1887

Journal of Medical Microbiology

Volume 62, Issue 10

Evaluation of LAMP assay using phenotypic tests and conventional PCR for detection of and genes among carbapenem-resistant clinical Gram-negative isolates Free

Abstract

Carbapenem-resistant pathogens cause infections associated with significant morbidity and mortality. This study evaluates the use of the loop-mediated isothermal amplification (LAMP) assay for rapid and cost-effective detection of and genes among carbapenem-resistant Gram-negative bacteria in comparison with conventional PCR and existing phenotypic methods. A total of 60 carbapenem-resistant clinical isolates [ (15), (22), (23)] were screened for the presence of carbapenemases ( and ) using phenotypic methods such as the modified Hodge test (MHT) and combined disc test (CDT) and molecular methods such as conventional PCR and LAMP assay. In all, 47/60 isolates (78.3 %) were MHT positive while 48 isolates were positive by CDT [46.6 % positive with EDTA, 30 % with 3′ aminophenylboronic acid (APB) plus EDTA and 1.6 % with APB alone]. Isolates showing CDT positivity with EDTA or APB contained and genes, respectively. was present as a lone gene in 28 isolates (46.7 %) and present together with the gene in 19 isolates (31.7 %). Only one isolate had a lone gene. The LAMP assay detected either or both and genes in four isolates that were missed by conventional PCR. Neither gene could be detected in 12 (20 %) isolates. The LAMP assay has greater sensitivity, specificity and rapidity compared to the phenotypic methods and PCR for the detection of and . With a turnaround time of only 2–3 h, the LAMP assay can be considered a point-of-care assay.

  • Received:
  • Accepted:
  • Published Online:
© 2013 SGM
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2013-10-01
2021-07-28
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References

