Skip to main content

Advertisement

SpringerLink
Log in

One-step sample preparation of positive blood cultures for the direct detection of methicillin-sensitive and -resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci within one hour using the automated GenomEra CDX™ PCR system

  • Article
  • Published:
European Journal of Clinical Microbiology & Infectious Diseases Aims and scope Submit manuscript

Abstract

A method for the rapid detection of methicillin-sensitive and -resistant Staphylococcus aureus (MSSA and MRSA, respectively) and methicillin-resistant coagulase-negative staphylococci (MRCoNS) with a straightforward sample preparation protocol of blood cultures using an automated homogeneous polymerase chain reaction (PCR) assay, the GenomEra™ MRSA/SA (Abacus Diagnostica Oy, Turku, Finland), is presented. In total, 316 BacT/Alert (bioMérieux, Marcy l’Etoile, France) and 433 BACTEC (Becton Dickinson, Sparks, MD, USA) blood culture bottles were analyzed, including 725 positive cultures containing Gram-positive cocci in clusters (n = 419) and other Gram stain forms (n = 361), as well as 24 signal- and growth-negative bottles. Detection sensitivities for MSSA, MRSA, and MRCoNS were 99.4 % (158/159), 100.0 % (9/9), and 99.3 % (132/133), respectively. One false-positive MRSA result was detected from a non-staphylococci-containing bottle, yielding a specificity of 99.8 %. The lowest detectable amount of viable cells in the blood culture sample was 4 × 104 CFU/mL. The results were available within one hour after microbial growth detection and the two-step, time-resolved fluorometric (TRF) measurement mode employed by the GenomEra CDX™ instrument showed no interference from blood, charcoal, or culture media. The method described lacks all sample purification steps and allows reliable and simplified pathogen detection also in clinical microbiology laboratory settings without specialized molecular microbiology competence.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Bauer KA, West JE, Balada-Llasat JM, Pancholi P, Stevenson KB, Goff DA (2010) An antimicrobial stewardship program’s impact with rapid polymerase chain reaction methicillin-resistant Staphylococcus aureus/S. aureus blood culture test in patients with S. aureus bacteremia. Clin Infect Dis 51:1074–1080. doi:10.1086/656623

    Article  PubMed  Google Scholar 

  2. Rahikka P, Syrjänen J, Vuento R, Laine J, Huttunen R (2011) Meticillin-resistant Staphylococcus aureus (MRSA) bacteraemia in Tampere University Hospital: a case–control study, Finland October 2002 to January 2010. Euro Surveill 16:pii=19958

  3. Shrestha NK, Tuohy MJ, Hall GS, Isada CM, Procop GW (2002) Rapid identification of Staphylococcus aureus and the mecA gene from BacT/ALERT blood culture bottles by using the LightCycler system. J Clin Microbiol 40:2659–2661. doi:10.1128/JCM.40.7.2659-2661.2002

    Article  PubMed  CAS  Google Scholar 

  4. Jukes L, Mikhail J, Bome-Mannathoko N, Hadfield SJ, Harris LG, El-Bouri K, Davies AP, Mack D (2010) Rapid differentiation of Staphylococcus aureus, Staphylococcus epidermidis and other coagulase-negative staphylococci and meticillin susceptibility testing directly from growth-positive blood cultures by multiplex real-time PCR. J Med Microbiol 59:1456–1461. doi:10.1099/jmm.0.023168-0

    Article  PubMed  Google Scholar 

  5. Klonoski J, Mondesire R, Rea L, Ward DC, Jenison RD (2010) Enhanced detection of staphylococcal genomes in positive blood cultures using a polymeric enzyme complex. Anal Biochem 396:284–289. doi:10.1016/j.ab.2009.09.035

    Article  PubMed  CAS  Google Scholar 

  6. Wolk DM, Struelens MJ, Pancholi P, Davis T, Della-Latta P, Fuller D, Picton E, Dickenson R, Denis O, Johnson D, Chapin K (2009) Rapid detection of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) in wound specimens and blood cultures: multicenter preclinical evaluation of the Cepheid Xpert MRSA/SA skin and soft tissue and blood culture assays. J Clin Microbiol 47:823–826. doi:10.1128/JCM.01884-08

    Article  PubMed  CAS  Google Scholar 

  7. Stamper PD, Cai M, Howard T, Speser S, Carroll KC (2007) Clinical validation of the molecular BD GeneOhm StaphSR assay for direct detection of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus in positive blood cultures. J Clin Microbiol 45:2191–2196. doi:10.1128/JCM.00552-07

    Article  PubMed  Google Scholar 

  8. Misawa Y, Yoshida A, Saito R, Yoshida H, Okuzumi K, Ito N, Okada M, Moriya K, Koike K (2007) Application of loop-mediated isothermal amplification technique to rapid and direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in blood cultures. J Infect Chemother 13:134–140. doi:10.1007/s10156-007-0508-9

