1Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Cente, 2Pasadena, CA, Southern California Permanente Medical Group, 3Detroit, Detroit Medical Center, 4Woburn, MA, AdvanDx
Morgan, M. A., Marlowe, E., Novak-Weekly, S., Miller, J., Painter, T., Salimnia, H., et al. A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures. J. Vis. Exp. (48), e2616, doi:10.3791/2616 (2011).
Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.
1. Specimen Collection and Preparation
2. Preparation of Reagents Prior to Staining
3. Gram Staining
4. PNA FISH Stain
5. Quality Control Material
One positive and one negative quality control slide must be tested with each batch of slides for staining.
The QC results should be able to monitor for appropriate testing conditions, particularly those affecting hybridization stringency and cell wall penetration, since PNA methodology is designed to optimize cell wall penetration
7. Stringent Wash
9. Interpretation of Results
The fluorescence microscope used for slide examination must be equipped with the AdvanDx Dual Band Filter and a 60x or 100x oil objective. The QC slides should be examined first to confirm that hybridization did occur. E. faecalis should appear as bright green fluorescent cocci in multiple fields of view and the E. faecium will appear as bright red cocci. Non-enterococci control slide should appear nonfluorescent. After confirming the system in controls, the patient slides can be examined. At first the blood film will appear reddish but the bright red and green cocci will be quite apparent.
Figure 1.Representative examples of green-positive E. faecalis (left), red positive E. faecium (middle), and negative (right) test results.
10. Representative Results
Three institutions were included in a multi-center clinical trial evaluating this PNA FISH stain and comparing a 2.5 hour protocol to the shortened 1.5 hour protocol that has just been reviewed. A total of 152 routine Gram positive cocci in pairs and chains (GPC) positive blood culture bottles were included in the studies. There was 100% (152/152) agreement between results of the modified and the original assay procedure for E. faecalis/OE PNA FISH: 41/41 E. faecalis; 33/33 other enterococci and 78/78 other GPC (Table 1). This staining method had exquisite sensitivity and specificity during this clinical trial (Table 2).
|Routine Methods||E. faecalis /OE PNA FISH Standard Procedure||E. faecalis/OE PNA FISH Short Procedure|
|E. faecalis||41||41 (Green Positive)||41 (Green Positive)|
|E. faecium||27||27 (Red Positive)||27 (Red Positive)|
|E. casseliflavus||2||2 (Red Positive)||2 (Red Positive)|
|E. gallinarum||2||2 (Red Positive)||2 (Red Positive)|
|Other Enterococcus spp.||2||2 (Red Positive)||2 (Red Positive)|
|S. pneumoniae||17||17 (Negative)||17 (Negative)|
|S. viridans||33||33 (Negative)||33 (Negative)|
|S. mitis||1||1 (Negative)||1 (Negative)|
|S. pyogenes||3||3 (Negative)||3 (Negative)|
|S. bovis||2||2 (Negative)||2 (Negative)|
|S. salivarius||1||1 (Negative)||1 (Negative)|
|S. sanguinis||2||2 (Negative)||2 (Negative)|
|S. agalagtae||7||7 (Negative)||7 (Negative)|
|Other Streptococcus spp.||7||7 (Negative)||7 (Negative)|
|Abiotrophia spp.||3||3 (Negative)||3 (Negative)|
|Peptostrepococcus spp.||2||2 (Negative)||2 (Negative)|
Table 1. Listing of bacteria tested using both the standard (2.5hr) and rapid (1.5 hr) protocol.
|Sensitivity E.faecalis||Sensitivity Other Enterococcus spp.||Specificity|
95% CI (93.0-100)
|100% (33/33) 33/33
95% CI (91.3-100)
95% CI (96.2-100)
Table 2. Sensitivity and Specificity of the 1.5 hour PNA FISH protocol.
The E. faecalis/OE PNA FISH assay for enterococcus can provide identification 2-3 days earlier than standard culture methods. The shortened procedure for the assay (1.5 hourr) provides rapid identification that can be used for a better approach to antimicrobial therapy and patient outcome. One study to support this was performed by Forrest, et al. (6). Over two consecutive years beginning in 2005 the microbiology laboratory identified Gram positive cocci in pairs and chains growing in blood culture bottles by conventional microbiological methods and in 2006 adding the E. faecalis/OE PNA FISH. In addition a treatment algorithm developed by the institutions antimicrobial team (AMT) to effectively use the PNA FISH data generated by the laboratory in timely manner. Primary outcome assessed was time from blood culture draw to the implementation of effective antimicrobial therapy before and after PNA FISH was instituted into the laboratories workflow. Severity of illness, patient location and empiric antimicrobial therapy were measured. A total of 224 patients with hospital acquired enterococcal bacteremia were evaluated with 129 in the pre-intervention period and 95 in the PNA FISH period. PNA FISH identified E. faecalis 3 days earlier than conventional cultures (1.1 vs. 4.1 days, p<0.001). PNA FISH identified E. faecium a median 2.3 days earlier (1.1 vs. 3.4 days, p<0.001) and was associated with statistically significant reductions in time to initiating effective therapy (1.3 vs. 3.1 days, p<0.001) and decreased 30 day mortality (26% vs. 45%, p=0.04). This group concluded that the E. faecalis/OE PNA FISH assay in conjunction with an AMT treatment algorithm resulted in earlier initiation of appropriate empiric antimicrobial therapy for patients with hospital acquired E. faecium bacteremia.
The production of this video-article was sponsored by AdvanDx. Benjamin Crystal is an employee of AdvanDx, who makes the tools and reagents used in this article.
Studies were sponsored by AdvanDx.
E. faecalis/– PNA FISH is comprised of the following kit components: