Filmarray® Blood Culture Identification Panel Quick Guide

® FilmArray Blood Culture Identification Panel Quick Guide

Testing on the FilmArray should be performed within 8 hours of the positive reading from a continuously monitoring blood culture instrument. To avoid contamination always wear gloves and work behind a protective shield.

Step 1: Prepare Pouch Once sample is ready for testing, remove pouch from packaging. Insert pouch into Pouch Loading Station. Place Sample Buffer vial into red well. OR Place Hydration Solution vial into blue well.

Step 2: Hydrate Pouch Draw 1 mL of Hydration Solution into Hydration Syringe. If bubbles are present in liquid, leave syringe tip in vial and gently tap side of syringe, releasing bubbles to float to surface. Insert Hydration Syringe tip into hydration port of pouch located OR directly below blue arrow. Holding the barrel of the Hydration Syringe, forcefully push down to puncture port seal. DO NOT PUSH THE SYRINGE PLUNGER. FilmArray Wait as Hydration Solution is drawn into pouch. ™ Panel Note: See FilmArray Operator's Manual Troubleshooting section if pouch fails to hydrate.

Step 3: Prepare Sample Mix Use a 28 gauge needle to withdraw 0.1 mL of sample from blood culture bottle. Transfer sample to Sample Buffer vial. Gently mix up and down using Transfer Pipette. Note: For alternate workflow, see package insert. FilmArray ™ Panel

Step 4: Load Sample Mix OR Draw 0.3 mL of Sample Mix using Sample Loading Syringe. Insert Sample Loading Syringe tip into sample port of pouch

FilmArray located directly below red arrow. ™ Panel Holding the barrel of the syringe, forcefully push down to puncture port seal. DO NOT PUSH THE SYRINGE PLUNGER. Wait as Sample Mix is drawn into pouch.

Step 5: Run Pouch Follow instructions on software screen.

FilmArray The pouch will click into place when properly seated. ™ Panel Note: If the pouch does not insert easily, ensure that the lid is opened completely.

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Section ®

6. 5. 4. 2. Run Summary Run Details is requiredforspeciesidentificationandsusceptibilitytestingofisolates. FilmArray antimicrobialresistancegeneassaysdoesnotindicatesusceptibility. Subculturing Ø Ø √ Results Summary-Interpretations √ √ Resistance Genes: | NOTE: Antimicrobial resistancecanoccurviamultiplemechanisms. A NotDetectedresultforthe 7. 390 Wakara Way,Salt LakeCity,Utah 84108,USA 1. N/A N/A Detected Not Detected Not Detected Not Detected Not Detected Not Detected Detected Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected BCID Panel Antimicrobial Run Status: Organisms Sample ID: Applicable Serial No.: Detected: Lot No.: Pouch: T M mecA Staphylococcusaureus Staphylococcus 8017366354 120228B 00169044 Completed BCID Panelv2.0 KPC (carbapenem-resistancegene) vanA/B mecA Candida tropicalis Candida parapsilosis Candida krusei Candida glabrata Candida albicans Pseudomonas aeruginosa Neisseria meningitidis Haemophilus influenzae Enterobacteriaceae Acinetobacter baumannii Streptococcus Staphylococcus Listeria monocytogenes Enterococcus Streptococcus pyogenes Streptococcus pneumoniae Streptococcus agalactiae Staphylococcus aureus Serratia marcescens Proteus Klebsiella pneumoniae Klebsiella oxytoca Escherichia coli Enterobacter cloacaecomplex -Detected (methicillin-resistancegene) Antimicrobial ResistanceGenes (vancomycin-resistancegenes) Gram NegativeBacteria Gram PositiveBacteria Yeast (Group A) (Group B) 3. Instrument: Run Date: Controls:

Operator: Protocol: Passed 19 Mar20123:36PM

ITI FA "FA1187" Operator BC v2.1 |

1-801-736-6354

RFIT-PRT-0056-04 RFIT-ASY-0114 RFIT-ASY-0109

FilmArray® Blood Culture Identification (BCID) Panel Instruction Booklet

For use with the syringe-based loading system

Customer and Technical Support for U.S. Customers

Reach Us on the Web Reach Us by Phone http://www.BioFireDX.com 1-800-735-6544 – Toll Free (801) 736-6354 – Utah

Reach Us by E-mail [email protected] Reach Us by Fax Reach Us by Mail (801) 588-0507 390 Wakara Way Salt Lake City, UT 84108 USA

Customer and Technical Support outside of the U.S. Contact your local sales representative or distributor for technical support

BioFire Diagnostics, LLC 390 Wakara Way Qarad b.v.b.a Cipalstraat 3 Salt Lake City, UT 84108 B-2440 Geel, USA Belgium

The information contained in this document is subject to change without notice. No part of this document may be reproduced or transmitted in any form or by any means, electronic or mechanical, for any purpose, without the express written permission of BioFire Diagnostics, LLC.

BioFire Diagnostics, BioFire, the BioFire logo, FilmArray and LCGreen are trademarks of BioFire Diagnostics, LLC or BioFire Defense, LLC and are registered trademarks in the United States.

All other names of products and brands appearing in this manual are trademarks or registered trademarks of their respective owners.

The purchase of this product includes a limited, nontransferable license under specific claims of one or more U.S. patents as listed on BioFire Diagnostics’ Web site (http://www.biofiredx.com/LegalNotices/) and owned by BioFire and the University of Utah Research Foundation.

BioFire Diagnostics, LLC FilmArray BCID Panel CE-IVD Instruction Booklet i

TABLE OF SYMBOLS

The following symbols can be found on FilmArray BCID Panel Kit components or throughout this Instruction Booklet. Use the definitions below as a guideline to interpreting the symbols.

Table of Symbols

Expiry Date Manufacturer Catalog Number YYYY-MM-DD

Consult Storage Temperature Lot Number Instructions for Use Limitations

European Union Contains Sufficient Serial Number Conformity n For Tests

In vitro Diagnostic Keep Away from Do Not Reuse Medical Device Sunlight

Acute toxicity, cat. Serious eye Do Not Use if 4 & Skin irritation, damage, cat. 1 Package is Damaged cat. 2

BioFire Diagnostics, LLC FilmArray BCID Panel CE-IVD Instruction Booklet ii

TABLE OF CONTENTS

TABLE OF SYMBOLS ...... II TABLE OF CONTENTS ...... III NAME AND INTENDED USE ...... 5

FILMARRAY BLOOD CULTURE IDENTIFICATION (BCID) PANEL ...... 5 SUMMARY AND EXPLANATION OF THE TEST ...... 5

BACKGROUND OF TARGETED ORGANISMS ...... 6 PRINCIPLE OF THE PROCEDURE ...... 11 MATERIALS PROVIDED ...... 12 MATERIALS REQUIRED BUT NOT PROVIDED ...... 13 WARNINGS AND PRECAUTIONS ...... 13

GENERAL PRECAUTIONS ...... 13 SAFETY PRECAUTIONS ...... 13 LABORATORY PRECAUTIONS ...... 14 PRECAUTION RELATED TO PUBLIC HEALTH REPORTING IN THE UNITED STATES ...... 15 REAGENT STORAGE, HANDLING AND STABILITY ...... 15 SAMPLE REQUIREMENTS ...... 16 PROCEDURE ...... 16

PREPARE POUCH ...... 16 HYDRATE POUCH ...... 17 PREPARE SAMPLE MIX ...... 17 LOAD SAMPLE MIX ...... 18 RUN POUCH ...... 18 QUALITY CONTROL ...... 19

PROCESS CONTROLS ...... 19 MONITORING TEST SYSTEM PERFORMANCE ...... 20 INTERPRETATION OF RESULTS ...... 20

ASSAY INTERPRETATION ...... 20 ORGANISM INTERPRETATION ...... 21 ANTIMICROBIAL RESISTANCE GENES INTERPRETATION ...... 28 FILMARRAY BCID TEST REPORT ...... 31 CONTROLS FIELD ...... 31 RESULTS SUMMARY – INTERPRETATIONS ...... 32 LIMITATIONS OF THE PROCEDURE ...... 34

BioFire Diagnostics, LLC FilmArray BCID Panel CE-IVD Instruction Booklet iii

EXPECTED VALUES ...... 37 PERFORMANCE CHARACTERISTICS...... 42

CLINICAL PERFORMANCE...... 42 GROWTH AND DETECTION ...... 57 ANALYTICAL REACTIVITY (INCLUSIVITY) ...... 59 ANALYTICAL SPECIFICITY (CROSS-REACTIVITY AND EXCLUSIVITY) ...... 73 CROSS-CONTAMINATION AND CARRYOVER ...... 76 REPRODUCIBILITY ...... 77 INTERFERENCE...... 82 PERFORMANCE CHARACTERISTICS ON THE FILMARRAY 2.0 ...... 82

CLINICAL COMPARISON...... 82 REPRODUCIBILITY ...... 84 REFERENCES ...... 87

BioFire Diagnostics, LLC FilmArray BCID Panel CE-IVD Instruction Booklet iv

NAME AND INTENDED USE

FilmArray Blood Culture Identification (BCID) Panel

The FilmArray Blood Culture Identification (BCID) Panel is a qualitative multiplexed nucleic acid-based in vitro diagnostic test intended for use with FilmArray systems. The FilmArray BCID Panel is capable of simultaneous detection and identification of multiple bacterial and yeast nucleic acids and select genetic determinants of antimicrobial resistance. The BCID assay is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system that demonstrate the presence of organisms as determined by Gram stain. The following gram-positive bacteria, gram-negative bacteria, and yeast are identified using the FilmArray BCID Panel: Enterococci, Listeria monocytogenes, Staphylococci (including specific differentiation of Staphylococcus aureus), Streptococci (with specific differentiation of Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes), Acinetobacter baumannii, Enterobacteriaceae (including specific differentiation of the Enterobacter cloacae complex, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus, and Serratia marcescens), Haemophilus influenzae, Neisseria meningitidis (encapsulated), Pseudomonas aeruginosa, Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis. The FilmArray BCID Panel also contains assays for the detection of genetic determinants of resistance to methicillin

(mecA), vancomycin (vanA and vanB), and carbapenems (blaKPC) to aid in the identification of potentially antimicrobial resistant organisms in positive blood culture samples. The antimicrobial resistance gene detected may or may not be associated with the agent responsible for disease. Negative results for these select antimicrobial resistance gene assays do not indicate susceptibility, as multiple mechanisms of resistance to methicillin, vancomycin, and carbapenems exist. FilmArray BCID is indicated as an aid in the diagnosis of specific agents of bacteremia and fungemia and results should be used in conjunction with other clinical and laboratory findings. Positive FilmArray results do not rule out co-infection with organisms not included in the FilmArray BCID Panel. FilmArray BCID is not intended to monitor treatment for bacteremia or fungemia. Subculturing of positive blood cultures is necessary to recover organisms for susceptibility testing and epidemiological typing, to identify organisms in the blood culture that are not detected by the FilmArray BCID Panel, and for species determination of some Staphylococci, Enterococci, Streptococci, and Enterobacteriaceae that are not specifically identified by the FilmArray BCID Panel assays.

SUMMARY AND EXPLANATION OF THE TEST Sepsis (defined as system inflammatory response syndrome in response to infection) is the 11th leading cause of death in the United States [1]. Life-threatening bacterial and fungal sepsis currently strikes approximately 240 out of 100,000 people per year in the U.S. (750,000 total cases), with severe sepsis (associated with acute organ dysfunction) in 95 out of 100,000 people [2]. Timely diagnosis and administration of effective treatment can significantly reduce mortality, duration of hospital stays, and costs due to sepsis. The FilmArray Blood Culture Identification (BCID) Panel simultaneously tests a single positive blood culture sample to provide results for 24 different organisms and organism groups that cause bloodstream infections and three genetic markers that are known to confer antimicrobial resistance (see Table 1). The test can be performed on blood culture bottles that are (1) flagged as positive by a continuously monitoring blood culture instrument and (2) positive by Gram stain examination. FilmArray BCID Panel results are available within about one hour. Rapid identification of the organism(s) in the blood culture, along with information about antimicrobial resistance gene status for select microorganisms, may aid the physician in making appropriate treatment decisions.

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Table 1. FilmArray BCID Panel Test Results. Gram-Positive Bacteria Gram-Negative Bacteria Yeast Enterococcus Acinetobacter baumannii Candida albicans Listeria monocytogenes Enterobacteriaceae Candida glabrata Staphylococcus Enterobacter cloacae complex Candida krusei Staphylococcus aureus Escherichia coli Candida parapsilosis Streptococcus Klebsiella oxytoca Candida tropicalis Streptococcus agalactiae Klebsiella pneumoniae Antimicrobial resistance genes Streptococcus pneumoniae Proteus mecA – methicillin resistance Streptococcus pyogenes Serratia marcescens vanA/B – vancomycin resistance Haemophilus influenzae KPC – carbapenem resistance

Neisseria meningitidis (encapsulated) Pseudomonas aeruginosa

Background of Targeted Organisms

Gram-Positive Bacteria

Enterococcus. Members of this genus are gram-positive facultative anaerobes that normally inhabit the alimentary tract of humans, but have recently become one of the most common nosocomial pathogens [3]. Enterococcal infections include urinary tract infections, hepatobiliary sepsis, endocarditis, surgical wound infection, bacteremia, and neonatal sepsis. In an evaluation of 49 US hospitals over a seven year period (1995 – 2002), enterococci were responsible for 9% of nosocomial bloodstream infections [4]. There are 28 species of Enterococcus [3]. While at least 12 species have been shown to cause human disease, E. faecalis (80-90%) and E. faecium (5-15%) cause the majority of clinical infections [5]. Enterococci can carry vancomycin resistance genes such as vanA/B. Infection with a vancomycin-resistant Enterococcus (VRE) increases the risk of death to 75%, compared with 45% for infection with a susceptible strain [3]. Listeria monocytogenes, the causative agent of listeriosis, is a gram-positive bacillus that is ubiquitous in soil and water and can be found in the gastrointestinal tract of up to 5% of healthy human adults [6-8]. Only three of the 12 known serovars of L. monocytogenes (1/2a, 1/2b and 4b) account for more than 90% of human cases of listeriosis [9]. Listeriosis is considered the most severe bacterial foodborne infection due to its high mortality rate despite early antibiotic treatment (11 – 60%) [8-9]. Populations at risk for developing invasive listeriosis include the immunosuppressed, pregnant women, neonates, fetuses and the elderly [6-7]. Invasive listeriosis can result in abortion, sepsis, and meningoencephalitis [6, 9]. Septicemia can account for greater than 50% of cases and can have a mortality rate up to 70% when associated with severe underlying conditions [8-9]. Staphylococcus, Staphylococcus aureus. Staphylococci are gram-positive cocci that are usually catalase positive and tend to form irregular, grape-like clusters. Nearly all species in the genus are facultative anaerobes. Staphylococcus spp. colonize the skin and mucous membranes. They are opportunistic pathogens that can cause infection following breaks in the cutaneous epithelial barrier through trauma or medical interventions [10]. Diagnostically, the genus is divided between coagulase-positive staphylococci and coagulase-negative staphylococci (CoNS). The most clinically-relevant Staphylococcus sp. is the coagulase-positive S. aureus. Other coagulase-positive species, such as S. intermedius, are isolated much less frequently from clinical specimens. S. aureus is the most common cause of nosocomial skin and soft tissue infections, and is second only to CoNS as a cause of primary bacteremia in hospitals [4, 11]. CoNS species are isolated very regularly from clinical specimens and care must be taken to assess clinical significance to differentiate

BioFire Diagnostics, LLC FilmArray BCID Panel CE-IVD Instruction Booklet 6

between contamination, colonization, and true infection. The most clinically-important CoNS species include S. epidermidis, S. haemolyticus, and S. saprophyticus. Many other CoNS species are isolated to lesser degrees. FilmArray BCID detects most but not all Staphylococcus spp. It is estimated that approximately 75% of CoNS isolates and 40% of S. aureus isolates may be methicillin resistant [4]. The primary mediator of methicillin resistance in staphylococci is acquisition of the mecA gene. Streptococcus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes. Streptococci are gram-positive, catalase negative cocci that grow in chains or pairs. They cause infections in a variety of species, including humans, mammals, and fish. Streptococcus species are frequently found as commensal bacteria on mucous membranes, and are occasionally present as transient skin microbiota [10]. Streptococci have historically been grouped as beta- hemolytic or non-beta-hemolytic, pyogenic (pus-forming) or non-pyogenic, and also divided according to presence of specific surface antigens (i.e., Lancefield grouping). Lancefield groups A, B, C, and G are pyogenic and most are also beta-hemolytic [10]. Of these, the Group A streptococci (represented primarily by S. pyogenes) and the Group B streptococci (S. agalactiae) are the most common in cases of septicemia. The non-pyogenic streptococci are subdivided into five groups (Mitis, Anginosus, Salivarius, Mutans, and Bovis groups). The Mitis, Anginosus, and Salivarius groups are also referred to as viridians streptococci; these bacteria produce no Lancefield antigens and are alpha-hemolytic or non- hemolytic. Viridans streptococci are also fairly common agents of septicemia causing 0.5% of sepsis cases in nonneutropenic patients and up to 2% in neutropenic patients [4]. S. pneumoniae has been classified into the Mitis group but is often considered as its own separate group. FilmArray BCID detects most but not all Streptococcus spp. Streptococcus pyogenes (Group A Streptococcus or GAS) colonizes the human skin and upper respiratory tract, with these sites serving as primary focal sites of infections and principal reservoirs of transmission [10]. S. pyogenes possesses complex virulence mechanisms to avoid host defenses [12-13] and is responsible for deep or invasive infections, especially bacteremia, sepsis, and deep soft tissue infections [10]. Streptococcus agalacticae (Group B Streptococcus or GBS) can cause both early onset neonatal disease, characterized by sepsis and pneumonia within the first seven days of life; and late onset disease with meningitis and sepsis between day seven and three months of age [10]. GBS infections emerged in the 1970s as the leading cause of early onset sepsis (EOS) and meningitis in neonates [14]. In adult patients, the spectrum of S. agalacticae infections includes bacteremia, pneumonia, meningitis, and endocarditis [10]. Streptococcus pneumoniae colonizes the upper respiratory tract, and is the most frequently isolated respiratory pathogen in community-acquired pneumonia. It is also a major cause of meningitis in pediatric and adult patients. S. pneumoniae was responsible for approximately 42,000 invasive infections in the U.S. in 2007, leading to an estimated 4,500 deaths [10].

Gram-Negative Bacteria

Acinetobacter baumannii is a ubiquitous, non-fermentative gram-negative coccobacillus that primarily acts as an opportunistic pathogen infecting critically-ill patients. While hospital-acquired pneumonia is the most common infection caused by A. baumannii, other nosocomial infections are increasing in frequency, including bacteremia, skin and soft tissue infections, urinary tract infections, and infections involving the central nervous system [15]. A. baumannii was the 10th most common cause of nosocomial bloodstream infection (1.3% of all infections) in a large U.S. study (1995-2002) [4]. The bacteria are capable of persisting for long periods on environmental surfaces, aiding the spread of infections in hospital settings. Additionally, the rapid emergence of multi-drug resistant (MDR) strains creates difficulties for treatment and infection control. MDR strains demonstrate resistance to most antibiotic classes, including carbapenems. Various carbapenem-hydrolyzing metallo-β-lactamases may be carried by the bacteria [16]. The specific carbapenemase, KPC, has also been infrequently identified in A. baumannii [17]. Several related Acinetobacter species cannot be reliably differentiated from A. baumannii by some manual or automated phenotypic microbial identification systems. These

BioFire Diagnostics, LLC FilmArray BCID Panel CE-IVD Instruction Booklet 7

species include A. calcoaceticus, A. pittii (genomospecies 3), and A. nosocomialis (genomospecies 13TU). Together with A. baumannii, these species have been grouped into the Acinetobacter calcoaceticus-baumannii (ACB) complex. It has been estimated that up to 25% of isolates from the ACB complex are routinely misidentified as A. baumannii [18]. Enterobacteriaceae. The family Enterobacteriaceae is composed of many genera and species of bacteria that share common features (e.g. gram-negative facultative anaerobic rods or coccobacilli). They are widely distributed in the environment and are found on plants, in soil and water, and within the gastrointestinal (GI) tract of many animals, including humans. While many organisms in this family are harmless, several are medically important and are associated with bacteremia and other illnesses, particularly gastroenteritis. Together, members of the Enterobacteriaceae family are among the most commonly recognized organisms seen in healthcare-associated infections. The FilmArray BCID Panel contains specific assays for the Enterobacteriaceae members that are most frequently associated with bacteremia and/or are associated with a higher frequency of antimicrobial resistance: Enterobacter cloacae complex, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus, and Serratia marcescens. However, many other Enterobacteriaceae can also cause bacteremia, though less frequently. These include Cedecea, Citrobacter, Salmonella, and Enterobacter aerogenes, among others [11, 19-20]. FilmArray BCID detects most commonly-encountered Enterobacteriaceae; however, not all genera or species in this family are detected. The emergence and spread of antimicrobial resistance in Enterobacteriaceae has increased the complexity of treating sepsis due to these organisms. Resistance to third and fourth-generation cephalosporins is mediated primarily by production of extended-spectrum β-lactamases (ESBLs) and overproduction of AmpC β-lactamases. While the majority of Enterobacteriaceae remain susceptible to carbapenems, KPC-type carbapenemases are emerging and spreading in Enterobacteriaceae (carbapenem-resistant Enterobacteriaceae; CRE) in certain locations within the United States and worldwide [21]. Enterobacter cloacae complex. Enterobacter spp. are facultatively-anaerobic gram-negative rods within the Enterobacteriaceae family. These bacteria are ubiquitous in the environment. Clinical significance is primarily limited to E. cloacae and E. aerogenes, which are common opportunistic nosocomial pathogens. The range of infections caused by Enterobacter spp. includes respiratory tract infections, urinary tract infections, soft tissue infections, septic arthritis, osteomyelitis, and bacteremia among others. Enterobacter spp. are estimated to be the fourth most common etiological agents in gram-negative bloodstream infections [22]. They often have the ability to overproduce AmpC β- lactamases and are increasingly found to carry extended spectrum β-lactamases [23]. Several species that are closely related to E. cloacae are grouped together with it in the E. cloacae complex; these include E. asburiae, E. hormaechei, E. kobei, E. ludwigii, and E. nimipressuralis. Clinical significance has been attributed to some but not all members of the complex [22]. Escherichia coli is an enteric organism most frequently isolated from the intestines of humans and animals. While most pathogenic E. coli infections are associated with gastrointestinal illness, certain strains may cause extraintestinal infections in healthy as well as immunocompromised individuals. These include urinary tract infections, bacteremia, and meningitis. Overall, E. coli are responsible for approximately 5.6% of bloodstream infections [4]. As with other Enterobacteriaceae, extended spectrum β-lactamases (ESBLs) pose a significant antibiotic resistance problem. Klebsiella oxytoca, Klebsiella pneumoniae. Klebsiella are gram-negative, rod-shaped bacteria in the Enterobacteriaceae family. Klebsiella spp. are ubiquitous in the environment, particularly in agricultural settings, and may colonize the skin, respiratory, and gastrointestinal (GI) tracts of humans. They are opportunistic pathogens and while colonization does not always result in illness, infection rates in carriers are four times higher than non-carriers [24]. Infection rates are higher in summer months, which is thought to be due to the greater prevalence of Klebsiella in the environment and thus in the GI tract [25]. K. pneumoniae and K. oxytoca are the species most frequently isolated from hospitalized patients [24]. Infections due to these bacteria include soft tissue infections, urinary tract infections, pneumonia, and septicemia [24]. Both K. pneumoniae and K. oxytoca can carry the Klebsiella pneumoniae

carbapenemase gene, blaKPC, which makes them resistant to carbapenem antibiotics. An increasing proportion of K.

BioFire Diagnostics, LLC FilmArray BCID Panel CE-IVD Instruction Booklet 8

oxytoca isolates from bacteremia infections demonstrate resistance to extended-spectrum β-lactams, especially when there is a history of prior antibiotic use [26]. Biochemical discrimination between species of Klebsiella is difficult: K. oxytoca isolates may be erroneously identified as K. pneumoniae by manual or automated biochemical detection algorithms [27]. Additionally, Raoultella ornithinolytica, which was recently separated from the genus Klebsiella, can also be misidentified as K. oxytoca by phenotypic identification systems [28]. Proteus. Members of the genus Proteus are commonly isolated in the clinical laboratory, with Proteus mirabilis being the most frequently seen species. Most infections (approximately 85%) are thought to be community acquired [29]; however, nosocomial outbreaks have also occurred [30]. The vast majority of P. mirabilis are isolated from complicated urinary tract infections involving abnormalities or indwelling catheters, while other species (e.g., P. vulgaris) are more commonly found in soft tissue infections [29]. Progression of these infections to septicemia is relatively uncommon [31]. A recent survey estimated the prevalence of P. mirabilis in bloodstream infection in hospitalized patients to be 1.6%; this resulted in a rank of 11 out of the top 15 most commonly isolated organisms from bloodstream infections in the study [32]. Antimicrobial resistance has become an increasing problem in Proteus infections, with approximately 32% of isolates producing extended-spectrum β-lactamases [32]. Serratia marcescens. Serratia are seldom a cause of primary infections, but are notorious nosocomial pathogens and colonizers. S. marcescens is the primary pathogenic species of the Serratia genus, responsible for 1.7% of nosocomial bloodstream infections [4]. It is of particular concern due to its emerging antibiotic resistance to commonly used agents like β-lactams, aminoglycosides, carbapenems, and fluoroquinolones. Non-pigmented S. marcescens are more resistant to antibiotics and are associated with most outbreaks [33]. Transmission may occur from person to person contact, via medical apparatus, intravenous fluids, or other solutions [34]. Patients with indwelling catheters, particularly those for urinary tract infections, serve as a primary reservoir for transmission via hospital personnel. In children, the gastrointestinal tract is a common source of infections. Haemophilus influenzae is a gram-negative coccobacillus that is isolated exclusively from humans [35]. Strains of H. influenzae are divided into two groups based on the presence or absence of a capsular polysaccharide [35-36]. Encapsulated strains are further divided into six serotypes (a through f). Prior to widespread use of the H. influenzae type b (Hib) conjugate vaccines, Hib caused >80% of invasive H. influenzae infections, predominantly in children under the age of five [35], with a mortality rate of 3 to 6% and a further 20 to 30% developing permanent sequelae ranging from mild hearing loss to mental retardation [36]. In areas of routine vaccination, the majority of invasive H. influenzae infection is caused by nontypeable strains and predominantly affects children under the age of one and the elderly [35], with a mortality rate of 13 to 20% [36]. Approximately 20 to 35% of isolated strains are resistant to amoxicillin [35]. Neisseria meningitidis (Encapsulated) is a fastidious, aerobic, gram-negative diplococcus that is spread by mucus or respiratory droplets often from asymptomatic carriers. Thirteen different serogroups of N. meningitidis (A, B, C, D, H, I, K, L, X, Y, Z, W135, and 29E) can be distinguished. Serogroups B, C, and Y are currently the most prevalent in developed countries and serogroup A is predominant in the rest of the world [37]. Serogroups W135 and X also cause epidemics in developing regions of the world. The serogroups are determined by a polysaccharide capsule that aids bacterial survival inside the human host. N. meningitidis is the only species of Neisseria to produce a capsule. Encapsulation inhibits bactericidal activity of human serum, thereby increasing survival of meningococci in the environment [38]. It is estimated that ~ 16% of N. meningitidis are not encapsulated [39]. Such strains are commonly found as commensal bacteria in the nasopharyngeal tract and are not considered to be virulent [40]. FilmArray BCID will not detect unencapsulated N. meningitidis. Meningococcal disease (spinal meningitis and/or meningococcemia) is rare in developed countries, but can occur in outbreaks. It is most common in infants, children, and young adults, and appears in places with crowded living conditions (e.g., college dormitories and military barracks). Seasonal incidence peaks in late winter and early spring [41]. Septicemia with N. meningitidis is associated with fever and a characteristic hemorrhagic rash that may be transient [42]. The disease

BioFire Diagnostics, LLC FilmArray BCID Panel CE-IVD Instruction Booklet 9

can progress extremely quickly (<24 hours) with hypotension, multiorgan dysfunction, shock, peripheral ischemia, and limb loss and has a mortality rate of approximately 5-10% [43]. Pseudomonas aeruginosa is a gram-negative opportunistic pathogen. It rarely causes disease in healthy individuals but can cause sepsis in patients with burn wounds, malignancies or immunodeficiency, or in preterm infants [44-45]. P. aeruginosa is a leading cause of nosocomial infections and is responsible for 10% of all hospital acquired infections [46]. Mortality rates due to P. aeruginosa bacteremia are greater than 20%, and may be as high as 50% for intensive care unit patients and burn victims [44-45]. P. aeruginosa is susceptible to a limited number of antibiotics (antipseudomonal penicillins and cephalosporins, carbapenems, fluorquinolones and ciprofloxacin) [44], and multi-drug resistant (MDR) P. aeruginosa infection is becoming an increasing problem in hospitals [46]. The carbapenemase, KPC, has recently been identified in isolates of P. aeruginosa [47].

Yeast

Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis. Candida species are yeasts that are ubiquitous in the environment and as members of the normal human microbiota, especially in the digestive tract and on mucous membranes. These fungi are important causative agents of opportunistic nosocomial infections ranging from superficial (e.g., oral thrush) to systemic (e.g., septicemia) [48]. Candida spp. are the 4th most common cause of nosocomial bloodstream infections, having been detected in approximately 9% of all cases and over 10% of cases from the ICU in a large U.S. surveillance study [4]. The mortality rate for Candida bloodstream infection is approximately 40% and they often occur in combination with bacteria or a second Candida sp. [49]. The five most common species causing bloodstream infections are C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei. Species distribution has changed in the past three decades so that non-C. albicans have become more frequent than C. albicans. This shift is significant due to non-C. albicans species, especially C. glabrata and C. krusei, having increased rates of resistance to fluconazole, the drug most often used to treat Candida bloodstream infections [50]. Less common Candida species may be misidentified as one of the five common species using phenotypic laboratory testing. In particular, C. dubliniensis can be misidentified as C. albicans; and C. orthopsilosis and C. metapsilosis (previously classified as Group II and Group III C. parapsilosis, respectively) can be misidentified as C. parapsilosis [51].

Antimicrobial Resistance Genes mecA – Methicillin resistance. Methicillin-resistant (MR) staphylococci are a concern in both hospital-acquired and community-acquired infections. Few options exist for treatment of these infections, as the bacteria are resistant to both natural and semi-synthetic β-lactam antibiotics (e.g., oxacillin/methicillin) [10]. The primary mechanism of methicillin resistance in staphylococci is through acquisition of the mecA gene that encodes a penicillin binding protein (PBP2a) that has low affinity for β-lactams. mecA is part of a gene complex carried on a chromosomally-integrated mobile genetic element called the staphylococcal cassette chromosome mec (SCCmec). Ten major SCCmec types (I - X) have been characterized [52]. In 2011, an SCCmec type XI cassette carrying a divergent mecA homologue (mecALGA251/mecC), which also confers methicillin resistance, was identified [53]. Although coagulase-negative staphylococci (CoNS) have higher rates of methicillin resistance (MR-CoNS) than S. aureus (MRSA), CoNS is less virulent than S. aureus and is primarily limited to infections in the immunocompromised or individuals with indwelling devices [54]. Other mechanisms, such as penicillin binding protein mutations and hyperproduction of staphylococcal β-lactamase, can facilitate reduced methicillin susceptibility in S. aureus in the absence of mecA [55]. vanA/B – Vancomycin resistance. The prevalence of vancomycin-resistant enterococcus (VRE) has increased rapidly, with VRE accounting for 60% of E. faecium and 2% of E. faecalis isolated from the bloodstream [3-4]. Infection with a VRE increases the risk of death to 75%, compared with 45% for infection with a susceptible strain [3]. Eight gene clusters

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associated with vancomycin resistance have been identified to date (vanA, vanB, vanC, vanD, vanE, vanG, vanL, and vanM), with vanA and vanB being the most common in clinical isolates [56-57]. Both the vanA and vanB gene clusters are borne on mobile genetic elements (transposons) and can be located either on the chromosome or carried on a plasmid. Enterococci carrying vanA or vanB are resistant to high levels of vancomycin. Isolates carrying vanA are also resistant to high levels of teicoplanin [57]. The next most commonly detected Enterococci, E. gallinarium and E. casseliflavus, display intrinsic low-level resistance (intermediate resistance, vanC phenotype) to vancomycin in the absence of vanA/B [58].

KPC (blaKPC) – Carbapenem resistance. Carbapenem-resistant Enterobacteriaceae (CRE) are increasingly important pathogens in the hospital setting. Limited treatment options exist for CRE and they are associated with high mortality rates. Those most at risk include patients receiving long courses of antibiotics and those with indwelling devices (e.g. ventilators, urinary catheters, or intravenous catheters). The most common mechanism of carbapenemase-resistance in

CRE in the United States is that conferred by Klebsiella pneumoniae carbapenemases (KPCs) genes (blaKPC), a family of carbapenemase enzymes first described in 2001 [59]. Since their emergence, KPCs have become endemic in several countries. Twelve variants have been identified to date, KPC-2 through KPC-13, which differ by one to three amino acids [60-61]. KPCs are frequently carried on mobile genetic elements with the potential to spread between organisms. KPCs have been identified in many Enterobacteriaceae, the most common being K. pneumoniae. In addition to CRE, non- Enterobacteriaceae such as Pseudomonas aeruginosa and Acinetobacter baumannii may also harbor KPCs [62]. However, KPCs are not the most common mechanisms of carbapenem resistance in these two organisms. P. aeruginosa and A. baumannii isolates frequently carry other β-lactamases that confer carbapenem resistance (e.g. VIM, IMP, SIM, OXAs) or have porin downregulation leading to carbapenem resistance [63-64]. Detection of KPCs using phenotypic susceptibility testing (e.g., MIC breakpoints or Modified Hodge Test) is very difficult, not only because other mechanisms of carbapenem-resistance exist, but also because KPC activity is regulated by multiple mechanisms that may not be accurately assessed in vitro resulting in incorrect susceptibility reporting [65-66]. Alternatively, molecular methods (e.g., PCR) are increasingly being used to specifically identify KPC genes in clinical isolates [67].

Principle of the Procedure

The FilmArray BCID pouch is a closed system disposable that houses all the chemistry required to isolate, amplify and detect nucleic acid from multiple bloodstream pathogens within a single blood culture sample. The rigid plastic component (fitment) of the FilmArray BCID pouch contains reagents in freeze-dried form. The flexible plastic portion of the pouch is divided into discrete segments (blisters) where the required chemical processes are carried out. The user of the FilmArray BCID Panel loads the sample into the FilmArray BCID pouch, places the pouch into the FilmArray instrument, and starts the run. All other operations are automated. The following is an overview of the testing procedure:

1. Remove the pouch from its vacuum-sealed package. Since solutions are drawn into the pouch by vacuum, it is important to keep pouches in their protective packaging until the time of use.

2. Place the pouch into the FilmArray Pouch Loading Station. The FilmArray Pouch Loading Station has been designed to prevent error by providing instructions and visual cues in the form of color-coded arrows to ensure that the pouch is properly loaded.

3. Load Hydration Solution into the pouch using the Pouch Hydration Syringe. The syringe is fitted with a blunt stainless steel cannula, which is used to deliver the solution into the pouch. Loading the pouch with Hydration Solution rehydrates the freeze-dried reagents contained in the pouch fitment.