    Article  PubMed  CAS  Google Scholar 

  9. Oliveira K, Procop GW, Wilson D, Coull J, Stender H (2002) Rapid identification of Staphylococcus aureus directly from blood cultures by fluorescence in situ hybridization with peptide nucleic acid probes. J Clin Microbiol 40:247–251. doi:10.1128/JCM.40.1.247-251.2002

    Article  PubMed  CAS  Google Scholar 

  10. Tissari P, Zumla A, Tarkka E, Mero S, Savolainen L, Vaara M, Aittakorpi A, Laakso S, Lindfors M, Piiparinen H, Mäki M, Carder C, Huggett J, Gant V (2010) Accurate and rapid identification of bacterial species from positive blood cultures with a DNA-based microarray platform: an observational study. Lancet 375:224–230. doi:10.1016/S0140-6736(09)61569-5

    Article  PubMed  CAS  Google Scholar 

  11. Ratnayake L, Olver WJ (2011) Rapid PCR detection of methicillin-resistant Staphylococcus aureus and methicillin-sensitive S. aureus samples from charcoal-containing blood culture bottles. J Clin Microbiol 49:2382. doi:10.1128/JCM.00106-11

    Article  PubMed  Google Scholar 

  12. Kiviniemi M, Ilonen J, Lövgren T (2009) A homogeneous HLA-B*27 genotyping assay using dried reagent mixtures. Dis Markers 27:85–91. doi:10.3233/DMA-2009-0653

    PubMed  CAS  Google Scholar 

  13. Karhunen U, Jaakkola L, Wang Q, Lamminmäki U, Soukka T (2010) Luminescence switching by hybridization-directed mixed lanthanide complex formation. Anal Chem 82:751–754. doi:10.1021/ac9020825

    Article  PubMed  CAS  Google Scholar 

  14. von Lode P, Rosenberg J, Pettersson K, Takalo H (2003) A europium chelate for quantitative point-of-care immunoassays using direct surface measurement. Anal Chem 75:3193–3201. doi:10.1021/ac0340051

    Article  Google Scholar 

  15. Hirvonen JJ, Nevalainen M, Tissari P, Salmenlinna S, Rantakokko-Jalava K, Kaukoranta SS (2012) Rapid confirmation of suspected methicillin-resistant Staphylococcus aureus colonies on chromogenic agars by a new commercial PCR assay, the GenomEra MRSA/SA Diagnose. Eur J Clin Microbiol Infect Dis. Jan 26 [Epub ahead of print]. doi: 10.1007/s10096-011-1527-0

  16. Soini E, Lövgren T, Reimer CB (1987) Time-resolved fluorescence of lanthanide probes and applications in biotechnology. CRC Crit Rev Anal Chem 18:105–154. doi:10.1080/10408348708542802

    Article  CAS  Google Scholar 

  17. Clinical and Laboratory Standards Institute (CLSI) (2007) Performance standards for antimicrobial susceptibility testing, 17th informational supplement. Approved standard MS100-S17. CLSI, Wayne

    Google Scholar 

  18. European Committee on Antimicrobial Susceptibility Testing (EUCAST) (2012) Breakpoint tables for interpretation of MICs and zone diameters, version 2.0. Available online at: http://eucast.org/clinical_breakpoints/. Last date accessed 2 January 2012

  19. Vainio A, Kardén-Lilja M, Ibrahem S, Kerttula AM, Salmenlinna S, Virolainen A, Vuopio-Varkila J (2008) Clonality of epidemic methicillin-resistant Staphylococcus aureus strains in Finland as defined by several molecular methods. Eur J Clin Microbiol Infect Dis 27:545–555. doi:10.1007/s10096-008-0470-1

    Article  PubMed  CAS  Google Scholar 

  20. Kumar A, Roberts D, Wood KE, Light B, Parrillo JE, Sharma S, Suppes R, Feinstein D, Zanotti S, Taiberg L, Gurka D, Kumar A, Cheang M (2006) Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med 34:1589–1596. doi:10.1097/01.CCM.0000217961.75225.E9

    Article  PubMed  Google Scholar 

  21. McDonald CP, Roy A, Lowe P, Robbins S, Hartley S, Barbara JA (2001) Evaluation of the BacT/Alert automated blood culture system for detecting bacteria and measuring their growth kinetics in leucodepleted and non-leucodepleted platelet concentrates. Vox Sang 81:154–160. doi:10.1046/j.0042-9007.2001.00104.x

    Article  PubMed  CAS  Google Scholar 

  22. Wellinghausen N, Wirths B, Franz AR, Karolyi L, Marre R, Reischl U (2004) Algorithm for the identification of bacterial pathogens in positive blood cultures by real-time LightCycler polymerase chain reaction (PCR) with sequence-specific probes. Diagn Microbiol Infect Dis 48:229–241. doi:10.1016/j.diagmicrobio.2003.11.005

