Use of real-time polymerase chain reaction for identification of methicillin-resistant Staphylococcus aureus directly from positive blood culture bottles

Stratidis, John; Bia, Frank J.; Edberg, Stephen C.

doi:10.1016/j.diagmicrobio.2006.12.017

Cited by 19 publications

(18 citation statements)

References 9 publications

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“…The analytical sensitivity in naïve Bactec bottles was similar to the analytical sensitivity of the singleplex bla KPC assay reported earlier (12). The assay is sensitive for concentrations 1 log unit below the limit of detection of the Bactec system (at least 10 4 CFU/ml); thus, it will detect the bla KPC -positive bacteria if they are present in the bottles (26). The analytical sensitivity of the multiplex bla KPC /RNase P assay was comparable to sensitivities achieved for detection of 10 CFU/reaction mixture of both E. coli and Klebsiella pneumoniae in blood culture bottles (10,17).…”

Section: Discussionsupporting

confidence: 77%

“…Automated blood culture systems take approximately 1 to 2 days, on average, to signal a positive blood culture and another 1 to 2 days to finalize bacterial identification and antimicrobial testing. With the advent of qPCR, the time to bacterial identification and detection of drug resistance has been reduced to 4 to 6 h after a positive blood culture has turned positive (22,24,26,28). However, the presence of PCR inhibitors in the blood culture bottles has thus far reduced the sensitivity of PCR assays (10).…”

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confidence: 99%

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Rapid Detection of bla _KPC Carbapenemase Genes by Internally Controlled Real-Time PCR Assay Using Bactec Blood Culture Bottles

et al. 2011

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Rapid detection of drug-resistant bacteria in clinical samples plays an instrumental role in patients'infection management and in implementing effective infection control policies. In the study described in this report, we validated a multiplex TaqMan real-time quantitative PCR (qPCR) assay for the detection of bla KPC genes and the human RNase P gene in Bactec blood culture bottles. The MagNA Pure LC (version 2.0) instrument was utilized to extract nucleic acids from the inoculated broth, while bovine serum albumin (BSA) was utilized as the PCR inhibitor reliever. The multiplex assay, which was specific for the detection of bla KPC genes, had a limit of detection of 19 CFU per reaction mixture with human blood-spiked Bactec bottles. Of the 323 Bactec blood culture sets evaluated, the same 55 (17%) blood cultures positive for carbapenem-resistant bacteria by culture were also positive by the validated qPCR assay. Thus, the sensitivity, specificity, positive predictive value, and negative predictive value of the qPCR assay compared to the results of culture were all 100%. bla KPC genes were also detected from the same Bactec bottle broth after manual extraction with a QIAamp DNA minikit; however, there was an average 3-threshold-cycle delay in the qPCR readings. With the limited therapeutic options available, the accurate and rapid detection of bla KPC -possessing bacteria by the described bla KPC /RNase P assay will be a crucial first step in ensuring optimal clinical outcomes and infection control.

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Section: Discussionsupporting

confidence: 77%

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confidence: 99%

Rapid Detection of bla _KPC Carbapenemase Genes by Internally Controlled Real-Time PCR Assay Using Bactec Blood Culture Bottles

et al. 2011

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“…With an increased demand for more rapid reporting of results in the laboratory, a shorter turnaround time for PNA-FISH and RT-PCR appears attractive (6,7,12,16). The PNA-FISH assay can be interpreted at 3 hours, a significant difference from TCT24.…”

Section: Discussionmentioning

confidence: 99%

Pharmacoeconomic Analysis of Microbiologic Techniques for Differentiating Staphylococci Directly from Blood Culture Bottles