4. Remove blood culture media from the bottle (using a 28-gauge syringe to prevent clogging from resin) and add it to the Sample Buffer vial. Mix with Transfer Pipette. The Sample Buffer contains reagents that promote binding of nucleic acids to magnetic beads for isolation.

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5. Load the sample/buffer mixture into the pouch using the Sample Loading Syringe. When the sample mixture is loaded, a process control contained in the fitment of the pouch is introduced into the sample. The process control monitors all of the critical processes that occur in the pouch.

6. Transfer the pouch to the instrument and initiate a run. The FilmArray software provides on-screen animations illustrating the steps needed to start the run.

7. View results on the report at the completion of the run.

The following is an overview of the operations and processes that occur during a FilmArray run:

1. Nucleic Acid Purification - Nucleic acid purification occurs in the first three blisters of the pouch. The sample is lysed by agitation (bead beating) and the liberated nucleic acid is captured, washed and eluted using magnetic bead technology. These steps require about ten minutes and the bead-beater apparatus can be heard as a high- pitched whine during the first minute of operation.

2. 1st Stage Multiplex PCR - The purified nucleic acid solution is combined with a preheated master mix to initiate thermocycling for multiplex PCR. The effect of 1st stage PCR is to enrich for the target nucleic acids present in the sample.

3. 2nd Stage PCR - The products of first stage PCR are diluted and mixed with fresh PCR reagents containing a double stranded DNA binding dye (LCGreen® Plus, BioFire Diagnostics, LLC). This solution is distributed over the 2nd stage PCR array. The individual wells of the array contain primers for different assays (each present in triplicate) that target specific nucleic acid sequences from each of the pathogens detected, as well as control template material. These primers are ‘nested’ or internal to the specific products of the 1st stage multiplex reaction, which enhances both the sensitivity and specificity of the reactions.

4. DNA Melting Analysis – After 2nd stage PCR, the temperature is slowly increased and fluorescence in each well of the array is monitored and analyzed to generate a melt curve. The temperature at which a specific PCR product melts (melting temperature or Tm) is consistent and predictable and the FilmArray software automatically evaluates the data from replicate wells for each assay to report results. For a description of data interpretation and reporting see the Interpretation of Results section of this booklet.

The FilmArray software controls the operation of the instrument, collects and analyzes data, and automatically generates a test report at the end of the run. The entire process takes about an hour. Additional details can be found in the FilmArray Operator’s Manual.

MATERIALS PROVIDED

Each kit contains sufficient reagents to test 30 samples (30 pouch kit) or 6 samples (6 pouch kit):

 Individually packaged FilmArray BCID Panel pouches

 Single-use (0.5 mL) Sample Buffer vials (red lid)

 Single-use (1.5 mL) Hydration Solution vials (blue lid)

 Individually packaged Transfer Pipettes

 Individually packaged Sample Loading Syringes with attached cannula (red cap)

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 Individually packaged Pouch Hydration Syringes with attached cannula (blue cap)

MATERIALS REQUIRED BUT NOT PROVIDED

FilmArray system including:

 FilmArray or FilmArray 2.0 instrument and software

 FilmArray Pouch Loading Station

Syringes with a 28-gauge needle capable of measuring 0.1 mL (100 µL) sample volume

 BD™ 1cc Insulin Syringe #329410, or equivalent

WARNINGS AND PRECAUTIONS

General Precautions

1. For in vitro diagnostic use only. 2. This device is restricted to sale by or on the order of a physician, or to a clinical laboratory; its use is restricted to, by, or on the order of a physician. 3. A trained healthcare professional should carefully interpret the results from the FilmArray BCID Panel in conjunction with a patient’s signs and symptoms and results from other diagnostic tests. 4. FilmArray BCID Panel pouches are only for use with FilmArray systems. 5. Clinical performance characteristics of the FilmArray BCID Panel have only been determined with positive blood culture samples using the BD BACTEC™ Plus Aerobic/F Medium that demonstrated the presence of organisms by Gram stain evaluation. Other blood culture bottle types were evaluated analytically only (see Interference Section). 6. FilmArray pouches are stored under vacuum in individually-wrapped canisters. To preserve the integrity of the pouch vacuum for proper operation, be sure that a FilmArray instrument will be available and operational before unwrapping any pouches for loading. 7. Always check the expiration date on the pouch and do not use a pouch after its expiration date.

Safety Precautions

1. Wear appropriate Personal Protective Equipment (PPE), including (but not limited to) disposable powder-free gloves and lab coats. Protect skin, eyes, and mucus membranes. Change gloves often when handling reagents or samples. 2. Handle all samples and waste materials as if they were capable of transmitting infectious agents. Observe safety guidelines such as those outlined in CDC/NIH Biosafety in Microbiological and Biomedical Laboratories [68], the CLSI Document M29 Protection of Laboratory Workers from Occupationally Acquired Infections [69], or other appropriate guidelines.

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3. Follow your institution's safety procedures for handling biological samples. 4. Dispose of materials used in this assay, including reagents, samples, and used buffer vials, according to federal, state, and local regulations. 5. Sample Buffer is assigned the following classifications: Acute toxicity (Category 4), Serious Eye damage (Category 1), and Skin irritation (Category 2). Please refer to the FilmArray Reagent Kit Safety Data Sheet (SDS) for more information. 6. Sample Buffer will form hazardous compounds and fumes when mixed with bleach or other disinfectants.

WARNING: Bleach should never be added to Sample Buffer or sample waste.

Laboratory Precautions

1. Preventing organism contamination Due to the sensitive nature of the FilmArray BCID Panel, it is important to guard against contamination of the work area by following these guidelines:

 Positive blood culture samples contain high concentrations of organisms and require careful adherence to the sample processing steps described in this booklet. To avoid possible contamination, samples should be processed in a biosafety cabinet. If a biosafety cabinet is not used, a dead air box (e.g., AirClean PCR workstation), a splash shield (e.g., Bel-Art Scienceware Splash Shields), or a face shield should be used when preparing samples.

 A biosafety cabinet that is used for performing bacterial culture should not be used for sample preparation or pouch loading.

 Prior to processing a sample, thoroughly clean both the work area and the FilmArray Pouch Loading Station using a suitable cleaner such as freshly prepared 10% bleach or a similar disinfectant. To avoid residue build- up and potential PCR inhibition, wipe disinfected surfaces with water.

 Samples and pouches should be handled one-at-a-time.

 Change gloves and clean the work area between each sample.

2. Preventing amplicon contamination A common concern with PCR-based assays is false positive results caused by contamination of the work area with PCR amplicon. Because the FilmArray BCID Panel pouch is a closed system, the risk of amplicon contamination is low provided that pouches remain intact after the test is completed. Adhere to the following guidelines to prevent amplicon contamination:  Discard used pouches in an appropriate biohazard container immediately after the run has completed.  Avoid excessive handling of pouches after test runs.  Avoid exposing pouches to sharp edges or anything that might cause a puncture.

WARNING: If liquid is observed on the exterior of a pouch, the liquid and pouch should be immediately contained and discarded in a biohazard container. The instrument and work space must be decontaminated as described in the FilmArray Operator’s Manual.

DO NOT PERFORM ADDITIONAL TESTING UNTIL THE AREA HAS BEEN DECONTAMINATED.

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3. Eliminating resin beads from the blood culture samples Some blood culture media contain resin beads. The presence of resin beads in the FilmArray BCID Panel test has been shown to cause pouch control failures and affect test performance. Blood culture samples should be collected in a manner that prevents resin beads from entering the sample and the pouch. Use of a 28-gauge needle has been shown to prevent resin beads from the BD BACTEC™ Plus Aerobic/F Medium from being drawn out of the bottle with the sample. USE OF SYRINGES WITH A LARGER NEEDLE BORE SIZE (SMALLER GAUGE) IS NOT RECOMMENDED. If the laboratory uses an alternate blood culture collection method (possibly to avoid the use of needles), the method should prevent resin beads from entering the FilmArray test.

4. Blood culture media may contain non-viable organisms and/or nucleic acids at levels that can be detected by the FilmArray BCID Panel. The presence of non-viable organisms and/or nucleic acids may lead to false positive test results. Typically, these false positives will present with more than one positive result because the BCID Panel will also detect the organism that is growing in the culture bottle.

 Do not use blood culture media that contains charcoal (e.g., BacT/ALERT FA FAN® Aerobic).

 There is an increased risk of false positive test results for Pseudomonas aeruginosa and Enterococcus when the FilmArray BCID Panel is used to test bioMérieux BacT/ALERT SN standard anaerobic blood culture bottles. If the FilmArray BCID Panel is used with these bottles, then positive results for Pseudomonas aeruginosa and Enterococcus should be confirmed by another method prior to reporting the test results.

 In rare cases, the Gram stain result and results of the FilmArray BCID Panel may be discrepant (for example, detection of a gram-positive cocci by FilmArray BCID when gram-positive cocci were not observed in the Gram stain). In these cases, the results should be used in conjunction with other clinical and laboratory findings.

Precaution Related to Public Health Reporting in the United States

Local, state, and federal regulations for notification of reportable disease are continually updated and include a number of organisms for surveillance and outbreak investigations [70-71]. Additionally, the Centers for Disease Control (CDC) recommends that when pathogens from reportable diseases are detected by a culture independent diagnostic test (CIDT), the laboratory should facilitate obtaining the isolate or clinical materials for submission to the appropriate public health laboratory to aid in outbreak detection and epidemiological investigations. Laboratories are responsible for following their state and/or local regulations and should consult their local and/or state public health laboratories for isolate and/or clinical sample submission guidelines.

REAGENT STORAGE, HANDLING AND STABILITY 1. Store the test kit, including reagent pouches and buffers, at room temperature (15–25 ºC). DO NOT REFRIGERATE. 2. Avoid storage of any materials near heating or cooling vents or in direct sunlight. 3. Always check the expiration date and do not use reagents beyond the expiration date printed on the pouch or kit. 4. All kit components should be stored and used together. Do not use components from one kit with those of another kit.

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5. Do not remove pouches from their packaging until a sample is ready to be tested. Once the pouch packaging has been opened, the pouch should be loaded as soon as possible (within approximately 30 minutes). 6. Once a pouch has been loaded, the test run should be started as soon as possible (within 60 minutes).

SAMPLE REQUIREMENTS The FilmArray BCID Panel is performed directly on positive blood culture samples that demonstrate the presence of organisms as determined by Gram stain.

Sample Volume – 0.1 mL

Sample Collection – Sample should be collected from the blood culture bottle using a syringe with a 28-gauge needle that is capable of measuring 0.1 mL. Needles with a larger bore size (e.g., 18-gauge) should not be used because they may allow resin beads from the blood culture media to enter the sample. THE PRESENCE OF RESIN BEADS IN THE SAMPLE WILL AFFECT TEST PERFORMANCE AND SHOULD BE AVOIDED. If an alternate collection method is used, the laboratory should ensure that the method adequately excludes resin beads from the sample.

Sample Age – Blood culture samples should be processed and tested as soon as possible after being flagged as positive by the culture instrument. However, samples may be stored for up to 8 hours at room temperature or in the culture instrument prior to testing.

PROCEDURE

Refer to the FilmArray Blood Culture Identification Panel Quick Guide, the FilmArray Training Video or the FilmArray Operator’s Manual for more details and pictorial representations of these instructions.

Gloves and other Personal Protective Equipment (PPE) should be used when handling pouches and samples. Only one FilmArray BCID pouch should be prepared at a time. Once sample is added to the pouch, it should be promptly transferred to the instrument to start the run. After the run is complete, the pouch should be discarded in a biohazard container.

Prepare Pouch

1. Thoroughly clean the work area and the FilmArray Pouch Loading Station with freshly prepared 10% bleach (or suitable disinfectant) followed by a water rinse. 2. Remove the pouch from its vacuum-sealed package by tearing or cutting the notched outer packaging and opening the protective aluminum canister.

NOTE: If the vacuum seal of the pouch is not intact, the pouch may still be used. Attempt to hydrate the pouch using the steps in the Hydrate Pouch section. If hydration is successful, continue with the run. If hydration fails, discard the pouch and use a new pouch to test the sample.

3. Slide the pouch into the FilmArray Pouch Loading Station so that the red and blue labels on the pouch align with the red and blue arrows on the FilmArray Pouch Loading Station.

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4. Place a blue-capped Hydration Solution vial in the blue well of the FilmArray Pouch Loading Station.

5. Place a red-capped Sample Buffer vial in the red well of the FilmArray Pouch Loading Station.

Hydrate Pouch

1. Remove the blue-labeled Pouch Hydration Syringe from the packaging. If the cannula/tip is not firmly attached to the syringe, hold the capped tip and rotate the syringe to tighten. 2. Using the Pouch Hydration Syringe, draw Hydration Solution to at least the 1 mL mark on the syringe, taking care to avoid the formation of bubbles. If you notice bubbles at the base of the syringe, leave the tip of the cannula in the Hydration Solution vial and dislodge the bubbles by gently tapping the side of the syringe with your finger. The bubbles will float up to the plunger.

NOTE: DO NOT remove air bubbles by inverting the syringe and expelling liquid. 3. Insert the cannula tip into the port in the pouch located directly below the blue arrow of the FilmArray Pouch Loading Station. While holding the body of the syringe, push down forcefully in a firm and quick motion until you hear a faint “pop” and feel an ease in resistance. The correct volume of liquid will be pulled into the pouch by vacuum; DO NOT push the syringe plunger. 4. Verify that the pouch has been hydrated. Most of the liquid will have been drawn out of the syringe. Flip the barcode label down and check to see that fluid has entered the reagent wells (located at the base of the rigid plastic part of the pouch). Small air bubbles may be seen. If the pouch fails to hydrate (dry reagents appear as white pellets), repeat Step 3 to verify that the seal of the port was broken or retrieve a new pouch and repeat from Step 2 of the Prepare Pouch section.

Prepare Sample Mix

1. Invert the positive blood culture bottle several times to mix. 2. Wipe the bottle septum with alcohol and air dry. 3. Tilt the bottle and hold in the tilted position to allow the bottle resin to settle (approximately 10 seconds). 4. Using a syringe with a 28-gauge needle, withdraw 0.1 mL of blood culture sample through the bottle septum, taking care to avoid drawing resin beads into the sample, or the formation of bubbles.

NOTE: DO NOT use a needle with a larger bore size (smaller gauge size, e.g., 18-gauge) as it may result in resin beads being introduced into the sample and the pouch. The presence of resin beads can cause control failures and interfere with test results.

5. Transfer sample directly to the Sample Buffer vial. Discard syringe in an appropriate biohazard sharps container. Alternately: Draw the desired amount of blood culture sample (>0.1 mL) from the bottle into the syringe and transfer to a sterile secondary container. Draw the blood culture sample from the secondary container to the first line of the Transfer Pipette (0.1 mL) and add the sample to the Sample Buffer vial. 6. Use the Transfer Pipette to mix the sample with the Sample Buffer by gently pipetting up and down.

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Load Sample Mix

1. Remove the red-labeled Sample Loading Syringe from the packaging. If the cannula/tip is not firmly attached to the syringe, hold the capped tip and rotate the syringe to tighten. 2. Using the Sample Loading Syringe, draw approximately 0.3 mL of sample/buffer mix (to the 0.3 mL/cc mark on the syringe), taking care to avoid the formation of bubbles. If you notice bubbles at the base of the syringe, leave the tip of the cannula in the Sample Buffer vial and dislodge the bubbles by gently tapping the side of the syringe with your finger. The bubbles will float up to the plunger. NOTE: To avoid contaminating the work area, DO NOT remove air bubbles by inverting the syringe and expelling liquid. 3. Insert the cannula tip into the port in the pouch fitment located directly below the red arrow of the FilmArray Pouch Loading Station. While holding the body of the syringe, push down forcefully in a firm and quick motion until you hear a faint “pop” and feel an ease in resistance. The correct volume of liquid will be pulled into the pouch by vacuum; DO NOT push the syringe plunger. 4. Verify that the sample has been loaded. Most of the liquid will have been drawn out of the syringe. Flip the barcode label down and check to see that fluid has entered the reagent well next to the sample loading port. If the pouch fails to pull sample from the Sample Loading Syringe, the pouch should be discarded. Retrieve a new pouch and repeat from Step 2 of the Prepare Pouch section.

NOTE: To reduce the risk of exposure to hazardous or potentially infectious material, DO NOT re-cap the syringes.

5. Dispose of syringes in an appropriate biohazard sharps container. 6. Record the Sample ID in the provided area on the pouch label (or affix a barcoded Sample ID) and remove the pouch from the FilmArray Pouch Loading Station.

Run Pouch

The FilmArray software includes a step-by-step on-screen tutor that shows each step of the test.

1. Ensure that the computer and FilmArray instrument(s) are on and the FilmArray software is launched.

2. Open the lid of an available instrument (if not already open).

NOTE: An available instrument is indicated by a constant green light on the front of the instrument.

3. Insert the pouch into the instrument. Position the pouch so that the array is on the right with the film directed downward into FilmArray instrument. The red and blue labels on the pouch should align with the red and blue arrows on the FilmArray instrument. The pouch will click into place. If inserted correctly, the barcode is visible and the label is readable on the top of the

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pouch. The instrument and software must detect that the pouch has been inserted correctly before continuing to the next step.

NOTE: If the pouch does not slide into the instrument easily, gently push the lid of the instrument back to be sure that it is completely open.

4. Scan the barcode on the FilmArray pouch using the barcode scanner. Pouch identification (Lot Number and Serial Number), Pouch Type and Protocol are preprogrammed in the rectangular barcode located on the FilmArray pouch. The information will be automatically entered when the barcode is scanned. If it is not possible to scan the barcode, the pouch Lot Number, Serial Number, Pouch Type and Protocol can be manually entered from the information provided on the pouch label into the appropriate fields. To reduce data entry errors, it is strongly recommended that the pouch information be entered by scanning the barcode.

NOTE: The barcode cannot be scanned prior to placing the pouch in the instrument.

5. Enter the Sample ID. The Sample ID can be entered manually or scanned in by using the barcode scanner when a barcoded Sample ID is used. 6. If necessary, select a protocol from the Protocol drop down list (the protocol is usually selected automatically). 7. Enter a user name and password in the Name and Password fields. 8. Close the FilmArray instrument lid. 9. Click the Start Run button on the screen. Once the run has started, the screen displays a list of the steps being performed by the instrument and the number of minutes remaining in the run.

NOTE: The bead-beater apparatus can be heard as a high-pitched noise (whine) during the first minute of operation.

10. When the run is finished, follow the on-screen instructions to open the instrument and remove the pouch. 11. Immediately discard the pouch in a biohazard container. 12. Results are automatically displayed in the report section of the screen. The run file is automatically saved in the FilmArray database and the report can be printed and/or saved as a PDF file.

QUALITY CONTROL

Process Controls

Two process controls are included in each pouch: 1. DNA Process Control

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The DNA Process Control assay targets DNA from the yeast Schizosaccharomyces pombe. The yeast is present in the pouch in a freeze-dried form and is hydrated and introduced into the test when the sample is loaded. The control material is carried through all stages of the test process, including lysis, nucleic acid purification, 1st stage PCR, dilution, 2nd stage PCR, and DNA melting. A positive control result indicates that all steps carried out in the pouch were successful. 2. PCR2 Control

The PCR2 Control assay detects a DNA target that is dried into the wells of the array along with the corresponding primers. A positive result indicates that 2nd stage PCR was successful. Both control assays must be positive for the test run to pass. When either control fails, the Controls field of the test report (upper right hand corner) will display Failed and all results will be listed as Invalid. If the controls fail, the sample should be retested using a new pouch.

Monitoring Test System Performance

The FilmArray software will automatically fail the run if the melting temperature (Tm) for either the DNA Process Control or the PCR2 Control is outside an acceptable range (77.6-81.6 for the DNA Process Control and 74.2-78.2 for the PCR2 Control). If required by local, state, or accrediting organization quality control requirements, users can monitor the system by trending Tm values for the control assays and maintaining records according to standard laboratory quality control practices [72-73]. The PCR2 Control is used in all pouch types and can therefore be used to monitor the system when multiple pouch types (e.g., RP and BCID) are used on the same FilmArray instrument. The Tm values for the DNA Process Control can only be evaluated for FilmArray runs with the BCID Panel.

Good laboratory practice recommends running external positive and negative controls regularly. Uninnoculated blood culture media can be used as an external negative control. Previously characterized positive blood culture samples or samples spiked with well characterized organisms can be used as external positive controls. External controls should be used in accordance with the appropriate accrediting organization requirements, as applicable.

INTERPRETATION OF RESULTS

The FilmArray software automatically analyzes and interprets assay results and displays the final results in a test report (see the FilmArray Blood Culture Identification Panel Quick Guide to view an example of a test report). The analyses performed by the FilmArray software and details of the test report are described below.

Assay Interpretation

When 2nd stage PCR is complete, the FilmArray instrument performs a high resolution DNA melting analysis on the PCR products and measures the fluorescence signal generated in each well (for more information see FilmArray Operator’s Manual). The FilmArray software then performs several analyses and assigns a final assay result. Analysis of melt curves. The FilmArray software evaluates the DNA melt curve for each well of the 2nd stage PCR array to determine if a PCR product was present in that well. If the melt profile indicates the presence of a PCR product, then the analysis software calculates the melting temperature (Tm) of the curve. The Tm value is then compared against the expected Tm range for the assay. If the software determines that the melt curve is positive and the Tm falls inside the

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assay-specific Tm range, the melt curve is called positive. If the software determines that the melt curve is negative or is not in the appropriate Tm range, the melt curve is called negative. Analysis of replicates. Once melt curves have been identified, the software evaluates the three replicates for each assay to determine the assay result. For an assay to be called positive, at least two of the three associated melt curves must be called positive, and the Tm for at least two of the three positive melt curves must be similar (within 1°C). Assays that do not meet these criteria are called negative.

Organism Interpretation

Interpretations for many of the organisms are based on the results of a single assay (see Table 2). Interpretations for Haemophilus influenzae and the Staphylococcus, Streptococcus, and Enterobacteriaceae groups rely on the results of several assays. The antimicrobial resistance genes are also based on the result of a single assay; however, the test results are only reported when specific organisms are also detected in the same sample. This section provides an explanation of the test results and guidelines for actions to be taken based on the test result. This section also contains information about known assay limitations (e.g., cross-reactivity, strains that are not detected) that may be important in the interpretation of the test result and in correlating the FilmArray results with the result of standard culture and biochemical identification. NOTE: Polymicrobial blood cultures with 3 or more distinct organisms are possible but rare. If Detected results are reported for 3 or more organisms in a sample, a retest of the sample is recommended to confirm the polymicrobial result. NOTE: In rare cases, the Gram stain result and FilmArray BCID Panel results may be discrepant (for example, detection of a gram-positive cocci by FilmArray BCID when gram-positive cocci were not observed in the Gram stain). In these cases, the results should be used in conjunction with other clinical and laboratory findings.

Single Assay Interpretations

Organisms listed in Table 2 are considered to be Detected if a single corresponding assay is positive. Table 2. FilmArray BCID Panel Single Assay Interpretations Bacteria Yeast Acinetobacter baumannii Candida albicans Enterococcus Candida glabrata Listeria monocytogenes Candida krusei Neisseria meningitidis Candida parapsilosis Pseudomonas aeruginosa Candida tropicalis

Details concerning assay reactivity (inclusivity and exclusivity) are provided here and also in the Analytical Reactivity (Inclusivity) and Analytical Specificity (Cross-Reactivity and Exclusivity) studies found in the Performance Characteristics section below. Acinetobacter baumannii

Acinetobacter baumannii is part of the Acinetobacter calcoaceticus-baumannii (ACB) complex. In addition to A. baumannii, the complex includes A. calcoaceticus, A. pittii (genomospecies 3), and A. nosocomialis (genomospecies 13TU). These species are genetically and phenotypically related and cannot be reliably differentiated from each other using current microbial identification methods. The BCID Panel Abaumannii assay detects A. baumannii; however, it also detects some strains of the non-baumannii species with varying sensitivity

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(see Analytical Specificity section for additional information). Discrepancies between the BCID Panel test result and microbial identification may be caused by misidentification of non-baumannii members of the ACB complex as A. baumannii. No cross-reactivity with other Acinetobacter species outside of the ACB complex is expected. Enterococcus

The Enterococcus assay detects the major species associated with Enterococcus bloodstream infections (E. faecium and E. faecalis) as well as several less common species of varying clinical relevance including: E. avium, E. casseliflavus, E. durans, E. gallinarum, and E. hirae. E. dispar and E. saccharolyticus are detected with reduced sensitivity and E. raffinosus, which is occasionally isolated from clinical specimens, will not be detected by the BCID Panel. NOTE: Limited cross-reactivity with coagulase-negative staphylococci has been observed. Sequence analysis and empirical testing suggest that this cross-reactivity may occur when select species (S. haemolyticus, S. epidermidis, and S. capitis) are in the blood culture at a very high concentration. NOTE: An increased risk of false positive test results for Enterococcus has been observed when the FilmArray BCID Panel is used to test bioMérieux BacT/ALERT SN standard anaerobic blood culture bottles. Listeria monocytogenes

There are 12 known serovars of L. monocytogenes, however; only three serovars (1/2a, 1/2b and 4b) account for more than 90% of human cases of listeriosis. The BCID Panel is designed to detect all known serovars. Sequence analysis predicts that cross-reactivity with some strains of atypical Listeria innocua is possible [74]. Neisseria meningitidis

The BCID Panel detects encapsulated strains of N. meningitidis. Unencapsulated strains are generally considered non-virulent species of the normal nasopharyngeal flora and are not detected by the BCID Panel. Pseudomonas aeruginosa

The Paeruginosa assay detects P. aeruginosa and does not cross-react with other Pseudomonas species or closely-related bacteria. NOTE: An increased risk of false positive test results for Pseudomonas aeruginosa has been observed when the FilmArray BCID Panel is used to test bioMérieux BacT/ALERT SN standard anaerobic blood culture bottles.

Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis

Species-specific assays are included in the BCID Panel for each of the five most common Candida species associated with candidemia (Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis). Based on in silico analysis and empirical testing (see Analytical Reactivity and Analytical Specificity sections for additional information), each assay is specific for detection of the indicated species with the following exceptions:  Candida albicans is closely related to Candida dubliniensis and misidentification of these species by laboratory methods does occur. Cross-reactivity between the Calbicans assay and C. dubliniensis has not been observed (see Analytical Specificity) but sequence analysis predicts that cross-reactivity with C. dubliniensis is possible.  The BCID Panel assay for detection of C. parapsilosis cross-reacts with Candida orthopsilosis. Prior to being designated as unique species, C. orthopsilosis and C. metapsilosis were classified as Group II and Group III Candida parapsilosis, respectively. Both are closely related to Candida parapsilosis and can be misidentified as Candida parapsilosis using standard identification methods. The BCID Panel assay for C. parapsilosis will not detect C. metapsilosis; however, amplification of C. orthopsilosis is predicted by

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sequence analysis and has been confirmed (see Analytical Specificity section for additional information). In silico analysis suggests that cross-reactivity with C. multigemmis may also be possible, though this cross-reactivity has not been observed. NOTE: Candida krusei is also known as Issatchenkia orientalis and Pichia kudriavzevkii, therefore reactivity with isolates identified as these species does not represent cross-reactivity. Multiple Assay Interpretations

Haemophilus influenzae

The BCID Panel contains two different assays (Hinfluenzae1 and Hinfluenzae2) for the detection of H. influenzae. If either or both of the assays are positive, the test result will be Haemophilus influenzae Detected. If both the Hinfluenzae1 and Hinfluenzae2 assays are negative, the test result will be Haemophilus influenzae Not Detected. Staphylococcus

The BCID Panel contains three assays for the detection of Staphylococcus species. The Staphylococcus aureus assay and two multi-species assays (Staphylococcus1 and Staphylococcus2). The Saureus assay detects all strains of S. aureus and does not cross-react with other organisms, including other species of Staphylococcus. The multi-species assays detect the most prevalent coagulase-negative Staphylococcus (CoNS) species encountered in blood culture specimens and can also react with high levels of S. aureus. The FilmArray software integrates the results of the three Staphylococcus assays into a final Staphylococcus test result. If all three assays are negative, the test result will be Staphylococcus Not Detected. If any of the three assays is positive, the result will be Staphylococcus Detected. Results for the Saureus assay (positive or negative) determine the Staphylococcus aureus test result (Detected or Not Detected, respectively). Table 7 (see Antimicrobial Resistance Genes Interpretation section below) provides a summary of the interpretation of these three assays as well as the results for the mecA assay. As stated above, the two multi-species assays (Staphlyococcus1 and Staphylococcus2) detect the most commonly encountered CoNS recovered for positive blood cultures. However, this is a large and diverse group and detection by BCID Panel assays is variable. The table below summarizes the ability of the BCID Panel to detect various species of Staphylococcus. The species in the detected column are expected to be reliably detected by the BCID Panel at organism concentrations observed in positive blood cultures (~ 5x106 CFU/mL). The species listed in the reduced sensitivity column have been shown to be detected at higher concentrations (~107 to 108 CFU/mL) but may not always be detected in positive blood cultures. The species listed in the Not Detected column are unlikely to be detected by the BCID Panel due to sequence mismatches with the BCID Panel assays. See Analytical Reactivity section for a more extensive description of Staphylococcus reactivity.

Table 3. Summary of BCID Panel Reactivity with Staphylococcia Detected with Detected Not Detected Reduced Sensitivity S. aureus S. capitis S. auricularis S. caprae S. pasteuri S. carnosus S. cohnii S. saprophyticus S. lentus S. epidermidis S. simulans S. pettenkoferi S. haemolyticus S. warneri S. pseudointermedius S. hominis S. schleiferi S. lugdunensis S. sciuri S. xylosus a See Analytical Reactivity (Inclusivity) section for additional in silico reactivity predictions.

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Note: CoNS are often contaminating organisms and may be present at lower concentrations in polymicrobial blood cultures than in single organism cultures. Note: Multiple Staphylococcus species may be present in a single sample. The presence of multiple Staphylococcus species cannot be determined by BCID Panel test results.

Streptococcus

The BCID Panel contains four assays for the detection of Streptococcus species. Species-specific assays are included for the detection of Group A Strep (Spyogenes), Group B Strep (Sagalactiae), and Spneumoniae. The fourth assay is a multi-species assay (Streptococcus) designed to react with select Viridans group and other Streptococcus species encountered in blood culture specimens. However, the BCID Panel may not detect all Streptococcus species. The FilmArray software integrates the results of all four Streptococcus assays into a final Streptococcus result as shown in the table below. If all of the assays are negative, the test result will be Streptococcus Not Detected. Alternatively, if any of the four assays are positive, the test result will be Streptococcus Detected. Results for each species-specific assay are also reported independently. Table 4. Possible Assay Results and Corresponding Streptococcus Test Results

FilmArray BCID Interpretations Streptococcus Assay Sagalactiae Assay Spneumoniae Assay Spyogenes Assay Description

Streptococcus Not Detected Streptococcus agalactiae Not Detected Negative Negative Negative Negative No Streptococcus species detected Streptococcus pneumoniae Not Detected Streptococcus pyogenes Not Detected

Streptococcus Detected Streptococcus species detected Streptococcus agalactiae Not Detected Positive Negative Negative Negative (not S. agalactiae, S. pneumoniae, or S. Streptococcus pneumoniae Not Detected pyogenes) Streptococcus pyogenes Not Detected

Streptococcus Detected Streptococcus agalactiae Detected Any result Positive Negative Negative Streptococcus agalactiae detected Streptococcus pneumoniae Not Detected Streptococcus pyogenes Not Detected

Streptococcus Detected Streptococcus agalactiae Not Detected Any result Negative Positive Negative Streptococcus pneumoniae detected Streptococcus pneumoniae Detected Streptococcus pyogenes Not Detected

Streptococcus Detected Streptococcus agalactiae Not Detected Any result Negative Negative Positive Streptococcus pyogenes detected Streptococcus pneumoniae Not Detected Streptococcus pyogenes Detected

Note: Multiple Streptococcus species assays may be positive in a single sample. If this occurs, the test result for each species with a positive assay will be reported as Detected.

Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes are three of the most important streptococci to identify from blood cultures and each is detected by the BCID Panel with a species-specific assay.

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The gene target for each assay is either found only in the species of interest or is highly conserved within the species of interest. No cross-reactivity with other streptococci is predicted for these assays. The multi-species Streptococcus assay reacts with the following species at levels observed in positive blood cultures: S. anginosus, S. bovis, S. constellatus, S. dysgalactiae, S. equinis, S. gallolyticus, S. gordonii, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. pseudopneumoniae, S. salivarius, and S. sanguinis. Streptococci that are not listed here are rare and have not been tested. The Streptococcus assay may demonstrate variable to no reactivity with those species.

Enterobacteriaceae

The BCID Panel includes seven assays to detect members of the Enterobacteriaceae family. Six genus/species specific assays are included for the detection of Enterobacter cloacae (and other E. cloacae complex species); Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus spp., and Serratia marcescens. A seventh assay (the Enteric assay) will react with some (not all) species detected by the other six assays; however, its primary function is to detect other less common, but clinically relevant members of the Enterobacteriaceae family. Combined, these seven assays will detect many, but not all Enterobacteriaceae (see Analytical Reactivity (Inclusivity) section below). As described for the other multi-assay interpretations, a positive result for any of the seven Enterobacteriaceae- associated assays will generate an Enterobacteriaceae Detected result. Each specific genus/species assay result will also be reported independently as shown in Table 5. Results for the Enteric assay are not reported independently, but are incorporated into the Enterobacteriaceae test result. Negative results for all seven assays will generate an Enterobacteriaceae Not Detected result.

Table 5. Possible Assay Results and the Corresponding Enterobacteriaceae Test Results

Assay

FilmArray BCID Interpretations Enteric Assay Assay Ecloacae Ecol Assay Koytoca Kpneumoniae Assay Assay Proteus Smarcescens Assay Description Enterobacteriaceae Not Detected Enterobacter cloacae complex Not Detected Escherichia coli Not Detected No Enterobacteriaceae species Klebsiella oxytoca Not Detected Neg Neg Neg Neg Neg Neg Neg detected Klebsiella pneumoniae Not Detected (See limitations below)a Proteus Not Detected Serratia marcescens Not Detected Enterobacteriaceae Detected Enterobacter cloacae complex Not Detected Enterobacteriaceae species Escherichia coli Not Detected detected Klebsiella oxytoca Not Detected Pos Neg Neg Neg Neg Neg Neg (not E. cloacae complex species, E. Klebsiella pneumoniae Not Detected coli, K. oxytoca, K. pneumoniae, Proteus Not Detected Proteus, or S. marcescens) Serratia marcescens Not Detected Enterobacteriaceae Detected Enterobacter cloacae complex Detected Escherichia coli Not Detected Any E. cloacae complex species Klebsiella oxytoca Not Detected Pos Neg Neg Neg Neg Neg result detected Klebsiella pneumoniae Not Detected Proteus Not Detected Serratia marcescens Not Detected

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Assay

FilmArray BCID Interpretations Enteric Assay Assay Ecloacae Ecol Assay Koytoca Kpneumoniae Assay Assay Proteus Smarcescens Assay Description Enterobacteriaceae Detected Enterobacter cloacae complex Not Detected Escherichia coli Detected Any Klebsiella oxytoca Not Detected Neg Pos Neg Neg Neg Neg E. coli detected result Klebsiella pneumoniae Not Detected Proteus Not Detected Serratia marcescens Not Detected Enterobacteriaceae Detected Enterobacter cloacae complex Not Detected Escherichia coli Not Detected Klebsiella oxytoca Detected Any Neg Neg Pos Neg Neg Neg K. oxytoca detected Klebsiella oxytoca Not Detected result Klebsiella pneumoniae Not Detected Proteus Not Detected Serratia marcescens Not Detected Enterobacteriaceae Detected Enterobacter cloacae complex Not Detected Escherichia coli Not Detected Any Klebsiella oxytoca Not Detected Neg Neg Neg Pos Neg Neg K. pneumoniae detected result Klebsiella pneumoniae Detected Proteus Not Detected Serratia marcescens Not Detected Enterobacteriaceae Detected Enterobacter cloacae complex Not Detected Escherichia coli Not Detected Any Klebsiella oxytoca Not Detected Neg Neg Neg Neg Pos Neg Proteus species detected result Klebsiella pneumoniae Not Detected Proteus Detected Serratia marcescens Not Detected Enterobacteriaceae Detected Enterobacter cloacae complex Not Detected Escherichia coli Not Detected Any Klebsiella oxytoca Not Detected Neg Neg Neg Neg Neg Pos S. marcescens detected result Klebsiella pneumoniae Not Detected Proteus Not Detected Serratia marcescens Detected Note: Multiple Enterobacteriaceae assays may be positive in a single sample. If this occurs, the test result for each species with a positive assay will be reported as Detected. a Morganella spp., Providencia spp., Rahnella spp., Yersinia spp., Tatumella spp., and some Serratia spp. are not detected by the Enterobacteriaceae assays.

