FilmArray Blood Culture Identification Panel Quick Guide

FilmArry Pnel FilmArry Blood Culture Identifiction Pnel Quick Guide Testing on the FilmArry should be performed within 8 hours of the positive reding from continuously monitoring blood culture instrument. To void contmintion lwys wer gloves nd work behind protective shield. Step 1: Prepre Pouch Once smple is redy for testing, remove pouch from pckging. Insert pouch into Pouch Loding Sttion. Plce Smple Buffer vil into red well. Plce Hydrtion Solution vil into blue well. Step 2: Hydrte Pouch Drw 1 ml of Hydrtion Solution into Hydrtion Syringe. If bubbles re present in liquid, leve syringe tip in vil nd gently tp side of syringe, relesing bubbles to flot to surfce. Insert Hydrtion Syringe tip into hydrtion port of pouch locted directly below blue rrow. Holding the brrel of the Hydrtion Syringe, forcefully push down to puncture port sel. DO NOT PUSH THE SYRINGE PLUNGER. Wit s Hydrtion Solution is drwn into pouch. Note: See FilmArry Opertor's Mnul Troubleshooting section if pouch fils to hydrte. OR Step 3: Prepre Smple Mix Use 28 guge needle to withdrw 0.1 ml of smple from blood culture bottle. Trnsfer smple to Smple Buffer vil. Gently mix up nd down using Trnsfer Pipette. Note: For lternte workflow, see pckge insert. Step 4: Lod Smple Mix Drw 0.3 ml of Smple Mix using Smple Loding Syringe. Insert Smple Loding Syringe tip into smple port of pouch locted directly below red rrow. Holding the brrel of the syringe, forcefully push down to puncture port sel. DO NOT PUSH THE SYRINGE PLUNGER. Wit s Smple Mix is drwn into pouch. FilmArry Pnel O Step 5: Run Pouch Follow instructions on softwre screen. The pouch will click into plce when properly seted. Note: If the pouch does not insert esily, ensure tht the lid is opened completely. FilmArry Pnel BioFire Dignostics, LLC 390 Wkr Wy, Slt Lke City, Uth 84108, USA 1-801-736-6354

FilmArry Blood Culture Identifiction Pnel Quick Guide TM BCID Pnel The Run Summry Section displys informtion bout the smple nd summry of the control nd test results. 1. Orgnisms Detected: Nmes of ny detected orgnisms If "None", no orgnisms were detected If " Invlid", RETEST SAMPLE 2. Applicble Antimicrobil Resistnce Genes: Displys results for ntimicrobil resistnce gene(s) ssocited with detected orgnism(s). 3. Controls: If "Pssed", results re vlid If " Filed" or " Invlid", RETEST SAMPLE 4. 2. Run Summry 1. Smple ID: 8017366354 Run Dte: 19 Mr 2012 3:36 PM Orgnisms 3. Controls: Pssed Detected: ureus Applicble Antimicrobil Resistnce Genes: meca - Detected Results Summry - Interprettions Antimicrobil Resistnce Genes Detected meca (methicillin-resistnce gene) Ø N/A vna/b (vncomycin-resistnce genes) Ø N/A KPC (crbpenem-resistnce gene) The Results Summry-Interprettions Section lists the test results for ech orgnism or ntimicrobil resistnce gene trgeted by the Blood Culture Identifiction Pnel. 4. If "N/A", ntimicrobil resistnce gene ws not reported becuse n orgnism ssocited with the gene ws not detected 5. If "Detected", orgnism or ntimicrobil resistnce gene ws detected 6. If "Not Detected", orgnism or ntimicrobil resistnce gene ws not detected If Invlid, RETEST SAMPLE Note: If repeted "Invlid" results re obtined, contct BioFire Dignostics, the locl biomérieux sles representtive, or n uthorized distributor. The Run Detils Section displys informtion bout the pouch, instrument, run sttus, nd opertor. 7. Run Sttus: If Completed, run is complete. If "Incomplete", "Aborted", "Instrument Communiction Error", "Instrument Error", or "Softwre Error", RETEST SAMPLE 5. 