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Application of loop-mediated isothermal amplification technique to rapid and direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in blood cultures

  • ORIGINAL ARTICLE
  • Published:
Journal of Infection and Chemotherapy

Abstract

Staphylococcus aureus is the most important pathogen in nosocomial infections, including bloodstream infections. Prompt identification of S. aureus from blood cultures and detection of methicillin resistance are essential in cases of suspected sepsis. A novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63°C) with high specificity, efficiency, and rapidity, was applied to detect methicillin-resistant S. aureus (MRSA) directly from positive blood culture bottles. MRSA-LAMP, which targets the spa gene, encoding S. aureus-specific protein A, and the mecA gene, encoding penicillin-binding protein-2′ for methicillin resistance, could detect MRSA within 2 h after the blood culture signal became positive. The diagnostic values of LAMP, compared to a duplex real-time polymerase chain reaction (Drt-PCR) assay, were 92.3% and 96.2% sensitivity, 100% and 100% specificity, 100% and 100% positive predictive value (PPV), and 96.9% and 98.4% negative predictive value (NPV), respectively. These two methods had almost the same results, but the LAMP method is more cost-effective and provides excellent availability for rapid examination in a hospital clinical laboratory. Therefore, the LAMP assay appears to be a sensitive and reliable new method to diagnose MRSA bloodstream infection for appropriate antibiotic therapy.

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References

  1. RS McClelland VG Fowler SuffixJr LL Sanders G Gottilieb LK Kong DJ Sexton et al. (1999) ArticleTitle Staphylococcus aureus bacteremia among elderly vs younger adult patients: comparison of clinical features and mortality Arch Intern Med 159 1244–7 Occurrence Handle10371233 Occurrence Handle10.1001/archinte.159.11.1244 Occurrence Handle1:STN:280:DyaK1M3pslKnsQ%3D%3D

    Article  PubMed  CAS  Google Scholar 

  2. CDC. MRSA in healthcare settings. http://www.cdc.gov/ncidod/dhqp/ar_MRSA_spotlight_2006.html

  3. TY Tan S Corden R Barnes B Cookson (2001) ArticleTitleRapid identification of methicillin-resistant Staphylococcus aureus from positive blood cultures by real-time fluorescence PCR J Clin Microbiol 39 4529–31 Occurrence Handle11724876 Occurrence Handle10.1128/JCM.39.12.4529-4531.2001 Occurrence Handle1:CAS:528:DC%2BD38XivFamsQ%3D%3D

    Article  PubMed  CAS  Google Scholar 

  4. GM Trenholme RL Kaplan PH Karakusis T Stine J Fuhrer W Landau et al. (1989) ArticleTitleClinical impact of rapid identification and susceptibility testing of bacterial blood culture isolates J Clin Microbiol 27 1342–5 Occurrence Handle2473995 Occurrence Handle1:STN:280:DyaL1MzislCqsA%3D%3D

    PubMed  CAS  Google Scholar 

  5. RI Jaffe JD Lane SV Albury DM Niemeyer (2000) ArticleTitleRapid extraction from and direct identification in clinical samples of methicillin-resistant staphylococci with the PCR J Clin Microbiol 38 3407–12 Occurrence Handle10970392 Occurrence Handle1:CAS:528:DC%2BD3cXmvFyhsLs%3D

    PubMed  CAS  Google Scholar 

  6. L Louie J Goodfellow P Mathieu A Glatt M Louie AE Simor (2002) ArticleTitleRapid detection of methicillin-resistant staphylococci from blood culture bottles by using a multiplex PCR assay J Clin Microbiol 40 2786–90 Occurrence Handle12149330 Occurrence Handle10.1128/JCM.40.8.2786-2790.2002 Occurrence Handle1:CAS:528:DC%2BD38Xms1Sgsr0%3D

    Article  PubMed  CAS  Google Scholar 

  7. N Maes J Magdalena S Rottiers Y De Gheldre MJ Struelens (2002) ArticleTitleEvaluation of a triplex PCR assay to discriminate Staphylococcus aureus from coagulase-negative Staphylococci and determine methicillin resistance from blood cultures J Clin Microbiol 40 1514–7 Occurrence Handle11923385 Occurrence Handle10.1128/JCM.40.4.1514-1517.2002 Occurrence Handle1:CAS:528:DC%2BD38XjtlCmsLk%3D

    Article  PubMed  CAS  Google Scholar 

  8. NK Shrestha MJ Tuohy GS Hall CM Isada GW Procop (2002) ArticleTitleRapid identification of Staphylococcus aureus and the mecA gene from BacT/ALERT blood culture bottles by using the LightCycler System J Clin Microbiol 40 2659–61 Occurrence Handle12089301 Occurrence Handle10.1128/JCM.40.7.2659-2661.2002 Occurrence Handle1:CAS:528:DC%2BD38XlvVSqsro%3D

    Article  PubMed  CAS  Google Scholar 

  9. M Hallin N Maes B Byl F Jacobs Y De Gheldre MJ Struelens (2003) ArticleTitleClinical impact of a PCR assay for identification of Staphylococcus aureus and determination of methicillin resistance directly from blood cultures J Clin Microbiol 41 3942–4 Occurrence Handle12904425 Occurrence Handle10.1128/JCM.41.8.3942-3944.2003 Occurrence Handle1:CAS:528:DC%2BD3sXnt1SktbY%3D

    Article  PubMed  CAS  Google Scholar 

  10. SM Paule AC Pasquariello RB Thomson SuffixJr KL Kaul LR Peterson (2005) ArticleTitleReal-time PCR can rapidly detect methicillin-susceptible and methicillin-resistant Staphylococcus aureus directly from positive blood culture bottles Am J Clin Pathol 124 404–7 Occurrence Handle16191508 Occurrence Handle10.1309/6EA3-U9V8-NCLL-GKQN Occurrence Handle1:CAS:528:DC%2BD2MXhtVCks73E

    Article  PubMed  CAS  Google Scholar 

  11. U Reischl HJ Linde M Metz B Leppmeier N Lehn (2000) ArticleTitleRapid identification of methicillin-resistant Staphylococcus aureus and simultaneous species confirmation using real-time fluorescence PCR J Clin Microbiol 38 2429–33 Occurrence Handle10835024 Occurrence Handle1:STN:280:DC%2BD3c3psFWnsw%3D%3D

    PubMed  CAS  Google Scholar 

  12. P Lem J Spiegelman B Toye K Ramotar (2001) ArticleTitleDirect detection of mecA, nuc and 16S rRNA genes in BacT/Alert blood culture bottles Diagn Microbiol Infect Dis 41 165–8 Occurrence Handle11750172 Occurrence Handle10.1016/S0732-8893(01)00301-7 Occurrence Handle1:CAS:528:DC%2BD3MXptFynurc%3D

    Article  PubMed  CAS  Google Scholar 

  13. T Notomi H Okayama H Masubuchi T Yonekawa K Watanabe N Amino et al. (2000) ArticleTitleLoop-mediated isothermal amplification of DNA Nucleic Acids Res 28 e63 Occurrence Handle10871386 Occurrence Handle10.1093/nar/28.12.e63 Occurrence Handle1:CAS:528:DC%2BD3cXmtlKqsr8%3D

    Article  PubMed  CAS  Google Scholar 

  14. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; sixteenth informational supplement. M100-16, Wayne, PA: Clinical and Laboratory Standards Institute; 2006

  15. H Fang G Hedin (2003) ArticleTitleRapid screening and identification of methicillin-resistant Staphylococcus aureus from clinical samples by selective-broth and real-time PCR assay J Clin Microbiol 41 2894–9 Occurrence Handle12843018 Occurrence Handle10.1128/JCM.41.7.2894-2899.2003 Occurrence Handle1:CAS:528:DC%2BD3sXmsVWms78%3D

    Article  PubMed  CAS  Google Scholar 

  16. MI Queipo-Ortuño JD Colmenero G Baeza P Morata (2005) ArticleTitleComparison between LightCycler real-time polymerase chain reaction (PCR) assay with serum and PCR enzyme-linked immunosorbent assay with whole blood samples for the diagnosis of human brucellosis Clin Infect Dis 40 260–4 Occurrence Handle15655745 Occurrence Handle10.1086/426818

    Article  PubMed  Google Scholar 

  17. RE Rothman MD Majmudar GD Kelen G Madico CA Gaydos T Walker et al. (2002) ArticleTitleDetection of bacteremia in emergency department patients at risk for infective endocarditis using universal 16S rRNA primers in a decontaminated polymerase chain reaction assay J Infect Dis 186 1677–81 Occurrence Handle12447747 Occurrence Handle10.1086/345367 Occurrence Handle1:CAS:528:DC%2BD38Xps1KrtL0%3D

    Article  PubMed  CAS  Google Scholar 

  18. K Makimura SY Murayama H Yamaguchi (1994) ArticleTitleDetection of a wide range of medically important fungi by the polymerase chain reaction J Med Microbiol 40 358–64 Occurrence Handle8176723 Occurrence Handle1:CAS:528:DyaK2MXislyrsg%3D%3D Occurrence Handle10.1099/00222615-40-5-358

    Article  PubMed  CAS  Google Scholar 

  19. F Martineau FJ Picard PH Roy M Ouellette MG Bergeron (1998) ArticleTitleSpecies-specific and ubiquitous-DNA-based assays for rapid identification of Staphylococcus aureus J Clin Microbiol 29 2240–4

    Google Scholar 

  20. JJ Wu AH Huang JH Dai TC Chang (1997) ArticleTitleRapid detection of oxacillin-resistant Staphylococcus aureus in blood cultures by an impedance method J Clin Microbiol 35 1460–4 Occurrence Handle9163462 Occurrence Handle1:STN:280:DyaK2szgsF2qtg%3D%3D

    PubMed  CAS  Google Scholar 

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Correspondence to Yoshiki Misawa.

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Misawa, Y., Yoshida, A., Saito, R. et al. 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 (2007). https://doi.org/10.1007/s10156-007-0508-9

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  • DOI: https://doi.org/10.1007/s10156-007-0508-9

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