Enterobacter cloacae complex The Enterobacter cloacae complex is comprised of six species (E. asburiae, E. cloacae, E. hormaechei, E. kobei, E. ludwigii, and E. nimipressuralis) that may all be identified as E. cloacae by phenotypic laboratory methods. Of the six complex species, the BCID Panel Ecloacae assay detects E. cloacae (subspecies cloacae and dissolvens), E. asburiae, and E. hormaechei. Detection of E. kobei, E. ludwigii, and E. nimipressuralis is not expected and the clinical significance of these species is uncertain. Cross-reactivity with the closely related Enterobacter cancerogenus (which has previously been described as a member of the E. cloacae complex; also known as E. taylorae) is possible. Cross-reactivity with the clinically important Enterobacter aerogenes and two former Enterobacter species (Cronobacter sakazakii and Pantoea agglomerans) has not been observed.

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Escherichia coli The BCID Panel Ecoli assay cross-reacts with Shigella species (S. boydii, S. dysenteriae, S. flexneri, and S. sonnei); which are practically indistinguishable from E. coli by both phenotypic and genetic analyses, but are only very rarely isolated from blood culture. Cross-reactivity has also been observed with Escherichia fergusonii, a rare but potentially emerging pathogen (see Analytical Specificity section).

Klebsiella oxytoca The BCID Panel Koxytoca assay does not cross-react with other Klebsiella or Enterobacteriaceae species. However, K. pneumoniae or Raoultella ornithinolytica can be misidentified as K. oxytoca by standard laboratory methods leading to instances of apparent false negative K. oxytoca results. Similarly, a few variant strains of K. oxytoca have been identified that will not be detected as K. oxytoca by the BCID Panel; however, these variants are detected by the Enteric assay and reported as Enterobacteriaceae Detected.

Klebsiella pneumoniae The BCID Panel Kpneumoniae assay detects K. pneumoniae (including three subspecies; ssp. pneumoniae, ssp. ozaenae, and ssp. rhinosclermatis) and K. variicola (see Analytical Reactivity section). K. variicola is a closely related species to K. pneumoniae that has been isolated from clinical specimens. The Kpneumoniae assay does not cross-react with Klebsiella oxytoca. However, the closely related Raoultella (formerly Klebsiella) ornithinolytica can be misidentified as K. oxytoca and exhibits cross-reactivity with the Kpneumoniae assay (see Analytical Specificity section).

Proteus The BCID Panel Proteus assay detects four of five characterized species within the genus (P. mirabilis, P. hauseri, P. penneri, and P. vulgaris). P. mirabilis, P. penneri, and P. vulgaris are considered opportunistic human pathogens, with P. mirabilis being the most common. The fifth species in the genus, P. myxofaciens, is not a known human pathogen and detection is not expected.

Serratia marcescens Serratia marcescens is the primary human pathogen within the Serratia genus, though rare cases of human infection with other Serratia species (S. plymuthica, S. liquefaciens, S. rubidaea, S. odorifera, S. ficaria and S. fonticola) have been described. The BCID Panel Smarcescens assay was designed to detect S. marcescens, but will exhibit variable reactivity with select Serratia species as well. Based on sequence analysis and empirical testing, S. ficaria and S. entomophila (non-human pathogen) can be reliably detected. Reactivity with S. odorifera and S. rubidaea (rare human pathogens) is also possible, depending on the amount of organism in the specimen. S. liquefaciens, S. plymuthica, S. fonticola, S. grimesii, and S. proteamaculans will not be detected. See Analytical Reactivity and Analytical Specificity sections below for more information. In addition to Serratia species, cross-reactivity has also been observed between the Smarcescens assay and a specific strain of Pseudomonas aeruginosa (ATCC25619), the soil bacterium Pseudomonas putida, some Pantoea species, such as P. calida and P. septica, and Raoultella ornithinolytica (commonly misidentified by standard laboratory methods as Klebsiella oxytoca).

Other Enterobacteriaceae If any of the species specific assays described above are positive, the Enterobacteriaceae result will be Detected. However, the Enterobacteriaceae family includes many additional species that are not covered by the species specific assays. The BCID Enteric assay detects many, but not all species of Enterobacteriaceae that may be associated with human infection, including Cedeceae davisiae, Citrobacter spp., Cronobacter (Enterobacter) sakazakii, Enterobacter spp. (including E. aerogenes), Escherichia spp., Kluyvera ascorbata, Leclercia adecarboxylata, Raoultella spp., Salmonella spp., Shigella spp., and Yokenella regensburgei. Detection of some other Enterobacteriaceae may only occur when the organism is present in a blood culture at high levels (>1x108 CFU/mL) including Hafnia alvei, and some Serratia spp. The BCID Enteric assay may also react with members of the Enterobacteriaceae family that are rare human pathogens (Edwardsiella spp. and

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Erwinia spp.) as well as species typically classified as plant pathogens (Brenneria spp., Dickeya spp., Pectobacterium spp., etc.). Sequence analysis and testing indicate that the BCID Enterobacteriaceae assays will not detect Morganella morganii, Providencia spp., Rahnella spp., Serratia liquefaciens, Tatumella ptyseos, Yersinia spp. and others (see Table 6 and Analytical Reactivity and Analytical Specificity sections below). Table 6. Summary of BCID Panel Reactivity with Enterobacteriaceaea Detected with Detected Not Detected Reduced Sensitivity Cedeceae spp. Edwardsiella spp. Morganella morganii Citrobacter spp. Enterobacter gergoviae Providencia spp. Cronobacter spp. Hafnia alvei Rahnella spp. Enterobacter spp. Pantoea spp. Serratia liquefaciens Escherichia spp. Salmonella bongori Serratia plymuthica Klebsiella spp. Serratia fonticola Tatumella ptyseos Kluyvera spp. Serratia odorifera Yersinia enterocolitica Leclercia adecarboxylata Serratia rubidaeae Proteus spp. Raoultella spp. Salmonella spp. Shigella spp. Serratia marcescens, S. ficaria, and S. entomophila b Yokenella regensbergei a See Analytical Reactivity (Inclusivity) section for additional in silico reactivity predictions. b Insect pathogen

Antimicrobial Resistance Genes Interpretation

The test results for antimicrobial resistance genes are only reported when an associated organism (as shown in Table 7) is detected in the same test. Tables 8, 9 and 10 provide detailed listing of the assay results and the corresponding BCID Panel test results.

Table 7. Antimicrobial Resistance Genes and Associated Organisms Antimicrobial Resistance Genes Associated Organism mecA Staphylococcus vanA/B* Enterococcus

Any Enterobacteriaceae, KPC A. baumannii, and/or P. aeruginosa

*NOT reported with Staphylococcus. Vancomycin-resistant Staphylococcus aureus (VRSA) is possible but extremely rare.

The results for each of the antimicrobial resistance genes will be listed as either: Detected – when an appropriate organism is detected and the antimicrobial resistance gene assay is positive. Not Detected – when an appropriate organism is detected and the antimicrobial resistance gene assay is negative. N/A – when NO appropriate organism is detected regardless of the result for the antimicrobial resistance gene assay.

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NOTE: A Not Detected result for any of the antimicrobial resistance genes does not indicate susceptibility, as resistance may occur by other mechanisms.

Table 8. Possible Assay Results and Corresponding Staphylococcus and mecA Test Results

FilmArray BCID Test Results Staphylococcus 1/2 Assays Assay Saureus Assay mecA Description

Staphylococcus Not Detected Any No Staphylococcus species detected; Staphylococcus aureus Not Detected Negative Negative result mecA results not applicable mecA N/A

Staphylococcus Detected Staphylococcus species detected Staphylococcus aureus Not Detected Positive Negative Negative AND mecA Not Detected mecA not detected

Staphylococcus species (not S. aureus) Staphylococcus Detected detected Staphylococcus aureus Not Detected Positive Negative Positive AND mecA Detected mecA detectedb Staphylococcus aureus detected OR Staphylococcus Detected Staphylococcus aureus and one or Any Staphylococcus aureus Detected Positive Negative more other Staphylococcus species result mecA Not Detected detected AND mecA not detected Staphylococcus aureus detected OR Staphylococcus Detected Staphylococcus aureus and one or Any Staphylococcus aureus Detected Positive Positive more other Staphylococcus species result mecA Detected detected AND mecA detectedb a The BCID Panel mecA assay will detect the mecA gene from all known SCCmec types, including the recently described mecALGA251/mecC variant (SCCmec type XI). b The mecA gene may not be from the Staphylococcus (or S. aureus) that was detected or may be from only one of multiple Staphylococcus spp. that were detected in the culture. Subculturing and AST testing is required in order to assign a resistant and/or susceptible phenotype to each isolate recovered from the blood culture sample.

Table 9. Possible Assay Results and Corresponding vanA/B Test Results

B Assay B

FilmArray BCID Test Result Enterococcus Assay vanA Description

Enterococcus Not Detected No Enterococcus species detected; Negative Any result vanA/B N/A vanA/B results not applicable

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B Assay B

FilmArray BCID Test Result Enterococcus Assay vanA Description Enterococcus species detected Enterococcus Detected Positive Negative AND vanA/B Not Detected vanA/B not detected

Enterococcus species detected Enterococcus Detected Positive Positive AND vanA/B Detected vanA/B detecteda a Subculturing and AST testing is required in order to assign a resistant and/or susceptible phenotype to isolates recovered from the blood culture sample.

Table 10. Possible Assay Results and the Corresponding KPC Test Results

FilmArray BCID Test Results Abaumannii Assay Enterobacteriaceae Assays associated Assay Paeruiginosa Assay KPC Description Acinetobacter baumannii Not Detected No A. baumannii, Enterobacteriaceae Enterobacteriaceae Not Detected Negative Negative Negative Any result species or P. aeruginosa detected; Pseudomonas aeruginosa Not Detected KPC results not applicable KPC N/A A. baumannii detected; no Acinetobacter baumannii Detected Enterobacteriaceae species or P. Enterobacteriaceae Not Detected Positive Negative Negative Negative aeruginosa detected Pseudomonas aeruginosa Not Detected AND KPC Not Detected KPC not detectedb A. baumannii detected; no Acinetobacter baumannii Detected Enterobacteriaceae species or P. Enterobacteriaceae Not Detected Positive Negative Negative Positive aeruginosa detected Pseudomonas aeruginosa Not Detected AND KPC Detected KPC detectedc Acinetobacter baumannii Not Detected Enterobacteriaceae species detected; no Enterobacteriaceae Detected Any A. baumannii or P. aeruginosa detected Negative Negative Negative Pseudomonas aeruginosa Not Detected Positive AND KPC Not Detected KPC not detectedb

Acinetobacter baumannii Not Detected Enterobacteriaceae species detected; no Enterobacteriaceae Detected Any A. baumannii or P. aeruginosa detected Negative Negative Positive Pseudomonas aeruginosa Not Detected Positive AND KPC Detected KPC detectedc

Acinetobacter baumannii Not Detected P. aeruginosa detected; no A. baumannii Enterobacteriaceae Not Detected or Enterobacteriaceae species detected Negative Negative Positive Negative Pseudomonas aeruginosa Detected AND KPC Not Detected KPC not detectedb

Acinetobacter baumannii Not Detected P. aeruginosa detected; no A. baumannii Enterobacteriaceae Not Detected or Enterobacteriaceae species detected Negative Negative Positive Positive Pseudomonas aeruginosa Detected AND KPC Detected KPC detectedc a The BCID Panel KPC assay will detect types 2-13 of the blaKPC gene

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b When KPC is Not Detected, the following warning statement will be printed on the test report in red text ‘WARNING: A Not Detected result for the KPC gene does not indicate susceptibility to carbapenems. Gram negative bacteria can be resistant to carbapenems by mechanisms other than carrying the KPC gene.’ This special warning has been added to the report because there are multiple mechanisms for resistance to carbapenems. In particular, Acinetobacter baumannii, and Pseudomonas aeruginosa are commonly resistant to carbapenems, but only rarely carry the KPC gene. c The KPC gene detected may not be from the A. baumannii, Enterobacteriaceae, and/or P. aeruginosa that was detected or may be from only one of multiple applicable organisms detected. Subculturing and AST testing is required in order to assign a resistant and/or susceptible phenotype to each isolate recovered from the blood culture sample.

FilmArray BCID Test Report

The FilmArray BCID test report is automatically displayed upon completion of a run and contains three sections, the Run Summary, the Results Summary, and the Run Details (see the FilmArray Blood Culture Identification Panel Quick Guide to view an example of a test report). The test report can be saved as a PDF or printed. The Run Summary section of the test report provides the Sample ID, time and date of the run, control results and an overall summary of the test results. Any organism with a Detected result will be listed in the corresponding field of the summary. If all of the tests were negative then None will be displayed in the Detected field. Antimicrobial resistance genes with a result of Detected or Not Detected will be listed in the corresponding field of the summary. Controls are listed as Passed, Failed or Invalid. See the Controls Field section below for detailed information about the interpretation of controls and appropriate follow-up in the case of control failures. The Results Summary – Interpretations section of the test report lists the result for each target tested by the panel. Possible results for each organism are Detected, Not Detected, or Invalid. Possible results for antimicrobial resistance genes are Detected, Not Detected, N/A, or Invalid. See Results Summary section below for detailed information about interpretation of test results and appropriate follow-up for Invalid results. The Run Details section provides additional information about the run including: pouch information (type, lot number, and serial number), Run Status (Completed, Incomplete, Aborted, Instrument Error, Instrument Communication Error, or Software Error), the protocol that was used to perform the test, the identity of the operator that performed the test, and the instrument used to perform the test. Once a run has completed, it is possible to edit the Sample ID. If this information has been changed, an additional section called Change History will be added to the test report. This Change History section lists the field that was changed, the original entry, the revised entry, the operator that made the change, and the date that the change was made. Sample ID is the only field of the report that can be changed.

Controls Field

The Controls field on the test report will display Passed, Failed, or Invalid. The Controls field will display Passed only if the run completed successfully (no instrument or software errors) and both of the pouch control assays (DNA Process Control and PCR2 Control) were successful. The Controls field will display Failed if the run was completed successfully (no instrument or software errors) but one or both of the pouch control assays failed (0 or 1 positive replicates for either of the controls, each of which is tested in triplicate). If the control result is Failed, then the result for all of the tests on the panel are displayed as Invalid and the sample will need to be retested with a new pouch. Table 11 provides a summary and explanation of the possible control results and follow-up actions.

Table 11. Interpretation of Controls Field on the FilmArray BCID Test Report

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Control Result Explanation Action Required Outcome

Passed The run was successfully completed None Report the results provided on the test report. AND Both pouch controls were successful.

Failed The run was successfully completed Repeat the test using a new pouch. Accept the results of the repeat testing. If the error BUT persists, contact Technical At least one of the pouch controls Support for further instruction. failed.

Invalid The controls are invalid because the Note any error codes displayed Accept the valid results of the run did not complete. during the run and the Run Status repeat testing. If the error field in the Run Details section of the persists, contact Technical (Typically this indicates a software report. Refer to the FilmArray Support for further instruction. or hardware error). Operator’s Manual or contact Technical Support for further instruction.

Once the error is resolved, repeat the test or repeat the test using another instrument.

Results Summary – Interpretations

The Results Summary – Interpretations section provides a complete list of the test results. Possible results for each organism include Detected, Not Detected, and Invalid. Possible results for antimicrobial resistance genes are Detected, Not Detected, N/A or Invalid. Table 12 provides an explanation for each interpretation and any follow-up necessary to obtain a final result.

Table 12. Interpretation of Results on the FilmArray BCID Test Report

Result Explanation Action

Detected The run was successfully completed Report results. AND NOTE: If Detected results are reported for 3 or more The pouch controls were successful (Passed) organisms in a sample, a AND retest of the sample is recommended to confirm the The assay(s) for the organism (or antimicrobial resistance gene) were polymicrobial result. POSITIVE

Not Detected The run was successfully completed Report results. AND The pouch controls were successful (Passed) AND The assay(s) for the organism (or antimicrobial resistance gene) were NEGATIVE

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Result Explanation Action

Invalid The pouch controls were not successful (Failed) See Table 11 Interpretation of Controls Field on the OR FilmArray Test Report for The run did not complete successfully instruction. (Run Status displayed as: Aborted, Incomplete, Instrument Error, Software Error, or Instrument Communication Error)

N/A The run was successfully completed Report results. (Antimicrobial AND Resistance Genes only) The pouch controls were successful (Passed) AND The assay(s) for the organism(s) associated with the antimicrobial resistance gene were NEGATIVE so the results of the antimicrobial resistance gene are not applicable to the test results.

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LIMITATIONS OF THE PROCEDURE • In mixed cultures, the FilmArray BCID Panel may not identify all detectable organisms in the specimen, depending upon the concentration of each target present.

• The FilmArray BCID Panel may not distinguish mixed cultures when two or more species of the same genus or organism group are present in a specimen (e.g., S. aureus and S. epidermidis).

• For prescription use only.

• FilmArray BCID Panel performance has only been established on the FilmArray and FilmArray 2.0 systems.

• This test is a qualitative test and does not provide a quantitative value for the organism(s) in the sample.

• The performance of this test has only been evaluated with the BD BACTEC Plus Aerobic/F blood culture bottle.

• This product should not be used to test blood culture media that contain charcoal. Charcoal containing media may contain non-viable organisms and/or nucleic acid at levels that can be detected by the FilmArray BCID Panel.

• Antimicrobial resistance can occur via multiple mechanisms. A Not Detected result for the FilmArray antimicrobial resistance gene assays does not indicate antimicrobial susceptibility. Subculturing and standard susceptibility testing of isolates is required to determine antimicrobial susceptibility.

• The results for the FilmArray antimicrobial resistance gene assays do not specifically link the resistance gene to the associated organism. In mixed growth, the FilmArray BCID Panel does not specifically attribute vanA/B- mediated vancomycin resistance to a specific Enterococcus sp.; mecA-mediated methicillin resistance to either S. aureus or other Staphylococcus sp.; or KPC mediated carbapenem-resistance to Enterobacteriaceae, Acinetobacter baumannii or Pseudomonas aeruginosa.

• Tables 41, 46, and 49 present predicted reactivity as per in silico analysis of available sequences for several Staphylococcus spp., Streptococcus spp., and members of the Enterobacteriaceae family. As empirical testing of these organisms was not conducted, assay performance has not been established for the described organisms.

• The FilmArray BCID Panel does not contain assays for obligate anaerobic organisms that might be recovered in blood culture.

• Resin beads contained in blood culture media have been shown to cause pouch control failures and affect assay performance. Blood culture samples must be collected in a manner that prevents resin beads from being introduced into the sample and FilmArray test.

• Blood culture samples must be tested within 8 hours of being flagged as positive by a continuously monitoring blood culture instrument.

• This test has not been validated for testing samples other than positive blood culture samples that demonstrate the presence of organisms by Gram stain evaluation.

• Results from this test must be correlated with the clinical history, epidemiological data, and other data available to the clinician evaluating the patient.

• The FilmArray BCID Panel does not detect all species in the Enterobacteriaceae family. Morganella spp., Providencia spp., Rahnella spp., and Yersinia spp. will not be detected. See Organism Interpretation and Reactivity/Specificity sections of this document for additional information.

• Based on sequence analysis, the BCID Panel may not detect S. pneumoniae serotypes 11A and 19, or may detect these serotypes with reduced sensitivity compared to other serotypes.

• The FilmArray BCID Panel will not detect encapsulated Neisseria meningitidis containing the variant ctrA gene sequences described in the cited references [75-76].

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• The FilmArray BCID Panel does not detect all species of Enterococcus, Proteus, Staphylococcus or Streptococcus. See Organism Interpretation and Reactivity/Specificity sections of this document for additional information.

• The FilmArray BCID Enterococcus assay may cross-react with high level of coagulase-negative staphylococci (CoNS). Cross-reactivity may occur when select species of CoNS (S. haemolyticus, S. epidermidis, and S. capitis) are in the blood culture at high concentrations.

• Discrepancies between the BCID Panel test result and microbial identification may be caused by the inability to reliability differentiate species based on standard phenotypic microbial identification methods. Examples include Acinetobacter baumannii, Klebsiella oxytoca, and Raoultella ornithinolytica. See Organism Interpretation section of this document for specific examples.

• The detection of bacterial, yeast, and antimicrobial resistance gene nucleic acid is dependent upon proper sample collection, handling, transportation, storage, and preparation. Failure to observe proper procedures in any one of these steps can lead to incorrect results. There is a risk of false positive or false negative values resulting from improperly collected, transported, or handled samples.

• A negative FilmArray BCID result does not exclude the possibility of bloodstream infection. Negative test results may occur from sequence variants in the region targeted by the assay, the presence of inhibitors, technical error, sample mix-up, or an infection caused by an organism not detected by the panel. Test results may also be affected by concurrent antibacterial/antifungal therapy or levels of organism in the sample that are below the limit of detection for the test. Negative results should not be used as the sole basis for diagnosis, treatment, or other management decisions.

• Organism and amplicon contamination may produce erroneous results for this test. Particular attention should be given to the Laboratory Precautions noted under the Warnings and Precautions section.

• There is a risk of false positive values resulting from cross-contamination by target organisms, their nucleic acids or amplified product, or from non-specific signals in the assay.

• If three or more distinct organisms are detected in a specimen, retesting is recommended to confirm the polymicrobial result.

• Cross-reactivity with organisms other than those listed in the Organism Interpretation section above or the Analytical Specificity section below may lead to erroneous results.

• The effect of interfering substances has only been evaluated for those listed in the labeling. Interference by substances other than those described in the Interference section below could lead to erroneous results.

• Detection of the following organisms which carry the KPC gene was demonstrated in the inclusivity studies: Enterobacter cloacae, Enterobacter hormaechei, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, and Serratia marcescens. Acinetobacter baumannii, Pseudomonas aeruginosa, and other Enterobacteriaceae that carry the KPC gene were not evaluated but are expected to be detected based on in silico data.

• Performance of the FilmArray BCID Panel for Listeria monocytogenes was performed using seeded specimens only.

• S. aureus strains containing the mecC gene (previously mecALG251) will be reported as containing mecA.

• Several organisms were shown to cross-react with FilmArray BCID assays. Please refer to the Organism Interpretation and Reactivity/Specificity sections of this document for additional information.

• The resistance determinant vanM was shown to cross-react with the vanA/B assay.

• Inclusivity testing and in silico analyses demonstrated that the FilmArray BCID Panel may have reduced sensitivity for some organisms detected by genus or family levels assays. The following species may not be detected at the level of organism present at the time of bottle positivity, but can be detected at higher concentrations that may be present in a positive bottle up to 8 hours after positivity: Enterococcus dispar, Enterococcus saccharolyticus, Staphylococcus capitis, Staphylococcus microti, Staphylococcus pasteuri,

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Staphylococcus saprophyticus, Staphylococcus simiae, Staphylococcus simulans, Staphylococcus succinus, Staphylococcus warneri, Streptococcus parauberis, Edwardsiella tarda, Enterobacter gergoviae, Hafnia alvei, Pantoea spp., Salmonella bongori, Serratia fonticola, Serratia odorifera, and Serratia rubidaea.

• Borderline oxacillin-resistant Staphylococcus aureus (BORSA) and moderately-resistant S. aureus (MODSA) strains demonstrate reduced susceptibility to oxacillin due to hyperproduction of -lactamases or modification of penicillin binding proteins respectively. BORSA and MODSA strains do not contain the mecA gene. A mecA Not Detected result will be reported by the FilmArray BCID Panel for these strains.

• The vanA/B result is not reported in the absence of Enterococcus detection and will therefore not be reported for blood cultures containing vancomycin-resistant Staphylococcus aureus (VRSA).

• Detection of KPC types 2, 3, and 4 was demonstrated in the inclusivity studies. KPC types 5-13 were not tested but are expected to be detected based on in silico data.

• In the clinical study, limited numbers of positive specimens were tested for less common species of the following genera/family: Staphylococcus, Streptococcus, Enterococcus, Enterobacteriaceae, and Proteus. Refer to the inclusivity testing and in silico analyses sections for additional information and predicted detection for specific species.

• In rare cases, the Gram stain result and results of the FilmArray BCID Panel may be discrepant (for example, detection of a gram-positive cocci by FilmArray BCID when gram-positive cocci were not observed in the Gram stain). In these cases, the results should be used in conjunction with other clinical and laboratory findings.

• Blood culture media may contain non-viable organisms and/or nucleic acid at levels that can be detected by the FilmArray BCID Panel leading to false positive test results. Typically, these false positives will present with more than one positive result because the BCID Panel will also detect the organism that is growing in the culture bottle.

• There is an increased risk of false positive test results for Pseudomonas aeruginosa and Enterococcus when the FilmArray BCID Panel is used to test bioMérieux BacT/ALERT SN standard anaerobic blood culture bottles. If the FilmArray BCID Panel is used with these bottles, then positive results for Pseudomonas aeruginosa and Enterococcus should be confirmed by another method prior to reporting the test results. Typically, these false positives will present with more than one positive result because the BCID Panel will also detect the organism that is growing in the culture bottle.

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EXPECTED VALUES In the prospective arm of the FilmArray BCID clinical study, 1568 eligible blood cultures were collected and tested at seven of eight study sites across the United States over eight months (July 2012 – February 2013). The number and percentage of positive results as determined by FilmArray BCID, stratified by study site or age group, are presented in the following tables. Overall, FilmArray BCID detected at least one organism in 88.1% (1382/1568) of prospective positive blood cultures.

Table 13. Expected Value (as Determined by FilmArray BCID) Summary by Study Site for the Prospective Arm of the Clinical Evaluation FilmArray BCID Site 1 Site 2 Site 4 Site 5 Site 6 Site 7 Site 8 Total Result (n = 94) (n = 611) (n = 225) (n = 193) (n = 122) (n = 178) (n = 145) (n = 1568)

Gram-Positive Bacteria 4 32 17 15 6 16 12 102 Enterococcus (4%) (5%) (8%) (8%) (5%) (9%) (8%) (7%) 0 0 0 0 0 0 0 0 Listeria monocytogenes (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) 47 314 108 87 74 85 65 780 Staphylococcus (50%) (51%) (48%) (45%) (61%) (48%) (45%) (50%) 19 84 34 35 30 34 21 257 Staphylococcus aureus (20%) (14%) (15%) (18%) (25%) (19%) (14%) (16%) 13 51 22 12 7 14 21 140 Streptococcus (14%) (8%) (10%) (6%) (6%) (8%) (14%) (9%) 1 5 4 2 3 1 2 18 Streptococcus agalactiae (1%) (1%) (2%) (1%) (2%) (1%) (1%) (1%) 1 8 5 2 3 4 3 26 Streptococcus pneumoniae (1%) (1%) (2%) (1%) (2%) (2%) (2%) (2%) 2 3 0 0 0 1 2 8 Streptococcus pyogenes (2%) (0%) (0%) (0%) (0%) (1%) (1%) (1%) Gram-Negative Bacteria 0 9 3 0 0 3 1 16 Acinetobacter baumannii (0%) (1%) (1%) (0%) (0%) (2%) (1%) (1%) 14 120 33 51 22 36 31 307 Enterobacteriaceae (15%) (20%) (15%) (26%) (18%) (20%) (21%) (20%) 3 9 3 3 1 2 3 24 Enterobacter cloacae complex (3%) (1%) (1%) (2%) (1%) (1%) (2%) (2%) 8 53 17 21 15 22 13 149 Escherichia coli (9%) (9%) (8%) (11%) (12%) (12%) (9%) (10%) 2 0 0 3 0 1 0 6 Klebsiella oxytoca (2%) (0%) (0%) (2%) (0%) (1%) (0%) (0%) 1 30 7 16 2 7 11 74 Klebsiella pneumoniae (1%) (5%) (3%) (8%) (2%) (4%) (8%) (5%) 0 14 2 1 3 1 1 22 Proteus (0%) (2%) (1%) (1%) (2%) (1%) (1%) (1%) 0 8 4 3 1 2 4 22 Serratia marcescens (0%) (1%) (2%) (2%) (1%) (1%) (3%) (1%) 3 2 1 1 0 0 1 8 Haemophilus influenzae (3%) (0%) (0%) (1%) (0%) (0%) (1%) (1%) 0 0 0 0 0 1 0 1 Neisseria meningitidis (0%) (0%) (0%) (0%) (0%) (1%) (0%) (0%) 3 19 8 11 3 6 2 52 Pseudomonas aeruginosa (3%) (3%) (4%) (6%) (2%) (3%) (1%) (3%) Yeast 1 7 2 3 1 3 3 20 Candida albicans (1%) (1%) (1%) (2%) (1%) (2%) (2%) (1%) 0 2 2 7 0 1 2 14 Candida glabrata (0%) (0%) (1%) (4%) (0%) (1%) (1%) (1%) 0 0 1 1 0 2 0 4 Candida krusei (0%) (0%) (0%) (1%) (0%) (1%) (0%) (0%) 0 5 0 0 0 2 1 8 Candida parapsilosis (0%) (1%) (0%) (0%) (0%) (1%) (1%) (1%) 0 2 0 1 0 0 0 3 Candida tropicalis (0%) (0%) (0%) (1%) (0%) (0%) (0%) (0%)

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FilmArray BCID Site 1 Site 2 Site 4 Site 5 Site 6 Site 7 Site 8 Total Result (n = 94) (n = 611) (n = 225) (n = 193) (n = 122) (n = 178) (n = 145) (n = 1568)

Antimicrobial Resistance Genes 28 201 70 56 43 56 37 491 mecA (30%) (33%) (31%) (29%) (35%) (32%) (26%) (31%) 0 13 8 4 0 5 6 36 vanA/B (0%) (2%) (4%) (2%) (0%) (3%) (4%) (2%) 0 2 0 1 0 1 2 6 KPC (0%) (<1%) (0%) (1%) (0%) (1%) (1%) (<1%) Note: Antimicrobial resistance genes are only reported when an applicable organism is detected in the same specimen.

Table 14. Expected Value (as Determined by FilmArray BCID) Summary by Age Group for the Prospective Arm of the Clinical Evaluation

FilmArray BCID <1 1-17 18-44 45-64 65-84 85+ Result (n = 57) (n = 92) (n = 281) (n = 583) (n = 442) (n = 113)

Gram-Positive Bacteria Enterococcus 1 (2%) 4 (4%) 17 (6%) 42 (7%) 30 (7%) 8 (7%) Listeria monocytogenes 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) Staphylococcus 34 (60%) 40 (43%) 141 (50%) 304 (52%) 201 (45%) 60 (53%) Staphylococcus aureus 7 (12%) 18 (20%) 43 (15%) 110 (19%) 62 (14%) 17 (15%) Streptococcus 8 (14%) 13 (14%) 33 (12%) 44 (8%) 31 (7%) 11 (10%) Streptococcus agalactiae (GBS) 2 (4%) 0 (0%) 3 (1%) 8 (1%) 3 (1%) 2 (2%) Streptococcus pneumoniae 0 (0%) 3 (3%) 5 (2%) 9 (2%) 5 (1%) 4 (4%) Streptococcus pyogenes (GAS) 1 (2%) 1 (1%) 2 (1%) 2 (0%) 2 (0%) 0 (0%) Gram-Negative Bacteria Acinetobacter baumannii 0 (0%) 1 (1%) 2 (1%) 6 (1%) 6 (1%) 1 (1%) Enterobacteriaceae 13 (23%) 14 (15%) 50 (18%) 112 (19%) 102 (23%) 16 (14%) Enterobacter cloacae complex 2 (4%) 2 (2%) 6 (2%) 8 (1%) 6 (1%) 0 (0%) Escherichia coli 10 (18%) 6 (7%) 25 (9%) 50 (9%) 48 (11%) 10 (9%) Klebsiella oxytoca 0 (0%) 1 (1%) 1 (0%) 3 (1%) 1 (0%) 0 (0%) Klebsiella pneumoniae 0 (0%) 5 (5%) 11 (4%) 32 (5%) 23 (5%) 3 (3%) Proteus 0 (0%) 0 (0%) 2 (1%) 9 (2%) 7 (2%) 4 (4%) Serratia marcescens 0 (0%) 1 (1%) 2 (1%) 9 (2%) 9 (2%) 1 (1%) Haemophilus influenzae 1 (2%) 2 (2%) 0 (0%) 2 (0%) 2 (0%) 1 (1%) Neisseria meningitidis 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (1%) Pseudomonas aeruginosa 0 (0%) 4 (4%) 9 (3%) 15 (3%) 18 (4%) 6 (5%) Yeast Candida albicans 0 (0%) 1 (1%) 3 (1%) 11 (2%) 1 (0%) 4 (4%) Candida glabrata 0 (0%) 0 (0%) 2 (1%) 7 (1%) 4 (1%) 1 (1%) Candida krusei 0 (0%) 1 (1%) 0 (0%) 2 (0%) 1 (0%) 0 (0%) Candida parapsilosis 0 (0%) 0 (0%) 2 (1%) 3 (1%) 3 (1%) 0 (0%) Candida tropicalis 0 (0%) 0 (0%) 2 (1%) 1 (0%) 0 (0%) 0 (0%) Antimicrobial Resistance Genes

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FilmArray BCID <1 1-17 18-44 45-64 65-84 85+ Result (n = 57) (n = 92) (n = 281) (n = 583) (n = 442) (n = 113)

mecA 24 (42%) 22 (24%) 97 (35%) 175 (30%) 133 (30%) 40 (35%) vanA/B 0 (0%) 0 (0%) 9 (3%) 14 (2%) 10 (2%) 3 (3%) KPC 0 (0%) 0 (0%) 1 (<1%) 0 (0%) 5 (1%) 0 (0%) Note: Antimicrobial resistance genes are only reported when an applicable organism is detected in the same specimen.

The antimicrobial resistance genes are only reported when an applicable organism is detected in the same specimen. The following tables present the proportion of specimens positive for an applicable organism in which a positive antimicrobial resistance gene result was also reported. The data are stratified by the associated organism result, by study site, and by age group.