6. NOTE: Antimicrobil resistnce cn occur vi multiple mechnisms. A Not Detected result for the FilmArry ntimicrobil resistnce gene ssys does not indicte ntimicrobil susceptibility. Subculturing is required for species identifiction nd susceptibility testing of isoltes. Not Detected Not Detected Enterococcus Grm Positive Bcteri Listeri monocytogenes Detected Detected ureus Not Detected Streptococcus Not Detected Streptococcus glctie (Group B) Not Detected Streptococcus pneumonie Not Detected Streptococcus pyogenes (Group A) 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 Grm Negtive Bcteri Acinetobcter bumnnii Enterobctericee Enterobcter cloce complex Escherichi coli Klebsiell oxytoc Klebsiell pneumonie Proteus Serrti mrcescens Hemophilus influenze Neisseri meningitidis Pseudomons eruginos Cndid lbicns Cndid glbrt Cndid krusei Cndid prpsilosis Cndid tropiclis Yest Note: If repeted "Error" messges re obtined, contct BioFire Dignostics, the locl biomérieux sles representtive, or n uthorized distributor. Run Detils 7. Pouch: Run Sttus: Seril No.: Lot No.: BCID Pnel v2.0 Completed 00169044 120228B Protocol: Opertor: Instrument: BC v 2.1 Opertor ITI FA "FA1187" Copyright 2015 BioFire Dignostics, LLC. All rights reserved. RFIT-PRT-0056-04 BioFire Dignostics, LLC 390 Wkr Wy, Slt Lke City, Uth 84108, USA 1-801-736-6354

RFIT-ASY-0114 RFIT-ASY-0109 FilmArry Blood Culture Identifiction (BCID) Pnel Instruction Booklet For use with the syringe-bsed loding system

Customer nd Technicl Support for U.S. Customers Rech Us on the Web http://www.biofiredx.com Rech Us by E-mil support@biofiredx.com Rech Us by Mil 390 Wkr Wy Slt Lke City, UT 84108 USA Rech Us by Phone 1-800-735-6544 Toll Free (801) 736-6354 Uth Rech Us by Fx (801) 588-0507 Customer nd Technicl Support outside of the U.S. Contct your locl sles representtive or distributor for technicl support BioFire Dignostics, LLC 390 Wkr Wy Slt Lke City, UT 84108 USA Qrd b.v.b. Ciplstrt 3 B-2440 Geel, Belgium Copyright 2007 2015, BioFire Dignostics, LLC. All rights reserved. RFIT-PRT-0101-04 Februry 2015 The informtion contined in this document is subject to chnge without notice. No prt of this document my be reproduced or trnsmitted in ny form or by ny mens, electronic or mechnicl, for ny purpose, without the express written permission of BioFire Dignostics, LLC. FilmArry Softwre, Detector, nd Metcll softwre modules 2002 2015 BioFire Dignostics, LLC. BioFire Dignostics, BioFire, the BioFire logo, FilmArry nd LCGreen re trdemrks of BioFire Dignostics, LLC or BioFire Defense, LLC nd re registered trdemrks in the United Sttes. All other nmes of products nd brnds ppering in this mnul re trdemrks or registered trdemrks of their respective owners. The purchse of this product includes limited, nontrnsferble license under specific clims of one or more U.S. ptents s listed on BioFire Dignostics Web site (http://www.biofiredx.com/leglnotices/) nd owned by BioFire nd the University of Uth Reserch Foundtion. BioFire Dignostics, LLC FilmArry BCID Pnel CE-IVD Instruction Booklet i

TABLE OF SYMBOLS The following symbols cn be found on FilmArry BCID Pnel Kit components or throughout this Instruction Booklet. Use the definitions below s guideline to interpreting the symbols. Tble of Symbols Mnufcturer Ctlog Number Expiry Dte YYYY-MM-DD Consult Instructions for Use Lot Number Storge Temperture Limittions Europen Union Conformity Seril Number n Contins Sufficient For <n> Tests In vitro Dignostic Medicl Device Keep Awy from Sunlight Do Not Reuse Serious eye dmge, ct. 1 Acute toxicity, ct. 4 & Skin irrittion, ct. 2 Do Not Use if Pckge is Dmged BioFire Dignostics, LLC FilmArry BCID Pnel 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 Dignostics, LLC FilmArry BCID Pnel 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 Dignostics, LLC FilmArry BCID Pnel CE-IVD Instruction Booklet iv

NAME AND INTENDED USE FilmArry Blood Culture Identifiction (BCID) Pnel The FilmArry Blood Culture Identifiction (BCID) Pnel is qulittive multiplexed nucleic cid-bsed in vitro dignostic test intended for use with FilmArry systems. The FilmArry BCID Pnel is cpble of simultneous detection nd identifiction of multiple bcteril nd yest nucleic cids nd select genetic determinnts of ntimicrobil resistnce. The BCID ssy is performed directly on blood culture smples identified s positive by continuous monitoring blood culture system tht demonstrte the presence of orgnisms s determined by Grm stin. The following grm-positive bcteri, grm-negtive bcteri, nd yest