Multiplex Identification of Gram-Positive Bacteria and Resistance Determinants Directly from Positive Blood Culture Broths: Evaluation of an Automated Microarray-Based Nucleic Acid Test

<div><p>Background</p><p>A multicenter study was conducted to evaluate the diagnostic accuracy (sensitivity and specificity) of the Verigene Gram-Positive Blood Culture Test (BC-GP) test to identify 12 Gram-positive bacterial gene targets and three genetic resistance determinants directly from positive blood culture broths containing Gram-positive bacteria.</p><p>Methods and Findings</p><p>1,252 blood cultures containing Gram-positive bacteria were prospectively collected and tested at five clinical centers between April, 2011 and January, 2012. An additional 387 contrived blood cultures containing uncommon targets (e.g., <i>Listeria</i> spp., <i>S. lugdunensis</i>, <i>vanB</i>-positive Enterococci) were included to fully evaluate the performance of the BC-GP test. Sensitivity and specificity for the 12 specific genus or species targets identified by the BC-GP test ranged from 92.6%–100% and 95.4%–100%, respectively. Identification of the <i>mecA</i> gene in 599 cultures containing <i>S. aureus</i> or <i>S. epidermidis</i> was 98.6% sensitive and 94.3% specific compared to cefoxitin disk method. Identification of the <i>vanA</i> gene in 81 cultures containing <i>Enterococcus faecium</i> or <i>E. faecalis</i> was 100% sensitive and specific. Approximately 7.5% (87/1,157) of single-organism cultures contained Gram-positive bacteria not present on the BC-GP test panel. In 95 cultures containing multiple organisms the BC-GP test was in 71.6% (68/95) agreement with culture results. Retrospective analysis of 107 separate blood cultures demonstrated that identification of methicillin resistant <i>S. aureus</i> and vancomycin resistant <i>Enterococcus</i> spp. was completed an average of 41.8 to 42.4 h earlier using the BC-GP test compared to routine culture methods. The BC-GP test was unable to assign <i>mecA</i> to a specific organism in cultures containing more than one <i>Staphylococcus</i> isolate and does not identify common blood culture contaminants such as <i>Micrococcus</i>, <i>Corynebacterium</i>, and <i>Bacillus</i>.</p><p>Conclusions</p><p>The BC-GP test is a multiplex test capable of detecting most leading causes of Gram-positive bacterial blood stream infections as well as genetic markers of methicillin and vancomycin resistance directly from positive blood cultures.</p><p><i>Please see later in the article for the Editors' Summary</i></p></div

Detection of resistance determinants <i>mecA, vanA, and vanB</i> in prospectively collected monomicrobial blood cultures by Verigene BC-GP (<i>n = </i>599 <i>S. aureus/S. epidermidis</i>, <i>n = </i>81 <i>E. faecalis/E. faecium</i>).

a<p>Total number of cultures with reported result.</p>b<p>95% confidence interval.</p>c<p><i>mecA</i> is reported only for cultures with positive <i>S. aureus</i> or <i>S. epidermidis</i> species targets. Inferred based upon resistance to cefoxitin using disk diffusion method.</p>d<p><i>mecA</i> specific PCR was positive for eight of 14 isolates. Final specificity of 97.5%.</p>e<p><i>mecA</i> specific PCR was negative for four of five isolates. Final sensitivity of 99.7%.</p>f<p>vanA and vanB are reported only for cultures with positive <i>E. feacium</i> or <i>E. faecalis</i> targets.</p>g<p><i>vanB</i> was not detected in any prospectively collected cultures.</p><p>FN, false negative; FP, false positive; TN, true negative; TP, true positive.</p

Detection of <i>E. faecalis</i>, <i>E. faecium</i>, and <i>Listeria</i> in prospectively collected monomicrobial blood cultures by Verigene BC-GP (<i>n = </i>1,157).

a<p>Total number of cultures with reported result.</p>b<p>95% confidence interval.</p><p>FN, false negative; FP, false positive; TN, true negative; TP, true positive.</p

Difference in time to final identification and antimicrobial susceptibility report (<i>n = </i>107).

a<p>Difference in time between BC-GP result and final culture-based identification and susceptibility results.</p>b<p>Includes 26 <i>S. epidermidis</i>.</p>c<p>Three cultures reported as “Not Detected” by BC-GP.</p>d<p>Based on eight cultures that met criteria for full identification and susceptibility testing. Average time to “CoNS” identification only was 27.5 h (range 14 h to 48 h) earlier using BC-GP, <i>n = </i>35.</p>e<p>One culture reported as <i>S. pneumoniae</i> by BC-GP.</p>f<p>Based on six cultures that met criteria for full identification and susceptibility testing.</p>g<p>Culture reported as “<i>Staphylococcus</i> spp.” by BC-GP.</p

Detection of <i>Staphylococcus</i> spp. in prospectively collected monomicrobial blood cultures by Verigene BC-GP (<i>n = </i>1,157).

a<p>Total number of cultures with reported result.</p>b<p>95% confidence interval.</p>c<p>Nucleic acid sequencing confirmed the identification of <i>S. epidermidis</i> in five of ten cultures.</p>d<p>Nucleic acid sequencing confirmed the identification of <i>S. epidermidis</i> in six of ten cultures.</p><p>FN, false negative; FP, false positive; TN, true negative; TP, true positive.</p

Detection of Gram positive identification and resistance determinants from contrived blood cultures by Verigene BC-GP (<i>n = </i>387).

a<p>Total number of cultures with reported result.</p>b<p>95% confidence interval.</p>c<p><i>mecA</i> is reported only for cultures with positive <i>S. aureus</i> or <i>S. epidermidis</i> species targets.</p>d<p><i>vanA</i> and <i>vanB</i> are reported only for cultures with positive <i>E. feacium</i> or <i>E. faecalis</i> targets.</p><p>FN, false negative; FP, false positive; TN, true negative; TP, true positive.</p

Detection of Gram-positive identification and resistance determinants from polymicrobial blood cultures by Verigene BC-GP (<i>n = </i>95).

a<p>Coagulase negative <i>Staphylococcus</i> species, not including <i>S. epidermidis</i> and <i>S. lugdunensis</i>.</p>b<p>Contrived culture.</p>c<p><i>E. faecalis</i> or <i>E. faecium</i>.</p>d<p><i>Gram-negative rod</i>.</p>e<p><i>Bacillus</i> spp. not <i>anthracis</i>.</p