Table 15. Expected Values (as Determined by FilmArray BCID) Summary for Antimicrobial Resistance Gene Detections in Presumptive Association with Applicable Organism Results, Stratified by Study Site for the Prospective Arm of the Clinical Evaluation

Applicable Organism Result Site 1 Site 2 Site 4 Site 5 Site 6 Site 7 Site 8 Total

mecA – Methicillin Resistance Gene

4/19 53/84 20/34 17/35 14/30 19/34 10/21 137/257 Staphylococcus, S. aureus (21%) (63%) (59%) (49%) (47%) (56%) (48%) (53%)

Staphylococcus 24/28 148/230 50/74 39/52 29/44 37/51 27/44 354/523 (S. aureus Not Detected) (86%) (64%) (68%) (75%) (66%) (73%) (61%) (68%)

Total Staphylococcus 28/47 201/314 70/180 56/87 43/74 56/85 37/65 491/780 (with or without S. aureus result) (60%) (64%) (65%) (64%) (58%) (66%) (57%) (63%)

vanA/B – Vancomycin Resistance Gene

0/4 13/32 8/17 4/15 0/6 5/16 6/12 36/102 Total Enterococcus (0%) (41%) (47%) (27%) (0%) (31%) (50%) (35%)

KPC – Carbapenem Resistance Gene (carbapenemase)

Enterobacteriaceae a 0/14 2/120 0/33 1/51 0/22 2/36 2/31 6/307 (with or without specific genus/species (0%) (2%) (0%) (2%) (0%) (3%) (6%) (2%) results)

A. baumannii and/or P. aeruginosa 0/3 0/27 0/9 0/10 0/3 0/8 0/3 0/63 (Enterobacteriaceae Not Detected) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%)

Total Acinetobacter baumannii, 0/17 2/147 0/42 1/61 0/25 1/44 2/34 6/370 Enterobacteriaceae, Pseudomonas (0%) (1%) (0%) (2%) (0%) (2%) (6%) (2%) aeruginosa a a All 6 KPC detections were in association with K. pneumoniae.

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Table 16. Expected Values (as Determined by FilmArray BCID) Summary for Antimicrobial Resistance Gene Detections in Presumptive Association with Applicable Organism Results, Stratified by Age Group for the Prospective Arm of the Clinical Evaluation

Applicable Organism Result <1 1-17 18-44 45-64 65-84 85+

mecA – Methicillin Resistance Gene

Staphylococcus, S. aureus 0/7 (0%) 6/18 (33%) 26/43 (60%) 60/110 (55%) 33/62 (53%) 12/17 (71%)

Staphylococcus 24/27 (89%) 16/22 (73%) 71/98 (72%) 115/194 (59%) 100/140 (71%) 28/43 (65%) (S. aureus Not Detected)

Total Staphylococcus 24/34 (71%) 22/40 (55%) 97/141 (69%) 175/304 (58%) 133/202 (66%) 40/60 (67%)

vanA/B – Vancomycin Resistance Gene

Total Enterococcus 0/1 (0%) 0/4 (0%) 9/17 (53%) 14/42 (33%) 10/30 (33%) 3/8 (38%)

KPC – Carbapenem Resistance Gene (Carbapenemase) Enterobacteriaceae (with or without specific 0/13 (0%) 0/14 (0%) 1/50 (2%) 0/112 (0%) 5/102 (5%) 0/16 (0%) genus/species results) A. baumannii and/or P. aeruginosa 0/0 (0%) 0/4 (0%) 0/10 (0%) 0/21 (0%) 0/21 (0%) 0/7 (0%) (Enterobacteriaceae Not Detected)

Total Acinetobacter baumannii, Enterobacteriaceae, 0/13 (0%) 0/18 (0%) 1/60 (2%) 0/133 (0%) 5/123 (4%) 0/23 (0%) Pseudomonas aeruginosa a All 6 KPC detections were in association with K. pneumoniae.

In the prospective arm of the clinical evaluation, FilmArray BCID reported a total of 81 specimens with discernible multiple organism detections (5.2% of all prospective specimens; 81/1568). The expected values for each FilmArray BCID organism result in polymicrobial specimens (as determined by FilmArray BCID) are presented in the following table.

Table 17. Expected Values for Organism Results in Polymicrobial Specimens (as Determined by FilmArray BCID) in the Prospective Arm of the Clinical Evaluation

Number of Specimens Prevalence in Specimens FilmArray BCID with Multiple with Mixed Detections Organism Result Detections Containing (n = 81) Result

Enterobacteriaceae 17 21.0% Staphylococcus 17 21.0% Enterococcus 11 13.6% Staphylococcus aureus 9 11.1% Streptococcus 9 11.1% Candida albicans 7 8.6% Klebsiella pneumoniae 6 7.4% Escherichia coli 5 6.2% Pseudomonas aeruginosa 4 4.9% Candida glabrata 4 4.9% Proteus 4 4.9% Acinetobacter baumannii 3 3.7% Enterobacter cloacae complex 2 2.5% Streptococcus agalactiae 2 2.5% Candida krusei 1 1.2% Candida parapsilosis 1 1.2% Klebsiella oxytoca 1 1.2%

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Number of Specimens Prevalence in Specimens FilmArray BCID with Multiple with Mixed Detections Organism Result Detections Containing (n = 81) Result

Streptococcus pneumoniae 1 1.2% Candida tropicalis 0 0.0% Haemophilus influenzae 0 0.0% Neisseria meningitidis 0 0.0% Serratia marcescens 0 0.0% Listeria monocytogenes 0 0.0% Streptococcus pyogenes 0 0.0%

The reference method detected 201 off-panel organism isolates (i.e., those not targeted by FilmArray BCID) from the 1568 prospective cultures. The majority of these isolates belong to groups of organisms commonly considered to be blood culture contaminants (49 Corynebacterium/Diphtheroids, 33 Bacillus sp., and 27 Micrococcus sp., among others). Occurrence of off-panel organisms in the prospective arm of the clinical evaluation is presented in the following table.

Table 18. Occurrence of Off-Panel Organisms as Determined by Reference/Comparator Methods Number Number Off-Panel Organism Off-Panel Organism Identified Identified Abiotrophia sp. or Granulicatella sp. (formerly 7 Flavobacterium species 1 nutritionally-deficient Streptococci) Achromobacter xylosoxidans 1 Fusarium species 1 Acinetobacter sp. (not A. baumannii) 23 Kocuria kristinae 1 Actinomyces odontolyticus 2 Lactobacillus acidophilus 1 Actinomyces species 1 Lactobacillus species 2 Aerococcus species 1 Micrococcus luteus 1 Aerococcus viridans 2 Micrococcus luteus/lylae 1 Aeromonas sobria 1 Micrococcus species 25 Bacillus cereus 19 Moraxella catarrhalis 1 Bacillus pumilus 1 Moraxella osloensis 1 Bacillus species 13 Moraxella species 1 Brevibacterium species 1 Mycobacterium fortuitum complex 1 Brevibacterium ensei 1 Mycobacterium species 1 Brevundimonas diminuta 1 Neisseria species 2 Brevundimonas vesicularis 1 Paenibacillus species 1 Burkholderia cepacia complex 2 Pasteurella multocida 2 Candida kefyr 1 Pasteurella species 1 Capnocytophaga species 1 Propionibacterium species 1 Chryseobacterium meningosepticum 1 Pseudomonas fluorescens/putida 2 (Elizabethkingia/Flavobacterium) Chryseobacterium indologenes 1 Pseudomonas species 3 Chryseomonas luteola 1 Rhizobium radiobacter 2 Corynebacterium jeikeium 1 Rothia (Stomatococcus) mucilaginosa 4 Corynebacterium mucifaciens 1 Sphingomonas mucosissima 1 Corynebacterium species/Diphtheroids 47 Stenotrophomonas maltophilia 10

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Number Number Off-Panel Organism Off-Panel Organism Identified Identified Cryptococcus neoformans 2 Weeksella virosa 1

PERFORMANCE CHARACTERISTICS

Clinical Performance

The clinical performance of the FilmArray BCID Panel was established during a two-armed clinical study conducted at 8 U.S. clinical sites over an 8 month time period. The study included a prospective residual blood culture arm and a seeded blood culture arm. In the prospective arm, 1635 prospectively-collected residual blood culture samples (pediatric and adult) were initially included in the study. Only blood cultures collected in BD BACTEC Plus Aerobic/F blood culture bottles were included in the study. Sixty-seven (67) specimens were excluded from the study. The most common reasons for exclusion were that the specimens were >8 hours past positivity, incomplete reference/comparator data were provided, or the specimen was from a subject who had a previous specimen included in the study. In the seeded culture arm, analytes proven to be of low prevalence in the prospective arm were evaluated by seeding previously characterized isolates into blood culture bottles and incubating until positivity. A total of 716 seeded cultures (grown in BD BACTEC Plus Aerobic/F blood culture bottles) were initiated for the study. Seventy-seven (77) cultures were excluded from the study. The most common reasons for exclusion were that the specimens were >8 hours past positivity, the seeded culture was not called positive by the automated blood culture system, or the culture was contaminated or inconsistent with the intended seed organism. The final specimen set consisted of 2207 blood cultures (1568 prospective and 639 seeded). Table 19 provides a summary of demographic information for the 1568 specimens included in the prospective study. Table 19. Demographic Summary for Prospective Arm of FilmArray BCID Clinical Evaluation Prospective Study Specimens Total Specimens 1568 Sex Number of Specimens Male 917 (58%) Female 651 (42%) Age Group Number of Specimens ≤ 1 year 57 (4%) 1 - 17 years 92 (6%) 18 - 44 years 281 (18%) 45 - 64 years 583 (37%) 65 - 84 years 442 (28%) ≥ 85 years 113 (7%)

Positive blood cultures (prospective and seeded) were tested with the FilmArray BCID Panel. The performance of FilmArray BCID was evaluated by comparing the FilmArray BCID test result for each panel member with the appropriate comparator/reference methods shown in Table 20.

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Table 20. Reference/Comparator Methods used to Assess FilmArray BCID Performance Test Result Reference/Comparator Method(s) All organism detections Standard manual and automated microbiological/biochemical identification except Acinetobacter baumannii methods a Standard manual and automated microbiological/biochemical identification methods Acinetobacter baumannii detection Plus 16S PCR with bi-directional sequencing of all A. calcoaceticus-baumannii complex isolates for characterization as A. baumannii or non-A. baumannii a Method 1: PCR with bi-directional sequencing for specific resistance gene direct from blood culture b

Antimicrobial resistance gene detections Method 2: PCR with bi-directional sequencing for specific resistance gene from in specimens in which an associated organism was detected appropriate cultured isolates b (mecA from Staphylococcus; vanA/B from Enterococcus,

KPC from Enterobacteriaceae, Acinetobacter baumannii, and Informational: Standard manual and automated phenotypic antimicrobial Pseudomonas aeruginosa) susceptibility testing of appropriate cultured isolates (methicillin resistance, vancomycin resistance, and carbapenem resistance (and/or carbapenemase production) according to current CLSI criteria) c a Performance of FilmArray BCID detecting all organisms was compared to standard manual and automated microbiological/biochemical identification methods. Additionally, isolates identified as being members of the A. calcoaceticus-baumannii complex were subjected to 16S PCR and bi-directional sequencing to categorize the isolate as being A. baumannii or non-A. baumannii for final comparison to the FilmArray BCID A. baumannii-specific results. Positive results required a sequencing result of adequate quality to match sequences of A. baumannii or non-A. baumannii organisms deposited in the National Center for Biotechnology Information (NCBI) GenBank database (www.ncbi.nlm.nih.gov), with an acceptable E-value. This was required due to the inability of phenotypic identification methods to adequately discriminate between members of the A. calcoaceticus-baumannii complex. b Performance of FilmArray BCID detecting antimicrobial resistance genes (mecA, vanA/B, and KPC) was compared to gene-specific PCR tests with bidirectional sequencing. The assays were designed to amplify different sequences than those targeted by FilmArray BCID. Positive results required a sequencing result of adequate quality to match a sequence of the expect gene deposited in the National Center for Biotechnology Information (NCBI) GenBank database (www.ncbi.nlm.nih.gov), with an acceptable E-value. c Performance of FilmArray BCID as compared to phenotypic antimicrobial susceptibility testing was performed for informational purposes. The phenotypic methods were performed in accordance with current CLSI criteria [77].

A total of 2207 blood culture specimens (1568 prospective and 639 seeded) were evaluated in the FilmArray BCID clinical evaluation. Specimens were tested by the FilmArray BCID Panel either fresh or from frozen aliquots. A total of 1240 specimens were tested fresh (821 prospective and 419 seeded) and 967 specimens were tested frozen (747 prospective and 220 seeded). Clinical sensitivity or positive percent agreement (PPA) was calculated as 100% x (TP/TP + FN). True positive (TP) indicates that both FilmArray BCID and the reference/comparator method had a positive result for a specific analyte, and false negative (FN) indicates that the FilmArray BCID result was negative while the reference/comparator method was positive. Clinical specificity or negative percent agreement (NPA) was calculated as 100% x (TN/TN + FP). True negative (TN) indicates that both FilmArray BCID and the reference/comparator method had a negative result for a specific analyte, and false positive (FP) indicates that the FilmArray BCID result was positive while the reference/comparator method was negative. The exact binomial two-sided 95% confidence interval was calculated. The results are summarized in Tables 21 – 25.

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Table 21. FilmArray BCID Clinical Performance Summary – Gram-Positive Organism Results (Comparator Method: Standard Manual/Automated Microbiological/Biochemical Identification) Sensitivity/PPA a Specificity/NPA a Gram-Positive Bacteria TP/TP + FN % 95% CI TN/TN + FP % 95% CI Prospective Fresh 55/55 100 93.5-100 762/766 99.5 98.7-99.9 Prospective Frozen 43/46 93.5 82.1-98.6 701/701 100 99.5-100 Enterococcus Seeded Fresh 12/12 100 73.5-100 407/407 100 99.1-100 Seeded Frozen 17/17 100 80.5-100 203/203 100 98.2-100 Overall 127/130 97.7 93.4-99.5 2073/2077 b 99.8 99.5-99.9 Prospective Fresh 0/0 - - 821/821 100 99.6-100 Prospective Frozen 0/0 - - 747/747 100 99.5-100 Listeria Seeded Fresh 23/23 100 85.2-100 396/396 100 99.1-100 monocytogenes Seeded Frozen 13/13 100 75.3-100 207/207 100 98.2-100 Overall 36/36 100 90.3-100 2171/2171 100 99.8-100 Prospective Fresh 405/418 96.9 94.7-98.3 401/403 99.5 98.2-99.9 Prospective Frozen 364/379 96.0 93.6-97.8 359/368 97.6 95.4-98.9 Staphylococcus Seeded Fresh 0/0 - - 418/419 99.8 98.7-100 Seeded Frozen 1/1 100 2.5-100 219/219 100 98.3-100 Overall 770/798 c 96.5 95.0-97.7 1397/1409 c 99.1 98.5-99.6 Prospective Fresh 133/136 97.8 93.7-99.5 685/685 100 99.5-100 Prospective Frozen 120/121 99.2 95.5-100 622/626 99.4 98.4-99.8 Staphylococcus Seeded Fresh 0/0 - - 419/419 100 99.1-100 aureus Seeded Frozen 0/0 - - 220/220 100 98.3-100 Overall 253/257 d 98.4 96.1-99.6 1946/1950 d 99.8 99.5-99.9 Prospective Fresh 73/77 94.8 87.2-98.6 740/744 99.5 98.6-99.9 Prospective Frozen 63/64 98.4 91.6-100 683/683 100 99.5-100 Streptococcus Seeded Fresh 18/18 100 81.5-100 401/401 100 99.1-100 Seeded Frozen 44/44 100 92.0-100 175/176 99.4 96.9-100 Overall 198/203 97.5 94.3-99.2 1999/2004 e 99.8 99.4-99.9 Prospective Fresh 8/8 100 63.1-100 813/813 100 99.5-100 Streptococcus Prospective Frozen 10/10 100 69.2-100 737/737 100 99.5-100 agalactiae Seeded Fresh 3/3 100 29.2-100 416/416 100 99.1-100 (Group B) Seeded Frozen 15/15 100 78.2-100 205/205 100 98.2-100 Overall 36/36 100 90.3-100 2171/2171 100 99.8-100 Prospective Fresh 15/15 100 78.2-100 805/806 99.9 99.3-100 Prospective Frozen 10/10 100 69.2-100 737/737 100 99.5-100 Streptococcus Seeded Fresh 4/5 80.0 28.4-99.5 413/414 99.8 98.7-100 pneumoniae Seeded Frozen 7/7 100 59.0-100 213/213 100 98.3-100 Overall 36/37 97.3 85.8-99.9 2168/2170 99.9 99.7-100 Prospective Fresh 5/5 100 47.8-100 815/816 99.9 99.3-100 Streptococcus Prospective Frozen 2/2 100 15.8-100 745/745 100 99.5-100 pyogenes Seeded Fresh 9/9 100 66.4-100 410/410 100 99.1-100 (Group A) Seeded Frozen 22/22 100 84.6-100 198/198 100 98.2-100 Overall 38/38 100 90.7-100 2168/2169 99.9 99.7-100 a Sensitivity and Specificity refer to performance with the prospective specimens only; Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) refer to performance with the seeded specimens. b 3/4 false positive Enterococcus specimens contained Staphylococcus; the false positive results may be due to cross-reactivity. c Isolates from 16/28 false negative Staphylococcus specimens were identified as the newly described species S. pettenkoferi by bi-directional sequencing. Bidirectional sequencing confirmed the presence of Staphylococcus in 10/12 false positive specimens; 2 were S. aureus, 6 were S. epidermidis, and 1 was S. haemolyticus. d Bidirectional sequencing identified 2 isolates from S. aureus false negative specimens as S. hominis and S. epidermidis; they were not S. aureus. Bidirectional sequencing confirmed the presence of S. aureus in 1/4 false positive specimens. One false positive and one false negative S. aureus were consecutively tested specimens and may be due to sample mix-up. e Bidirectional sequencing confirmed the presence of S. mitis in 1/5 false positive Streptococcus specimens.

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Table 22. FilmArray BCID Clinical Performance Summary – Gram-Negative Organism Results (Comparator Method: Standard Manual/Automated Microbiological/Biochemical Identification plus 16S Sequencing for Speciation for A. baumannii) Sensitivity/PPA a Specificity/NPA a Gram-Negative Bacteria TP/TP + FN % 95% CI TP/TP + FP % 95% CI Prospective Fresh 7/7 100 59.0-100 813/814 99.9 99.3-100 Prospective Frozen 7/7 100 59.0-100 739/740 99.9 99.2-100 Acinetobacter Seeded Fresh 20/20 100 83.2-100 397/399 99.5 98.2-99.9 baumannii Seeded Frozen 17/17 100 80.5-100 202/203 99.5 97.3-100 Overall 51/51 100 93.0-100 2151/2156 b 99.8 99.5-99.9 Prospective Fresh 153/156 98.1 94.5-99.6 665/665 100 99.4-100 Prospective Frozen 150/154 97.4 93.5-99.3 589/593 99.3 98.3-99.8 Enterobacteriaceae Seeded Fresh 93/93 100 96.1-100 326/326 100 98.9-100 Seeded Frozen 94/95 98.9 94.3-100 125/125 100 97.1-100 Overall 490/498 c 98.4 96.9-99.3 1705/1709 c 99.8 99.4-99.9 Prospective Fresh 10/11 90.9 58.7-99.8 809/810 99.9 99.3-100 Prospective Frozen 11/11 100 71.5-100 734/736 99.7 99.0-100 Enterobacter cloacae Seeded Fresh 8/8 100 63.1-100 411/411 100 99.1-100 complex Seeded Frozen 9/9 100 66.4-100 211/211 100 98.3-100 Overall 38/39 97.4 86.5-99.9 2165/2168 99.9 99.6-100 Prospective Fresh 77/79 97.5 91.2-99.7 742/742 100 99.5-100 Prospective Frozen 68/69 98.6 92.2-100 674/678 99.4 98.5-99.8 Escherichia coli Seeded Fresh 4/4 100 39.8-100 414/415 99.8 98.7-100 Seeded Frozen 1/1 100 2.5-100 219/219 100 98.3-100 Overall 150/153 d 98.0 94.4-99.6 2049/2054 d 99.8 99.4-99.9 Prospective Fresh 4/4 100 39.8-100 817/817 100 99.5-100 Prospective Frozen 1/2 50 1.3-98.7 744/745 99.9 99.3-100 Klebsiella oxytoca Seeded Fresh 32/36 88.9 73.9-96.9 383/383 100 99.0-100 Seeded Frozen 22/22 100 84.6-100 198/198 100 98.2-100 Overall 59/64 e 92.2 82.7-97.4 2142/2143 99.9 99.7-100 Prospective Fresh 33/34 97.1 84.7-99.9 786/787 99.9 99.3-100 Prospective Frozen 35/37 94.6 81.8-99.3 705/710 99.3 98.4-99.8 Klebsiella Seeded Fresh 13/13 100 75.3-100 403/406 99.3 97.9-99.8 pneumoniae Seeded Frozen 21/21 100 83.9-100 199/199 100 98.2-100 Overall 102/105 f 97.1 91.9-99.4 2093/2102 f 99.6 99.2-99.8 Prospective Fresh 11/11 100 71.5-100 810/810 100 99.5-100 Prospective Frozen 11/11 100 71.5-100 736/736 100 99.5-100 Proteus Seeded Fresh 2/2 100 15.8-100 417/417 100 99.1-100 Seeded Frozen 15/15 100 78.2-100 205/205 100 98.2-100 Overall 39/39 100 91.0-100 2168/2168 100 99.8-100 Prospective Fresh 14/14 100 76.8-100 807/807 100 99.5-100 Prospective Frozen 8/8 100 63.1-100 739/739 100 99.5-100 Serratia marcescens Seeded Fresh 28/28 100 87.7-100 390/391 99.7 98.6-100 Seeded Frozen 26/27 96.3 81.0-99.9 193/193 100 98.1-100 Overall 76/77 g 98.7 93.0-100 2129/2130 g 99.9 99.7-100 Prospective Fresh 5/5 100 47.8-100 816/816 100 99.5-100 Prospective Frozen 3/3 100 29.2-100 744/744 100 99.5-100 Haemophilus Seeded Fresh 29/29 100 88.1-100 390/390 100 99.1-100 influenzae Seeded Frozen 6/6 100 54.1-100 214/214 100 98.3-100 Overall 43/43 100 91.8-100 2164/2164 100 99.8-100 Prospective Fresh 1/1 100 2.5-100 820/820 100 99.6-100 Prospective Frozen 0/0 - - 747/747 100 99.5-100 Neisseria Seeded Fresh 30/30 100 88.4-100 389/389 100 99.1-100 meningitidis Seeded Frozen 5/5 100 47.8-100 215/215 100 98.3-100 Overall 36/36 100 90.3-100 2171/2171 100 99.8-100 Prospective Fresh 19/19 100 82.4-100 802/802 100 99.5-100 Prospective Frozen 32/33 97 84.2-99.9 713/714 99.9 99.2-100 Pseudomonas Seeded Fresh 0/0 - - 419/419 100 99.1-100 aeruginosa Seeded Frozen 0/0 - - 220/220 100 98.3-100 Overall 51/52h 98.1 89.7-100 2154/2155 99.9 99.7-100 a Sensitivity and Specificity refer to performance with the prospective specimens only; Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) refer to performance with the seeded specimens. b Bidirectional sequencing identified isolates from 4 false positive specimens as A. pittii (genomospecies 3); this species cross-reacts with the A. baumannii assay. These four isolates were identified as A. baumannii by phenotypic methods. 6 other isolates originally identified as A. baumannii by

BioFire Diagnostics, LLC FilmArray BCID Panel CE-IVD Instruction Booklet 45

phenotypic methods were identified by bidirectional sequencing as A. nosocomialis (genomospecies 13; 4 isolates), A. bereziniae, and A. radioresistens; these 6 isolates did not cross-react with the A. baumannii assay. c One false positive and one false negative Enterobacteriaceae were consecutively tested specimens and may be due to sample mix-up. One isolate from another false negative specimen, identified as E. coli by phenotypic methods, was identified as Pasteurella, and not E. coli, by bidirectional sequencing. d One false positive and one false negative E. coli were consecutively tested specimens and may be due to sample mix-up. e Bidirectional sequencing identified 4/5 isolates from false negative K. oxytoca specimens as the closely related species, Raoultella ornithinolytica, and not K. oxytoca. The misidentification is a known limitation of phenotypic testing methods for this species. f The isolate from one false negative K. pneumoniae specimen was identified as the closely related organism, Raoultella planticola and not K. pneumoniae. 6/9 false positive K. pneumoniae results appear to be due to cross-reactivity with Enterobacter aerogenes and Raoultella ornithinolytica (misidentified as K. oxytoca by phenotypic methods). g Bidirectional sequencing identified the isolate from the one false negative S. marcescens specimen as being in the S. proteomaculans/grimesii group and not S. marcescens. The one false positive S. marcescens result appears to be due to cross-reactivity with Raoultella ornithinolytica (misidentified as K. oxytoca by phenotypic methods). h Bidirectional sequencing identified the isolate from the one false negative P. aeruginosa specimen as the closely related species Pseudomonas stutzeri and not P. aeruginosa.

Table 23. FilmArray BCID Clinical Performance Summary – Yeast Organism Results (Comparator Method: Standard Manual/Automated Microbiological/Biochemical Identification) Sensitivity/PPA a Specificity/NPA a Yeast TP/TP + FN % 95% CI TN/TN + FP % 95% CI Prospective Fresh 12/12 100 73.5-100 808/809 99.9 99.3-100 Prospective Frozen 4/4 100 39.8-100 740/743 99.6 98.8-99.9 Candida albicans Seeded Fresh 47/47 100 92.5-100 372/372 100 99.0-100 Seeded Frozen 1/1 100 2.5-100 219/219 100 98.3-100 Overall 64/64 100 94.4-100 2139/2143 99.8 99.5-99.9 Prospective Fresh 6/6 100 54.1-100 813/815 99.8 99.1-100 Prospective Frozen 6/6 100 54.1-100 741/741 100 99.5-100 Candida glabrata Seeded Fresh 32/32 100 89.1-100 387/387 100 99.1-100 Seeded Frozen 5/5 100 47.8-100 215/215 100 98.3-100 Overall 49/49 100 92.7-100 2156/2158 99.9 99.7-100 Prospective Fresh 2/2 100 15.8-100 819/819 100 99.6-100 Prospective Frozen 2/2 100 15.8-100 745/745 100 99.5-100 Candida krusei Seeded Fresh 28/28 100 87.7-100 391/391 100 99.1-100 Seeded Frozen 5/5 100 47.8-100 215/215 100 98.3-100 Overall 37/37 100 90.5-100 2170/2170 100 99.8-100 Prospective Fresh 3/3 100 29.2-100 818/818 100 99.6-100 Prospective Frozen 4/4 100 39.8-100 742/743 99.9 99.3-100 Candida Seeded Fresh 47/49 95.9 86.0-99.5 370/370 100 99.0-100 parapsilosis Seeded Frozen 5/5 100 47.8-100 214/215 99.5 97.4-100 Overall 59/61 b 96.7 88.7-99.6 2144/2146 99.9 99.7-100 Prospective Fresh 0/0 - - 821/821 100 99.6-100 Prospective Frozen 3/3 100 29.2-100 744/744 100 99.5-100 Candida tropicalis Seeded Fresh 31/31 100 88.8-100 388/388 100 99.1-100 Seeded Frozen 5/5 100 47.8-100 215/215 100 98.3-100 Overall 39/39 100 91.0-100 2168/2168 100 99.8-100 a Sensitivity and Specificity refer to performance with the prospective specimens only; Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) refer to performance with the seeded specimens. b Bidirectional sequencing identified the isolates from the two false negative C. parapsilosis specimens as being the closely related species C. metapsilosis. This misidentification is a known limitation of phenotypic identification methods.

Comparator testing for the antimicrobial resistance genes was performed with both the blood culture sample and with isolates recovered from the blood culture. The results are presented in Tables 24 and 25 below. The NPA for mecA and vanA/B are lower when comparing to PCR/sequencing from bacterial isolates than to PCR/sequencing direct from blood culture primarily due to the reference methods not isolating a resistant clone of an applicable organism. This may be due to heterogeneous resistance within a population of cultured organisms or co-culturing of multiple indistinguishable applicable organisms with different resistance profiles (e.g., culturing a resistant Staphylococcus along with a sensitive Staphylococcus).

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Table 24. FilmArray BCID Clinical Performance Summary – Antimicrobial Resistance Genes (Comparator Method: PCR/Sequencing Direct from Blood Culture) Sensitivity /PPA a Specificity /NPA a Antimicrobial Resistance Genes TP/TP + FN % 95% CI TN/TN + FP % 95% CI mecA - Methicillin Resistance Gene Prospective Fresh 253/257 98.4% 96.1-99.6% 147/150 98.0% 94.3-99.6% mecA Prospective Frozen 233/237 98.3% 95.7-99.5% 134/136 98.5% 94.8-99.8% All Staphylococcus Seeded Fresh 1/1 100% n/a 0/0 - - Detected Seeded Frozen 1/1 100% n/a 0/0 - - Overall 488/496 98.4% 96.8-99.3% 281/286 98.3% 96.0-99.4% Prospective Fresh 67/69 97.1% 89.9-99.6% 64/64 100% 94.4-100% mecA Prospective Frozen 70/70 100% 94.9-100% 54/54 100% 93.4-100% Staphylococcus Seeded Fresh 0/0 - - 0/0 - - Detected; Seeded Frozen 0/0 - - 0/0 - - S. aureus Detected Overall 137/139 98.6% 94.9-99.8% 118/118 100% 96.9-100% mecA Prospective Fresh 186/188 98.9% 96.2-99.9% 83/86 96.5% 90.1-99.3% Staphylococcus Prospective Frozen 163/167 97.6% 94.0-99.3% 80/82 97.6% 91.5-99.7% Detected; Seeded Fresh 1/1 100% n/a 0/0 - - S. aureus Seeded Frozen 1/1 100% n/a 0/0 - - Not Detected Overall 351/357 98.3% 96.4-99.4% 163/168 97.0% 93.2-99.0% vanA/B - Vancomycin Resistance Genes Prospective Fresh 23/23 100% 85.2-100% 36/36 100% 90.3-100% vanA/B Prospective Frozen 13/13 100% 75.3-100% 30/30 100% 88.4-100% Enterococcus Seeded Fresh 12/12 100% 73.5-100% 0/0 - - Detected Seeded Frozen 16/16 100% 79.4-100% 1/1 100% n/a Overall 64/64 100% 94.4-100% 67/67 100% 94.6-100% KPC - Carbapenem Resistance Gene (Carbapenemase) KPC Prospective Fresh 3/3 100% 29.2-100% 177/177 100% 97.9-100% Enterobacteriaceae Prospective Frozen 3/3 100% 29.2-100% 187/187 100% 98.0-100% and/or Seeded Fresh 10/10 100% 69.2-100% 105/105 100% 96.5-100% A. baumannii and/or Seeded Frozen 23/23 100% 85.2-100% 89/89 100% 95.9-100% P. aeruginosa Detected Overall 39/39 100% 91.0-100% 558/558 100% 99.3-100% Prospective Fresh 3/3 100% 29.2-100% 150/150 100% 97.6-100% KPC Prospective Frozen 3/3 100% 29.2-100% 151/151 100% 97.6-100% Enterobacteriaceae Seeded Fresh 10/10 100% 69.2-100% 83/83 100% 95.7-100% Detected Seeded Frozen 23/23 100% 85.2-100% 71/71 100% 94.9-100% Overall 39/39 100% 91.0-100% 455/455 100% 99.2-100% KPC Prospective Fresh 0/0 - - 27/27 100% 87.4-100% Enterobacteriaceae Prospective Frozen 0/0 - - 36/36 100% 90.3-100% Not Detected; Seeded Fresh 0/0 - - 22/22 100% 84.6-100% A. baumannii and/or Seeded Frozen 0/0 - - 18/18 100% 81.5-100% P. aeruginosa Detected Overall 0/0 - - 103/103 100% 96.5-100% a Sensitivity and Specificity refer to performance with the prospective specimens only; Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) refer to performance with the seeded specimens.

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Table 25. FilmArray BCID Clinical Performance Summary – Antimicrobial Resistance Genes (Comparator Method: PCR/Sequencing of Cultured Isolates) Positive Percent Agreement Negative Percent Agreement a ANTIMICROBIAL RESISTANCE GENES TP/TP + FN % 95% CI TN/TN + FP % 95% CI mecA - Methicillin Resistance Gene Prospective Fresh 234/236 99.2% 97.0-99.9% 149/172 86.7% 80.6-91.3% mecA Prospective Frozen 219/222 98.6% 96.1-99.7% 135/151 89.4% 83.4-93.8% All Staphylococcus Seeded Fresh 0/0 - - 0/0 - - Detected Seeded Frozen 1/1 100% n/a 0/0 - - Overall 454/459 98.9% 97.5-99.6% 284/323 87.9% 83.9-91.3% Prospective Fresh 64/65 98.5% 91.7-100% 65/68 95.6% 87.6-99.1% mecA Prospective Frozen 66/66 100% 94.6-100% 54/58 93.1% 83.3-98.1% Staphylococcus Seeded Fresh 0/0 - - 0/0 - - Detected; Seeded Frozen 0/0 - - 0/0 - - S. aureus Detected Overall 130/131 99.2% 95.8-100% 119/126 94.4% 88.9-97.7% mecA Prospective Fresh 170/171 99.4% 96.8-100% 84/104 80.8% 71.9-87.8% Staphylococcus Prospective Frozen 153/156 98.1% 94.5-99.6% 81/93 87.1% 78.6-93.2% Detected; Seeded Fresh 0/0 - - 0/0 - - S. aureus Seeded Frozen 1/1 100% n/a 0/0 - - Not Detected Overall 324/328 98.8% 96.9-99.7% 165/197 83.8% 77.9-88.6% vanA/B - Vancomycin Resistance Genes Prospective Fresh 20/20 100% 83.2-100% 36/39 92.3% 79.1-98.4% vanA/B Prospective Frozen 12/12 100% 73.5-100% 30/31 96.8% 83.3-99.9% Enterococcus Seeded Fresh 12/12 100% 73.5-100% 0/0 - - Detected Seeded Frozen 16/16 100% 79.4-100% 1/1 100% n/a Overall 60/60 100% 94.0-100% 67/71 94.4% 86.2-98.4% KPC - Carbapenem Resistance Gene (Carbapenemase) KPC Prospective Fresh 3/3 100% 29.2-100% 177/177 100% 97.9-100% Enterobacteriaceae Prospective Frozen 3/3 100% 29.2-100% 187/187 100% 98.1-100% and/or Seeded Fresh 10/10 100% 69.2-100% 105/105 100% 96.5-100% A. baumannii and/or Seeded Frozen 23/23 100% 85.2-100% 89/89 100% 95.9-100% P. aeruginosa Detected Overall 39/39 100% 91.0-100% 558/558 100% 99.3-100% Prospective Fresh 3/3 100% 29.2-100% 151/151 100% 97.6-100% KPC Prospective Frozen 3/3 100% 29.2-100% 152/152 100% 97.6-100% Enterobacteriaceae Seeded Fresh 10/10 100% 69.2-100% 83/83 100% 95.7-100% Detected Seeded Frozen 23/23 100% 85.2-100% 71/71 100% 94.9-100% Overall 39/39 100% 91.0-100% 457/457 100% 99.2-100% KPC Prospective Fresh 0/0 - - 26/26 100% 86.8-100% Enterobacteriaceae Prospective Frozen 0/0 - - 35/35 100% 90.0-100% Not Detected; Seeded Fresh 0/0 - - 22/22 100% 84.6-100% A. baumannii and/or Seeded Frozen 0/0 - - 18/18 100% 81.5-100% P. aeruginosa Detected Overall 0/0 - - 101/101 100% 96.4-100% a Isolates for 12 Staphylococci, 4 Enterococci, and 7 Enterobacteriaceae/A. baumannii/P. aeruginosa did not grow from the subcultured blood culture and could therefore not be tested with the PCR/bi-directional sequencing comparator method. These blood cultures were considered negative for the antimicrobial resistance genes by comparator method, and FilmArray performance has been calculated as True Negative (when FilmArray is negative for the analyte) or False Positive (when FilmArray is positive for the analyte) for each of these isolates.