re identified using the FilmArry BCID Pnel: Enterococci, Listeri monocytogenes, Stphylococci (including specific differentition of ureus), Streptococci (with specific differentition of Streptococcus glctie, Streptococcus pneumonie, nd Streptococcus pyogenes), Acinetobcter bumnnii, Enterobctericee (including specific differentition of the Enterobcter cloce complex, Escherichi coli, Klebsiell oxytoc, Klebsiell pneumonie, Proteus, nd Serrti mrcescens), Hemophilus influenze, Neisseri meningitidis (encpsulted), Pseudomons eruginos, Cndid lbicns, Cndid glbrt, Cndid krusei, Cndid prpsilosis, nd Cndid tropiclis. The FilmArry BCID Pnel lso contins ssys for the detection of genetic determinnts of resistnce to methicillin (meca), vncomycin (vna nd vnb), nd crbpenems (blkpc) to id in the identifiction of potentilly ntimicrobil resistnt orgnisms in positive blood culture smples. The ntimicrobil resistnce gene detected my or my not be ssocited with the gent responsible for disese. Negtive results for these select ntimicrobil resistnce gene ssys do not indicte susceptibility, s multiple mechnisms of resistnce to methicillin, vncomycin, nd crbpenems exist. FilmArry BCID is indicted s n id in the dignosis of specific gents of bcteremi nd fungemi nd results should be used in conjunction with other clinicl nd lbortory findings. Positive FilmArry results do not rule out co-infection with orgnisms not included in the FilmArry BCID Pnel. FilmArry BCID is not intended to monitor tretment for bcteremi or fungemi. Subculturing of positive blood cultures is necessry to recover orgnisms for susceptibility testing nd epidemiologicl typing, to identify orgnisms in the blood culture tht re not detected by the FilmArry BCID Pnel, nd for species determintion of some Stphylococci, Enterococci, Streptococci, nd Enterobctericee tht re not specificlly identified by the FilmArry BCID Pnel ssys. SUMMARY AND EXPLANATION OF THE TEST Sepsis (defined s system inflmmtory response syndrome in response to infection) is the 11th leding cuse of deth in the United Sttes [1]. Life-thretening bcteril nd fungl sepsis currently strikes pproximtely 240 out of 100,000 people per yer in the U.S. (750,000 totl cses), with severe sepsis (ssocited with cute orgn dysfunction) in 95 out of 100,000 people [2]. Timely dignosis nd dministrtion of effective tretment cn significntly reduce mortlity, durtion of hospitl stys, nd costs due to sepsis. The FilmArry Blood Culture Identifiction (BCID) Pnel simultneously tests single positive blood culture smple to provide results for 24 different orgnisms nd orgnism groups tht cuse bloodstrem infections nd three genetic mrkers tht re known to confer ntimicrobil resistnce (see Tble 1). The test cn be performed on blood culture bottles tht re (1) flgged s positive by continuously monitoring blood culture instrument nd (2) positive by Grm stin exmintion. FilmArry BCID Pnel results re vilble within bout one hour. Rpid identifiction of the orgnism(s) in the blood culture, long with informtion bout ntimicrobil resistnce gene sttus for select microorgnisms, my id the physicin in mking pproprite tretment decisions. BioFire Dignostics, LLC FilmArry BCID Pnel CE-IVD Instruction Booklet 5

Tble 1. FilmArry BCID Pnel Test Results. Grm-Positive Bcteri Grm-Negtive Bcteri Yest Enterococcus Acinetobcter bumnnii Cndid lbicns Listeri monocytogenes Enterobctericee Cndid glbrt Enterobcter cloce complex Cndid krusei ureus Escherichi coli Cndid prpsilosis Streptococcus Klebsiell oxytoc Cndid tropiclis Streptococcus glctie Klebsiell pneumonie Antimicrobil resistnce genes Streptococcus pneumonie Proteus meca methicillin resistnce Streptococcus pyogenes Serrti mrcescens vna/b vncomycin resistnce Hemophilus influenze KPC crbpenem resistnce Neisseri meningitidis (encpsulted) Pseudomons eruginos Bckground of Trgeted Orgnisms Grm-Positive Bcteri Enterococcus. Members of this genus re grm-positive fculttive nerobes tht normlly inhbit the limentry trct of humns, but hve recently become one of the most common nosocomil pthogens [3]. Enterococcl infections include urinry trct infections, heptobiliry sepsis, endocrditis, surgicl wound infection, bcteremi, nd neontl sepsis. In n evlution of 49 US hospitls over seven yer period (1995 2002), enterococci were responsible for 9% of nosocomil bloodstrem infections [4]. There re 28 species of Enterococcus [3]. While t lest 12 species hve been shown to cuse humn disese, E. feclis (80-90%) nd E. fecium (5-15%) cuse the mjority of clinicl infections [5]. Enterococci cn crry vncomycin resistnce genes such s vna/b. Infection with vncomycin-resistnt Enterococcus (VRE) increses the risk of deth to 75%, compred with 45% for infection with susceptible strin [3]. Listeri monocytogenes, the custive gent of listeriosis, is grm-positive bcillus tht is ubiquitous in soil nd wter nd cn be found in the gstrointestinl trct of up to 5% of helthy humn dults [6-8]. Only three of the 12 known serovrs of L. monocytogenes (1/2, 1/2b nd 4b) ccount for more thn 90% of humn cses of listeriosis [9]. Listeriosis is considered the most severe bcteril foodborne infection due to its high mortlity rte despite erly ntibiotic tretment (11 60%) [8-9]. Popultions t risk for developing invsive listeriosis include the immunosuppressed, pregnnt women, neontes, fetuses nd the elderly [6-7]. Invsive listeriosis cn result in bortion, sepsis, nd meningoencephlitis [6, 9]. Septicemi cn ccount for greter thn 50% of cses nd cn hve mortlity rte up to 70% when ssocited with severe underlying conditions [8-9]., ureus. Stphylococci re grm-positive cocci tht re usully ctlse positive nd tend to form irregulr, grpe-like clusters. Nerly ll species in the genus re fculttive nerobes. spp. colonize the skin nd mucous membrnes. They re opportunistic pthogens tht cn cuse infection following breks in the cutneous epithelil brrier through trum or medicl interventions [10]. Dignosticlly, the genus is divided between cogulse-positive stphylococci nd cogulse-negtive stphylococci (CoNS). The most cliniclly-relevnt sp. is the cogulse-positive S. ureus. Other cogulse-positive species, such s S. intermedius, re isolted much less frequently from clinicl specimens. S. ureus is the most common cuse of nosocomil skin nd soft tissue infections, nd is second only to CoNS s cuse of primry bcteremi in hospitls [4, 11]. CoNS species re isolted very regulrly from clinicl specimens nd cre must be tken to ssess clinicl significnce to differentite BioFire Dignostics, LLC FilmArry BCID Pnel CE-IVD Instruction Booklet 6

between contmintion, coloniztion, nd true infection. The most cliniclly-importnt CoNS species include S. epidermidis, S. hemolyticus, nd S. sprophyticus. Mny other CoNS species re isolted to lesser degrees. FilmArry BCID detects most but not ll spp. It is estimted tht pproximtely 75% of CoNS isoltes nd 40% of S. ureus isoltes my be methicillin resistnt [4]. The primry meditor of methicillin resistnce in stphylococci is cquisition of the meca gene. Streptococcus, Streptococcus glctie, Streptococcus pneumonie, Streptococcus pyogenes. Streptococci re grm-positive, ctlse negtive cocci tht grow in chins or pirs. They cuse infections in vriety of species, including humns, mmmls, nd fish. Streptococcus species re frequently found s commensl bcteri on mucous membrnes, nd re occsionlly present s trnsient skin microbiot [10]. Streptococci hve historiclly been grouped s bethemolytic or non-bet-hemolytic, pyogenic (pus-forming) or non-pyogenic, nd lso divided ccording to presence of specific surfce ntigens (i.e., Lncefield grouping). Lncefield groups A, B, C, nd G re pyogenic nd most re lso bet-hemolytic [10]. Of these, the Group A streptococci (represented primrily by S. pyogenes) nd the Group B streptococci (S. glctie) re the most common in cses of septicemi. The non-pyogenic streptococci re subdivided into five groups (Mitis, Anginosus, Slivrius, Mutns, nd Bovis groups). The Mitis, Anginosus, nd Slivrius groups re lso referred to s viridins streptococci; these bcteri produce no Lncefield ntigens nd re lph-hemolytic or nonhemolytic. Viridns streptococci