Multi-centre evaluation of real-time multiplex PCR for detection of carbapenemase genes OXA-48, VIM, IMP, NDM and KPC

Zee, Anneke van der; Roorda, Lieuwe; Bosman, Gerda; Fluit, Ad C.; Hermans, Mirjam H. A.; Smits, Paul H.M.; Zanden, Adri G. M. van der; Witt, René te; Coppenraet, L.E.S. Bruijnesteijn van; Stuart, James Cohen; Ossewaarde, Jacobus M.

doi:10.1186/1471-2334-14-27

Cited by 82 publications

(59 citation statements)

References 14 publications

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“…Single and multiplex polymerase chain reaction (PCR) assays and microarray assays are available for common carbapenemases, including KPC, VIM, IMP, NDM, and OXA-48, with sensitivity and specificity of these tests approaching 100% [66–68]. Clear advantages of these molecular methods include identification of specific carbapenemase genes with superior sensitivity and specificity as well as rapid turn around time.…”

Section: Diagnosis Of Crementioning

confidence: 99%

Carbapenem-Resistant Enterobacteriaceae Infections in Children

Chiotos

Han

Tamma

2015

Curr Infect Dis Rep

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Carbapenem-resistant Enterobacteriaceae (CRE) are an emerging global public health threat. Infections due to CRE are associated with significant morbidity and mortality. Few therapeutic options are available for treatment of these infections, and optimal antibiotic treatment regimens are unclear. Along with the rapidly increasing prevalence of CRE in the United States and worldwide, several studies have described the epidemiology of CRE in the adult population. While CRE is now also reported sporadically in children, there is a significant lack of data on the epidemiology, risk factors, treatment, and outcomes in this population. This article provides a comprehensive review of what is known to date about CRE, including clinical and molecular epidemiology, microbiologic diagnosis, antibiotic treatment options, and outcomes. In particular, this review will focus on the available data on CRE in the pediatric population.

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Section: Diagnosis Of Crementioning

confidence: 99%

Carbapenem-Resistant Enterobacteriaceae Infections in Children

Chiotos

Han

Tamma

2015

Curr Infect Dis Rep

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“…These included 39 isolates that were previously characterized as carrying bla KPC , bla NDM , bla VIM , bla IMP , and bla OXA-48 . Real-time PCR was performed to detect bla KPC , bla NDM , and bla VIM as described elsewhere (6,7). bla IMP , and bla OXA-48 were detected using conventional PCR with primers as follows: for bla IMP , 5=-GTTTATGT TCATACWTCG-3= (forward) and 5=-GGTTTAAYAAAACAA CCAC-3= (reverse); for bla OXA-48 , 5=-ATGCGTGTATTAGCCT TATCGGCTG-3= (forward) and 5=-CTAGGGAATAATTTTTT CCTGTTTG-3= (reverse).…”

mentioning

confidence: 99%

Performance of the CLSI Carba NP and the Rosco Carb Screen Assays Using North American Carbapenemase-Producing Enterobacteriaceae and Pseudomonas aeruginosa Isolates