Performance of FilmArray BCID as compared to phenotypic antimicrobial susceptibility testing (AST) results was calculated for informational purposes. Results stratified by AST method are presented in Tables 26-28. Some PPA are lower when comparing results from bacterial isolates than to PCR/sequencing direct from blood culture because phenotypic AST testing is capable of detecting antimicrobial resistance due to mechanisms other than acquisition of mecA, vanA/B, or KPC.

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Table 26. mecA Performance – Comparison to Phenotypic Antimicrobial Susceptibility Testing (AST) Methods Note: AST results were not provided for several isolates. Positive Percent Agreement Negative Percent Agreement PHENOTYPIC METHODS TP/TP + FN % (95%CI) TN/TN + FP % (95%CI) Cefoxitin Disc Diffusion 22/22 100% 15/15 100% Chromogenic Agar 42/46 91.3% 25/32 78.1% Prospective Automated Antimicrobial All Staphylococcus 366/380 96.3% 226/262 86.3% Susceptibility Testing 96.0% 86.1% All Methods 430/448 266/309 (93.7 - 97.6%) (81.7 - 89.7%) Chromogenic Agar 10/11 90.9% 8/8 100% Prospective Automated Antimicrobial 117/119 98.3% 108/112 96.4% Staphylococcus, Susceptibility Testing S. aureus Detected 97.7% 96.7% All Methods 127/130 116/120 (93.4 - 99.5%) (91.7 - 99.1%) Seeded Automated Antimicrobial 1/1 100% 0/0 - Staphylococcus Susceptibility Testing

Table 27. vanA/B Performance – Comparison to Phenotypic Vancomycin AST Methods Positive Percent Agreement Negative Percent Agreement PHENOTYPIC METHODS TP/TP + FN % (95%CI) TN/TN + FP % (95%CI) Vancomycin Screen Agar 3/3 100% 5/5 100% Vancomycin Disc Diffusion 0/1 0.0% - - Prospective Automated Antimicrobial Enterococcus 29/30 96.7% 55/58 94.8% Susceptibility Testing 94.1% 95.2% All Methods 32/34 a 60/63 (80.3 - 99.3%) (86.7 - 99.0%) Vancomycin Disc Diffusion 14/14 100% 1/1 100% Seeded Vancomycin Screen Agar 14/14 100% - - Enterococcus 100% All Methods 28/28 1/1 100% (n/a) (87.7 - 100%)

Combined Prospective and 96.8% 95.3% All Methods 60/62 a 61/64 Seeded (88.8 - 99.6%) (86.9 - 99.0% Enterococcus a Two isolates (one E. gallinarum and one E. faecalis) that were vancomycin resistant by phenotypic AST testing were negative for the vanA/B genes by bi-directional sequence analysis.

Table 28. KPC Performance – Comparison to Phenotypic Carbapenem AST Methods Note: AST results were not provided for several isolates. Note: Acinetobacter baumannii and Pseudomonas aeruginosa are commonly resistant to carbapenems due to mechanisms other than acquisition of the KPC gene (blaKPC). These bacteria very rarely carry the KPC gene. Positive Percent Agreement Negative Percent Agreement PHENOTYPIC METHODS TP/ TN/ % (95%CI) % (95%CI) TP + FN TN + FP Prospective Automated Antimicrobial 0/10 0% 4/4 100% A. baumannii Susceptibility Testing Seeded Meropenem Disc Diffusion 0/30 0% 9/9 100% A. baumannii 100% A. baumannii – All Methods 0/40 0% (n/a) 13/13 (75.3-100%) Automated Antimicrobial 0/10 0% 32/32 100% Susceptibility Testing Prospective Meropenem Disc Diffusion - - 6/6 100% P. aeruginosa Meropenem/Ertapenem Disc 0/1 0% 2/2 100% Diffusion

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Positive Percent Agreement Negative Percent Agreement PHENOTYPIC METHODS TP/ TN/ % (95%CI) % (95%CI) TP + FN TN + FP 100% P. aeruginosa – All Methods 0/11 0% (n/a) 40/40 (91.2-100%) Prospective Automated Antimicrobial 6/6 100% 64/64 100% K. pneumoniae Susceptibility Testing Meropenem Disc Diffusion 19/19 100% 1/1 100% Seeded Modified Hodge Test K. pneumoniae 11/11 100% 1/1 100% (Meropenem) 100% 100% K. pneumoniae – All Methods 36/36 66/66 (90.3-100%) (94.6-100%) Prospective Automated Antimicrobial - - 22/22 100% E. cloacae Susceptibility Testing Automated Antimicrobial - - 3/3 100% Susceptibility Testing Seeded Meropenem Disc Diffusion 0/1 0% - - E. cloacae Modified Hodge Test 2/2 100% 11/11 100% (Meropenem) 66.7% 100% E. cloacae – All Methods 2/3 a 36/36 (9.4-99.2%) (90.3-100%) Prospective Automated Antimicrobial - - 144/144 100% E. coli Susceptibility Testing Seeded Modified Hodge Test 1/1 100% 4/4 100% E. coli (Meropenem) 100% E. coli – All Methods 1/1 100% (n/a) 148/148 (97.5-100%) Prospective Automated Antimicrobial - - 21/21 100% P. mirabilis Susceptibility Testing Meropenem Disc Diffusion - - 4/4 100% Seeded Modified Hodge Test P. mirabilis 0/1 0% 11/11 100% (Meropenem) 100% P. mirabilis – All Methods 0/1 a 0% (n/a) 36/36 (90.3-100%) Prospective Automated Antimicrobial All Other - - 43/43 100% Susceptibility Testing Enterobacteriaceae Automated Antimicrobial - - 42/42 100% Seeded Susceptibility Testing All Other Meropenem Disc Diffusion - - 13/13 100% Enterobacteriaceae Modified Hodge Test - - 61/61 100% (Meropenem) 100% All Other Enterobacteriaceae – All Methods - - 159/159 (97.7-100%) a Two isolates (one E. cloacae and one P. mirabilis) that were carbapenem resistant by phenotypic AST testing were negative for the KPC gene by bidirectional sequence analysis.

FilmArray BCID reports genus or family level results for Enterococcus, Staphylococcus, Streptococcus, Enterobacteriaceae, and Proteus. Standard identification methods identified various genera/species within each of these groups during the clinical evaluation. Stratification of performance by species within the groups is presented below.

Table 29. Stratification of Enterococcus Clinical Performance by Species (Comparator Method: Standard Manual/Automated Microbiological/Biochemical Identification) Positive Agreement Enterococcus species Prospective Seeded E. avium 2/2 (100%) - E. casseliflavus 1/2 (50%) 1/1 (100%) E. durans 1/1 (100%) - E. faecalis 55/56 (98.2%) 8/8 (100%) E. faecalis + E. faecium 1/1 (100%) - E. faecium 36/37 (97.3%) 9/9 (100%) E. gallinarum 2/2 (100%) 1/1 (100%)

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Positive Agreement Enterococcus species Prospective Seeded Enterococcus sp. (not speciated) - 10/10 (100%) 98/101 (97.0%) 29/29 (100%) Overall Enterococcus 95%CI = 91.6-99.4% 95%CI = 88.1-100%

Table 30. Stratification of Staphylococcus Clinical Performance by Species (Comparator Method: Standard Manual/Automated Microbiological/Biochemical Identification) Positive Agreement Staphylococcus species Prospective Seeded S. aureus 256/257 (99.6%) - S. auricularis 0/1 (0%) - S. capitis 15/17 (88.2%) - S. capitis + S. epidermidis 1/1 (100%) - S. capitis + S. hominis 1/1 (100%) - S. capitis + S. lugdunensis 1/1 (100%) - S. carnosus 0/1 (0%) - S. cohnii 1/1 (100%) - S. cohnii + S. hominis 1/1 (100%) - S. epidermidis 200/201 (99.5%) 1/1 (100%) S. epidermidis + S. hominis 4/4 (100%) - S. epidermidis + Staphylococcus sp. 2/2 (100%) - (not speciated) S. haemolyticus 19/19 (100%) - S. haemolyticus + S. hominis 1/1 (100%) - S. hominis 65/65 (100%) - S. hominis + Staphylococcus sp. (not 1/1 (100%) - speciated) S. intermedius 2/2 (100%) - S. intermedius + Staphylococcus sp. 1/1 (100%) - (not speciated) S. lentus 1/1 (100%) - S. lugdunensis 5/5 (100%) - S. saprophyticus 2/2 (100%) - S. sciuri 0/1 (0%) - S. simulans 3/3 (100%) - S. warneri 4/5 (80%) - Staphylococcus sp. (not speciated)a 180/200 (90%) - 769/797 (96.5%) 1/1 (100%) Overall Staphylococcus 95%CI = 95.0-97.7% 95%CI = n/a a Of the 20 unspeciated staphylococci not detected by FilmArray BCID, 16 were identified as S. pettenkoferi, 2 as S. epidermidis, 1 as S. capitis, and 1 as S. caprae by 16S sequence analysis. The 180 unspeciated Staphylococci that were detected by FilmArray BCID were not sequenced.

Table 31. Stratification of Streptococcus Clinical Performance by Species (Comparator Method: Standard Manual/Automated Microbiological/Biochemical Identification) Positive Agreement Streptococcus species Prospective Seeded Group A (Pyogenic) S. pyogenes 7/7 (100%) 31/31 (100%) Group B (Pyogenic) S. agalactiae 18/18 (100%) 18/18 (100%) Group C/G (Pyogenic) S. canis 1/1 (100%) - S. equi/S. dysgalactiae 1/1 (100%) - Streptococcus group C 2/2 (100%) - Streptococcus group G 2/2 (100%) - Group D (Bovis Group) S. bovis 3/3 (100%) - S. equinus 1/1 (100%) - Group F (Anginosus Group)

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Positive Agreement Streptococcus species Prospective Seeded S. anginosus 4/4 (100%) - S. anginosus group 1/1 (100%) - S. intermedius 3/3 (100%) - S. constellatus 2/2 (100%) - Mitis Group S. gordonii 1/1 (100%) - S. mitis 8/9 (88.9%) - S. mitis + viridans streptococci 1/1 (100%) - S. mitis/S. oralis 2/2 (100%) - S. mitis/S. oralis + viridans 1/1 (100%) - streptococci S. oralis 5/5 (100%) - S. parasanguinis 1/1 (100%) - S. parasanguinis + viridans 1/1 (100%) - streptococci S. pneumoniae 25/25 (100%) 12/12 (100%) S. sanguinis 2/2 (100%) - Salivarius Group S. salivarius 1/2 (50%) - S. salivarius + S. sanguinis group 1/1 (100%) - Other S. vestibularis 1/1 (100%) - Viridans streptococci 40/43 (93.0%) 1/1 (100%) (not further speciated) Streptococcus sp. (not speciated) 1/1 (100%) - 136/141 (96.5) 62/62 (100%) Overall Streptococcus 95%CI = 91.9-98.8% 95%CI = 94.2-100%

Table 32. Stratification of Enterobacteriaceae Clinical Performance by Genus/Species. (Comparator Method: Standard Manual/Automated Microbiological/Biochemical Identification) Positive Agreement Enterobacteriaceae genus/species Prospective Seeded Citrobacter freundii 2/2 (100%) - Citrobacter freundii + Escherichia coli 1/1 (100%) - Citrobacter koseri 1/2 (50%) - Enterobacter aerogenes 5/5 (100%) 2/2 (100%) Enterobacter aerogenes + Klebsiella 1/1 (100%) - oxytoca Enterobacter cloacae 19/19 (100%) 17/17 (100%) Enterobacter cloacae complex 3/3 (100%) - Enterobacter gergoviae 1/1 (100%) - Enterobacter sakasaki 1/1 (100%) - Enterobacter sp. 1/1 (100%) - Escherichia coli 141/144 (98%) 5/5 (100%) Escherichia coli + Klebsiella 2/2 (100%) - pneumoniae Escherichia coli + Providencia stuartii a 1/1 (100%) - Escherichia hermannii 1/1 (100%) - Klebsiella oxytoca 5/5 (100%) 58/58 (100%) Klebsiella pneumoniae 67/68 (99%) 34/34 (100%) Klebsiella pneumoniae + Pantoea 1/1 (100%) - agglomerans Leclercia adacarboxylata 1/1 (100%) - Morganella morganii b + Proteus 1/1 (100%) - mirabilis Pantoea agglomerans 1/1 (100%) - Pantoea sp. 0/2 (0%) - Proteus mirabilis 21/21 (100%) 15/15 (100%) Proteus vulgaris - 2/2 (100%) Salmonella group B 1/1 (100%) - Salmonella group C 1/1 (100%) -

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Positive Agreement Enterobacteriaceae genus/species Prospective Seeded Salmonella sp. 1/1 (100%) - Salmonella typhi 1/1 (100%) - Serratia marcescens 22/22 (100%) 54/55 (98%) 303/310 (97.7%) 187/188 (99.5%) Overall Enterobacteriaceae 95%CI = 95.4-99.1% 95%CI = 97.1-100% a FilmArray BCID does not detect Providenicia stuartii; the positive Enterobacteriaceae result is likely due to the presence of Escherichia coli in the blood culture. b FilmArray BCID does not detect Morganella morganii; the positive Enterobacteriaceae result is likely due to the presence of Proteus mirabilis in the blood culture.

Table 33. Stratification of Proteus Clinical Performance by Species. (Comparator Method: Standard Manual/Automated Microbiological/Biochemical Identification) Positive Agreement Proteus species Prospective Seeded Proteus mirabilis 22/22 (100%) 15/15 (100%) Proteus vulgaris - 2/2 (100%) 22/22 (100%) 17/17 (100%) Overall Proteus 95%CI = 84.6-100% 95%CI = 80.5-100%

FilmArray BCID reported a total of 81 prospective specimens with discernible multiple organism detections (5.2% of all prospective specimens; 81/1568). The majority of multiple detections (74/81; 91.3%) contained two discernible organisms, while 6.2% (5/81) contained three discernible organisms, and 2.5% (2/81) contained four discernible organisms. The most prevalent multiple detection was Enterococcus with Staphylococcus (S. aureus not detected) (1.3% of all specimens; 20/1568). Out of the 81 polymicrobial specimens, 29 contained one or more analytes that had not been detected with the reference/comparator methods, i.e., discrepant result.

Table 34. Discernible Multiple Detection Combinations as Determined by FilmArray BCID

Distinct Multiple Detection Combinations Discrepant Result(s) as Determined by FilmArray BCID (Organism Not Detected by

Discrepant Discrepant Specimens Reference Method) Organism 1 Results Organism 2 Results Organism 3 Results Organism 4 Results

Total Specimens Total Enterobacter cloacae Klebsiella Escherichia coli, Klebsiella oxytoca, E. cloacae, E. coli, K. complex, pneumoniae, 1 1 Enterobacteriaceae Enterobacteriaceae oxytoca Enterobacteriaceae Enterobacteriaceae Candida albicans Candida glabrata Staphylococcus Streptococcus 1 1 C. albicans Candida albicans Candida parapsilosis Enterococcus 1 1 C. parapsilosis Staphylococcus Pseudomonas Enterococcus aureus, 1 0 aeruginosa Staphylococcus Proteus, Enterococcus Staphylococcus 1 0 Enterobacteriaceae Enterococcus Staphylococcus Streptococcus 1 1 Streptococcus Candida albicans Staphylococcus Streptococcus 1 0 Streptococcus Staphylococcus agalactiae, 1 0 Streptococcus Staphylococcus Proteus, Staphylococcus, aureus, 1 1 Enterobacteriaceae S. aureus Staphylococcus Staphylococcus Streptococcus aureus, agalactiae, 1 0 Staphylococcus Streptococcus Staphylococcus Streptococcus Streptococcus, aureus, pneumoniae, 1 1 S. pneumoniae Staphylococcus Streptococcus

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Distinct Multiple Detection Combinations Discrepant Result(s) as Determined by FilmArray BCID (Organism Not Detected by

Discrepant Discrepant Specimens Reference Method) Organism 1 Results Organism 2 Results Organism 3 Results Organism 4 Results

Total Specimens Total Staphylococcus Escherichia coli, aureus, 3 0 Enterobacteriaceae Staphylococcus Staphylococcus Staphylococcus, Enterococcus aureus, 3 1 S. aureus Staphylococcus Staphylococcus Candida albicans aureus, 1 1 C. albicans Staphylococcus Staphylococcus Acinetobacter aureus, 1 0 baumannii Staphylococcus Staphylococcus Pseudomonas aureus, 1 1 P. aeruginosa aeruginosa Staphylococcus Staphylococcus aureus, Streptococcus 4 0 Staphylococcus Escherichia coli, Enterobacteriaceae, E. Enterococcus 1 1 Enterobacteriaceae coli Acinetobacter Klebsiella pneumoniae, 2 1 A. baumannii baumannii Enterobacteriaceae Enterobacter cloacae Klebsiella pneumoniae, 1 1 E. cloacae complex complex Enterobacteriaceae Klebsiella pneumoniae, K. pneumoniae, Enterococcus 3 1 Enterobacteriaceae Enterobacteriaceae Klebsiella pneumoniae, Escherichia coli, E. coli, 5 3 Enterobacteriaceae Enterobacteriaceae K. pneumoniae (2) Proteus, Candida glabrata 1 1 C. glabrata Enterobacteriaceae Proteus, Enterococcus 1 1 Enterobacteriaceae Staphylococcus (3), Enterococcus Staphylococcus 20 6 Enterococcus (3) Pseudomonas Staphylococcus 1 1 Staphylococcus aeruginosa Escherichia coli, Streptococcus 2 1 Streptococcus Enterobacteriaceae Klebsiella pneumoniae, Streptococcus 1 0 Enterobacteriaceae Staphylococcus Streptococcus 7 0 Candida albicans Enterococcus 2 0 Candida krusei Enterococcus 1 0

Candida glabrata Enterococcus 1 0

Enterococcus Staphylococcus 1 0 C. glabrata Candida albicans Candida glabrata 1 1 C. albicans Candida albicans Enterococcus 1 1

Enterobacteriaceae Enterococcus 1 0 Acinetobacter Pseudomonas 2 0 baumannii aeruginosa Pseudomonas Enterobacteriaceae 1 0 aeruginosa Staphylococcus Enterobacteriaceae Staphylococcus 1 1

Total Specimens with Multiple Detections 81 29

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Table 35. Additional Specimens with Multiple Isolates Identified by Reference/Comparator Methods Note: Organisms shaded gray are not targeted by FilmArray BCID (i.e., off-panel organisms). This list does not include multiple detection combinations already represented in the previous table of FilmArray BCID multiple detections.

Discrepant Result(s) Distinct Multiple Detections by Reference/Comparator methods (Targeted Organisms Not Detected by

Discrepant Discrepant Specimens FilmArray BCID) Isolate 1 Isolate 2 Isolate 3 Isolate 4

Total Specimens Total Pseudomonas Aeromonas sobria Pantoea agglomerans Pantoea agglomerans 1 0 aeruginosa Enterococcus faecalis Flavobacterium species Klebsiella pneumoniae Staphylococcus species 1 1 Staphylococcus Staphylococcus, Klebsiella pneumoniae Staphylococcus species Staphylococcus species Viridans streptococci 1 1 Streptococcus Neisseria species Viridans streptococci Viridans streptococci Viridans streptococci 1 0 Corynebacterium Staphylococcus Acinetobacter lwoffii 1 0 species epidermidis Coryneform bacterium Staphylococcus aureus Streptococcus oralis 1 0 species Enterococcus Escherichia coli Staphylococcus aureus 1 1 Enterococcus casseliflavus Streptococcus Klebsiella pneumoniae Klebsiella pneumoniae 1 0 mitis/oralis Staphylococcus Pantoea species Staphylococcus species 1 1 Enterobacteriaceae intermedius Staphylococcus Streptococcus Staphylococcus aureus 1 0 haemolyticus parasanguis Staphylococcus Staphylococcus Staphylococcus capitis 1 0 epidermidis lugdunensis Streptococcus Viridans streptococci Viridans streptococci 1 0 mitis/oralis Viridans streptococci Viridans streptococci Viridans streptococci 1 0 Abiotrophia defectiva Staphyloccocus species 1 1 Staphylococcus Acinetobacter Acinetobacter baumannii (seq. = A. baumannii (seq. = A. 1 0 nosocomialis/calcoaceti nosocomialis/calcoaceti cus) cus) Acinetobacter lwoffi Klebsiella pneumoniae 1 0 Acinetobacter lwoffi Viridans streptococci 1 1 Streptococcus Acinetobacter lwoffi Staphylococcus species 1 1 Staphylococcus K. pneumoniae, Aerococcus viridans Klebsiella pneumoniae 1 1 Enterobacteriaceae Staphylococcus Aerococcus species 1 1 Staphylococcus epidermidis Pseudomonas Bacillus pumilus 1 0 fluorescens/putida Brevundimonas Weeksella virosa 1 0 diminuta Candida parapsilosis Kocuria kristinae 1 0 Citrobacter freundii Escherichia coli 1 0 Citrobacter koseri Enterococcus faecium 1 0 Corynebacterium Corynebacterium 1 0 jeikeium species Corynebacterium Corynebacterium 1 0 species species Corynebacterium Enterococcus faecalis 1 0 species

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Discrepant Result(s) Distinct Multiple Detections by Reference/Comparator methods (Targeted Organisms Not Detected by

Discrepant Discrepant Specimens FilmArray BCID) Isolate 1 Isolate 2 Isolate 3 Isolate 4

Total Specimens Total Corynebacterium Micrococcus species 1 0 species Corynebacterium Staphylococcus aureus 2 0 species Corynebacterium Staphylococcus 2 0 species haemolyticus Corynebacterium Staphylococcus hominis 2 0 species Corynebacterium Staphylococcus species 3 1 Staphylococcus species Diphtheroids Staphylococcus species 1 0 Enterobacter aerogenes Klebsiella oxytoca 1 0 Enterococcus faecalis Enterococcus faecium 1 0 Stenotrophomonas Enterococcus faecalis 1 0 maltophilia Enterococcus faecalis Viridans streptococci 1 1 Enterococcus Enterococcus faecium Enterococcus faecium 1 0 Escherichia coli Escherichia coli 3 0 E. coli, Escherichia coli Pasteurella multocida 1 1 Enterobacteriaceae Escherichia coli Providencia stuartii 1 0 Stenotrophomonas Escherichia coli 1 0 maltophilia Haemophilus influenzae Moraxella catarrhalis 1 0 Klebsiella pneumoniae Pantoea agglomerans 1 1 K. pneumoniae Lactobacillus Streptococcus species 1 0 acidophilus Staphylococcus Micrococcus species 1 0 epidermidis Morganella morganii Proteus mirabilis 1 0 Neisseria species Staphylococcus hominis 1 0 Rhodococcus species Staphylococcus warneri 1 1 Staphylococcus Staphylococcus aureus Staphylococcus aureus 2 0 Staphylococcus aureus Staphylococcus caprae 1 0 Staphylococcus aureus Staphylococcus species 2 0 Streptococcus Staphylococcus aureus 1 1 Streptococcus salivarius Staphylococcus capitis Staphylococcus capitis 1 0 Staphylococcus Staphylococcus capitis 1 0 epidermidis Staphylococcus capitis Staphylococcus hominis 1 0 Streptococcus Staphylococcus capitis 1 1 Staphylococcus pneumoniae Staphylococcus cohnii Staphylococcus hominis 1 0 Staphylococcus Staphylococcus hominis 4 0 epidermidis Staphylococcus Staphylococcus species 2 0 epidermidis Staphylococcus Staphylococcus hominis 1 0 haemolyticus Staphylococcus hominis Staphylococcus hominis 1 0 Staphylococcus hominis Staphylococcus species 1 0

Staphylococcus species Staphylococcus species 3 0 Stenotrophomonas Staphylococcus species 1 0 maltophilia Streptococcus Viridans streptococci 1 0 parasanguinis Streptococcus Streptococcus sanguis 1 0 salivarius group

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Discrepant Result(s) Distinct Multiple Detections by Reference/Comparator methods (Targeted Organisms Not Detected by

Discrepant Discrepant Specimens FilmArray BCID) Isolate 1 Isolate 2 Isolate 3 Isolate 4

Total Specimens Total

Viridans streptococci Streptococcus mitis 1 0

Viridans streptococci Viridans streptococci 3 0 Total 86 16

A total of 2207 specimens were tested in the clinical evaluation, of which 99% (2185/2207) were successful on the first test. Six (6) initial tests were incomplete due to instrument/software errors (5 tests) or a user aborted run (1 test). Sixteen (16) of the completed runs were invalid due to a pouch control failure. Valid results were achieved after a single retest for the 22 incomplete/invalid specimens, resulting in a final successful testing rate of 100%.

Growth and Detection

Blood cultures should be processed and tested with the FilmArray BCID Panel as soon as possible after being indicated as positive for growth by a continuously monitoring blood culture system and gram stain. However, samples can be tested up to 8 hours after bottle positivity. This study established the range of expected organism concentrations in blood cultures that would be tested with the FilmArray BCID Panel, from the time of positivity up to 8 hours after positivity.

All organism growth and testing was performed using seeded blood culture bottles (BACTEC™ Plus Aerobic/F Medium) incubated in the BACTEC™ 9050 continuously monitoring blood culture instrument. Each microorganism was mixed with human whole blood and seeded directly into blood culture bottles for growth. At the time of positivity (and/or eight hours after positivity), the blood culture was removed from the instrument for plate enumeration (determination of CFU/mL) and FilmArray BCID testing. Three independent positive cultures (bottles) were evaluated for each organism at each time point and FilmArray testing was performed in triplicate for each bottle.

Table 36 summarizes the concentration of organism (CFU/mL) determined for a representative panel of 30 isolates. The number and percent of correct positive BCID Panel test results is provided for each isolate and overall (% Detected). A correct result means that both the correct organism and antimicrobial resistance gene (where applicable) were detected in the sample. The correct organism and antimicrobial resistance gene results were reported for all 540 samples tested (540/540, 100%).

Table 36. Summary of Organism Concentration (CFU/mL) in Positive Blood Cultures and Correct Detection of Organisms in Positive Blood Cultures by the FilmArray BCID Panel At Positivity 8 Hours After Positivity Per Bottle Mean # Detected/Total Per Bottle Mean # Detected/Total Species/Isolate(s) Tested (CFU/mL) (CFU/mL) (% Detected) (CFU/mL) (CFU/mL) (% Detected) Gram-Positive Bacteria 4.60E+08 7.25E+08 Enterococcus faecalis [vanB] 9/9 9/9 1.80E+08 3.01E+08 8.90E+08 8.95E+08 JMI 368 (100%) (100%) 2.62E+08 1.07E+09 1.47E+08 2.23E+08 Enterococcus faecium [vanA] 9/9 9/9 1.53E+08 1.53E+08 1.64E+08 1.81E+08 JMI 475 (100%) (100%) 1.59E+08 1.55E+08 1.26E+08 8.00E+08 Enterococcus hirae 9/9 9/9 2.76E+08 2.42E+08 6.60E+08 7.27E+08 ATCC 49135 (100%) (100%) 3.25E+08 7.20E+08 Listeria monocytogenes 4.50E+08 9.50E+08 9/9 1.76E+09 1.91E+09 9/9

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At Positivity 8 Hours After Positivity Per Bottle Mean # Detected/Total Per Bottle Mean # Detected/Total Species/Isolate(s) Tested (CFU/mL) (CFU/mL) (% Detected) (CFU/mL) (CFU/mL) (% Detected) CDC F2380 (ATCC 43256) 1.22E+09 (100%) 2.31E+09 (100%) 1.18E+09 1.67E+09 1.48E+08 8.75E+08 Staphylococcus aureus 9/9 9/9 2.00E+07 6.45E+07 9.80E+08 6.59E+08 ATCC 11632 (100%) (100%) 2.56E+07 1.21E+08 1.41E+07 5.70E+07 Staphylococcus aureus [MRSA/mecA] 9/9 9/9 5.65E+06 8.60E+06 3.85E+07 6.43E+07 ATCC BAA-1747 (100%) (100%) 6.05E+06 9.75E+07 1.38E+08 2.12E+08 Staphylococcus epidermidis 9/9 9/9 9.85E+07 1.18E+08 3.95E+08 7.22E+08 ATCC 12228 (100%) (100%) 1.16E+08 1.56E+09 3.60E+07 1.35E+09 Staphylococcus epidermidis [MRSE/mecA] 9/9 9/9 3.75E+07 7.65E+07 6.80E+08 1.44E+09 ATCC 29887 (100%) (100%) 1.56E+08 2.29E+09 4.50E+08 3.15E+08 Streptococcus agalactiae 9/9 9/9 1.22E+08 4.96E+08 5.80E+08 4.42E+08 ATCC 13813 (100%) (100%) 9.15E+08 4.30E+08 1.57E+08 1.50E+09 Streptococcus mitis 9/9 9/9 1.51E+09 7.86E+08 2.03E+09 2.15E+09 ATCC 15914 (100%) (100%) 6.90E+08 2.91E+09 3.45E+08 1.03E+09 Streptococcus pneumoniae 9/9 9/9 2.67E+08 6.41E+08 6.00E+08 1.00E+09 ATCC BAA-255 (100%) (100%) 1.31E+09 1.37E+09 2.53E+08 2.38E+08 Streptococcus pyogenes 9/9 9/9 2.44E+08 2.92E+08 5.70E+08 5.66E+08 ATCC 19615 (100%) (100%) 3.80E+08 8.90E+08 Gram-Negative Bacteria 2.17E+08 4.85E+08 Acinetobacter baumannii 9/9 9/9 1.44E+08 2.02E+08 3.85E+08 4.35E+08 ATCC 9955 (100%) (100%) 2.45E+08 4.35E+08 4.20E+08 2.23E+09 Enterobacter cloacae 9/9 9/9 3.95E+08 3.22E+08 1.46E+09 1.96E+09 ATCC 13047 (100%) (100%) 1.50E+08 2.19E+09 9.80E+07 1.17E+09 Escherichia coli 9/9 9/9 6.10E+07 1.17E+08 1.39E+09 8.79E+08 ATCC 43888 (100%) (100%) 1.93E+08 7.70E+07 7.40E+08 3.05E+09 Klebsiella oxytoca 9/9 9/9 6.85E+08 6.03E+08 1.86E+09 2.04E+09 ATCC 13182 (100%) (100%) 3.85E+08 1.20E+09 6.15E+07 1.96E+09 Klebsiella oxytoca [KPC] 9/9 9/9 9.15E+07 6.12E+07 2.00E+09 1.70E+09 JMI 7818 (100%) (100%) 3.05E+07 1.13E+09 4.35E+08 1.60E+09 Klebsiella pneumoniae 9/9 9/9 2.10E+08 5.20E+08 1.65E+09 1.61E+09 ATCC 13883 (100%) (100%) 9.15E+08 1.58E+09 1.21E+08 1.14E+09 Klebsiella pneumoniae [KPC] 9/9 9/9 2.50E+08 1.92E+08 9.10E+08 9.40E+08 JMI 766 (100%) (100%) 2.05E+08 7.70E+08 3.25E+07 1.04E+09 Proteus mirabilis 9/9 9/9a 1.04E+08 7.58E+07 9.80E+08 9.17E+08 ATCC 29906 (100%) (100%) 9.10E+07 7.30E+08 8.35E+08 1.05E+09 Serratia marcescens 9/9 9/9 1.46E+09 9.28E+08 1.37E+09 1.15E+09 ATCC 27137 (100%) (100%) 4.90E+08 1.02E+09 4.90E+08 2.19E+09 Serratia marcescens [KPC] 9/9 9/9 3.90E+08 3.27E+08 1.40E+09 1.28E+09 JMI 697 (100%) (100%) 1.02E+08 2.42E+08 2.80E+08 3.25E+09 Haemophilus influenzae (type b) 9/9 9/9 3.60E+08 2.88E+08 3.35E+09 3.11E+09 ATCC 10211 (100%) (100%) 2.23E+08 2.74E+09 Neisseria meningitidis 2.07E+08 9/9 6.65E+08 9/9 2.51E+08 7.38E+08 ATCC 43744 3.90E+08 (100%) 7.65E+08 (100%)

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At Positivity 8 Hours After Positivity Per Bottle Mean # Detected/Total Per Bottle Mean # Detected/Total Species/Isolate(s) Tested (CFU/mL) (CFU/mL) (% Detected) (CFU/mL) (CFU/mL) (% Detected) 1.55E+08 7.85E+08 1.34E+08 1.35E+09 Pseudomonas aeruginosa 9/9 9/9 1.76E+08 1.36E+08 1.39E+08 1.08E+09 ATCC 27853 (100%) (100%) 9.75E+07 1.76E+09 Yeast 9.05E+03 8.80E+04 Candida albicans 9/9 9/9 8.00E+04 3.12E+04 1.03E+05 9.70E+04 ATCC 10231 (100%) (100%) 4.65E+03 1.00E+05 1.26E+06 1.47E+07 Candida glabrata 9/9 9/9 1.11E+06 1.45E+06 2.65E+07 2.01E+07 ATCC 15545 (100%) (100%) 1.97E+06 1.91E+07 5.65E+06 2.68E+07 Candida krusei 9/9 9/9 2.47E+06 4.82E+06 3.55E+07 3.16E+07 ATCC 90878 (100%) (100%) 6.35E+06 3.25E+07 2.56E+06 6.70E+07 Candida parapsilosis 9/9 9/9 3.60E+06 3.12E+06 3.80E+07 5.35E+07 ATCC 90875 (100%) (100%) 3.20E+06 5.55E+07 1.50E+06 1.10E+07 Candida tropicalis 9/9 9/9 7.45E+05 9.70E+05 2.04E+07 1.36E+07 ATCC 66029 (100%) (100%) 6.65E+05 9.45E+06 Overall Correct Detectiona 270/270 8 Hours After 270/270 (Organism and Antimicrobial At Positivity: (100%) Positivity: (100%) Resistance Genes) a In addition to the correct results, 5 false positive results (Streptococcus, Streptococcus agalactiae, Haemophilus influenzae, Neisseria meningitidis, and Candida krusei) were observed in a single run (1/540; 0.2%). The correct results were obtained when the sample was retested.

Analytical Reactivity (Inclusivity)

The analytical reactivity (inclusivity) of the FilmArray BCID Panel was evaluated with a collection of 303 bacterial and yeast isolates that represent the diversity of the FilmArray BCID Panel analytes, including antimicrobial resistance genes. Isolates were selected to represent relevant subspecies or serotypes and selection was biased toward more common species and known human pathogens. When possible, in silico analysis of sequence data was used to make predictions of assay reactivity for less common species that were not tested but that may be detected by the FilmArray BCID Panel. Each isolate was initially tested in blood culture matrix at a concentration consistent with the levels of organism enumerated from blood cultures at the time of positivity (see Growth and Detection section above). If the expected result was obtained at the initial test level, no further testing was performed. If an isolate was not detected initially, additional testing was performed at 10-100 fold higher concentrations. If detected at the higher concentration(s), the species/isolate is indicated as detected with reduced sensitivity and the concentration of organism that was detected is indicated. If not detected at the highest concentration the isolate is listed as not detected by the FilmArray BCID Panel. Results are provided below for each FilmArray BCID Panel test result.