re lso firly common gents of septicemi cusing 0.5% of sepsis cses in nonneutropenic ptients nd up to 2% in neutropenic ptients [4]. S. pneumonie hs been clssified into the Mitis group but is often considered s its own seprte group. FilmArry BCID detects most but not ll Streptococcus spp. Streptococcus pyogenes (Group A Streptococcus or GAS) colonizes the humn skin nd upper respirtory trct, with these sites serving s primry focl sites of infections nd principl reservoirs of trnsmission [10]. S. pyogenes possesses complex virulence mechnisms to void host defenses [12-13] nd is responsible for deep or invsive infections, especilly bcteremi, sepsis, nd deep soft tissue infections [10]. Streptococcus glctice (Group B Streptococcus or GBS) cn cuse both erly onset neontl disese, chrcterized by sepsis nd pneumoni within the first seven dys of life; nd lte onset disese with meningitis nd sepsis between dy seven nd three months of ge [10]. GBS infections emerged in the 1970s s the leding cuse of erly onset sepsis (EOS) nd meningitis in neontes [14]. In dult ptients, the spectrum of S. glctice infections includes bcteremi, pneumoni, meningitis, nd endocrditis [10]. Streptococcus pneumonie colonizes the upper respirtory trct, nd is the most frequently isolted respirtory pthogen in community-cquired pneumoni. It is lso mjor cuse of meningitis in peditric nd dult ptients. S. pneumonie ws responsible for pproximtely 42,000 invsive infections in the U.S. in 2007, leding to n estimted 4,500 deths [10]. Grm-Negtive Bcteri Acinetobcter bumnnii is ubiquitous, non-fermenttive grm-negtive coccobcillus tht primrily cts s n opportunistic pthogen infecting criticlly-ill ptients. While hospitl-cquired pneumoni is the most common infection cused by A. bumnnii, other nosocomil infections re incresing in frequency, including bcteremi, skin nd soft tissue infections, urinry trct infections, nd infections involving the centrl nervous system [15]. A. bumnnii ws the 10 th most common cuse of nosocomil bloodstrem infection (1.3% of ll infections) in lrge U.S. study (1995-2002) [4]. The bcteri re cpble of persisting for long periods on environmentl surfces, iding the spred of infections in hospitl settings. Additionlly, the rpid emergence of multi-drug resistnt (MDR) strins cretes difficulties for tretment nd infection control. MDR strins demonstrte resistnce to most ntibiotic clsses, including crbpenems. Vrious crbpenem-hydrolyzing metllo-β-lctmses my be crried by the bcteri [16]. The specific crbpenemse, KPC, hs lso been infrequently identified in A. bumnnii [17]. Severl relted Acinetobcter species cnnot be relibly differentited from A. bumnnii by some mnul or utomted phenotypic microbil identifiction systems. These BioFire Dignostics, LLC FilmArry BCID Pnel CE-IVD Instruction Booklet 7

species include A. clcoceticus, A. pittii (genomospecies 3), nd A. nosocomilis (genomospecies 13TU). Together with A. bumnnii, these species hve been grouped into the Acinetobcter clcoceticus-bumnnii (ACB) complex. It hs been estimted tht up to 25% of isoltes from the ACB complex re routinely misidentified s A. bumnnii [18]. Enterobctericee. The fmily Enterobctericee is composed of mny gener nd species of bcteri tht shre common fetures (e.g. grm-negtive fculttive nerobic rods or coccobcilli). They re widely distributed in the environment nd re found on plnts, in soil nd wter, nd within the gstrointestinl (GI) trct of mny nimls, including humns. While mny orgnisms in this fmily re hrmless, severl re mediclly importnt nd re ssocited with bcteremi nd other illnesses, prticulrly gstroenteritis. Together, members of the Enterobctericee fmily re mong the most commonly recognized orgnisms seen in helthcre-ssocited infections. The FilmArry BCID Pnel contins specific ssys for the Enterobctericee members tht re most frequently ssocited with bcteremi nd/or re ssocited with higher frequency of ntimicrobil resistnce: Enterobcter cloce complex, Escherichi coli, Klebsiell oxytoc, Klebsiell pneumonie, Proteus, nd Serrti mrcescens. However, mny