et al. 2008

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Differentiating staphylococci in blood cultures is a critical issue, particularly when only one of two cultures is positive by Gram staining for staphylococci. New tests for the identification of Staphylococcus aureus allow faster results and definitive treatment compared to the tube coagulase test interpreted at 24 h (TCT24). These newer tests, peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) and real-time PCR (RT-PCR), offer improved sensitivity at higher cost. Data suggest that the tube coagulase test may be interpreted at 4 h (TCT4) with little loss of sensitivity. The impact of variability in turnaround time, sensitivity, specificity, and cost on comparative cost-effectiveness is unknown. Our aim was to establish the cost-effectiveness of TCT24, PNA-FISH, RT-PCR, and TCT4 for direct identification of staphylococci in blood cultures. Decision analysis comparing these strategies was done from the institutional perspective. Besides test variables, other variables included patient risk factors, empirical treatment, and follow-up cultures. Probability and cost estimates came from the literature and institutional data. Base case estimates were derived from institutional rates of 73% contamination when coagulase-negative staphylococci were identified, 67.6% prevalence of risk factors, and 12.4% prevalence of S. aureus when one of two cultures yielded staphylococci. Sensitivity analysis was done across a range of probabilities and costs. In the base case, TCT4 and TCT24 were more cost-effective than RT-PCR and PNA-FISH ($78 versus $120 versus $165 per patient, respectively). The advantage of TCT4 and TCT24 remained robust upon sensitivity analysis. TCT4 should be further evaluated as a rapid, cost-effective means for identification of S. aureus in blood cultures.

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“…Several mechanisms for determining Staphylococcus species and/or oxacillin results directly from blood culture bottles include selective differentiation chromogenic agar (17), direct identification methods such as the BBL Crystal MRSA ID test (Becton Dickinson Microbiology Systems, Cockeysville, MD) (11), and molecular methods, including the BD GeneOhm StaphSR assay (Becton Dickinson, Sparks, MD), Xpert MRSA/SA blood culture test from Cepheid (Sunnyvale, CA), multiplex detection of mecA and the nuc genes (12), detection of mecA and orfX genes by real-time PCR (21), and the IDI real-time assay (Infectio Diagnostic, Sainte-Foy, Quebec, Canada) using the SmartCycler (Cepheid, Sunnyvale, CA) (7). In addition, the PBP2a MRSA latex agglutination test (Oxoid Ltd., Basingstoke, United Kingdom) could be used the morning following subculture of positive samples if sufficient growth occurs (incubation time dependent).…”

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confidence: 99%

Rapid Differentiation of Methicillin-Resistant Staphylococcus aureus and Methicillin-Susceptible Staphylococcus aureus from Blood Cultures by Use of a Direct Cefoxitin Disk Diffusion Test

Bennett

Sharp

2008

J Clin Microbiol

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Use of real-time polymerase chain reaction for identification of methicillin-resistant Staphylococcus aureus directly from positive blood culture bottles

Cited by 19 publications

References 9 publications

Rapid Detection of bla _KPC Carbapenemase Genes by Internally Controlled Real-Time PCR Assay Using Bactec Blood Culture Bottles

Rapid Detection of bla _KPC Carbapenemase Genes by Internally Controlled Real-Time PCR Assay Using Bactec Blood Culture Bottles

Pharmacoeconomic Analysis of Microbiologic Techniques for Differentiating Staphylococci Directly from Blood Culture Bottles

Rapid Differentiation of Methicillin-Resistant Staphylococcus aureus and Methicillin-Susceptible Staphylococcus aureus from Blood Cultures by Use of a Direct Cefoxitin Disk Diffusion Test

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Use of real-time polymerase chain reaction for identification of methicillin-resistant Staphylococcus aureus directly from positive blood culture bottles

Cited by 19 publications

References 9 publications

Rapid Detection of bla KPC Carbapenemase Genes by Internally Controlled Real-Time PCR Assay Using Bactec Blood Culture Bottles

Rapid Detection of bla KPC Carbapenemase Genes by Internally Controlled Real-Time PCR Assay Using Bactec Blood Culture Bottles

Pharmacoeconomic Analysis of Microbiologic Techniques for Differentiating Staphylococci Directly from Blood Culture Bottles

Rapid Differentiation of Methicillin-Resistant Staphylococcus aureus and Methicillin-Susceptible Staphylococcus aureus from Blood Cultures by Use of a Direct Cefoxitin Disk Diffusion Test

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Rapid Detection of bla _KPC Carbapenemase Genes by Internally Controlled Real-Time PCR Assay Using Bactec Blood Culture Bottles

Rapid Detection of bla _KPC Carbapenemase Genes by Internally Controlled Real-Time PCR Assay Using Bactec Blood Culture Bottles