Enterococcus

Table 37. Results of Enterococcus Inclusivity Testing Enterococcus Detected with Enterococcus Detected Enterococcus Reduced Sensitivity [~1x108 CFU/mL] Not Detecteda [~1x109 CFU/mL] Enterococcus Enterococcus Enterococcus ATCC 49463 ATCC 43076 ATCC 49372 avium saccharolyticus pseudoavium

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Enterococcus Detected with Enterococcus Detected Enterococcus Reduced Sensitivity [~1x108 CFU/mL] Not Detecteda [~1x109 CFU/mL] Enterococcus Enterococcus Enterococcus ATCC 700668 ATCC 51266 ATCC 49427 casseliflavus dispar raffinosus Enterococcus ATCC 43198 cecorum Enterococcus ATCC 11576 durans ATCC 49532 ATCC 49533 Enterococcus JMI 12536 faecalis ATCC 51299 ATCC 700802 JMI 368 ATCC 27270 ATCC 35667 Enterococcus ATCC BAA-2127 faecium JMI 536 ATCC 700221 JMI 475 Enterococcus ATCC 49996 flavescens Enterococcus ATCC 49608 gallinarum Enterococcus ATCC 8043 hirae Enterococcus ATCC 43197 malodoratus Enterococcus ATCC 43187 mundtii a Not detected at the highest test concentrations ~1x109-1x1010 CFU/mL.

Listeria monocytogenes

Table 38. Results of Listeria monocytogenes Inclusivity Testing Listeria monocytogenes Detecteda Species Serotype Isolate ID Listeria monocytogenes 1/2a FSL-C1-056b Listeria monocytogenes 1/2a FSL-J2-020 b Listeria monocytogenes 1/2b FSL-J2-064 b Listeria monocytogenes 1/2b HUM-2009042206c Listeria monocytogenes 4b ATCC 43256 Listeria monocytogenes 4b ATCC 13932 a Estimated concentration in a positive blood culture is ~5x108 CFU/mL. b Isolates obtained from Cornell University. c Isolates obtained from the Colorado Department of Public Health (CDPH).

Staphylococcus (including Staphylococcus aureus)

Table 39. Results of Staphylococcus aureus Inclusivity Testing

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Staphylococcus/Staphylococcus aureus Detecteda Species Isolate ID Strain Information PFGE Type Methicillin-sensitive S. aureus (MSSA) Staphylococcus aureus ATCC BAA-1749 96:308 USA 900 Staphylococcus aureus ATCC BAA-1759 N7129 USA 900 Staphylococcus aureus ATCC BAA-1765 102-04 USA 1200 Staphylococcus aureusb ATCC 12600 NCTC 8532 Type strain Unknown Staphylococcus aureusb ATCC 11632 S13 Unknown Staphylococcus aureus ATCC BAA-2419 Mass/2010 Unknown Staphylococcus aureus ATCC BAA-2420 Mass/2010 Unknown Staphylococcus aureus ATCC BAA-2421 Mass/2010 Unknown Staphylococcus aureus 1060728 n/ac Unknown Staphylococcus aureus Ant1 n/ac Unknown Staphylococcus aureus Lem8 n/ac Unknown Staphylococcus aureus MAL8134 n/ac Unknown Staphylococcus aureus MAQ n/ac Unknown Staphylococcus aureus Per2 n/ac Unknown Staphylococcus aureus RAR n/ac Unknown Staphylococcus aureus S313 n/ac Unknown Staphylococcus aureus Sal3 n/ac Unknown Staphylococcus aureus Ver2 n/ac Unknown Staphylococcus aureus ssp. aureusb ATCC 10832 Wood 46 Unknown Staphylococcus aureus ssp. aureusb ATCC 14154 Rose Unknown Staphylococcus aureus ssp. aureus ATCC 25923 Seattle/1945 Unknown Borderline Oxacillin-resistant S. aureus (BORSA) Staphylococcus aureus SUN1d n/a Unknown Staphylococcus aureus SUN2d n/a Unknown Staphylococcus aureus SUN3d n/a Unknown Staphylococcus aureus SUN4d n/a Unknown Staphylococcus aureus SUN5d n/a Unknown Staphylococcus aureus SUN6d n/a Unknown Methicillin-resistant S. aureus (MRSA) Staphylococcus aureus ssp. aureus ATCC BAA-38 E2125 Denmark Unknown Staphylococcus aureus ssp. aureus ATCC 43300 F-182 Kansas Unknown Staphylococcus aureus ssp. aureus ATCC 700698 Mu3 Japan/1996 Unknown Staphylococcus aureus ssp. aureus ATCC BAA-1720 MRSA252 UK Unknown Staphylococcus aureus ssp. aureus ATCC BAA-39 HUSA304 Hungary/1993 Unknown Staphylococcus aureus NARSA NRS705 NY-12 New York/2005 USA 100 Staphylococcus aureus NARSA NRS701 MN-082 Minn/2006 USA 200 Staphylococcus aureus ssp. aureus ATCC BAA-1717 TCH1516 Texas USA 300 Staphylococcus aureus NARSA NRS703 MN-095 Minn/2006 USA 300 Staphylococcus aureus NARSA NRS683 GA-298 Georgia/2005 USA 300 Staphylococcus aureus NARSA NRS662 CO-34 Colorado/2005 USA 300 Staphylococcus aureus NARSA NRS707 NY-155 New York/2005 USA 300 Staphylococcus aureus ATCC BAA-1707 MW2 N. Dakota/1998 USA 400 Staphylococcus aureus NARSA NRS691 GA-62 Georgia/2005 USA 500 Staphylococcus aureus NARSA NRS648 CA-347 California/2005 USA 600 Staphylococcus aureus NARSA NRS689 GA-442 Georgia/2006 USA 700 Staphylococcus aureus ssp. aureus ATCC BAA-42 HDE288 Portugal/1996 USA 800 Staphylococcus aureus NARSA NRS668 CO-72 Colorado/2005 USA 800 Staphylococcus aureus ATCC BAA-1747 94:1013 Vermont/1993 USA 1000 Staphylococcus aureus NARSA NRS676 CT-19 Conn/2005 USA 1000 Staphylococcus aureus NARSA NRS745 CA-629 California/2006 USA 1000 Staphylococcus aureus ATCC BAA-1764 7031 Alaska USA 1100 Staphylococcus aureus ATCC BAA-1691 HFH-30137 Michigan/2003 Not 100-1100 Staphylococcus aureus ATCC BAA-1700 HFH-33798 Illinois/2004 Not 100-1100 Staphylococcus aureus ATCC BAA-2312 M10/0061 Ireland/2010 Unknown Staphylococcus aureus ATCC BAA-2313 M10/0148 Ireland/2010 CC130 Staphylococcus aureus (VRSA)e NARSA VRS5 HIP15178 Michigan/2005 Unknown

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a Detected at the initial test concentration of 5x106CFU/mL. b Initial test concentration was 5x105 CFU/mL. c Isolates obtained from University of Rennes, France. d Isolates obtained from Sunnybrook Research Institute, affiliated with the University of Toronto. e Tested as a seeded blood culture at the time of positivity.

Table 40. Results of Staphylococcus (non-S. aureus) Inclusivity Testinga Staphylococcus Detected with Staphylococcus Detected Staphylococcus Reduced Sensitivity [~5x106 CFU/mL] Not Detectedb [~5x107 CFU/mL] Coagulase-positive staphylococci (non-S.aureus) Staphylococcus Staphylococcus ATCC 700373 ATCC 29663 lutrae intermediusc Staphylococcus ATCC 49444 pseudointermedius Staphylococcus schleiferi ATCC 49545 subsp. coagulans Coagulase-negative staphylococci (CoNS) Staphylococcus Staphylococcus Staphylococcus Clinical ATCC 51548 ATCC 27842 caprae capitis subsp. capitis auricularis isolated Staphylococcus Staphylococcus Staphylococcus ATCC 29972 ATCC 51127 ATCC 51365 cohnii pasteuri carnosus Staphylococcus Staphylococcus ATCC 12228 ATCC 15305 ATCC 700403 saprophyticus lentuse Staphylococcus Clinical Staphylococcus 5 clinical ATCC 29886 simulans isolatesf pettenkoferi isolates Staphylococcus Staphylococcus schleiferi Staphylococcus ATCC 55133 ATCC 25614 ATCC 43808 epidermidis warneri subsp. schleiferi Staphylococcus ATCC 29887 ATCC 29060 sciuri ATCC 51625 ATCC 35984 Staphylococcus ATCC 43958 equorum Staphylococcus ATCC 29968 haemolyticus Staphylococcus hominis ATCC 25615 ssp. hominis Staphylococcus ATCC 43809 lugdunensis Staphylococcus ATCC 29966 xylosus a All 54 S. aureus isolates (Table 39 above) received Staphylococcus Detected results. b Not detected when tested at a concentration of ≥5x108 CFU/mL. c Isolates identified as Staphylococcus intermedius by automated identification systems were detected in two clinical specimens. d Staphylococcus auricularis was not tested in analytic studies, but was not detected in a clinical blood culture. e An isolate identified as Staphylococcus lentus by an automated identification system was detected in one clinical specimen. f Staphylococcus simulans was not tested in analytic studies, but was detected in three clinical blood cultures at unknown concentration.

Based on inclusivity testing results for staphylococci and in silico analysis of available sequences, the following predictions of reactivity are provided for less common CoNS species that were not tested.

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Please note that performance of the FilmArray BCID Panel for these organisms has not been established.

Table 41. In silico Predictions of Staphylococcus Reactivity Detection Predicted with Detection Predicteda Detection Not Predictedc Reduced Sensitivityb Staphylococcus gallinarum Staphylococcus microti Staphylococcus arlettae Staphylococcus kloosii Staphylococcus simiae Staphylococcus chromogenes Staphylococcus succinus Staphylococcus condimenti Staphylococcus fleurettii Staphylococcus piscifermentans Staphylococcus pulvereri

Staphylococcus rostri Staphylococcus saccharolyticus Staphylococcus vitulinus a Predicted result of Staphylococcus Detected when present in a blood culture sample at a concentration of ≥5x106 CFU/mL. b Predicted result of Staphylococcus Detected when present in a blood culture sample at a concentration of ≥ 5x107 CFU/mL. c Predicted result of Staphylococcus Not Detected at relevant concentrations.

Streptococcus (including Streptococcus agalactiae, Streptococcus pneumoniae and Streptococcus pyogenes)

Table 42. Results of Streptococcus Inclusivity Testing Streptococcus Detecteda Species Isolate ID Strain Information Streptococcus pyogenes ATCC 19615 Streptococcus pyogenes PCMC 20100107CI02 Streptococcus pyogenes ATCC 49399 Group A (Pyogenic group) Streptococcus pyogenes ATCC 12344 Streptococcus pyogenes ATCC 12384 ATCC 13813 Streptococcus agalactiae Type strain – Serotype 1a/c PCMC 20100107CI03 Streptococcus agalactiae Untyped clinical isolate ATCC 12403 Streptococcus agalactiae Group B (Pyogenic group) Type III ATCC BAA-611 Streptococcus agalactiae Serotype V NCTC 8017 Streptococcus agalactiae Unknown serotype Streptococcus dysgalactiae ssp. equisimilis ATCC 12388 Group C/G (Pyogenic group) Streptococcus bovis ATCC 33317 Group D (Bovis group) Streptococcus equinis ATCC 9812 Streptococcus mutans ATCC 25175 Group E (Mutans group) Streptococcus anginosus ATCC 33397 Group F (Anginosus group)

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Streptococcus Detecteda Species Isolate ID Strain Information Streptococcus intermedius ATCC 27335 Streptococcus constellatus ATCC 27513 Streptococcus gordonii ATCC 10558 Streptococcus parasanguinis ATCC 31412 Streptococcus sanguinis ATCC 10556 Streptococcus mitis ATCC 15914 Streptococcus oralis ATCC 10557 Streptococcus pseudopneumoniae ATCC BAA-960 ATCC BAA-255 Streptococcus pneumoniae Strain R6 (no capsule) Mitis group ATCC 700672 Streptococcus pneumoniae Serotype 14 ATCC BAA-334 Streptococcus pneumoniae Serotype 4 ATCC 700673 Streptococcus pneumoniae Serotype 19A ATCC BAA-341 Streptococcus pneumoniae Serotype 5 Streptococcus salivarius ATCC 13419 Salivarius group Streptococcus gallolyticus ATCC BAA-2069 Uncertain grouping a Detected at the initial test concentration of ~1x108 CFU/mL.

Table 43. Results of Streptococcus agalactiae Inclusivity Testing Streptococcus/Streptococcus agalactiae (Group B) Detecteda Species Isolate ID Strain Information ATCC 13813 Streptococcus agalactiae Type strain – Serotype 1a/c PCMC 20100107CI03 Streptococcus agalactiae Untyped clinical isolate ATCC 12403 Streptococcus agalactiae Group B (Pyogenic group) Type III ATCC BAA-611 Streptococcus agalactiae Serotype V NCTC 8017 Streptococcus agalactiae Unknown serotype a Detected at the initial test concentration of ~1x108 CFU/mL.

Table 44. Results of Streptococcus pneumoniae Inclusivity Testing Streptococcus/Streptococcus pneumoniae Detecteda,b Species Isolate ID Strain Information ATCC BAA-255 Streptococcus pneumoniae Strain R6 (no capsule) ATCC 700672 Streptococcus pneumoniae Serotype 14 ATCC BAA-334 Streptococcus pneumoniae Mitis group Serotype 4 ATCC 700673 Streptococcus pneumoniae Serotype 19A ATCC BAA-341 Streptococcus pneumoniae Serotype 5 a Detected at the initial test concentration of ~1x108 CFU/mL. b Based on sequence analysis, the BCID Panel may not detect S. pneumoniae serotypes 11A and 19, or may detect these serotypes with reduced sensitivity compared to other serotypes.

Table 45. Results of Streptococcus pyogenes Inclusivity Testing

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Streptococcus/Streptococcus pyogenes (Group A) Detecteda Species Isolate ID Strain Information Streptococcus pyogenes ATCC 19615 Streptococcus pyogenes PCMC 20100107CI02 Streptococcus pyogenes ATCC 49399 Group A (Pyogenic group) Streptococcus pyogenes ATCC 12344 Streptococcus pyogenes ATCC 12384 a Detected at the initial test concentration of ~1x108 CFU/mL.

Based on results of inclusivity testing and in silico analysis of available sequences, the following predictions of reactivity are provided for less common Streptococcus species that were not tested. As shown in Table 46 below, the analysis predicts that many species will be detected at concentrations expected in positive blood cultures (108-109 CFU/mL), and others (particularly Mutans group species) will likely not be detected due to sequence mismatches with the assay primers.

Please note that performance of the FilmArray BCID Panel for these organisms has not been established.

Table 46. In silico Predictions of Streptococcus Reactivity Detection Predicted with Detection Predicteda Detection Not Predicted Reduced Sensitivityb Streptococcus australis Streptococcus parauberis Streptococcus cricetic Streptococcus equi Streptococcus downeic Streptococcus ictaluri Streptococcus macacaec Streptococcus infantis Streptococcus porcinus Streptococcus infantarius Streptococcus urialis Streptococcus pasteurianus Streptococcus perois Streptococcus suis Streptococcus thermophilus Streptococcus vestibularis a Predicted result of Streptococcus Detected when present in a blood culture sample at a concentration of ~1x108 CFU/mL. b Predicted result of Streptococcus Detected when present in a blood culture sample at a concentration of ≥1x109 CFU/mL. c Mutans group streptococci.

Acinetobacter baumannii

Table 47. Results of Acinetobacter baumannii Inclusivity Testing Acinetobacter baumannii Detecteda Species Isolate ID Acinetobacter baumannii ATCC 9955 Acinetobacter baumannii ATCC BAA-1605 Acinetobacter baumannii ATCC 17961 Acinetobacter baumannii ATCC 19003 Acinetobacter baumannii ATCC BAA-2093 Acinetobacter baumannii ATCC 15308 a Detected at the initial test concentration of ~1x108 CFU/mL.

Enterobacteriaceae (including Enterobacter cloacae complex, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus and Serratia marcescens)

Table 48. Results of Enterobacteriaceae Inclusivity Testing

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Enterobacteriaceae Detected with Enterobacteriaceae Detected Enterobacteriaceae Reduced Sensitivity [~5×107 CFU/mL or 1×108 CFU/mL] Not Detecteda [~5×108-1×109 CFU/mL] Morganella morganii Cedeceae davisiae ATCC 43023 Edwardsiella tarda ATCC 15947 ATCC 25829 subsp. morganii Enterobacter Pantoea (Enterobacter) Citrobacter freundii ATCC 43864 ATCC 33028 ATCC 27155 gergoviae agglomeransb Providencia (Proteus) Citrobacter koseri ATCC 29223 Hafnia alvei ATCC 51815 ATCC 51902 acalifaciens Providencia (Proteus) Cronobacter muytjensii ATCC 51329 Salmonella bongori SGSC 3041 ATCC 9250 rettgeri Cronobacter ATCC 29544 Serratia fonticola ATCC 29844 Providencia stuarti ATCC 33672 (Enterobacter) sakazakii Enterobacter aerogenes ATCC 13048 Serratia odorifera ATCC 33077 Rahnella aquatilis ATCC 33071 Enterobacter aerogenes ATCC 29751 Serratia rubidaea ATCC 27593 Serratia liquefaciens ATCC 27592 Enterobacter amnigenus ATCC 51816 Serratia plymuthica ATCC 183

Enterobacterasburiae ATCC 35953 Tatumella ptyseos ATCC 33301 Enterobacter cloacae 9 isolatesc Yersinia enterocolitica ATCC 6025

Enterobacter hormaechei ATCC 49162 ATCC Enterobacter kobei BAA-260d Enterobacter ATCC 9912d nimipressuralis Escherichia coli 5 isolatese

Escherichia fergusonii ATCC 35469 Escherichia hermanii ATCC 33650

Escherichia vulneris ATCC 33821 Klebsiella oxytoca 11 isolatesf

Klebsiella pneumoniae 10 isolatesg

ATCC Klebsiella variicola BAA-830 Kluyvera ascorbata ATCC 33433 Kluyvera (Enterobacter) ATCC 33110 intermedius Leclercia adecarboxylata ATCC 23216 h Proteus species 10 isolates Raoultella ornithinolytica ATCC 31898

Raoultella planticola ATCC 31900 Raoultella terrigena ATCC 33257

Salmonella ATCC 10708 enterica-cholerasius Salmonella ATCC 8326 enterica-heidelberg Salmonella SGSC 3222 enterica-paratyphi Salmonella ATCC 13311 enterica-typhimurium Serratia marcescens 6 isolatesi

Serratia entomophila ATCC 43705

Serratia ficaria ATCC 33105 Shigella boydiij ATCC 8700

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Yokenella regensburgei ATCC 35313

a Not Detected at the highest test concentration of 1×109-1×1010CFU/mL. b Not Detected in this study, but Pantoea agglomerans was detected by the BCID Panel in a clinical blood culture. c See Enterobacter cloacae complex table. d Tested as purified nucleic acid at a concentration of 0.63µg/mL (equivalent to ~1.0×108 CFU/mL). e See Escherichia coli table. f See Klebsiella oxytoca table. g See Klebsiella pneumoniae table. h See Proteus table. i See Serratia marcescens table. j Tested as a seeded blood culture within 1 hour of positivity.

Based on results of inclusivity testing and in silico analysis of available sequences, the following predictions of reactivity are provided for less common Enterobacteriaceae that were not tested.

Please note that performance of the FilmArray BCID Panel for these organisms has not been established.

Table 49. In silico Predictions of Enterobacteriaceae Reactivity Detection Predicted with Detection Not Predicted Unknown Reactivityb Reduced Sensitivitya Brenneria spp. Photorhabdus spp. Buttiauxella spp. Dickeya spp. Serratia grimesii Ewingella americana Erwinia spp. Serratia proteamaculans Leminorella spp. Pectobacterium spp. Xenorhabdus spp. Moellerella spp. Yersinia spp. a Predicted result of Enterobacteriaceae Detected when present in a blood culture sample at a concentration of ≥ 1x108 CFU/mL b Sequence data not available for in silico reactivity predictions

Table 50. Results of Enterobacter cloacae complex Inclusivity Testing Enterobacter cloacae complex Detected Enterobacter cloacae complex [~1×108 CFU/mL] Not Detecteda Enterobacter asburiae ATCC 35953 Enterobacter nimipressuralisb ATCC 9912 Enterobacter cloacae ATCC ATCC Enterobacter kobei subsp. cloacae BAA-1143 BAA-260 Enterobacter cloacae ATCC 13047 subsp. cloacae Enterobacter cloacae NCTC 10005 subsp. cloacae Enterobacter cloacae ATCC 49141 subsp. cloacae Enterobacter cloacae ATCC 23373 subsp. dissolvensb Enterobacter hormaechei ATCC 49162 a Not Detected at highest test concentration of 1×1010 CFU/mL. b Tested as purified nucleic acid at a concentration of 0.63µg/mL (equivalent to ~1×108 CFU/mL). Detected by Enterobacteriaceae assay.

Table 51. Results of Escherichia coli Inclusivity Testing Escherichia coli Detecteda

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Species Isolate ID Strain Info Escherichia coli ATCC 43888 CDC B6914-MS1 serotype O157:H7 Escherichia coli ATCC 49105 7482-1-1 serotype O15 Escherichia coli ATCC 25922 FDA-Seattle1946 Escherichia coli ATCC 35401 H10407 serotype O78:H11 Escherichia coli ATCC BAA-201 Produces ESBL TEM-3 a Detected at the initial test concentration of 5×107 CFU/mL.

Table 52. Results of Klebsiella oxytoca Inclusivity Testing Klebsiella oxytoca Detecteda Klebsiella oxytoca Not Detected Species Isolate ID Strain Info Species Isolate ID Strain Info Klebsiella oxytoca ATCC 13182 n/a Klebsiella oxytocab,c JMI 10678 MY/2011 Klebsiella oxytoca ATCC 49131 n/a Klebsiella oxytoca ATCC 700324 n/a Klebsiella oxytoca ATCC 43086 n/a Klebsiella oxytoca ATCC 8724 n/a Klebsiella oxytoca JMI 14611 AR/2011 Klebsiella oxytoca JMI 12707 MA/2011 Klebsiella oxytoca JMI 7818 AR/2004 Klebsiella oxytoca JMI 2661 NY/2003 Klebsiella oxytoca JMI 2523 n/a a Detected at the initial test concentration of 5×107 CFU/mL. b Detected as Enterobacteriaceae at the initial test concentration of 5×107 CFU/mL but Not Detected for Klebsiella oxytoca at the highest test concentration of 1×1010 CFU/mL. c Sequence analysis confirmed this isolate as a variant K. oxytoca that will not be detected by the FilmArray BCID Panel Koxytoca assay.

Table 53. Results of Klebsiella pneumoniae Inclusivity Testing Klebsiella pneumoniae Detecteda Species Isolate ID Strain Information Klebsiella pneumoniae ATCC BAA-1706 n/a Klebsiella pneumoniae ssp. pneumoniae ATCC 13883 Type strain Klebsiella pneumoniae ssp. ozaenae ATCC 11296 NCTC 5050 Klebsiella pneumoniae ssp. rhinoscleromatis ATCC 13884 NCTC 5046 Type strain Klebsiella pneumoniae ATCC 700603 n/a Klebsiella pneumoniae ATCC BAA-1705 n/a Klebsiella pneumoniae JMI 766 n/a Klebsiella pneumoniae JMI 328 n/a Klebsiella pneumoniae JMI 8091 n/a Klebsiella pneumoniae JMI 438 n/a Klebsiella variicolab ATCC BAA-830 F2R9/ 2001 Type strain a Detected at the initial test concentration of 1×108 CFU/mL. b Identical sequence to K. pneumoniae variant 342. Both K. pneumoniae variant 342 and Klebsiella variicola have been recovered from clinical specimens and will be identified by the BCID Panel and most standard laboratory methods as Klebsiella pneumoniae.

Table 54. Results of Proteus Inclusivity Testing Proteus Detecteda Species Isolate ID ATCC 29906 JMI 10793 Proteus mirabilis ATCC 25933 ATCC 33583 ATCC 7002 ATCC 13315 Proteus hauseri ATCC 700826 Proteus penneri ATCC 33519 ATCC 33420 Proteus vulgaris ATCC 27973

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a Detected at the initial test concentration of 1×107 CFU/mL.

Table 55. Results of Serratia marcescens Inclusivity Testing Serratia marcescens Detecteda Species Isolate ID Strain Information Serratia marcescens ATCC 13880 Type strain Serratia marcescens ATCC 14756 n/a Serratia marcescens ATCC 27137 n/a Serratia marcescens ATCC 43297 n/a Serratia marcescens JMI 697 CT/2009 Serratia marcescens JMI 8089 TX/2004 a Detected at the initial test concentration of 1×108CFU/mL. Haemophilus influenzae

Table 56. Results of Inclusivity Testing for Haemophilus influenzae Haemophilus influenzae Detecteda Species Isolate ID Strain Information Haemophilus influenzae ATCC 33929 Non-typeable Haemophilus influenzae ATCC 51907 Non-typeable Haemophilus influenzae ssp. aegyptus ATCC 11116 Non-typeable Haemophilus influenzae ATCC 9006 Type a Haemophilus influenzae ATCC 31512 Type b Haemophilus influenzae ATCC 10211 Type b Haemophilus influenzae ATCC 49699 Type c Haemophilus influenzae ATCC 9008 Type d Haemophilus influenzae ATCC 8142 Type e Haemophilus influenzae ATCC 700223 Type f a Detected as seeded positive blood cultures tested within 1 hour of positivity. The concentration of H. influenzae in a positive blood culture at the time of positivity is estimated to be ~1×108 CFU/mL.

Neisseria meningitidis (encapsulated)

Table 57. Results of Neisseria meningitidis Inclusivity Testing Neisseria meningitidis Detecteda Neisseria meningitidis Not Detectedb,c Species Isolate ID Serogroup Species Isolate ID Serogroup Neisseria meningitidis Neisseria meningitidis ATCC 43744 W135 Clinical isolatec None (unencapsulated) Neisseria meningitidis Neisseria meningitidis ATCC 13077 A Clinical isolatec None (unencapsulated) Neisseria meningitidis Neisseria meningitidis ATCC 13090 B Clinical isolatec None (unencapsulated) Neisseria meningitidis Neisseria meningitidis ATCC 13102 C Clinical isolatec None (unencapsulated) Neisseria meningitidis ATCC 13113 D Neisseria meningitidis Clinical isolated B Neisseria meningitidis ATCC 35561 Y a Detected in a seeded blood culture tested within 1 hour of positivity (estimated concentration ~1×108 CFU/mL). b Not Detected in a seeded blood culture tested 1-5 hours after positivity. c Clinical isolates of unencapsulated N. meningitidis were tested from seeded positive blood cultures to confirm that they would not be detected by the BCID Panel. d DNA from a clinical isolate with a variant ctrA gene was tested and not detected at a concentration equivalent to 2.5×109 CFU/mL. DNA obtained from University of Lausanne, Institute of Microbiology, Switzerland [75].

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Pseudomonas aeruginosa

Table 58. Results of Pseudomonas aeruginosa Inclusivity Testing Pseudomonas aeruginosa Detecteda Species Isolate ID Pseudomonas aeruginosa ATCC 27853 Pseudomonas aeruginosa ATCC 10145 Pseudomonas aeruginosa ATCC 19429 Pseudomonas aeruginosa ATCC 25619 Pseudomonas aeruginosa ATCC BAA-1744 Pseudomonas aeruginosa ATCC 35554 a Detected at the initial test concentration of 1×108CFU/mL.

Candida albicans

Table 59. Results of Candida albicans Inclusivity Testing Candida albicans Detecteda Species Isolate ID Strain Info Candida albicans ATCC 10231 Serotype A - 3147 Candida albicans ATCC MYA-427 A39 [DUMC 136.97] Candida albicans ATCC MYA-2876 SC5314 Candida albicans ATCC 11651 171D Candida albicans ATCC 22972 M 97 Candida albicans ATCC 90028 NCCLS 11 a Detected at the initial test concentration of 1×104 CFU/mL.

Candida glabrata

Table 60. Results of Candida glabrata Inclusivity Testing Candida glabrata Detecteda Species Isolate ID Strain Info Candida glabrata ATCC 15545 NRRL YB-4025 Candida glabrata ATCC 32554 26247-1 Candida glabrata ATCC 2001 CBS138 Candida glabrata ATCC 15126 CBS15126 Candida glabrata ATCC MYA-2950 n/a a Detected at the initial test concentration of 1×106 CFU/mL.

Candida krusei

Table 61. Results of Candida krusei Inclusivity Testing Candida krusei Detecteda Species Isolate ID Strain Info Candida krusei ATCC 90878 B74 Candida krusei ATCC 201748 89-08-008 Candida krusei ATCC 14243 n/a Candida krusei/Issatchenkia orientalisb ATCC 28870 CBS 2052 Issatchenkia orientalisb ATCC 6258 NRRL Y-413 a Detected at the initial test concentration of 1×106 CFU/mL. b Issatchenkia orientalis and Pichia kudriavzevii are anamorphs of C. krusei.

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Candida parapsilosis

Table 62. Results of Candida parapsilosis Inclusivity Testing Candida parapsilosis Detecteda Candida parapsilosis Detected with Reduced Sensitivityb Species Isolate ID Strain Info Species Isolate ID Strain Info MCO462 Candida parapsilosis ATCC 90875 B78 Candida parapsilosis ATCC 96142 [UTHSC R-648] Candida parapsilosis ATCC 34136 ST-89 Candida parapsilosis ATCC 96138 MCO433 Candida parapsilosis ATCC 22019 CBS604 a Detected at the initial test concentration of 1×106 CFU/mL. b Detected at a test concentration of 1×107 CFU/mL.

Candida tropicalis

Table 63. Results of Candida tropicalis Inclusivity Testing Candida tropicalis Detecteda Species Isolate ID Strain Info Candida tropicalis ATCC 66029 AmMS 227 Candida tropicalis ATCC 750 Type Strain Candida tropicalis ATCC 90874 B79 Candida tropicalis ATCC MYA-2734 508-12.1 Candida tropicalisb ATCC 201380 API 90 01 105 (Vitek QC) a Detected at the initial test concentration of 1×105 CFU/mL. b Target concentration was 5×105 CFU/mL, final test concentration was 1×106 CFU/mL (2×). mecA

Table 64. Results of mecA Inclusivity Testing mecA Detecteda,b Species Isolate ID Strain Information SCCmec Type Methicillin-sensitive S. aureus (MSSA) with SCCmec cassette (mecA positive) Staphylococcus aureus ATCC BAA-2419 Mass/2010 II Staphylococcus aureus ATCC BAA-2420 Mass/2010 II Staphylococcus aureus ATCC BAA-2421 Mass/2010 II Methicillin-resistant S. epidermidis (MRSE) (mecA positive) Staphylococcus epidermidis ATCC 29887 255-01B Staphylococcus epidermidisc ATCC 51625 CCF 15990 Unknown Staphylococcus epidermidis ATCC 35984 RP62A Methicillin-resistant S. aureus (MRSA) (mecA positive) Staphylococcus aureus ssp. aureus ATCC BAA-38 E2125 Denmark I Staphylococcus aureus ssp. aureus ATCC 43300 F-182 Kansas II Staphylococcus aureus ssp. aureus ATCC 700698 Mu3 Japan/1996 II Staphylococcus aureus ssp. aureus ATCC BAA-1720 MRSA252 UK II Staphylococcus aureus NARSA NRS705 NY-12 New York/2005 II Staphylococcus aureus NARSA NRS701 MN-082 Minn/2006 II Staphylococcus aureus NARSA NRS648 CA-347 California/2005 II Staphylococcus aureus ssp. aureus ATCC BAA-39 HUSA304 Hungary/1993 III 3A&5 Staphylococcus aureus NARSA NRS703 MN-095 Minnesota/2006 IV

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mecA Detecteda,b Species Isolate ID Strain Information SCCmec Type Staphylococcus aureus NARSA NRS683 GA-298 Georgia/2005 IV Staphylococcus aureus NARSA NRS662 CO-34 Colorado/2005 IV Staphylococcus aureus NARSA NRS707 NY-155 New York/2005 IV Staphylococcus aureus ATCC BAA-1707 MW2 N. Dakota/1998 IV Staphylococcus aureus NARSA NRS691 GA-62 Georgia/2005 IV Staphylococcus aureus NARSA NRS689 GA-442 Georgia/2006 IV Staphylococcus aureus NARSA NRS668 CO-72 Colorado/2005 IV Staphylococcus aureus ATCC BAA-1747 94:1013 Vermont/1993 IV Staphylococcus aureus NARSA NRS676 CT-19 Conn/2005 IV Staphylococcus aureus ATCC BAA-1764 7031 Alaska IV Staphylococcus aureus ATCC BAA-1691 HFH-30137 Michigan/2003 IV Staphylococcus aureus ATCC BAA-1700 HFH-33798 Illinois/2004 IV Staphylococcus aureus ssp. aureus ATCC BAA-1717 TCH1516 Texas IVa Staphylococcus aureus NARSA NRS745 CA-629 California/2006 V Staphylococcus aureus ssp. aureus ATCC BAA-42 HDE288 Portugal/1996 VI Methicillin-resistant S. aureus with mecALGA251/mecC variant Staphylococcus aureus ATCC BAA-2312 M10/0061 Ireland/2010 XI Staphylococcus aureus ATCC BAA-2313 M10/0148 Ireland/2010 XI

a Detected at the initial test concentration of 5×106CFU/mL. b Staphylococcus Detected and/or Staphylococcus aureus Detected results also reported, as appropriate. c Initial test concentration was 5 x105 CFU/mL. vanA/B

Table 65. Results of vanA/B Inclusivity Testing vanA/B Detecteda,b Species Isolate ID Strain Information Enterococcus faecium [vanA] JMI 536 TX/2006 Enterococcus faecium [vanA] ATCC 700221 Connecticut Enterococcus faecium [vanA] JMI 475 IN/2003 Enterococcus faecalis [vanA] JMI 12536 Mass/2002 Enterococcus faecalis [vanB] ATCC 51299 Missouri Enterococcus faecalis [vanB] ATCC 700802 Missouri/1987 Enterococcus faecalis [vanB] JMI 368 VA/2003 a Detected at the initial test concentration of 1×108 CFU/mL. b Enterococcus Detected results also reported.

KPC

Table 66. Results of KPC Inclusivity Testing KPC Detecteda, b Speciesc Isolate ID KPC Type Strain Information Enterobacter cloacae BAA-2341 Unknown 1101152 Enterobacter hormaechei BAA-2082 Unknown n/a Escherichia coli BAA-2340 Unknown 1101362 Klebsiella oxytoca JMI 2523 Unknown n/a Escherichia coli Clinical Isolate KPC-2 n/a Enterobacter cloacae Clinical Isolate KPC-2 n/a Klebsiella oxytoca JMI 7818 KPC-2 AR/2004 Klebsiella pneumoniae JMI 328 KPC-2 n/a Klebsiella pneumoniae ATCC BAA-1705 KPC-2 Modified Hodge Test Control Serratia marcescens JMI 697 KPC-2 CT/2009 Enterobacter cloacae Clinical Isolate KPC-3 n/a

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KPC Detecteda, b Speciesc Isolate ID KPC Type Strain Information Klebsiella oxytoca JMI 2661 KPC-3 NY/2003 Klebsiella pneumoniae JMI 766 KPC-4 n/a Klebsiella pneumoniae JMI 8091 KPC-4 n/a Klebsiella pneumoniae JMI 438 KPC-4 n/a a Detected at the initial test concentration of 5×107 CFU/mL for K. oxytoca isolates and 1×108 CFU/mL for K. pneumoniae and S. marcescens isolates. Detected in a seeded blood culture tested within 1 hour of positivity for Enterobacter spp. and E. coli. b Enterobacteriaceae and corresponding species specific Detected results also reported. c Other isolates which carry the KPC gene (i.e. Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae other than those listed above) were not evaluated.