other Enterobctericee cn lso cuse bcteremi, though less frequently. These include Cedece, Citrobcter, Slmonell, nd Enterobcter erogenes, mong others [11, 19-20]. FilmArry BCID detects most commonly-encountered Enterobctericee; however, not ll gener or species in this fmily re detected. The emergence nd spred of ntimicrobil resistnce in Enterobctericee hs incresed the complexity of treting sepsis due to these orgnisms. Resistnce to third nd fourth-genertion cephlosporins is medited primrily by production of extended-spectrum β-lctmses (ESBLs) nd overproduction of AmpC β-lctmses. While the mjority of Enterobctericee remin susceptible to crbpenems, KPC-type crbpenemses re emerging nd spreding in Enterobctericee (crbpenem-resistnt Enterobctericee; CRE) in certin loctions within the United Sttes nd worldwide [21]. Enterobcter cloce complex. Enterobcter spp. re fculttively-nerobic grm-negtive rods within the Enterobctericee fmily. These bcteri re ubiquitous in the environment. Clinicl significnce is primrily limited to E. cloce nd E. erogenes, which re common opportunistic nosocomil pthogens. The rnge of infections cused by Enterobcter spp. includes respirtory trct infections, urinry trct infections, soft tissue infections, septic rthritis, osteomyelitis, nd bcteremi mong others. Enterobcter spp. re estimted to be the fourth most common etiologicl gents in grm-negtive bloodstrem infections [22]. They often hve the bility to overproduce AmpC β- lctmses nd re incresingly found to crry extended spectrum β-lctmses [23]. Severl species tht re closely relted to E. cloce re grouped together with it in the E. cloce complex; these include E. sburie, E. hormechei, E. kobei, E. ludwigii, nd E. nimipressurlis. Clinicl significnce hs been ttributed to some but not ll members of the complex [22]. Escherichi coli is n enteric orgnism most frequently isolted from the intestines of humns nd nimls. While most pthogenic E. coli infections re ssocited with gstrointestinl illness, certin strins my cuse extrintestinl infections in helthy s well s immunocompromised individuls. These include urinry trct infections, bcteremi, nd meningitis. Overll, E. coli re responsible for pproximtely 5.6% of bloodstrem infections [4]. As with other Enterobctericee, extended spectrum β-lctmses (ESBLs) pose significnt ntibiotic resistnce problem. Klebsiell oxytoc, Klebsiell pneumonie. Klebsiell re grm-negtive, rod-shped bcteri in the Enterobctericee fmily. Klebsiell spp. re ubiquitous in the environment, prticulrly in griculturl settings, nd my colonize the skin, respirtory, nd gstrointestinl (GI) trcts of humns. They re opportunistic pthogens nd while coloniztion does not lwys result in illness, infection rtes in crriers re four times higher thn non-crriers [24]. Infection rtes re higher in summer months, which is thought to be due to the greter prevlence of Klebsiell in the environment nd thus in the GI trct [25]. K. pneumonie nd K. oxytoc re the species most frequently isolted from hospitlized ptients [24]. Infections due to these bcteri include soft tissue infections, urinry trct infections, pneumoni, nd septicemi [24]. Both K. pneumonie nd K. oxytoc cn crry the Klebsiell pneumonie crbpenemse gene, blkpc, which mkes them resistnt to crbpenem ntibiotics. An incresing proportion of K. BioFire Dignostics, LLC FilmArry BCID Pnel CE-IVD Instruction Booklet 8

oxytoc isoltes from bcteremi infections demonstrte resistnce to extended-spectrum β-lctms, especilly when there is history of prior ntibiotic use [26]. Biochemicl discrimintion between species of Klebsiell is difficult: K. oxytoc isoltes my be erroneously identified s K. pneumonie by mnul or utomted biochemicl detection lgorithms [27]. Additionlly, Roultell ornithinolytic, which ws recently seprted from the genus Klebsiell, cn lso be misidentified s K. oxytoc by phenotypic identifiction systems [28]. Proteus. Members of the genus Proteus re commonly isolted in the clinicl lbortory, with Proteus mirbilis being the most frequently seen species. Most infections (pproximtely 85%) re