    Article  PubMed  CAS  Google Scholar 

  23. Ferroni A, Suarez S, Beretti JL, Dauphin B, Bille E, Meyer J, Bougnoux ME, Alanio A, Berche P, Nassif X (2010) Real-time identification of bacteria and Candida species in positive blood culture broths by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol 48:1542–1548. doi:10.1128/JCM.02485-09

    Article  PubMed  CAS  Google Scholar 

  24. Snyder JW, Munier GK, Heckman SA, Camp P, Overman TL (2009) Failure of the BD GeneOhm StaphSR assay for direct detection of methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates in positive blood cultures collected in the United States. J Clin Microbiol 47:3747–3748. doi:10.1128/JCM.01391-09

    Article  PubMed  Google Scholar 

  25. Benedik MJ, Strych U (1998) Serratia marcescens and its extracellular nuclease. FEMS Microbiol Lett 165:1–13. doi:10.1111/j.1574-6968.1998.tb13120.x

    Article  PubMed  CAS  Google Scholar 

  26. Chang A, Khemlani A, Kang H, Proft T (2011) Functional analysis of Streptococcus pyogenes nuclease A (SpnA), a novel group A streptococcal virulence factor. Mol Microbiol 79:1629–1642. doi:10.1111/j.1365-2958.2011.07550.x

    Article  PubMed  CAS  Google Scholar 

  27. Stamper PD, Louie L, Wong H, Simor AE, Farley JE, Carroll KC (2011) Genotypic and phenotypic characterization of methicillin-susceptible Staphylococcus aureus isolates misidentified as methicillin-resistant Staphylococcus aureus by the BD GeneOhm MRSA assay. J Clin Microbiol 49:1240–1244. doi:10.1128/JCM.02220-10

    Article  PubMed  CAS  Google Scholar 

  28. Blanc DS, Basset P, Nahimana-Tessemo I, Jaton K, Greub G, Zanetti G (2011) High proportion of wrongly identified methicillin-resistant Staphylococcus aureus carriers by use of a rapid commercial PCR assay due to presence of staphylococcal cassette chromosome element lacking the mecA gene. J Clin Microbiol 49:722–724. doi:10.1128/JCM.01988-10

    Article  PubMed  Google Scholar 

  29. Kassem II, Esseili MA, Sigler V (2008) Occurrence of mecA in nonstaphylococcal pathogens in surface waters. J Clin Microbiol 46:3868–3869. doi:10.1128/JCM.01035-08

    Article  PubMed  Google Scholar 

  30. Sarvikivi E, Lyytikäinen O, Vaara M, Saxén H (2008) Nosocomial bloodstream infections in children: an 8-year experience at a tertiary-care hospital in Finland. Clin Microbiol Infect 14:1072–1075. doi:10.1111/j.1469-0691.2008.02079.x

    Article  PubMed  CAS  Google Scholar 

  31. Frye AM, Baker CA, Rustvold DL, Heath KA, Hunt J, Leggett JE, Oethinger M (2012) Clinical impact of a real-time PCR assay for rapid identification of staphylococcal bacteremia. J Clin Microbiol 50:127–133. doi:10.1128/JCM.06169-11

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

Dr. P. von Lode is employed by Abacus Diagnostica Oy, the manufacturer of the PCR test. The other investigators have no conflicts of interest to declare. The results were presented, in part, at the 1st International Conference on Prevention and Infection Control (ICPIC), June/July 2011, Geneva, Switzerland, poster no. 204.

Author information

Authors and Affiliations

  1. Department of Clinical Microbiology, Vaasa Central Hospital, Hietalahdenkatu 2-4 B2, 65130, Vaasa, Finland

    J. J. Hirvonen & S.-S. Kaukoranta

  2. Abacus Diagnostica Oy, Tykistökatu 4D, 20520, Turku, Finland

    P. von Lode

  3. Fimlab Laboratories, Pirkanmaa Hospital District, Biokatu 4, 33520, Tampere, Finland

    M. Nevalainen

  4. Clinical Microbiology Laboratory, Turku University Hospital, Kiinamyllynkatu 4-8, 20521, Turku, Finland

    K. Rantakokko-Jalava

Authors
  1. J. J. Hirvonen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P. von Lode
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Nevalainen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. K. Rantakokko-Jalava
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. S.-S. Kaukoranta
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J. J. Hirvonen.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hirvonen, J.J., von Lode, P., Nevalainen, M. et al. One-step sample preparation of positive blood cultures for the direct detection of methicillin-sensitive and -resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci within one hour using the automated GenomEra CDX™ PCR system. Eur J Clin Microbiol Infect Dis 31, 2835–2842 (2012). https://doi.org/10.1007/s10096-012-1637-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10096-012-1637-3

Keywords

Search

Navigation