10). The assay demonstrated a sensitivity of 100% and specificity of 95.8% for the detection of blaOXA-48 genes, with one false positive blaOXA-48 result, which was duplicated on repeat testing. The BD MAX CRE assay detected the OXA-48-like enzyme group including blaOXA-232 (n=4), blaOXA-181 (n=1) and blaOXA-48 (n=1) genes, but not other OXA-type carbapenemases as expected. There was no assay inhibition detected in any of the tests performed. In this study, we describe the diagnostic performance of the Xpert Carba-R and BD MAX CRE assays, which permit genotypic detection of CPE directly from positive blood culture broths. Overall, both assays performed well and had excellent sensitivity and specificity for the detection of blaKPC, blaNDM and blaOXA-48 genes. The lack of detection of OXA-181-producing isolates had previously been recognised as a short coming of the Xpert Carba-R assay and the assay has subsequently been modified.6Lafeuille E. Laouira S. Sougakoff W. et al.Detection of OXA-48-like carbapenemase genes by the Xpert® Carba-R test: room for improvement.Int J Antimicrob Agents. 2015; 45: 441-442Crossref PubMed Scopus (19) Google Scholar One discordant result was recorded between the BD MAX CRE assay and sequencing, with the detection of an additional blaOXA-48 gene by the BD MAX CRE assay. This may represent a problem with probe specificity or an issue with the sensitivity of the sequencing method. The Xpert Carba-R detected one of four samples harbouring blaVIM (VIM-2). Of interest, a second isolate harbouring blaVIM-2 was not detected. It is possible that pathogens may have lost their resistance genes as a result of repeated subculture, however there were too few samples to make any formal conclusions. For every hour that effective antimicrobial therapy is delayed in septic shock there is an associated increase in mortality.7Kumar A. Roberts D. Wood K.E. et al.Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock.Crit Care Med. 2006; 34: 1589-1596Crossref PubMed Scopus (4114) Google Scholar Unless there is a high index of suspicion, empiric treatment for CPE is rarely embarked upon in a septic patient due to the toxicity of some of the agents that remain active, such as colistin. The turnaround time for confirmation of antimicrobial resistance from blood culture isolates can take up to 48 hours. Genotypic and phenotypic assays for detecting CPE directly from positive blood culture broths have been previously described.8Sfeir M.M. Satlin M.J. Fauntleroy K.A. et al.Blood-modified carbapenem inactivation method: a phenotypic method for detecting carbapenemase-producing Enterbacteriaceae directly from positive blood culture broths.J Clin Microbiol. 2020; 58: e01377-19Crossref PubMed Scopus (6) Google Scholar, 9Jonasson E. Matuschek E. Kahlmeter G. The EUCAST rapid disc diffusion method for antimicrobial susceptibility testing directly from positive blood culture bottles.J Antimicrob Chemother. 2020; 75: 968-978Crossref PubMed Scopus (54) Google Scholar, 10Hogan C.A. Watz N. Budvytiene I. et al.Rapid antimicrobial susceptibility testing by VITEK®2 directly from blood cultures in patients with Gram-negative rod bacteremia.Diagn Microbiol Infect Dis. 2019; 94: 116-121Crossref PubMed Scopus (17) Google Scholar, 11Takissian J. Bonnin R.A. Naas T. et al.NG-Test Carba 5 for rapid detection of carbapenemase-producing Enterobacterales from positive blood cultures.Antimicrob Agents Chemother. 2019; 63: e00011-19Crossref PubMed Scopus (43) Google Scholar, 12Cointe A. Walewski V. Hobson C.A. et al.Rapid carbapenemase detection with Xpert Carba-R V2 directly on positive blood vials.Infect Drug Resist. 2019; 12: 3311-3316Crossref PubMed Scopus (12) Google Scholar There are a number of benefits to phenotypic methods of CPE detection such as blood-modified carbapenem inactivation method (Blood-mCIM) and rapid antimicrobial susceptibility testing (RAST) directly from positive blood culture bottles. These methods are cost efficient as they utilise inexpensive materials for testing. Phenotypic methods are not restricted to the detection of known carbapenemases, in comparison to genotypic methods, which are limited to the detection of genes that are specifically targeted by the assay. Blood-mCIM has recently reported promising performance characteristics, but there still remains a 22–28 hour delay following a blood culture signalling positive.8Sfeir M.M. Satlin M.J. Fauntleroy K.A. et al.Blood-modified carbapenem inactivation method: a phenotypic method for detecting carbapenemase-producing Enterbacteriaceae directly from positive blood culture broths.J Clin Microbiol. 2020; 58: e01377-19Crossref PubMed Scopus (6) Google Scholar RAST has a shorter turnaround time of 4–8 hours, but is unable to differentiate between carbapenemase production and other mechanisms of resistance.9Jonasson E. Matuschek E. Kahlmeter G. The EUCAST rapid disc diffusion method for antimicrobial susceptibility testing directly from positive blood culture bottles.J Antimicrob Chemother. 2020; 75: 968-978Crossref PubMed Scopus (54) Google Scholar In addition, 20% of Escherichia coli and 19% of Klebsiella pneumoniae isolates return uninterpretable results by RAST. The use of genotypic methods, such as real-time PCR (RT-PCR), can yield rapid results and reduce the time to initiation of CPE appropriate antibiotic therapy to 1 hour from the time the blood culture signals positive. Despite these benefits, there are a number of drawbacks. Whilst the detection of carbapenemase genes provides some information regarding potential resistance patterns, it is not a substitute for antimicrobial susceptibility testing. Cost of RT-PCR assays prohibits routine use in most diagnostic laboratories, however cost efficiency may be improved by careful patient selection. For example, RT-PCR methods could be restricted to positive blood cultures from patients with risk factors for CPE, such as prolonged length of hospital stay, history of central venous catheter and prior multidrug resistant organism colonisation.4Cronin K.M. Poy Lorenzo Y.S. Olenski M.E. et al.Risk factors for KPC acquisition and infection in a healthcare setting with possible local transmission: a case control study.J Hosp Infect. 2017; 96: 111-115Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar This study has several limitations. The number of resistance genes surveyed in the assay is limited. In addition, discordant results were not subjected to resequencing, therefore it remains unknown whether false negatives were a result of organisms losing their resistance genes due to repeated subculture. In conclusion, we describe the diagnostic performance of the Xpert Carba-R and BD MAX CRE assays for the detection of CPE genes direct from positive blood cultures broths. Both assays demonstrated excellent performance characteristics for the detection of blaKPC, blaNDM and blaOXA-48 genes without the need for manipulation of the sample and without inhibition of the assay.