Analytical Specificity (Cross-Reactivity and Exclusivity)

The potential for cross-reactivity between assays contained in the BCID Panel was evaluated by testing high concentrations of organism in contrived or seeded blood culture samples. The test concentration was equal to or greater than the level of organism estimated to be in a blood culture sample 8 hours after positivity (approximately 109-1010 CFU/mL for bacteria and 107-108 CFU/mL for yeast), or the highest concentration possible based on the organism stock. The selection of organisms focused on species that may be found in positive blood cultures (clinically relevant) and/or those that are closely related to target organisms (nearest neighbors). Organisms were also selected based on antimicrobial resistance phenotypes and the presence or absence of the antimicrobial resistance genes identified by the BCID Panel. The tested organisms were divided into two categories: on-panel organisms and off- panel organisms. On-panel organisms were tested to verify that they only react with the appropriate assays on the panel. On-panel exclusivity testing included a total of 141 isolates of gram-positive bacteria, gram-negative bacteria and yeast, representing 29 genera and 98 individual species. Off-panel organisms were expected to have negative test results for all of the assays on the FilmArray BCID Panel (or positive organism results but negative results for the antimicrobial resistance genes detected by the FilmArray BCID Panel). Off-panel testing included a total of 82 isolates of gram-positive bacteria, gram-negative bacteria, yeast, viruses and Mycoplasmataceae. Results are presented for all organisms that were tested and received the expected FilmArray BCID Panel test result(s) (no cross-reactivity, Tables 67–71), followed by a summary of species or isolates with which cross-reactivity that was observed (Table 72).

Table 67. Non-Cross-Reactive Gram-Positive Bacteria ON PANEL Coagulase-Negative Enterococcus Species Staphylococcus aureus Streptococcus Species Staphylococci E. avium MSSA (18 isolates) S. capitis ssp. capitis S. agalactiae E. casseliflavus Resistant S. aureus – S. caprae S. anginosus (2 isolates) BORSA (6 isolates) S. cohnii S. bovis E. cecorum MRSA (mecA) S. epidermidis S. dysgalactiae E. dispar VRSA (mecA, vanA) (2 isolates) S. gallolyticus E. durans S. haemolyticus S. mitis E. faecalis (3 isolates) S. hominis S. mutans E. faecium (2 isolates)a S. lugdunensis S. parasanguinis E. gallinarium S. pasteuri S. pneumoniae Coagulase-Positive (2 isolates) S. saprophyticus S. pseudopneumoniae Staphylococci

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E. hirae S. intermedius S. schleiferi ssp. S. pyogenes E. raffinosus S. lutrae schleiferi S. salivarius Listeria monocytogenes S. pseudointermedius S. sciuri L. monocytogenes S. schleiferi ssp. S. warneri coagulans S. xylosus

OFF PANEL Gram-positive Cocci Gram-positive Bacilli Listeria Species Gram-positive Anaerobes Granulicatella adiacensb Actinomyces L. (murrayi) grayi Clostridium perfringens Gemella morbillorum odontolyticus L. innocuad Peptostreptococcus Lactococcus lactis Bacillus cereus L. ivanovii anaerobius Macrococcus Corynebacterium ssp. londoniensis Propionibacterium acnes caseolyticus jeikeium L. seeligeri Micrococcus luteus Lactobacillus acidophilus L. welshimeri Vagococcus fluvialis Mycobacterium tuberculosisc Rhodococcus equi Rothia mucilaginosa a One isolate was tested at a concentration of 5ng/mL Extracted DNA; ~1×106 CFU/mL. b A false positive Streptococcus result was observed in the initial test of this isolate. The expected negative results were observed in multiple subsequent tests. No cross-reactivity between G. adiacens and the BCID Panel Streptococcus assays is predicted by sequence analysis. c Tested at a concentration of 7.33×106 CFU/mL. d In silico analysis predicts that cross-reactivity between the Lmonocytogenes assay and some atypical strains of L. innocua is possible, however, no cross-reactivity was observed in this testing.

Table 68. Non-Cross-Reactive Gram-Negative Bacteria ON PANEL Acinetobacter baumannii Enterobacteriaceae Isolatesa A. baumannii (2 isolates) Cedeceae davisiae Escherichia hermanii Providencia acalifaciens Citrobacter fruendi Escherichia vulneris Providencia rettgeri Haemophilus influenzae Citrobacter koseri Hafnia alvei Providencia stuarti Cronobacter muytjensi Klebsiella oxytoca (3 isolates) Rahnella aquatilis H. influenzae (type b) Cronobacter sakazakii Klebsiella pneumoniae Raoultella terrigena Enterobacter amnigenus (6 isolates) Raoultella planticola Neisseria meningitidis Enterobacter asburiae Kluyvera ascorbata Salmonella enterica Enterobacter cancerogenus Kluyvera intermedius Serratia liquefaciens N. meningitidis Enterobacter cloacae Leclercia adecarboxylata Serratia fonticola

Pseudomonas Enterobacter hormaechei Morganella morganii Serratia marcescens (2 isolates) b aeruginosa Enterobacter gergoviae Pantoea agglomerans Serratia plymuthica Escherichia coli (2 isolates) Proteus mirabilis Tatumella ptyseos P. aeruginosa Proteus penneri Yersinia enterocolitica Proteus vulgaris Yokenella regensburgei OFF PANEL Acinetobacter Species Haemophilus Species Pseudomonas Species Gram-negative Bacilli A. calcoaceticus H. parahaemolyticus P. fluorescens Aeromonas hydrophila A. haemolyticus H. parainfluenzae P. luteola Brevundimonasdiminuta A. johnsonii H. parasuis P. nitroreducens Moraxella catarrhalis (3 isolates) A. junii H. somnus P. oryzihabitans Stenotrophomonas maltophila A. lwoffii P. pertucinogena Vibrio parahaemolyticus A. radioresistens Neisseria Species P. stutzeri A. schindleri A. ursingii N. sicca A. nosocomialis N. elongate N. perflava (genomospecies Gram-negative Anaerobes Gram-negative Coccobacilli 13TU; 2 isolates ) N. mucosa N. lactamica Bacteroides fragilis Bordetella pertussis Veillonella parvula Campylobacter fetus Chlamydia trachomatis Legionella pneumophiliac

a Some isolates were not detected by the FilmArray BCID Panel and are discussed in the inclusivity evaluation. b In silico analysis indicates that cross-reactivity between the Enterobacter cloacae complex assay and Pantoea (Enterobacter) agglomerans may be possible. However, no cross-reactivity was observed in this study. c Tested at a concentration of 2.63×108 CFU/mL.

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Table 69. Non-Cross-Reactive Fungi ON PANEL OFF PANEL Candida Species Candida Species Non-Candida Fungi C. albicans C. dubliniensis C. sojae Aspergillus fumigatus C. glabrata C. lusitaniae C. viswanathii Debaryomyces hansenii C. krusei C. metapsilosis C. guilliermondii Kluyveromyces lactis C. parapsilosis C. multigemmisa Saccharomyces cerevisiae C. tropicalis Schizosaccharomyces pombe a In silico analysis predicts that cross-reactivity between the Cparapsilosis assay and C. multigemmis is possible, however, no cross-reactivity was observed in this testing.

Table 70. Non-Cross-Reactive Viruses and Mycoplasmataceae OFF PANEL Mycoplasmataceae Isolates Viruses 7 4 Mycoplasma hominis (3.16×10 CFU/mL ) Cytomegalovirus (1.67×10 TCID50/mL) 6 5 Ureaplasma urealyticum (1.57×10 CFU/mL) Epstein Barr Virus (1.00×10 TCID50/mL) Herpes Simplex Virus - Type 1 (1:30 dilution of stock) 3 Varicella Zoster Virus (8.17×10 TCID50/mL)

Table 71. Non-cross-reactive with Antimicrobial Resistance Gene Assays ON PANEL OFF PANELa mecA Methicillin Resistant Staphylococci (mecA) Borderline Oxacillin Resistant S. aureus (BORSA) Staphylococcus epidermidis-MRSE mecA Staphylococcus aureus-BORSA (6 isolates) Staphylococcus aureus-MRSA mecA Methicillin Sensitive Staphylococci Staphylococcus aureus-VRSA mecA/vanA Staphylococcus aureus-MSSA (18 isolates)b Staphylococcus epidermidis-MRSE (1 isolate) Staphylococcus spp. (16 isolates) vanA/B Vancomycin Resistant Enterococci (vanA/B) Vancomycin Resistant Enterococci (non-vanA/B) Enterococcus faecalis vanB Enterococcus casseliflavus vanC Enterococcus faecium vanA Enterococcus casseliflavus vanC Enterococcus gallinarium vanC Enterococcus gallinarium vanC Vancomycin Sensitive Enterococci Enterococcus spp. (8 isolates) KPC Carbapenem Resistant Enterobacteriaceae (KPC) Carbapenem Resistant Enterobacteriaceae (non-KPC) Klebsiella oxytoca KPC-2 Klebsiella pneumoniae Unknown Klebsiella pneumoniae KPC-4 Klebsiella pneumoniae NDM Serratia marcescens KPC-2 Carbapenem Sensitive/Beta-lactam Resistant Isolates Klebsiella pneumoniae AmpC Klebsiella pneumoniae SHV Escherichia coli TEM-3/CTX-1 Acinetobacter baumannii blaOXA Moraxella catarrhalis blaOXA Moraxella catarrhalis BRO-1(bla)/orf3

Carbapenem Sensitive Isolates Enterobacteriaceae (51 Isolates) Acinetobacter baumannii (1 isolate) Pseudomonas aeruginosa (2 isolates) a Off-panel refers to the antimicrobial resistance gene. Organisms may be positive for organism assay(s).

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b Ten Isolates known to harbor remnants of SCCmec cassette.

Table 72. Predicted and Observed Cross-Reactivity with On-Panel or Off-Panel Organisms BCID Panel Result Cross-Reactive Organism(s)/Isolate(s)/Gene Gram-positive Bacteria Enterococcus Some coagulase-negative staphylococcia Gram-negative Bacteria Acinetobacter calcoaceticus-baumannii (ACB) complex species: Acinetobacter baumannii Acinetobacter calcoaceticus (ssp. anitratus) b Acinetobacter pittii (formerly genomospecies 3) b Shigella species: Shigella boydii Escherichia coli/ Shigella dysenteriae Enterobacteriaceae Shigella flexneri Shigella sonnei Escherichia fergusonnii Klebsiella variicola (aka Klebsiella pneumoniae variant 342) Klebsiella pneumoniae/ Enterobacter aerogenes Enterobacteriaceae Raoultella ornithinolyticac Serratia species (S. entomophilae, S. ficaria, S. odoriferad, and S. rubidaead) Raoultella ornithinolyticac Serratia marcescens/ Pseudomonas aeruginosa (ATCC 25619)f Enterobacteriaceae Pseudomonas putidae Pantoea calida Pantoea septica Haemophilus influenzae Haemophilus haemolyticusg Yeast Candida parapsilosis Candida orthopsilosis (Group III Candida parapsilosis) h Antimicrobial Resistance Genes vanA/B vanM i a Cross-reactivity was not observed in this study but is predicted by in silico analysis to occur only with some species (i.e. S. epidermis, S. capitis and S. haemolyticus) when present in a sample at very high levels. This cross-reactivity was observed infrequently in pre-analytical studies and the clinical evaluation (estimated occurrence of ~0.25% of all Staphylococcus positive patient samples). b Acinetobacter calcoaceticus-baumannii (ACB) complex species are often mis-identified as A. baumannii by automated and manual microbial identification methods. c Cross-reactivity was not observed when ATCC 31898 was tested in the inclusivity study at a concentration ~1x108 CFU/mL, but cross- reactivity was observed in clinical cultures containing R. ornithinolytica. d Cross-reactivity was observed only at high organism concentration (≥109 CFU/mL); rare human pathogens. e Pseudomonas putida is a rare opportunistic pathogen. f No cross-reactivity observed with five other Pseudomonas aeruginosa isolates tested at ≥108 CFU/mL. g Haemophilus haemolyticus is a commensal organism of the respiratory tract that is rarely isolated from blood culture. h Candida orthopsilosis is mis-identified as C. parapsilosis by automated and manual microbial identification methods. i Vancomycin-resistant Enterococcus faecium isolated in Asia, 2011; vanB resistance phenotype.

Cross-Contamination and Carryover

The potential for run-to-run carryover was evaluated by determining whether a high positive organism tested in one pouch would cause false positive results in subsequently tested pouches. No false positive results were observed during consecutive testing of 10 high positive samples alternately with negative samples, demonstrating that recommended sample handling and testing practices are effective in preventing false positive results due to carryover or cross-contamination between samples.

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Reproducibility

A multicenter reproducibility study was performed to determine between-site and overall reproducibility of the BCID Panel. Reproducibility testing occurred at three test sites using a panel of six simulated blood culture specimens, each spiked with various combinations of two different organisms (analytes). To best represent the composition of specimens likely to be tested by the BCID Panel, half of the analytes were at a concentration consistent with the level of organism in a blood culture bottle at the time of positivity, and half of the analytes were at a concentration similar to that observed in bottles 8 hours after positivity (see Growth and Detection above). Negative results for each assay were obtained from samples that were not spiked with a corresponding organism (analyte not in the sample). The data incorporate a range of potential variation introduced by seven different operators, three different pouch lots, and 10 different FilmArray instruments. Over the course of four weeks, every specimen was tested on eight different days, for a total of 90 replicates per analyte. A summary of results (percent (%) agreement with the expected result) for each analyte is provided in the following tables.

Table 73. Summary of Reproducibility Results – Organism Assays Results BCID Panel Organism Tested Not % Agreement with Test Result Test Concentration Test Site Detected Detected Expected Result Site A 30/30 0/30 Enterococcus faecium [vanA] Site B 30/30 0/30 JMI475 Site C 30/30 0/30 1.50E+08 CFU/mL 180/180 All Sites 90/90 0/90 100% Site A 30/30 0/30 Enterococcus faecalis [vanB] [98.0% - 100%] Site B 30/30 0/30 Enterococcus JMI 368 Site C 30/30 0/30 8.95E+08 CFU/mL All Sites 90/90 0/90 Site A 0/120 120/120 360/360 Site B 0/120 120/120 Negative 100% Site C 0/120 120/120 [99.0% - 100%] All Sites 0/360 360/360 Site A 0/180 180/180 540/540 Listeria Site B 0/180 180/180 Negative 100% monocytogenes Site C 0/180 180/180 [99.3% - 100%] All Sites 0/540 540/540 Site A 30/30 0/30 Staphylococcus aureus [MRSA] 90/90 Site B 30/30 0/30 ATCC BAA-1747 100% Site C 30/30 0/30 8.60E+06 CFU/mL [96.0% - 100%] All Sites 90/90 0/90 Staphylococcus Site A 0/150 150/150 449/450a Site B 1/150 a 149/150 Negative 99.8% Site C 0/150 150/150 [98.8% - 100%] All Sites 1/450 449/450 Site A 30/30 0/30 Staphylococcus aureus [MRSA] 90/90 Site B 30/30 0/30 ATCC BAA-1747 100% Site C 30/30 0/30 8.60E+06 CFU/mL [96.0% - 100%] Staphylococcus All Sites 90/90 0/90 aureus Site A 0/150 150/150 450/450 Site B 0/150 150/150 Negative 100% Site C 0/150 150/150 [99.2% - 100%] All Sites 0/450 450/450 Site A 30/30 0/30 Streptococcus pyogenes 90/90 Site B 30/30 0/30 ATCC 19615 100% Site C 30/30 0/30 5.70E+08 CFU/mL [96.0% - 100%] All Sites 90/90 0/90 Streptococcus Site A 0/150 150/150 450/450 Site B 0/150 150/150 Negative 100% Site C 0/150 150/150 [99.2% - 100%] All Sites 0/450 450/450 Site A 0/180 180/180 540/540 Streptococcus Negative Site B 0/180 180/180 100% agalactiae Site C 0/180 180/180 [99.3% - 100%]

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Results BCID Panel Organism Tested Not % Agreement with Test Result Test Concentration Test Site Detected Detected Expected Result All Sites 0/540 540/540 Site A 0/180 180/180 540/540 Streptococcus Site B 0/180 180/180 Negative 100% pneumoniae Site C 0/180 180/180 [99.3% - 100%] All Sites 0/540 540/540 Site A 30/30 0/30 Streptococcus pyogenes 90/90 Site B 30/30 0/30 ATCC 19615 100% Site C 30/30 0/30 5.70E+08 CFU/mL [96.0% - 100%] Streptococcus All Sites 90/90 0/90 pyogenes Site A 0/150 150/150 450/450 Site B 0/150 150/150 Negative 100% Site C 0/150 150/150 [99.2% - 100%] All Sites 0/450 450/450 Site A 30/30 0/30 Acinetobacter baumannii 90/90 Site B 30/30 0/30 ATCC 9955 100% Site C 30/30 0/30 2.00E+08 CFU/mL [96.0% - 100%] Acinetobacter All Sites 90/90 0/90 baumannii Site A 0/150 150/150 450/450 Site B 0/150 150/150 Negative 100% Site C 0/150 150/150 [99.2% - 100%] All Sites 0/450 450/450 Site A 30/30 0/30 Klebsiella pneumoniae [KPC] Site B 30/30 0/30 JMI 766 Site C 30/30 0/30 9.40E+08 CFU/mL 180/180 All Sites 90/90 0/90 100% Site A 30/30 0/30 Proteus mirabilis [98.0% - 100%] Site B 30/30 0/30 Enterobacteriaceae ATCC 29906 Site C 30/30 0/30 9.20E+08 CFU/mL All Sites 90/90 0/90 Site A 0/120 120/120 360/360 Site B 0/120 120/120 Negative 100% Site C 0/120 120/120 [99.0% - 100%] All Sites 0/360 360/360 Site A 0/180 180/180 540/540 Enterobacter Site B 0/180 180/180 Negative 100% cloacae complex Site C 0/180 180/180 [99.3% - 100%] All Sites 0/540 540/540 Site A 0/180 180/180 540/540 Site B 0/180 180/180 Escherichia coli Negative 100% Site C 0/180 180/180 [99.3% - 100%] All Sites 0/540 540/540 Site A 0/180 180/180 540/540 Site B 0/180 180/180 Klebsiella oxytoca Negative 100% Site C 0/180 180/180 [99.3% - 100%] All Sites 0/540 540/540 Site A 30/30 0/30 Klebsiella pneumoniae [KPC] 90/90 Site B 30/30 0/30 JMI 766 100% Site C 30/30 0/30 9.40E+08 CFU/mL [96.0% - 100%] Klebsiella All Sites 90/90 0/90 pneumoniae Site A 0/150 150/150 450/450 Negative Site B 0/150 150/150 100% Site C 0/150 150/150 [99.2% - 100%] All Sites 0/450 450/450 Site A 30/30 0/30 Proteus mirabilis 90/90 Site B 30/30 0/30 ATCC 29906 100% Site C 30/30 0/30 9.20E+08 CFU/mL [96.0% - 100%] All Sites 90/90 0/90 Proteus Site A 0/150 150/150 450/450 Site B 0/150 150/150 Negative 100% Site C 0/150 150/150 [99.2% - 100%] All Sites 0/450 450/450 Serratia Site A 0/180 180/180 Negative marcescens Site B 0/180 180/180

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Results BCID Panel Organism Tested Not % Agreement with Test Result Test Concentration Test Site Detected Detected Expected Result Site C 0/180 180/180 540/540 100% All Sites 0/540 540/540 [99.3% - 100%] Site A 0/180 180/180 539/540 a Haemophilus Site B 1/180 a 179/180 Negative 98.0% influenzae Site C 0/180 180/180 [99.0% - 100%] All Sites 1/540 539/540 Site A 0/180 180/180 540/540 Neisseria Site B 0/180 180/180 Negative 100% meningitidis Site C 0/180 180/180 [99.3% - 100%] All Sites 0/540 540/540 Site A 30/30 0/30 Pseudomonas aeruginosa 90/90 Site B 30/30 0/30 ATCC 27853 100% Site C 30/30 0/30 1.40E+08 CFU/mL [96.0% - 100%] Pseudomonas All Sites 90/90 0/90 aeruginosa Site A 0/150 150/150 450/450 Site B 0/150 150/150 Negative 100% Site C 0/150 150/150 [99.2% - 100%] All Sites 0/450 450/450 Site A 30/30 0/30 Candida albicans 90/90 Site B 30/30 0/30 ATCC 10231 100% Site C 30/30 0/30 3.10E+04 [96.0% - 100%] All Sites 90/90 0/90 Candida albicans Site A 0/150 150/150 450/450 Site B 0/150 150/150 Negative 100% Site C 0/150 150/150 [99.2% - 100%] All Sites 0/450 450/450 Site A 30/30 0/30 Candida glabrata 90/90 Site B 30/30 0/30 ATCC 15545 100% Site C 30/30 0/30 2.00E+07 [96.0% - 100%] All Sites 90/90 0/90 Candida glabrata Site A 0/150 150/150 450/450 Site B 0/150 150/150 Negative 100% Site C 0/150 150/150 [99.2% - 100%] All Sites 0/450 450/450 Site A 30/30 0/30 Candida krusei 90/90 Site B 30/30 0/30 ATCC 90878 100% Site C 30/30 0/30 3.20E+07 [96.0% - 100%] All Sites 90/90 0/90 Candida krusei Site A 0/150 150/150 450/450 Site B 0/150 150/150 Negative 100% Site C 0/150 150/150 [99.2% - 100%] All Sites 0/450 450/450 Site A 0/180 180/180 539/540 a Candida Site B 1/180 a 179/180 Negative 99.8% parapsilosis Site C 0/180 180/180 [99.0% - 100%] All Sites 1/540 539/540 Site A 30/30 0/30 Candida tropicalis 90/90 Site B 30/30 0/30 ATCC 66029 100% Site C 30/30 0/30 9.70E+05 [96.0% - 100%] All Sites 90/90 0/90 Candida tropicalis Site A 0/150 150/150 450/450 Site B 0/150 150/150 Negative 100% Site C 0/150 150/150 [99.2% - 100%] All Sites 0/450 450/450 a A single pouch run at Site B generated four false positive results: Staphylococcus, mecA (see below), Haemophilus influenzae, and Candida parapsilosis.

Table 74. Summary of Reproducibility Results – Antimicrobial Resistance Gene Assays

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Results % Agreement BCID Panel Organism Tested Not with Expected Test Result Test Concentration Test Site Detected Detected N/A Test Result Site A 30/30 0/30 0/30 Enterococcus faecium [vanA] Site B 30/30 0/30 0/30 JMI475 Site C 30/30 0/30 0/30 1.50E+08 CFU/mL 180/180 All Sites 90/90 0/90 0/90 100% Site A 30/30 0/30 0/30 Enterococcus faecalis [vanB] [98.0% - 100%] Site B 30/30 0/30 0/30 vanA/B JMI 368 Site C 30/30 0/30 0/30 8.95E+08 CFU/mL All Sites 90/90 0/90 0/90 Site A 0/120 0/120 120/120 360/360 Site B 0/120 0/120 120/120 No Associated Organism 100% Site C 0/120 0/120 120/120 [99.0% - 100%] All Sites 0/360 0/360 360/360 Site A 30/30 0/30 0/30 Staphylococcus aureus [MRSA] 90/90 Site B 30/30 0/30 0/30 ATCC BAA-1747 100% Site C 30/30 0/30 0/30 8.60E+06 CFU/mL [96.0% - 100%] All Sites 90/90 0/90 0/90 mecA Site A 0/150 0/150 150/150 449/450a Site B 1/150a 0/150 149/150 No Associated Organism 99.8% Site C 0/150 0/150 150/150 [98.8% - 100%] All Sites 1/450 0/450 449/450 Site A 30/30 0/30 0/30 Klebsiella pneumoniae [KPC] 90/90 Site B 30/30 0/30 0/30 JMI 766 100% Site C 30/30 0/30 0/30 9.40E+08 [96.0% - 100%] All Sites 90/90 0/90 0/90 Proteus mirabilis Site A 0/90 90/90 0/90 ATCC 29906 Site B 0/90 90/90 0/90 270/270 KPC and Site C 0/90 90/90 0/90 100% Pseudomonas aeruginosa [98.6% - 100%] ATCC 27853 All Sites 0/270 270/270 0/270 Site A 0/60 0/60 60/60 180/180 Site B 0/60 0/60 60/60 No Associated Organism 100% Site C 0/60 0/60 60/60 [98.0% - 100%] All Sites 0/180 0/180 180/180 a A single pouch run at Site B generated a false positive mecA result.

The reproducibility of Tm for each analyte and positive assay was also evaluated and a summary is provided in the following tables.

Table 75. Summary of Tm Analysis for Positive Organism Assays Reproducibility of Tm Observed Organism Tested Tm Tm Tm Tm Range BCID Panel Assay Test Concentration Test Site Mean Std Dev Minimum Maximum (Max-Min) Gram-Positive Bacteria Site A 82.5 0.4 81.9 84.0 2.1 Enterococcus faecium [vanA] Site B 82.6 0.2 82.3 83.0 0.7 JMI475 Site C 82.3 0.2 81.9 82.8 0.9 1.50E+08 CFU/mL All Sites 82.5 0.3 81.9 84.0 2.1 Enterococcus Site A 82.0 0.3 81.5 82.4 0.9 Enterococcus faecalis [vanB] Site B 82.2 0.2 81.8 82.8 1.0 JMI 368 Site C 81.6 0.4 81.0 82.4 1.4 8.95E+08 CFU/mL All Sites 81.9 0.4 81.0 82.8 1.8 Site A 77.1 0.3 76.6 77.8 1.2 Staphylococcus aureus [MRSA] Site B 77.3 0.3 76.8 77.8 1.0 Saureus ATCC BAA-1747 Site C 76.9 0.2 76.5 77.5 1.0 8.60E+06 CFU/mL All Sites 77.1 0.3 76.5 77.8 1.3 Site A 81.9 0.4 81.5 83.6 2.1 Streptococcus pyogenes Site B 82.1 0.1 81.8 82.3 0.5 Streptococcus ATCC 19615 Site C 81.8 0.2 81.5 82.1 0.6 5.70E+08 CFU/mL All Sites 81.9 0.3 81.5 83.6 2.1

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Reproducibility of Tm Observed Organism Tested Tm Tm Tm Tm Range BCID Panel Assay Test Concentration Test Site Mean Std Dev Minimum Maximum (Max-Min) Site A 79.0 0.4 78.5 79.8 1.3 Streptococcus pyogenes Site B 79.2 0.3 78.7 79.8 1.1 Spyogenes ATCC 19615 Site C 78.8 0.3 78.5 79.5 1.0 5.70E+08 CFU/mL All Sites 79.0 0.3 78.5 79.8 1.3 Gram-Negative Bacteria Site A 80.6 0.4 80.0 81.2 1.2 Acinetobacter baumannii Site B 80.8 0.2 80.4 81.2 0.8 Abaumannii ATCC 9955 Site C 80.3 0.4 79.5 80.9 1.4 2.00E+08 CFU/mL All Sites 80.5 0.4 79.5 81.2 1.7 Site A 88.6 0.3 88.1 89.1 1.0 Klebsiella pneumoniae [KPC] Site B 88.8 0.1 88.6 89.2 0.5 Enteric JMI 766 Site C 88.3 0.3 87.8 88.8 1.0 9.40E+08 CFU/mL All Sites 88.6 0.3 87.8 89.2 1.4 Site A 87.9 0.3 87.3 88.5 1.2 Klebsiella pneumoniae [KPC] Site B 88.1 0.2 87.8 88.4 0.6 Kpneumoniae JMI 766 Site C 87.6 0.3 86.7 88.1 1.5 9.40E+08 CFU/mL All Sites 87.8 0.4 86.7 88.5 1.8 Site A 81.2 0.3 80.6 81.8 1.2 Proteus mirabilis Site B 81.4 0.2 81.2 81.9 0.7 Proteus ATCC 29906 Site C 81.2 0.2 80.7 81.6 0.9 9.20E+08 CFU/mL All Sites 81.3 0.3 80.6 81.9 1.2 Site A 87.9 0.3 87.3 88.5 1.2 Pseudomonas aeruginosa Site B 88.2 0.3 87.8 89.5 1.7 Paeruginosa ATCC 27853 Site C 88.5 0.2 88.1 89.1 1.0 1.40E+08 CFU/mL All Sites 88.2 0.4 87.3 89.5 2.2 Yeast Site A 79.8 0.3 79.3 80.3 1.0 Candida albicans Site B 80.1 0.2 79.7 80.5 0.8 Calbicans ATCC 10231 Site C 79.5 0.3 78.9 80.2 1.3 3.10E+04 All Sites 79.8 0.4 78.9 80.5 1.7 Site A 75.3 0.3 74.7 76.1 1.3 Candida glabrata Site B 75.4 0.3 74.9 76.4 1.5 Cglabrata ATCC 15545 Site C 75.7 0.2 75.4 76.1 0.7 2.00E+07 All Sites 75.5 0.3 74.7 76.4 1.7 Site A 84.5 0.4 84.1 85.2 1.2 Candida krusei Site B 84.7 0.3 84.3 85.3 1.1 Ckrusei ATCC 90878 Site C 85.0 0.3 84.6 85.8 1.3 3.20E+07 All Sites 84.8 0.4 84.1 85.8 1.8 Site A 79.1 0.3 78.6 80.1 1.6 Candida tropicalis Site B 79.2 0.2 78.8 79.6 0.8 Ctropicalis ATCC 66029 Site C 79.5 0.2 79.3 80.0 0.7 9.70E+05 All Sites 79.3 0.3 78.6 80.1 1.6

Table 76. Summary of Tm Analysis for Positive Antimicrobial Resistance Gene Assays Reproducibility of Tm Observed BCID Panel Organism Tested Tm Tm Tm Tm Range Test Result Test Concentration Test Site Mean Std Dev Minimum Maximum (Max-Min) Site A 85.7 0.4 85.1 86.7 1.6 Enterococcus faecium [vanA] Site B 86.0 0.3 85.5 86.5 1.0 JMI475 Site C 85.6 0.3 85.1 86.3 1.2 1.50E+08 CFU/mL All Sites 85.7 0.4 85.1 86.7 1.6 vanA/B Site A 86.0 0.3 85.3 86.6 1.3 Enterococcus faecalis [vanB] Site B 86.3 0.2 85.9 86.9 1.0 JMI 368 Site C 85.7 0.4 85.1 86.6 1.5 8.95E+08 CFU/mL All Sites 86.0 0.4 85.1 86.9 1.8 Site A 73.6 0.3 73.1 74.4 1.3 Staphylococcus aureus [MRSA] Site B 73.7 0.3 73.2 74.2 1.0 mecA ATCC BAA-1747 Site C 73.4 0.3 72.8 74.1 1.3 8.60E+06 CFU/mL All Sites 73.6 0.3 72.8 74.4 1.7 KPC Site A 86.2 0.3 85.5 86.7 1.1

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Reproducibility of Tm Observed BCID Panel Organism Tested Tm Tm Tm Tm Range Test Result Test Concentration Test Site Mean Std Dev Minimum Maximum (Max-Min) Klebsiella pneumoniae [KPC] Site B 86.4 0.2 86.1 86.7 0.5 JMI 766 Site C 85.9 0.3 85.2 86.4 1.2 9.40E+08 All Sites 86.1 0.4 85.2 86.7 1.5

Interference

Substances that could be present in blood culture samples or introduced during sample handling were evaluated for their potential to interfere with assay performance. A potentially interfering substance (see Table 77) was added to a simulated positive blood culture sample which contained simulated blood culture matrix (human whole blood that had been incubated in a blood culture bottle) and one of six different organism mixes. Each organism mix contained two live pathogens at a concentration equivalent to the level determined to be present when a blood culture bottle is detected as positive by the blood culture instrument. None of the substances tested were found to compete or interfere with the assays in the BCID Panel. Table 77. Potentially Interfering Substances Tested (No Interference Observed) Technique-Specific Endogenous Substances Exogenous Substances Substances Hemoglobin Fluconazole Ceftriaxone Bleach Triglycerides Vancomycin Tetracycline Ethanol Bilirubin Ciprofloxacin Amoxicillin/Clavulanate γ-globulin Gentamicin sulfate Heparin Human Genomic DNA Imipenem SodiumPolyanetholesulfonate (SPS) On-Panel Competing Microorganisms Off-Panel Competing Microorganisms Staphylococcus epidermidis Corynebacterium jeikeium Escherichia coli Bacillus cereus Streptococcus mitis Micrococcus luteus Clostridium perfringens Propionibacterium acnes Blood Culture Media/Bottle Types BACTEC Plus Aerobic/F BacT/ ALERT SA Standard Aerobic VersaTREK REDOX 1 BACTEC Standard Aerobic BacT/ ALERT SN Standard Anaerobic VersaTREK REDOX 2 BACTEC Standard Anaerobic BacT/ ALERT FA Aerobic FAN BACTEC Plus Anaerobic/F BacT/ ALERT FN Anaerobic FAN BACTEC Pediatric Plus BacT/ ALERT PF Pediatric FAN BACTEC Lytic/10 Anaerobic/F BacT/ALERT FA Plus Aerobic Note: While not shown to interfere in this evaluation, the BacT/ALERT blood culture bottles that contain charcoal have the potential to generate false positive results presumably due to the presence of nucleic acids from non-viable organisms and are listed as contraindicated for use with the FilmArray BCID system.

PERFORMANCE CHARACTERISTICS ON THE FILMARRAY 2.0

Clinical and non-clinical studies were carried out to establish that the performance characteristics of the FilmArray BCID Panel are equivalent on the FilmArray and FilmArray 2.0 systems.

Clinical Comparison

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The clinical comparison study was performed using archived specimens that were originally obtained and characterized during the FilmArray BCID prospective clinical evaluation. A total of 100 specimens were selected such that each analyte (and antibiotic resistance marker) was represented 3-5 times. Each specimen was thawed and tested using the FilmArray and FilmArray 2.0systems. Overall positive percent agreement (PPA) between systems was 100% with the lower bound of the two-sided 95% confidence interval (95% CI) at 96.8%. Overall negative percent agreement (NPA) was 99.9% with the lower bound of the two-sided 95% CI at 99.6%.

