Abstract
Background: The Verigene Gram-Positive Blood Culture (BC-GP) nucleic acid assay (Nanosphere, Inc., Northbrook, IL, USA) is a newly developed microarray-based test with which 12 Gram-positive bacterial genes and three resistance determinants can be detected using blood culture broths. We evaluated the performance of this assay and investigated the signal characteristics of the microarray images.
Methods: At the evaluation stage, we tested 80 blood cultures that were positive for various bacteria (68 bacteria covered and 12 not covered by the BC-GP panel) collected from the blood of 36 patients and 44 spiked samples. In instances where the automated system failed and errors were called, we manually inspected microarray images, measured the signal intensities of target spots, and reclassified the results.
Results: With the manual analysis of the microarray images of 14 samples for which error calls were reported, we could obtain correct identification results for 12 samples without the need for retesting, because strong signals in the target spots were clearly discriminable from background noise. With our interpretation strategy, we could obtain 97.1% sensitivity and 100% specificity for bacterial identification by using the BC-GP assay. The two unidentified bacteria were viridans group streptococci, which produced weaker target signals. During the application stage, among 25 consecutive samples positive for Gram-positive bacteria, we identified two specimens with error calls as Streptococcus spp. by using manual analysis.
Conclusions: With help of the manual review of the microarray images, the BC-GP assay could successfully identify species and resistance markers for many clinically important Gram-positive bacteria.
References
1. Pittet D, Tarara D, Wenzel RP. Nosocomial bloodstream infection in critically ill patients. Excess length of stay, extra costs, and attributable mortality. J Am Med Assoc 1994;271:1598–601.10.1001/jama.1994.03510440058033Search in Google Scholar
2. Levy MM, Artigas A, Phillips GS, Rhodes A, Beale R, Osborn T, et al. Outcomes of the Surviving Sepsis Campaign in intensive care units in the USA and Europe: a prospective cohort study. Lancet Infect Dis 2012;12:919–24.10.1016/S1473-3099(12)70239-6Search in Google Scholar
3. Grozdanovski K, Milenkovic Z, Demiri I, Spasovska K. Prediction of outcome from community-acquired severe sepsis and septic shock in tertiary-care university hospital in a developing country. Crit Care Res Pract 2012;2012:182324.10.1155/2012/182324Search in Google Scholar PubMed PubMed Central
4. Buchan BW, Ginocchio CC, Manii R, Cavagnolo R, Pancholi P, Swyers L, et al. Multiplex identification of gram-positive bacteria and resistance determinants directly from positive blood culture broths: evaluation of an automated microarray-based nucleic acid test. PLoS Med 2013;10:e1001478.10.1371/journal.pmed.1001478Search in Google Scholar PubMed PubMed Central
5. Dellinger RP, Levy MM, Rhodes A, Annane D, Gerlach H, Opal SM, et al. Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012. Intensive Care Med 2013;39:165–228.10.1007/s00134-012-2769-8Search in Google Scholar PubMed PubMed Central
6. Stevenson LG, Drake SK, Murray PR. Rapid identification of bacteria in positive blood culture broths by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol 2010;48:444–7.10.1128/JCM.01541-09Search in Google Scholar PubMed PubMed Central
7. Buchan BW, Riebe KM, Ledeboer NA. Comparison of the MALDI Biotyper system using Sepsityper specimen processing to routine microbiological methods for identification of bacteria from positive blood culture bottles. J Clin Microbiol 2012;50:346–52.10.1128/JCM.05021-11Search in Google Scholar PubMed PubMed Central
8. Vlek AL, Bonten MJ, Boel CH. Direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry improves appropriateness of antibiotic treatment of bacteremia. PLoS One 2012;7:e32589.10.1371/journal.pone.0032589Search in Google Scholar PubMed PubMed Central
9. Ratnayake L, Olver WJ. Rapid PCR detection of methicillin-resistant Staphylococcus aureus and methicillin-sensitive S. aureus samples from charcoal-containing blood culture bottles. J Clin Microbiol 2011;49:2382.10.1128/JCM.00106-11Search in Google Scholar PubMed PubMed Central