et al. 2015

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fThis study compared the performance of the Carba NP assay, published by the Clinical and Laboratory Standards Institute, and the Rosco Rapid Carb Screen kit. Carba NP had superior sensitivity, but both assays required an increased inoculum to detect carbapenemase production in isolates with bla NDM , bla IMP , and bla OXA-48 . Rapid detection of carbapenemase production in multidrugresistant organisms may help to mitigate institutional outbreaks by expediting initiation of infection control procedures (1). Nordmann et al. first described the Carba NP assay and reported sensitivity and specificity of 100% (2). Carbapenemase activity is detected using a pH indicator (phenol red) that changes color with the hydrolysis of the ␤-lactam ring of imipenem. Dortet et al. (3) published a modified procedure in 2014. The bacterial inoculum was reduced, testing was streamlined using whole lysed bacterial cells instead of supernatant, and the concentration of imipenem was raised from 3 to 6 mg/ml (3). Reported sensitivity and specificity were 100% (3). In 2015, based on the work of Vasoo et al. (4), the Clinical and Laboratory Standards Institute (CLSI) published a similar Carba NP procedure using a bacterial inoculum that was further reduced to 1 l (5).Carba NP is labor-intensive. Reagents need to be prepared inhouse, and some have shelf lives as short as 72 h. In contrast, the Rapid Carb Screen kit (98021; Rosco Diagnostica A/S, Taastrup, Denmark) provides tablets containing a pH indicator, with and without imipenem. No reagent preparation is required, and the shelf life is Ͼ12 months. In this study, we evaluated the performance of the Carba NP assay (CNP) as published by CLSI and the Rapid Carb Screen (RCS).In total, 49 organisms were tested. These included 39 isolates that were previously characterized as carrying bla KPC , bla NDM , bla VIM , bla IMP , and bla . Real-time PCR was performed to detect bla KPC , bla NDM , and bla VIM as described elsewhere (6, 7). bla IMP , and bla OXA-48 were detected using conventional PCR with primers as follows: for bla IMP , 5=-GTTTATGT TCATACWTCG-3= (forward) and 5=-GGTTTAAYAAAACAA CCAC-3= (reverse); for bla OXA-48 , 5=-ATGCGTGTATTAGCCT TATCGGCTG-3= (forward) and 5=-CTAGGGAATAATTTTTT CCTGTTTG-3= (reverse). In addition, we included 10 isolates that tested negative for all targets. Imipenem, meropenem, and ertapenem MICs were produced using GN4F Sensititre Gramnegative MIC plates (Thermo Fisher Scientific, Oakwood Village, OH) and interpreted using CLSI interpretive breakpoints (5). Organisms were identified to the species level using the Vitek 2 system (bioMérieux, Durham, NC). Prior to testing, isolates were subcultured and incubated twice in ambient air at 37°C on Trypticase soy agar with 5% sheep's blood (Remel, Lenexa, KS). All testing runs included positive (ATCC BAA-1705) and negative (ATCC BAA-1706) controls. CNP also had a reagent-only control.CNP was performed strictly as stated by CLSI (3). Briefly, solution A with 0.5% phenol red solution (made with phenol red indicator po...

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“…PCR-based methods that detect carbapenem resistance genes have recently been developed [14,15]. These tests have proved to be extremely sensitive and specific in the detection of colonization in rectal swabs.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the Xpert Carba-R (Cepheid) Assay Using Contrived Bronchial Specimens from Patients with Suspicion of Ventilator-Associated Pneumonia for the Detection of Prevalent Carbapenemases

et al. 2016

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There is a critical need for rapid diagnostic methods for multidrug-resistant (MDR) pathogens in patients with a suspicion of ventilator-associated pneumonia (VAP). The Xpert Carba-R detects 5 targets for carbapenemase-producing organisms (blaKPC, blaNDM, blaVIM, blaOXA-48, and blaIMP-1). Our objective was to evaluate the performance of this assay directly on bronchial aspirates and to correlate the cycle number for a positive result (Ct) with the bacterial count. Bronchial aspirates from patients with a suspicion of VAP were spiked with a dilution of 1 of 4 MDR organisms carrying the resistance genes detected by the test prepared to a final concentration of 102−105 cfu/mL. We used a ROC curve and provided areas under the curve (AUC) with their 95% confidence intervals (CI). A point of maximum sensitivity (Se) and specificity (Sp) was derived and validity indices were calculated. One hundred contrived tests were performed. Se and Sp were 100% for all bacterial counts. A positive sample with a Ct ≤24.7 corresponded to a count ≥105 cfu/mL; if the Ct was within the range >24.7-≤26.9, this corresponded to a count ≥104 cfu/mL. When the Ct was >26.9, this corresponded to a count <104 cfu/mL. The Xpert Carba-R detects carbapenemase-producing organisms directly in contrived bronchial aspirates. Still, an important issue to consider is that the number of gene copies may vary according to many factors in vivo. If confirmed in further studies, the strong correlation observed between Ct values and the results of semiquantitative cultures suggests this test could serve to differentiate between infection and colonization in routine clinical practice.

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Multi-centre evaluation of real-time multiplex PCR for detection of carbapenemase genes OXA-48, VIM, IMP, NDM and KPC

Cited by 82 publications

References 14 publications

Carbapenem-Resistant Enterobacteriaceae Infections in Children

Carbapenem-Resistant Enterobacteriaceae Infections in Children

Performance of the CLSI Carba NP and the Rosco Carb Screen Assays Using North American Carbapenemase-Producing Enterobacteriaceae and Pseudomonas aeruginosa Isolates

Evaluation of the Xpert Carba-R (Cepheid) Assay Using Contrived Bronchial Specimens from Patients with Suspicion of Ventilator-Associated Pneumonia for the Detection of Prevalent Carbapenemases

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