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Home , Enterococcus faecalis, Enterococcus faecium, Staphylococcus, Staphylococcus aureus

20-Minute Identification of Enterococci and aureus Causing Bloodstream by QuickFISH™, A Novel Assay Based on PNA Technology F. Wu 1, S. Whittier1, M. Fiandaca2, B. Crystal2, P. Della-Latta1* 1Columbia University Medical Center, NewYork-Presbyterian Medical Center, New York NY, 2AdvanDx, Inc. Woburn, MA.

Background Methods Clinical samples Identification of is responsible for 750,000 hospitalizations annually Patient blood samples were collected in BD BACTEC Plus Positive BC were subcultured onto appropriate media following and is the tenth leading cause of mortality in the US. Rapid Aerobic, BACTEC Peds Plus, and BACTEC Lytic Anaerobic routine protocol, and bacteria were identified utilizing MicroScan diagnosis of (BSI) from newly positive bottles (Becton, Dickinson and Company, Sparks, MD). From WalkAway (Dade Behring Inc., Deerfield, IL). Staphylococcal bottles (BC) is critical to improving patient care July to October 2011, 182 BC bottle collected from 171 cultures were also speciated by Staphaureux test (Murex by supporting strong stewardship programs and patients, newly positive for gram-positive cocci in clusters Biotech Ltd. Dartford, England), and -resistance was impacting the prudent selection of appropriate empiric (GPCC) or pairs and chains (GPCPCH) were used to evaluate confirmed by the PBP’2a latex agglutination test (Oxoid, therapy. Most bacteria recovered from BC are gram-positive the performance characteristics of two rapid PNA FISH Hampshire, UK). Discordant culture/FISH results were resolved cocci, namely, -negative staphylococci (CoNS), assays, the Staphylococcus QuickFISH BC and the using 16S sequencing technology. and enterococci, in order of QuickFISH BC (AdvanDx) for identification of prevalence. A daunting challenge is to rapidly differentiate staphylococci and enterococci. from BC the , S. aureus from CoNS (usual contaminants) and (usually Figure 1: QuickFISH Procedure (Blood Culture Bottles) and susceptible) from non-E. faecalis (often vancomycin resistant ) in order to Fix Hybridize Examine impact patient management and therapeutic decisions.

Culture methods for pathogen identification require 1 – 3 days and currently there are no PCR assays to identify CoNS or enterococci directly from BC. This study evaluated the accuracy of Staphylococcus QuickFISH and Enterococcus QuickFISH (AdvanDx), in differentiating S. aureus from CoNS and E. faecalis from non-E. faecalis Add ~10 drops of Transfer 10 μL to Add 1 drop Add PNA Blue, Flip coverslip and Read Results enterococci, respectively, directly from BC in ~20 minutes. blood culture slide on 55ºC heat QuickFix-1. Mix then PNA Yellow place on wells. These assays have the potential to improve appropriate use sample to vial. block. and Dry. to coverslip. Mix Hybridize at 55ºC of and patient care. until green. Hands-on Time Add 2 drops for 15 min. QuickFix-2. Dry. 5 Minutes

Results Of 182 newly positive BC bottles recovered from 171 patients, Of 58 GPCPCH, 17 were E. faecalis,12non-E. faecalis Enterococci comprised 50% of the GPCPCH samples, 58.6% 124 were GPCC, identified as CoNS (87), S. aureus (34) (10 enterococci, 1 gasseri, 1 Lactococcus garvieae, of which were E. faecalis (vancomycin susceptible) and 31% methicillin-resistant) and Micrococcus spp (3) (Table 2). and 27 streptococcal species (Table 3). One discordant BC vancomycin resistant E. faecium. The sensitivity, specificity, QuickFISH accurately identified both S. aureus and CoNS in isolate was misidentified as E. faecium by MicroScan, identified positive and negative predictive values for both FISH assays one mixed BC. The staphylococci isolated were CoNS (72%), by 16S sequencing as L. garvieae and was accurately reported compared to culture were 100%. followed by S. aureus (28%). by QuickFISH as negative for non-E. faecalis enterococci.

Table 1: Comparison of 1st & 2nd Generation PNA FISH Assays Table 2: Staphylococcus QuickFISH Assay Results Table 3: Enterococcus QuickFISH Assay Results

1st Generation 2nd Generation QuickFISH Results QuickFISH Results Assay Features Culture Identification (PNA FISH® Assay) (QuickFISHTM Assay) Green Red Culture Identification Red (# of Bottles) Negative Green (S. aureus) (CoNS) (# of Bottles) (Enterococci Negative Blood Culture (E. faecalis) 40 μL 10 μL Non-E. faecalis) Volume S. aureus (33) 33 0 0 S. aureus + S. epidermidis (1) 110E. faecalis (17) 17 0 0 Target specific PNA probes & quencher- Target Detection S. epidermidis (53) 0530E. faecium (9) 090 PNA probes labeled complements S. hominis (16) 0160E. gallinarum (3) 030 Fixation Step ~20 minutes ~3 minutes S. capitis (7) 070Lactobacillus gasseri* (1) 001 S. haemolyticus (3) 030Lactococcus garvieae* (1) 001 Hybridization Step 30 minutes 15 minutes S. auricularis (2) 020Streptococcus agalactiae (4) 004 S. caprae* (1) 010Streptococcus constellatus (2) 002 Remove unbound probe Wash Step Step eliminated 30 minutes S. hyicus (1) 010Streptococcus gordonii (4) 004 S. intermedius (1) 010Streptococcus intermedius (1) 001 Positive and negative Controls Separate control slides controls on one slide S. saccharolyticus (1) 010Streptococcus mitis (7) 007 S. schleiferi (1) 010Streptococcus parasanguinis (4) 004 Run time ~90 minutes ~20 minutes S. xylosus (1) 010Streptococcus pneumoniae (4) 004 Micrococcus spp. (3) 003 Technologist pyogenes (1) 001 10 minutes 5 minutes Hands-on Time Total (125 isolates/124 BC) 34 88 3 Total (58) 17 12 29 *Identification using 16S rDNA sequencing *Identification using 16S rDNA sequencing

Conclusions Figure 2: QuickFISH Results Reporting • Staphylococcus QuickFISH and Enterococcus Positive QuickFISH™ Critical Antibiotic QuickFISH assays results were 100% accurate Blood Culture (10 min.) (20 min.) Values Call Change • QuickFISH results were available in 20 min of the Gram stain from newly positive blood culture bottles • Clinicians can receive reports in a timeframe that can influence therapeutic decisions (Figure 2).

• Chart review of study patients: BC with GPCPCH S. aureus resulted in empiric vancomycin treatment, later changed to narrower antibiotics when E. faecalis was identified and to when with E. faecium was identified. Gram-Positive Call clinician Cocci in Clusters with Gram stain • Early CoNS identification alerts the clinician to and QuickFISH either skin contamination or true , results which can occur in up to 20% of bacteremic CoNS patients. • Although S. aureus is accurately identified, the Staphylococcus QuickFISH must be coupled References with a rapid test such as the PBP2’ to detect Munson E, Diekema D, Beekmann S, Chapin K, Doern G. Detection and Ly T, Gulia J, Pyrogos V, Wago M, Shoham S. Impact upon clinical outcomes of methicillin resistance. treatment of bloodstream infection: Laboratory reporting and translation of PNA FISH-generated laboratory data from the clinical microbiology management. J Clin Microbiol. 2003 Jan;41(1):495-7. bench to bedside in real time. Ther Clin Risk Manag. 2008 Jun;4(3):637-40.