10. Davies J, Gordon CL, Tong SY, Baird RW, Davis JS. Impact of results of a rapid Staphylococcus aureus diagnostic test on prescribing of antibiotics for patients with clustered Gram-positive cocci in blood cultures. J Clin Microbiol 2012;50:2056–8.10.1128/JCM.06773-11Search in Google Scholar PubMed PubMed Central
11. Frye AM, Baker CA, Rustvold DL, Heath KA, Hunt J, Leggett JE, et al. Clinical impact of a real-time PCR assay for rapid identification of staphylococcal bacteremia. J Clin Microbiol 2012;50:127–33.10.1128/JCM.06169-11Search in Google Scholar
12. Biendo M, Mammeri H, Pluquet E, Guillon H, Rousseau F, Canarelli B, et al. Value of Xpert MRSA/SA blood culture assay on the Gene Xpert(R) Dx System for rapid detection of Staphylococcus aureus and coagulase-negative staphylococci in patients with staphylococcal bacteremia. Diagn Microbiol Infect Dis 2013;75:139–43.10.1016/j.diagmicrobio.2012.11.005Search in Google Scholar
13. Wang B, Jessamine P, Desjardins M, Toye B, Ramotar K. Direct mecA polymerase chain reaction testing of blood culture bottles growing Gram-positive cocci and the clinical potential in optimizing antibiotic therapy for staphylococcal bacteremia. Diagn Microbiol Infect Dis 2013;75:37–41.10.1016/j.diagmicrobio.2012.09.014Search in Google Scholar
14. Peleg AY, Tilahun Y, Fiandaca MJ, D’Agata EM, Venkataraman L, Moellering RC, Jr., et al. Utility of peptide nucleic acid fluorescence in situ hybridization for rapid detection of Acinetobacter spp. and Pseudomonas aeruginosa. J Clin Microbiol 2009;47:830–2.10.1128/JCM.01724-08Search in Google Scholar
15. Holtzman C, Whitney D, Barlam T, Miller NS. Assessment of impact of peptide nucleic acid fluorescence in situ hybridization for rapid identification of coagulase-negative staphylococci in the absence of antimicrobial stewardship intervention. J Clin Microbiol 2011;49:1581–2.10.1128/JCM.02461-10Search in Google Scholar
16. Lindholm L, Sarkkinen H. Direct identification of gram-positive cocci from routine blood cultures by using AccuProbe tests. J Clin Microbiol 2004;42:5609–13.10.1128/JCM.42.12.5609-5613.2004Search in Google Scholar
17. Jarvinen AK, Laakso S, Piiparinen P, Aittakorpi A, Lindfors M, Huopaniemi L, et al. Rapid identification of bacterial pathogens using a PCR- and microarray-based assay. BMC Microbiol 2009;9:161.10.1186/1471-2180-9-161Search in Google Scholar
18. Tissari P, Zumla A, Tarkka E, Mero S, Savolainen L, Vaara M, et al. Accurate and rapid identification of bacterial species from positive blood cultures with a DNA-based microarray platform: an observational study. Lancet 2010;375:224–30.10.1016/S0140-6736(09)61569-5Search in Google Scholar
19. Scott LJ. Verigene(R) gram-positive blood culture nucleic acid test. Mol Diagn Ther 2013;17:117–22.10.1007/s40291-013-0021-zSearch in Google Scholar PubMed
20. Wojewoda CM, Sercia L, Navas M, Tuohy M, Wilson D, Hall GS, et al. Evaluation of the Verigene Gram-positive blood culture nucleic acid test for rapid detection of bacteria and resistance determinants. J Clin Microbiol 2013;51:2072–6.10.1128/JCM.00831-13Search in Google Scholar PubMed PubMed Central
21. Cleven BE, Palka-Santini M, Gielen J, Meembor S, Kronke M, Krut O. Identification and characterization of bacterial pathogens causing bloodstream infections by DNA microarray. J Clin Microbiol 2006;44:2389–97.10.1128/JCM.02291-05Search in Google Scholar PubMed PubMed Central
22. Fishbain JT, Sinyavskiy O, Riederer K, Hujer AM, Bonomo RA. Detection of extended-spectrum beta-lactamase and Klebsiella pneumoniae Carbapenemase genes directly from blood cultures by use of a nucleic acid microarray. J Clin Microbiol 2012;50:2901–4.10.1128/JCM.01023-12Search in Google Scholar PubMed PubMed Central
23. Samuel LP, Tibbetts RJ, Agotesku A, Fey M, Hensley R, Meier FA. Evaluation of a microarray-based assay for rapid identification of Gram-positive organisms and resistance markers in positive blood cultures. J Clin Microbiol 2013;51:1188–92.10.1128/JCM.02982-12Search in Google Scholar PubMed PubMed Central
24. Sullivan KV, Turner NN, Roundtree SS, Young S, Brock-Haag CA, Lacey D, et al. Rapid detection of gram-positive organisms using the Verigene gram-positive blood culture nucleic acid test and the BacT/ALERT pediatric FAN system in a multi-centered pediatric evaluation. J Clin Microbiol 2013;51:3579–84.10.1128/JCM.01224-13Search in Google Scholar PubMed PubMed Central
25. Mancini N, Infurnari L, Ghidoli N, Valzano G, Clementi N, Burioni R, et al. Potential impact of a microarray-based nucleic acid assay for rapid detection of Gram-negative bacteria and resistance markers in positive blood cultures. J Clin Microbiol 2014;52:1242–5.10.1128/JCM.00142-14Search in Google Scholar PubMed PubMed Central
26. Sullivan KV, Deburger B, Roundtree SS, Ventrola CA, Blecker-Shelly DL, Mortensen JE. Pediatric multicenter evaluation of the Verigene gram-negative blood culture test for rapid detection of inpatient bacteremia involving gram-negative organisms, extended-spectrum beta-lactamases, and carbapenemases. J Clin Microbiol 2014;52:2416–21.10.1128/JCM.00737-14Search in Google Scholar PubMed PubMed Central
27. Hackett JL, Arche KJ, Gaigalas AK, Garrett CT, Joseph LJ, Koch WH, et al. MM12-A diagnostic nucleic acid microarays – approved guideline. Wayne PA: The Clinical and Laboratory Standards Institute (CLSI), 2006.Search in Google Scholar
28. Nanosphere Inc. Verigene gram-positive blood culture nucleic acid test (BC-GP) package insert. Nanosphere, Inc.: Northbrook, IL, 2012.Search in Google Scholar
29. van Veen SQ, Claas EC, Kuijper EJ. High-throughput identification of bacteria and yeast by matrix-assisted laser desorption ionization-time of flight mass spectrometry in conventional medical microbiology laboratories. J Clin Microbiol 2010;48:900–7.10.1128/JCM.02071-09Search in Google Scholar PubMed PubMed Central
30. Kim M, Heo SR, Choi SH, Kwon H, Park JS, Seong MW, et al. Comparison of the MicroScan, VITEK 2, and Crystal GP with 16S rRNA sequencing and MicroSeq 500 v2.0 analysis for coagulase-negative Staphylococci. BMC Microbiol 2008;8:233.10.1186/1471-2180-8-233Search in Google Scholar PubMed PubMed Central
31. Hwang SM, Kim MS, Park KU, Song J, Kim EC. Tuf gene sequence analysis has greater discriminatory power than 16S rRNA sequence analysis in identification of clinical isolates of coagulase-negative staphylococci. J Clin Microbiol 2011;49:4142–9.10.1128/JCM.05213-11Search in Google Scholar PubMed PubMed Central
32. Zbinden A, Kohler N, Bloemberg GV. recA-based PCR assay for accurate differentiation of Streptococcus pneumoniae from other viridans streptococci. J Clin Microbiol 2011;49:523–7.10.1128/JCM.01450-10Search in Google Scholar PubMed PubMed Central
33. Francois P, Pittet D, Bento M, Pepey B, Vaudaux P, Lew D, et al. Rapid detection of methicillin-resistant Staphylococcus aureus directly from sterile or nonsterile clinical samples by a new molecular assay. J Clin Microbiol 2003;41:254–60.10.1128/JCM.41.1.254-260.2003Search in Google Scholar PubMed PubMed Central
34. Jukes L, Mikhail J, Bome-Mannathoko N, Hadfield SJ, Harris LG, El-Bouri K, et al. 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 2010;59:1456–61.10.1099/jmm.0.023168-0Search in Google Scholar PubMed
35. Patel R, Uhl JR, Kohner P, Hopkins MK, Cockerill FR, 3rd. Multiplex PCR detection of vanA, vanB, vanC-1, and vanC-2/3 genes in enterococci. J Clin Microbiol 1997;35:703–7.10.1128/jcm.35.3.703-707.1997Search in Google Scholar PubMed PubMed Central
36. Kim TS, Kwon HL, Song SH, Song KH, Kim HB, Park KU, et al. Real-time PCR surveillance of vanA for vancomycin-resistant Enterococcus faecium. Mol Med Report 2012;6:488–92.10.3892/mmr.2012.952Search in Google Scholar PubMed
37. Hussain Z, Stoakes L, Massey V, Diagre D, Fitzgerald V, El Sayed S, et al. Correlation of oxacillin MIC with mecA gene carriage in coagulase-negative staphylococci. J Clin Microbiol 2000;38:752–4.10.1128/JCM.38.2.752-754.2000Search in Google Scholar PubMed PubMed Central
38. Carroll KC. Rapid diagnostics for methicillin-resistant Staphylococcus aureus: current status. Mol Diagn Ther 2008;12:15–24.10.1007/BF03256265Search in Google Scholar PubMed
39. Loonen AJ, Jansz AR, Kreeftenberg H, Bruggeman CA, Wolffs PF, van den Brule AJ. Acceleration of the direct identification of Staphylococcus aureus versus coagulase-negative staphylococci from blood culture material: a comparison of six bacterial DNA extraction methods. Eur J Clin Microbiol Infect Dis 2011;30:337–42.10.1007/s10096-010-1090-0Search in Google Scholar PubMed PubMed Central
40. Regan JF, Furtado MR, Brevnov MG, Jordan JA. A sample extraction method for faster, more sensitive PCR-based detection of pathogens in blood culture. J Mol Diagn 2012;14:120–9.10.1016/j.jmoldx.2011.10.001Search in Google Scholar PubMed PubMed Central
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