  1. Anderson K. F., Lonsway D. R., Rasheed J. K., Biddle J., Jensen B., McDougal L. K., Carey R. B., Thompson A., Stocker S. other authors 2007; Evaluation of methods to identify the Klebsiella pneumoniae carbapenemase in Enterobacteriaceae. J Clin Microbiol 45:2723–2725 [View Article][PubMed]
     [Google Scholar]
  2. CLSI 2012; Performance Standards for Antimicrobial Susceptibility Testing; CLSI M100–S22; Update January 2012. Wayne, PA: Clinical and Laboratory Standards Institute;
     [Google Scholar]
  3. Cole J. M., Schuetz A. N., Hill C. E., Nolte F. S. 2009; Development and evaluation of a real-time PCR assay for detection of Klebsiella pneumoniae carbapenemase genes. J Clin Microbiol 47:322–326 [View Article][PubMed]
     [Google Scholar]
  4. Deshpande P., Rodrigues C., Shetty A., Kapadia F., Hedge A., Soman R. 2010; New Delhi Metallo-beta lactamase (NDM-1) in Enterobacteriaceae: treatment options with carbapenems compromised. J Assoc Physicians India 58:147–149[PubMed]
     [Google Scholar]
  5. European Centre for Disease Prevention and Control 2011; Risk assessment on the spread of carbapenemase-producing Enterobacteriaceae (CPE) through patient transfer between healthcare facilities, with special emphasis on cross-border transfer. http://www.ecdc.europa.eu/en/press/press%20releases/110913_press_release_cpe.pdf
  6. Kumarasamy K. K., Toleman M. A., Walsh T. R., Bagaria J., Butt F., Balakrishnan R., Chaudhary U., Doumith M., Giske C. G. other authors 2010; Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis 10:597–602 [View Article][PubMed]
     [Google Scholar]
  7. Liu W., He X., Wei X., Shao C., Zou D., Li X. other authors 2012; Sensitive and rapid diagnostic method for detection of New Delhi metallo-beta-lactamase gene by loop-mediated isothermal amplification. J Clin Microbiol 50:1580–1585 [View Article][PubMed]
     [Google Scholar]
  8. Lonsway D.R., Wong B.K., Rasheed J.K. 2010; Detection of the blaKPC gene encoding Klebsiella pneumoniae carbapenemase by real-time PCR. In Clinical Microbiology Procedures Handbook pp. 12.5.4.1–12.5.4.10 Edited by Garcia L. Washington, DC: ASM Press;
     [Google Scholar]
  9. Manchanda V., Rai S., Gupta S., Rautela R. S., Chopra R., Rawat D. S., Verma N., Singh N. P., Kaur I. R., Bhalla P. 2011; Development of TaqMan real-time polymerase chain reaction for the detection of the newly emerging form of carbapenem resistance gene in clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii.. Indian J Med Microbiol 29:249–253 [View Article][PubMed]
     [Google Scholar]
  10. Notomi T., Okayama H., Masubuchi H., Yonekawa T., Watanabe K., Amino N., Hase T. 2000; Loop-mediated isothermal amplification of DNA.. Nucleic Acids Res 28:E63 [View Article][PubMed]
     [Google Scholar]
  11. Pasteran F., Mendez T., Rapoport M., Guerriero L., Corso A. 2010; Controlling false-positive results obtained with the Hodge and Masuda assays for detection of class a carbapenemase in species of Enterobacteriaceae by incorporating boronic acid. J Clin Microbiol 48:1323–1332 [View Article][PubMed]
     [Google Scholar]
  12. Reddy A. K., Balne P. K., Reddy R. K., Mathai A., Kaur I. 2010; Development and evaluation of loop-mediated isothermal amplification assay for rapid and inexpensive detection of cytomegalovirus DNA in vitreous specimens from suspected cases of viral retinitis. J Clin Microbiol 48:2050–2052 [View Article][PubMed]
     [Google Scholar]
  13. Robledo I. E., Aquino E. E., Vázquez G. J. 2011; Detection of the KPC gene in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii during a PCR-based nosocomial surveillance study in Puerto Rico. Antimicrob Agents Chemother 55:2968–2970 [View Article][PubMed]
     [Google Scholar]
  14. Tenover F. C., Kalsi R. K., Williams P. P., Carey R. B., Stocker S., Lonsway D., Rasheed J. K., Biddle J. W., McGowan J. E. Jr, Hanna B. 2006; Carbapenem resistance in Klebsiella pneumoniae not detected by automated susceptibility testing. Emerg Infect Dis 12:1209–1213 [View Article][PubMed]
     [Google Scholar]
  15. Walsh T. R., Weeks J., Livermore D. M., Toleman M. A. 2011; Dissemination of NDM-1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study. Lancet Infect Dis 11:355–362 [View Article][PubMed]
     [Google Scholar]
  16. Wattal C., Goel N., Oberoi J. K., Raveendran R., Datta S., Prasad K. J. 2010; Surveillance of multidrug resistant organisms in tertiary care hospital in Delhi, India. J Assoc Physicians India 58:Suppl32–36[PubMed]
     [Google Scholar]
  17. Woodford N., Tierno P. M. Jr, Young K., Tysall L., Palepou M. F., Ward E., Painter R. E., Suber D. F., Shungu D. other authors 2004; Outbreak of Klebsiella pneumoniae producing a new carbapenem-hydrolyzing class A β-lactamase, KPC-3, in a New York Medical Center. Antimicrob Agents Chemother 48:4793–4799 [View Article][PubMed]
     [Google Scholar]
  18. Yong D., Lee K., Yum J. H., Shin H. B., Rossolini G. M., Chong Y. 2002; Imipenem-EDTA disk method for differentiation of metallo-beta-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 40:3798–3801 [View Article][PubMed]
     [Google Scholar]
  19. Yong D., Toleman M. A., Giske C. G., Cho H. S., Sundman K., Lee K., Walsh T. R. 2009; Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother 53:5046–5054 [View Article][PubMed]
     [Google Scholar]
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Evaluation of LAMP assay using phenotypic tests and conventional PCR for detection of blaNDM-1 and blaKPC genes among carbapenem-resistant clinical Gram-negative isolates
J. Med. Microbiol. 62, 1540 (2013); https://doi.org/10.1099/jmm.0.059907-0
/content/journal/jmm/10.1099/jmm.0.059907-0
/content/journal/jmm/10.1099/jmm.0.059907-0
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