Table 78. Analyte Results from FilmArray System Clinical Comparison Study FilmArray 2.0 / FilmArray Analyte PPA % 95% CI NPA % 95% CI Gram Positive Bacteria Enterococcus 6/6 100% 54.1-100% 93/94 98.9% 94.2-100% Listeria monocytogenes 4/4 100% 39.8-100% 96/96 100% 96.2-100% Staphylococcus 10/10 100% 69.2-100% 89/90 98.9% 94-100% Staphylococcus aureus 5/5 100% 47.8-100% 95/95 100% 96.2-100% Streptococcus 15/15 100% 78.2-100% 85/85 100% 95.8-100% Streptococcus agalactiae 3/3 100% 29.2-100% 97/97 100% 96.3-100% Streptococcus pneumoniae 3/3 100% 29.2-100% 97/97 100% 96.3-100% Streptococcus pyogenes 3/3 100% 29.2-100% 97/97 100% 96.3-100% Gram Negative Bacteria Acinetobacter baumannii 5/5 100% 47.8-100% 95/95 100% 96.2-100% Enterobacteriaceae 23/23 100% 85.2-100% 77/77 100% 95.3-100% Enterobacter cloacae complex 3/3 100% 29.2-100% 97/97 100% 96.3-100% Escherichia coli 3/3 100% 29.2-100% 97/97 100% 96.3-100% Klebsiella oxytoca 3/3 100% 29.2-100% 97/97 100% 96.3-100% Klebsiella pneumoniae 4/4 100% 39.8-100% 96/96 100% 96.2-100% Proteus 3/3 100% 29.2-100% 97/97 100% 96.3-100% Serratia marcescens 3/3 100% 29.2-100% 97/97 100% 96.3-100% Haemophilus influenzae 4/4 100% 39.8-100% 96/96 100% 96.2-100% Neisseria meningitidis 4/4 100% 39.8-100% 96/96 100% 96.2-100% Pseudomonas aeruginosa 4/4 100% 39.8-100% 96/96 100% 96.2-100% Yeast Candida albicans 5/5 100% 47.8-100% 95/95 100% 96.2-100% Candida glabrata 5/5 100% 47.8-100% 95/95 100% 96.2-100% Candida krusei 5/5 100% 47.8-100% 95/95 100% 96.2-100% Candida parapsilosis 5/5 100% 47.8-100% 95/95 100% 96.2-100% Candida tropicalis 5/5 100% 47.8-100% 95/95 100% 96.2-100% Antimicrobial Resistance Genes mecA 5/5 100% 47.8-100% 5/5 100% 47.8-100% vanA/B 3/3 100% 29.2-100% 3/3 100% 29.2-100% KPC 4/4 100% 39.8-100% 28/28 100% 87.7-100% Overall agreement 113/113 100% 96.8-100% 1885/1887 99.9% 99.6-100%

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System performance for testing of 100 specimens on each platform was calculated. For the FilmArray, a total of 102 runs were attempted, 100 of which were completed (98.8%; 100/102). There were two run failures for software errors (2.0%), both of which were completed following a single retest. No control failures were observed. For the FilmArray 2.0, a total of 100 FilmArray runs were attempted, 100 of which were completed (100%; 100/100). No control or software errors were observed.

Reproducibility

A multicenter reproducibility study was performed to determine between-site and overall reproducibility of the BCID Panel on multi-instrument FilmArray 2.0 systems. Reproducibility testing occurred at three sites using a panel of two simulated blood culture specimens, each spiked with various combinations of three different organisms (analytes). The organisms tested include representative strains of two gram-positive bacteria, two gram-negative bacteria and two species of Candida as well as one representative strain for each of the antimicrobial resistance genes detected by the panel.

To best represent the composition of specimens likely to be tested by the BCID Panel, half of the analytes were at a concentration consistent with the level of organism in a blood culture bottle at the time of positivity, and half of the analytes were at a concentration similar to that observed in bottles 8 hours after positivity (see Growth and Detection above). Negative results for each assay were obtained from samples that were not spiked with a corresponding organism (analyte not in the sample).

The data include 90 replicates per analyte and incorporate a range of potential variation introduced by 10 different operators, 3 different pouch lots, and 14 different FilmArray 2.0 instruments configured on 3 different multi-instrument systems. Similar to the reproducibility of the FilmArray BCID Panel on the FilmArray (Tables 73-76 above), percent (%) agreement with the expected Detected, Not Detected or N/A result was 97.8% or better and the standard deviation in Tm was 0.5˚C or less for all assays.

Table 79. Summary of Reproducibility Results on the FilmArray 2.0 % Agreement with Expected Result BCID Panel Organism Tested and Expected Site/System Test Result Test Concentration Test Result Total A B C [95% Confidence Interval] 89/90 Enterococcus faecalis [vanB] 30/30 30/30 29/30 JMI 368 Detected 98.9% 100% 100% 96.7% 8.95E+08 CFU/mL (94.0%-100%) Enterococcus 90/90 30/30 30/30 30/30 N/A Not Detected 100% 100% 100% 100% (96.0%-100%) 180/180 60/60 60/60 60/60 Listeria monocytogenes N/A Not Detected 100% 100% 100% 100% (98.0%-100%) 90/90 Staphylococcus aureus [MRSA] 30/30 30/30 30/30 ATCC BAA-1747 Detected 100% 100% 100% 100% 8.60E+06 CFU/mL (96.0%-100%) Staphylococcus 90/90 30/30 30/30 30/30 N/A Not Detected 100% 100% 100% 100% (96.0%-100%) 90/90 Staphylococcus aureus [MRSA] 30/30 30/30 30/30 ATCC BAA-1747 Detected 100% 100% 100% 100% 8.60E+06 CFU/mL (96.0%-100%) Staphylococcus aureus 90/90 30/30 30/30 30/30 N/A Not Detected 100% 100% 100% 100% (96.0%-100%)

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% Agreement with Expected Result BCID Panel Organism Tested and Expected Site/System Test Result Test Concentration Test Result Total A B C [95% Confidence Interval] 180/180 60/60 60/60 60/60 Streptococcus N/A Not Detected 100% 100% 100% 100% (98.0%-100%) 180/180 60/60 60/60 60/60 Streptococcus agalactiae N/A Not Detected 100% 100% 100% 100% (98.0%-100%) 180/180 Streptococcus 60/60 60/60 60/60 N/A Not Detected 100% pneumoniae 100% 100% 100% (98.0%-100%) 180/180 60/60 60/60 60/60 Streptococcus pyogenes N/A Not Detected 100% 100% 100% 100% (98.0%-100%) 180/180 60/60 60/60 60/60 Acinetobacter baumannii N/A Not Detected 100% 100% 100% 100% (98.0%-100%) 90/90 Klebsiella pneumoniae [KPC] 30/30 30/30 30/30 JMI 766 Detected 100% 100% 100% 100% 9.40E+08 CFU/mL (96.0%-100%) Enterobacteriaceae 90/90 30/30 30/30 30/30 N/A Not Detected 100% 100% 100% 100% (96.0%-100%) 180/180 Enterobacter cloacae 60/60 60/60 60/60 N/A Not Detected 100% complex 100% 100% 100% (98.0%-100%) 180/180 60/60 60/60 60/60 Escherichia coli N/A Not Detected 100% 100% 100% 100% (98.0%-100%) 180/180 60/60 60/60 60/60 Klebsiella oxytoca N/A Not Detected 100% 100% 100% 100% (98.0%-100%) 90/90 Klebsiella pneumoniae [KPC] 30/30 30/30 30/30 JMI 766 Detected 100% 100% 100% 100% 9.40E+08 CFU/mL (96.0%-100%) Klebsiella pneumoniae 90/90 30/30 30/30 30/30 N/A Not Detected 100% 100% 100% 100% (96.0%-100%) 180/180 60/60 60/60 60/60 Proteus N/A Not Detected 100% 100% 100% 100% (98.0%-100%) 180/180 60/60 60/60 60/60 Serratia marcescens N/A Not Detected 100% 100% 100% 100% (98.0%-100%) 180/180 60/60 60/60 60/60 Haemophilus influenzae N/A Not Detected 100% 100% 100% 100% (98.0%-100%) 180/180 60/60 60/60 60/60 Neisseria meningitidis N/A Not Detected 100% 100% 100% 100% (98.0%-100%) 90/90 Pseudomonas aeruginosa 30/30 30/30 30/30 ATCC 27853 Detected 100% 100% 100% 100% Pseudomonas 1.40E+08 CFU/mL (96.0%-100%) aeruginosa 90/90 30/30 30/30 30/30 N/A Not Detected 100% 100% 100% 100% (96.0%-100%)

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% Agreement with Expected Result BCID Panel Organism Tested and Expected Site/System Test Result Test Concentration Test Result Total A B C [95% Confidence Interval] 90/90 Candida albicans 30/30 30/30 30/30 ATCC 10231 Detected 100% 100% 100% 100% 3.10E+04 (96.0%-100%) Candida albicans 90/90 30/30 30/30 30/30 N/A Not Detected 100% 100% 100% 100% (96.0%-100%) 180/180 60/60 60/60 60/60 Candida glabrata N/A Not Detected 100% 100% 100% 100% (98.0%-100%) 88/90 Candida krusei 29/30 30/30 29/30 ATCC 90878 Detected 97.8% 96.7% 100% 96.7% 3.20E+07 (92.2%-99.7%) Candida krusei 90/90 30/30 30/30 30/30 N/A Not Detected 100% 100% 100% 100% (96.0%-100%) 180/180 60/60 60/60 60/60 Candida parapsilosis N/A Not Detected 100% 100% 100% 100% (98.0%-100%) 180/180 60/60 60/60 60/60 Candida tropicalis N/A Not Detected 100% 100% 100% 100% (98.0%-100%) 89/90 Enterococcus faecalis [vanB] 30/30 30/30 29/30 JMI 368 Detected 98.9% 100% 100% 96.7% 8.95E+08 CFU/mL (94.0%-100%) vanA/B 90/90 30/30 30/30 30/30 N/A N/A 100% 100% 100% 100% (96.0%-100%) 90/90 Staphylococcus aureus [MRSA] 30/30 30/30 30/30 ATCC BAA-1747 Detected 100% 100% 100% 100% 8.60E+06 CFU/mL (96.0%-100%) mecA 90/90 30/30 30/30 30/30 N/A N/A 100% 100% 100% 100% (96.0%-100%) 90/90 Klebsiella pneumoniae [KPC] 30/30 30/30 30/30 JMI 766 Detected 100% 100% 100% 100% 9.40E+08 CFU/mL (96.0%-100%) KPC 90/90 30/30 30/30 30/30 N/A Not Detected a 100% 100% 100% 100% (96.0%-100%) a The expected KPC test result for samples not spiked with a KPC containing organism was Not Detected (rather than N/A) due to the presence of P. aeruginosa in the sample.

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REFERENCES

1. National Vital Statistics Reports, Deaths: Preliminary Data for 2010.; Available from: http://www.cdc.gov/nchs/data/nvsr/nvsr60/nvsr60_04.pdf. 2. Angus, D.C., et al., Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med, 2001. 29(7): p. 1303-10. 3. Fisher, K. and C. Phillips, The ecology, epidemiology and virulence of Enterococcus. Microbiology, 2009. 155(Pt 6): p. 1749-57. 4. Wisplinghoff, H., et al., Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis, 2004. 39(3): p. 309-17. 5. Sood, S., et al., Enterococcal infections & antimicrobial resistance. Indian J Med Res, 2008. 128(2): p. 111-21. 6. Shahban, S.A., N. Manjula, and S. Siddiqui, Listeria septicaemia following insertion of a dynamic hip screw: A case report and literature review. Int J Surg Case Rep, 2012. 3(9): p. 448-50. 7. Wing, E.J. and S.H. Gregory, Listeria monocytogenes: clinical and experimental update. J Infect Dis, 2002. 185 Suppl 1: p. S18-24. 8. Disson, O. and M. Lecuit, Targeting of the central nervous system by Listeria monocytogenes. Virulence, 2012. 3(2): p. 213-21. 9. Vazquez-Boland, J.A., et al., Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev, 2001. 14(3): p. 584-640. 10. Versalovic, J., Manual of clinical microbiology. 10th ed, ed. A.S.f. Microbiology. 2011, Washington DN: ASM Press. 11. Hidron, A.I., et al., NHSN annual update: antimicrobial-resistant pathogens associated with healthcare-associated infections: annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006-2007. Infect Control Hosp Epidemiol, 2008. 29(11): p. 996-1011. 12. Cunningham, M.W., Pathogenesis of group A streptococcal infections. Clin Microbiol Rev, 2000. 13(3): p. 470- 511. 13. Efstratiou, A., Group A streptococci in the 1990s. J Antimicrob Chemother, 2000. 45 Suppl: p. 3-12. 14. Stoll, B.J., et al., Early onset neonatal sepsis: the burden of group B Streptococcal and E. coli disease continues. Pediatrics, 2011. 127(5): p. 817-26. 15. Peleg, A.Y., H. Seifert, and D.L. Paterson, Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev, 2008. 21(3): p. 538-82. 16. Dijkshoorn, L., A. Nemec, and H. Seifert, An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat Rev Microbiol, 2007. 5(12): p. 939-51. 17. Robledo, I.E., et al., Detection of KPC in Acinetobacter spp. in Puerto Rico. Antimicrob Agents Chemother, 2010. 54(3): p. 1354-7. 18. Higgins, P.G., et al., A PCR-based method to differentiate between Acinetobacter baumannii and Acinetobacter genomic species 13TU. Clin Microbiol Infect, 2007. 13(12): p. 1199-201. 19. Fedler, K.A., D.J. Biedenbach, and R.N. Jones, Assessment of pathogen frequency and resistance patterns among pediatric patient isolates: report from the 2004 SENTRY Antimicrobial Surveillance Program on 3 continents. Diagn Microbiol Infect Dis, 2006. 56(4): p. 427-36. 20. Lockhart, S.R., et al., Antimicrobial resistance among Gram-negative bacilli causing infections in intensive care unit patients in the United States between 1993 and 2004. J Clin Microbiol, 2007. 45(10): p. 3352-9. 21. Paterson, D.L., Resistance in gram-negative bacteria: Enterobacteriaceae. Am J Infect Control, 2006. 34(5 Suppl 1): p. S20-8; discussion S64-73. 22. Diekema, D.J., et al., Survey of bloodstream infections due to gram-negative bacilli: frequency of occurrence and antimicrobial susceptibility of isolates collected in the United States, Canada, and Latin America for the SENTRY Antimicrobial Surveillance Program, 1997. Clin Infect Dis, 1999. 29(3): p. 595-607. 23. Mezzatesta, M.L., F. Gona, and S. Stefani, Enterobacter cloacae complex: clinical impact and emerging antibiotic resistance. Future Microbiol, 2012. 7: p. 887-902. 24. Abbott, S.L., Klebsiella, enterobacter, citrobacter, serratia, plesiomonas, and other enterobacteriaceae, in Manual of Clinical Microbiology, 10th Edition, J. Versalovic, Carroll, K. C., Funke, G., Jorgensen, J. H., Landry, M. L., and Warnock, D. W., Editor. 2011, ASM press: Washington, D. C. p. 639 - 657. 25. Anderson, D.J., et al., Seasonal variation in Klebsiella pneumoniae bloodstream infection on 4 continents. J Infect Dis, 2008. 197(5): p. 752-6.

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26. Kim, B.N., et al., Retrospective analysis of clinical and microbiological aspects of Klebsiella oxytoca bacteremia over a 10-year period. Eur J Clin Microbiol Infect Dis, 2002. 21(6): p. 419-26. 27. Kovtunovych, G., et al., Identification of Klebsiella oxytoca using a specific PCR assay targeting the polygalacturonase pehX gene. Res Microbiol, 2003. 154(8): p. 587-92. 28. Park, J.S., et al., Evaluation of three phenotypic identification systems for clinical isolates of Raoultella ornithinolytica. J Med Microbiol, 2011. 60(Pt 4): p. 492-9. 29. Laupland, K.B., et al., Population-based laboratory surveillance for tribe Proteeae isolates in a large Canadian health region. Clin Microbiol Infect, 2007. 13(7): p. 683-8. 30. Nagano, N., et al., Nosocomial outbreak of infections by Proteus mirabilis that produces extended-spectrum CTX- M-2 type beta-lactamase. J Clin Microbiol, 2003. 41(12): p. 5530-6. 31. Chen, C.Y., et al., Proteus mirabilis urinary tract infection and bacteremia: risk factors, clinical presentation, and outcomes. J Microbiol Immunol Infect, 2012. 45(3): p. 228-36. 32. Luzzaro, F., et al., Prevalence and epidemiology of microbial pathogens causing bloodstream infections: results of the OASIS multicenter study. Diagn Microbiol Infect Dis, 2011. 69(4): p. 363-9. 33. Aucken, H.M. and T.L. Pitt, Antibiotic resistance and putative virulence factors of Serratia marcescens with respect to O and K serotypes. J Med Microbiol, 1998. 47(12): p. 1105-13. 34. Janda, J.M.a.S.L.A., The enterobacteria. 2nd ed. 2005, Washington DC: ASM Press. 35. Agrawal, A. and T.F. Murphy, Haemophilus influenzae infections in the H. influenzae type b conjugate vaccine era. J Clin Microbiol, 2011. 49(11): p. 3728-32. 36. Ladhani, S., et al., Invasive Haemophilus influenzae Disease, Europe, 1996-2006. Emerg Infect Dis, 2010. 16(3): p. 455-63. 37. Bilukha, O.O. and N. Rosenstein, Prevention and control of meningococcal disease. Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep, 2005. 54(RR-7): p. 1-21. 38. Dolan-Livengood, J.M., et al., Genetic basis for nongroupable Neisseria meningitidis. J Infect Dis, 2003. 187(10): p. 1616-28. 39. Claus, H., et al., Many carried meningococci lack the genes required for capsule synthesis and transport. Microbiology, 2002. 148(Pt 6): p. 1813-9. 40. Sadler, F., et al., Genetic analysis of capsular status of meningococcal carrier isolates. Epidemiol Infect, 2003. 130(1): p. 59-70. 41. Munro, R., Meningococcal disease: treatable but still terrifying. Intern Med J, 2002. 32(4): p. 165-9. 42. Milonovich, L.M., Meningococcemia: epidemiology, pathophysiology, and management. J Pediatr Health Care, 2007. 21(2): p. 75-80. 43. Pollard, A.J., et al., Emergency management of meningococcal disease: eight years on. Arch Dis Child, 2007. 92(4): p. 283-6. 44. Yang, M.A., et al., Pseudomonas aeruginosa bacteremia in children over ten consecutive years: analysis of clinical characteristics, risk factors of multi-drug resistance and clinical outcomes. J Korean Med Sci, 2011. 26(5): p. 612-8. 45. Haynes, A., 3rd, et al., Syndecan 1 shedding contributes to Pseudomonas aeruginosa sepsis. Infect Immun, 2005. 73(12): p. 7914-21. 46. Morales, E., et al., Hospital costs of nosocomial multi-drug resistant Pseudomonas aeruginosa acquisition. BMC Health Serv Res, 2012. 12: p. 122. 47. Correa, A., et al., First report of a Pseudomonas aeruginosa isolate coharboring KPC and VIM carbapenemases. Antimicrob Agents Chemother, 2012. 56(10): p. 5422-3. 48. Pfaller, M.A. and D.J. Diekema, Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev, 2007. 20(1): p. 133-63. 49. Klotz, S.A., et al., Polymicrobial bloodstream infections involving Candida species: analysis of patients and review of the literature. Diagn Microbiol Infect Dis, 2007. 59(4): p. 401-6. 50. Lockhart, S.R., et al., Species identification and antifungal susceptibility testing of Candida bloodstream isolates from population-based surveillance studies in two U.S. cities from 2008 to 2011. J Clin Microbiol, 2012. 50(11): p. 3435-42. 51. Cornet, M., et al., Molecular identification of closely related Candida species using two ribosomal intergenic spacer fingerprinting methods. J Mol Diagn, 2011. 13(1): p. 12-22. 52. Bouchami, O., et al., Molecular epidemiology of methicillin-resistant Staphylococcus hominis (MRSHo): low clonality and reservoirs of SCCmec structural elements. PLoS One, 2011. 6(7): p. e21940.

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53. Shore, A.C., et al., Detection of staphylococcal cassette chromosome mec type XI carrying highly divergent mecA, mecI, mecR1, blaZ, and ccr genes in human clinical isolates of clonal complex 130 methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother, 2011. 55(8): p. 3765-73. 54. Ruppe, E., et al., Diversity of staphylococcal cassette chromosome mec structures in methicillin-resistant Staphylococcus epidermidis and Staphylococcus haemolyticus strains among outpatients from four countries. Antimicrob Agents Chemother, 2009. 53(2): p. 442-9. 55. Jorgensen, J.H., Mechanisms of methicillin resistance in Staphylococcus aureus and methods for laboratory detection. Infect Control Hosp Epidemiol, 1991. 12(1): p. 14-9. 56. Xu, X., et al., vanM, a new glycopeptide resistance gene cluster found in Enterococcus faecium. Antimicrob Agents Chemother, 2010. 54(11): p. 4643-7. 57. Zirakzadeh, A. and R. Patel, Vancomycin-resistant enterococci: colonization, infection, detection, and treatment. Mayo Clin Proc, 2006. 81(4): p. 529-36. 58. Clark, N.C., et al., Detection and differentiation of vanC-1, vanC-2, and vanC-3 glycopeptide resistance genes in enterococci. J Clin Microbiol, 1998. 36(8): p. 2294-7. 59. Yigit, H., et al., Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob Agents Chemother, 2001. 45(4): p. 1151-61. 60. Rapp, R.P. and C. Urban, Klebsiella pneumoniae carbapenemases in Enterobacteriaceae: history, evolution, and microbiology concerns. Pharmacotherapy, 2012. 32(5): p. 399-407. 61. Chen, L., et al., Multiplex real-time PCR for detection of an epidemic KPC-producing Klebsiella pneumoniae ST258 clone. Antimicrob Agents Chemother, 2012. 56(6): p. 3444-7. 62. Arnold, R.S., et al., Emergence of Klebsiella pneumoniae carbapenemase-producing bacteria. South Med J, 2011. 104(1): p. 40-5. 63. Poirel, L. and P. Nordmann, Carbapenem resistance in Acinetobacter baumannii: mechanisms and epidemiology. Clin Microbiol Infect, 2006. 12(9): p. 826-36. 64. Wolter, D.J., et al., Surveillance of carbapenem-resistant Pseudomonas aeruginosa isolates from Puerto Rican Medical Center Hospitals: dissemination of KPC and IMP-18 beta-lactamases. Antimicrob Agents Chemother, 2009. 53(4): p. 1660-4. 65. Landman, D., et al., Accuracy of carbapenem nonsusceptibility for identification of KPC-possessing Enterobacteriaceae by use of the revised CLSI breakpoints. J Clin Microbiol, 2011. 49(11): p. 3931-3. 66. Nordmann, P., G. Cuzon, and T. Naas, The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria. Lancet Infect Dis, 2009. 9(4): p. 228-36. 67. Bratu, S., et al., Detection of KPC carbapenem-hydrolyzing enzymes in Enterobacter spp. from Brooklyn, New York. Antimicrob Agents Chemother, 2005. 49(2): p. 776-8. 68. Biosafety in Microbiological and Biomedical Laboratories (BMBL) 2009. 69. Institute, C.L.S., Protection of Laboratory Workers From Occupationally Acquired Infections; Approved Guideline - Third Edition M29-A3. 2005. 70. Summary of Notifiable Diseases. MMWR available at http://www.cdc.gov 71. CIFOR Analysis of State Legal Authorities available at http://www.cifor.us 72. CLSI, Statistical Quality Control for Quantitative Measurement Procedures: Principles and Definitions; Approved Guideline. CLSI Document C24-A3. 2006: Clinical Laboratory Standards Institute: Wayne Pennsylvania. 73. CLSI, User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline. CLSI Document E12- A2. 2008: Clinical Laboratory Standards Institute: Wayne Pennsylvania. 74. Johnson, J., et al., Natural atypical Listeria innocua strains with Listeria monocytogenes pathogenicity island 1 genes. Appl Environ Microbiol, 2004. 70(7): p. 4256-66. 75. Jaton, K., et al., False-negative PCR result due to gene polymorphism: the example of Neisseria meningitidis. J Clin Microbiol, 2010. 48(12): p. 4590-1. 76. Cavrini, F., et al., Multiple nucleotide substitutions in the Neisseria meningitidis serogroup C ctrA gene cause false-negative detection by real-time PCR. J Clin Microbiol, 2010. 48(8): p. 3016-8. 77. CLSI, Performance Standards for Antimicrobial Susceptibility Testing; 23rd Informational Supplement. CLSI Document M10-S23. 2013: Clinical Laboratory Standards Institute: Wayne Pennsylvania.

BioFire Diagnostics, LLC FilmArray BCID Panel CE-IVD Instruction Booklet 89

For additional information regarding our products and applications, please contact BioFire Diagnostics Customer Support Department, local bioMérieux sales representative or an authorized distributor.

Safety Data Sheet (SDS/MSDS) (According to regulation (EC) 1907/2006.) ™ Date SDS established : 05.2014 FilmArray Reagent Kit Revision number : 04 // ASAY-PRT-0643-04

1. IDENTIFICATION OF THE SUBSTANCE / MIXTURE AND OF THE COMPANY / UNDERTAKING 1.1 PRODUCT IDENTIFIER Product Name: FilmArray™ Reagent Kit RFIT-ASY-0002, RFIT-ASY-0007 (RUO) or RFIT-ASY-0114 (IVD), RFIT-ASY-0008 Catalog #: (RUO) or RFIT-ASY-0116 (IVD), RFIT-ASY-0094, RFIT-ASY-0104, RFIT-ASY-0105 (IVD) OR RFIT-ASY-0115 (RUO), RFIT-ASY-0107, RFIT-ASY-0109, RFIT-ASY-0118 ™ Kit Components: FilmArray Respiratory Panel Pouch, BioThreat Pouch, BCID Pouch, Hydration Solution, Sample Buffer, GI Panel Pouch, ME Panel Pouch 1.2 RELEVANT IDENTIFIED USES OF THE SUBSTANCE OR MIXTURE AND USES ADVICES AGAINST In vitro diagnostic use and for research use. 1.3 DETAILS OF THE SUPPLIER OF THE SAFETY DATA SHEET Address: BioFire Diagnostics, LLC, 390 Wakara Way, Salt Lake City, Utah 84108, USA Telephone Number: 1-801-736-6354 E-mail Address: [email protected] 1.4 EMERGENCY TELEPHONE NUMBER Call your local emergency center. 2. HAZARDOUS IDENTIFICATION 2.1 CLASSIFICATION OF THE MIXTURE Acute toxicity (Category 4) Sample Buffer: Serious Eye damage (Category 1) Skin irritation (Category 2) The other kit components are not classified as dangerous mixtures according to regulation 1272/2008. 2.2 LABEL ELEMENTS Labeling according Regulation (EC) No 1272/2008 [CLP]

Pictogram/Signal Word: Danger

Hazardous Statements H302 Harmful if swallowed. H318 Causes serious eye damage. H315 Causes skin irritation. Precautionary Statements P280 Wear protective gloves/eye protection/face protection. P305 + P351 + P338 IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. P301 + P312 IF SWALLOWED: Call a POISON CENTER or doctor/physician if you feel unwell. Supplemental Hazard Statements None 2.3 OTHER HAZARDS None

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3. COMPOSITION/INFORMATION ON INGREDIENTS

Component Name/ Hazardous Ingredient EC nr. Cas# Classification acc. 1272/2008 Concentration Acute toxicity (Cat. 4) Sample Buffer: 200-0 02-3 50-01-1 Eye irritation (Cat. 2) 50-60% w/w Guanidinium Chloride Skin irritation (Cat. 2)

Sample Buffer: Acute toxicity, Oral (Cat. 4) Triton X-100 / 9002-93-1 Serious eye damage (Cat. 1) 10-20% w/w Chronic aquatic toxicity (Cat. 2) 4. FIRST AID MEASURES 4.1 DESCRIPTION OF FIRST AID MEASURES If Inhaled: Remove to fresh air. Seek medical attention. Immediately flush skin with plenty of water for at least 15 minutes. Cover In Case of Skin Contact: irritated skin with an emollient. Remove contaminated clothing. Seek medical attention. Check for and remove any contact lenses and immediately flush eyes with In Case of Eye Contact: copious amounts of water. Seek medical attention. Immediately seek medical attention. If swallowed, induce vomiting as directed to If Swallowed: do so by medical personnel. Loosen tight clothing. 4.2 MOST IMPORTANT SYMPTOMS AND EFFECTS, BOTH ACUTE AND DELAYED To the best of our knowledge, the chemical, physical, and toxicological properties have not been thoroughly investigated. 4.3 INDICATION OF ANY IMMEDIATE MEDICAL ATTENTION AND SPECIAL TREATMENT NEEDED No data available 5. FIREFIGHTING MEASURES 5.1 EXTINGUISHING MEDIA Suitable Extinguishing Use foam, carbon dioxide, water spray or dry chemical powder. Foam and water Media: spray may cause frothing, but are still effective. Unsuitable Extinguishing None Media: 5.2 SPECIAL HAZARDS ARISING FROM THE SUBSTANCE OR MIXTURE Carbon oxides, nitrogen oxides (NOx), Hydrogen chloride gas 5.3 ADVICE FOR FIREFIGHTERS Wear self-contained breathing apparatus for fire fighting if necessary.

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6. ACCIDENTAL RELEASE MEASURES 6.1 PERSONAL PRECAUTIONS, PROTECTIVE EQUIPMENT, EMERGENCY PROCEDURES Use personal protective equipment. Avoid dust formation. Avoid breathing vapors, mist or gas. Ensure adequate ventilation. 6.2 ENVIRONMENTAL PRECAUTIONS Prevent further leakage or spillage if safe to do so. Do not let product enter drains. Discharge into the environment must be avoided. 6.3 METHODS AND MATERIAL FOR CONTAINMENT AND CLEANING UP Soak up with inert absorbent material and dispose of as hazardous waste. Keep in suitable, closed containers for disposal. 6.4 REFERENCE TO OTHER SECTIONS For disposal, see section 13. 7. HANDLING AND STORAGE 7.1 PRECAUTIONS FOR SAFE HANDLING Avoid contact with skin and eyes. Avoid formation of dust and aerosols. 7.2 CONDITIONS FOR SAFE STORAGE, INCLUDING ANY INCOMPATIBILITIES Store at room temperature. Keep container closed and away from direct sunlight. 7.3 SPECIFIC END USE(S) No data available 8. EXPOSURE CONTROLS/PERSONAL PROTECTION 8.1 CONTROL PARAMETERS Components with workplace control parameters: no data available. 8.2 EXPOSURE CONTROLS Respiratory Protection: Exhaust ventilation or other engineering controls. Hand Protection: Compatible chemical resistant gloves Eye Protection: Chemical safety goggles or face shield Body Protection: Lab coat Other Information: Change contaminated clothing. Wash hands after working with substances. 9. PHYSICAL AND CHEMICAL PROPERTIES 9.1 INFORMATION ON BASIC PHYSICAL AND CHEMICAL PROPERTIES Physical State/Form: Colorless Liquid Solubility in Water: Soluble 9.2 OTHER INFORMATION None

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10. STABILITY AND REACTIVITY 10.1 REACTIVITY No data available 10.2 CHEMICAL STABILITY No data available 10.3 HAZARDOUS REACTIONS No data available 10.4 CONDITIONS TO AVOID No data available 10.5 INCOMPATIBLE MATERIALS Oxidizing agents 10.6 HAZARDOUS DECOMPOSITION PRODUCTS COx and some metallic oxides 11. TOXICOLOGICAL INFORMATION 11.1 INFORMATION ON TOXICOLOGICAL EFFECTS Guanidinium Chloride Triton X-100

LD50 Oral - rat - 475 mg/kg LD50 Oral - rat - male - 500 mg/kg Remarks: Behavioral: Altered sleep time LD50 Dermal - rabbit – 8,000 mg/kg

(including change in righting reflex).

Behavioral: Excitement. Diarrhea LD50 Oral - mouse - 571 mg/kg Acute Remarks: Behavioral: Altered sleep time Toxicity: (including change in righting reflex). Behavioral: Muscle contraction or spasticity. Behavioral: Irritability. LD50 Oral - rat – 1,120 mg/kg LC50 Inhalation - rat - 4 h - 5,3 mg/l Skin Corrosion/Irritation: Skin - rabbit - Skin irritation No data available Serious Eye Damage/ Eyes - rabbit - Irritating to eyes Eyes - rabbit - Severe eye irritation Eye Irritation: Respiratory or Skin Buehler Test - guinea pig - Did not cause No data available Sensitization: sensitization on laboratory animals. Germ Cell Not mutagenic in Ames Test. No data available Mutagenicity: IARC: No component of this product present at levels greater than or equal to Carcinogenicity: 0.1% is identified as probable, possible or confirmed human carcinogen by IARC. Reproductive toxicity, Specific target organ toxicity - single exposure, Specific target organ toxicity - repeated exposure, Aspiration hazard: No data available Inhalation: May be harmful if inhaled. May cause respiratory tract irritation.

Potential Health Effects: Ingestion: Harmful if swallowed. Skin: May be harmful if absorbed through skin. May cause skin irritation. Eyes: Causes eye burns. Signs and Symptoms of To the best of our knowledge, the chemical, physical, and toxicological properties Exposure: have not been thoroughly investigated. Additional Information: RTECS: MF4300000 RTECS: MD0907700

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12. ECOLOGICAL INFORMATION 12.1 TOXICITY Guanidinium Chloride: Toxicity to fish LC50 - Leuciscus idus (Golden orfe) – 1,759 mg/l

Toxicity to fish LC50 - Pimephales promelas (fathead minnow) - 8,9 mg/l - 96,0 h Triton X-100: Toxicity to daphnia and other aquatic invertebrates. EC50 - Daphnia - 26 mg/l - 48 h 12.2 PERSISTENCE AND DEGRADABILITY Guanidinium Chloride: Result: - Not readily biodegradable.

Biodegradability Biotic/Aerobic Biochemical oxygen demand - Exposure time 28 d Triton X-100: Result: 36 % - Not readily biodegradable. Method: Closed Bottle test 12.3 BIOACCUMULATIVE POTENTIAL No data available 12.4 MOBILITY IN SOIL No data available 12.5 RESULTS OF PBT AND VPVB ASSESSMENT No data available 12.6 OTHER ADVERSE EFFECTS

Triton X-100: Toxic to aquatic life with long lasting effects. Chemical Oxygen Demand (COD) 2,19 mg/g 13. DISPOSAL CONSIDERATIONS 13.1 WASTE TREATMENT METHODS Recommendation: Chemicals must be disposed of in compliance with the respective national regulations.

Uncleaned packaging: Recommendation: Disposal must be made according to official regulations. Packagings that may not be cleansed are to be disposed of in the same manner as the product. Recommended cleansing agents: Water, if necessary together with cleansing agents. 14. TRANSPORT INFORMATION No restrictions apply due to the low volumes. 15. REGULATORY INFORMATION This safety datasheet complies with the requirements of Regulation (EC) No. 1907/2006. 15.1 SAFETY, HEALTH AND ENVIRONMENTAL REGULATIONS/LEGISLATION SPECIFIC FOR THE SUBSTANCE OR MIXTURE. U.S. Federal Regulations: TSCA 8(a) PAIR; TSCA 8(b) inventory; TSCA 8(d) H and S data reporting

(1996). SARA 311/312 MSDS distribution – chemical inventory – hazard Triton X-100: Identification: Immediate (Acute) and Delayed (Chronic) Health Hazard. SARA 302/304/311/312 hazardous chemicals Guanidine: SARA 311/312:; Acute: Yes; Chronic: No 15.2 CHEMICAL SAFETY ASSESSMENT No data available

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16. OTHER INFORMATION Classification and procedure used to derive the classification of the sample buffer: Calculation method.

Not for food, drug, household, agricultural or cosmetic use. The above information is correct to the best of our knowledge. The user should make independent decisions regarding completeness of the information based on all sources available. BioFire Diagnostics shall not be held liable for any damage resulting from handling or from contact with the above product.

It remains the user’s own responsibility to make sure that the information is appropriate and complete for his specific use of this product. The user is also responsible for observing any laws and applicable guidelines.

Changes to previous version of the SDS: Revision date: 05.01.2014 Revised for compliance with Regulation (EC) 1907/2006 (REACH)

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