thought to be community cquired [29]; however, nosocomil outbreks hve lso occurred [30]. The vst mjority of P. mirbilis re isolted from complicted urinry trct infections involving bnormlities or indwelling ctheters, while other species (e.g., P. vulgris) re more commonly found in soft tissue infections [29]. Progression of these infections to septicemi is reltively uncommon [31]. A recent survey estimted the prevlence of P. mirbilis in bloodstrem infection in hospitlized ptients to be 1.6%; this resulted in rnk of 11 out of the top 15 most commonly isolted orgnisms from bloodstrem infections in the study [32]. Antimicrobil resistnce hs become n incresing problem in Proteus infections, with pproximtely 32% of isoltes producing extended-spectrum β-lctmses [32]. Serrti mrcescens. Serrti re seldom cuse of primry infections, but re notorious nosocomil pthogens nd colonizers. S. mrcescens is the primry pthogenic species of the Serrti genus, responsible for 1.7% of nosocomil bloodstrem infections [4]. It is of prticulr concern due to its emerging ntibiotic resistnce to commonly used gents like β-lctms, minoglycosides, crbpenems, nd fluoroquinolones. Non-pigmented S. mrcescens re more resistnt to ntibiotics nd re ssocited with most outbreks [33]. Trnsmission my occur from person to person contct, vi medicl pprtus, intrvenous fluids, or other solutions [34]. Ptients with indwelling ctheters, prticulrly those for urinry trct infections, serve s primry reservoir for trnsmission vi hospitl personnel. In children, the gstrointestinl trct is common source of infections. Hemophilus influenze is grm-negtive coccobcillus tht is isolted exclusively from humns [35]. Strins of H. influenze re divided into two groups bsed on the presence or bsence of cpsulr polyscchride [35-36]. Encpsulted strins re further divided into six serotypes ( through f). Prior to widespred use of the H. influenze type b (Hib) conjugte vccines, Hib cused >80% of invsive H. influenze infections, predominntly in children under the ge of five [35], with mortlity rte of 3 to 6% nd further 20 to 30% developing permnent sequele rnging from mild hering loss to mentl retrdtion [36]. In res of routine vccintion, the mjority of invsive H. influenze infection is cused by nontypeble strins nd predominntly ffects children under the ge of one nd the elderly [35], with mortlity rte of 13 to 20% [36]. Approximtely 20 to 35% of isolted strins re resistnt to moxicillin [35]. Neisseri meningitidis (Encpsulted) is fstidious, erobic, grm-negtive diplococcus tht is spred by mucus or respirtory droplets often from symptomtic crriers. Thirteen different serogroups of N. meningitidis (A, B, C, D, H, I, K, L, X, Y, Z, W135, nd 29E) cn be distinguished. Serogroups B, C, nd Y re currently the most prevlent in developed countries nd serogroup A is predominnt in the rest of the world [37]. Serogroups W135 nd X lso cuse epidemics in developing regions of the world. The serogroups re determined by polyscchride cpsule tht ids bcteril survivl inside the humn host. N. meningitidis is the only species of Neisseri to produce cpsule. Encpsultion inhibits bctericidl ctivity of humn serum, thereby incresing survivl of meningococci in the environment [38]. It is estimted tht ~ 16% of N. meningitidis re not encpsulted [39]. Such strins re commonly found s commensl bcteri in the nsophryngel trct nd re not considered to be virulent [40]. FilmArry BCID will not detect unencpsulted N. meningitidis. Meningococcl disese (spinl meningitis nd/or meningococcemi) is rre in developed countries, but cn occur in outbreks. It is most common in infnts, children, nd young dults, nd ppers in plces with crowded living conditions (e.g., college dormitories nd militry brrcks). Sesonl incidence peks in lte winter nd erly spring [41]. Septicemi with N. meningitidis is ssocited with fever nd chrcteristic hemorrhgic rsh tht my be trnsient [42]. The disese BioFire Dignostics, LLC FilmArry BCID Pnel CE-IVD Instruction Booklet 9

cn progress extremely quickly (<24 hours) with hypotension, multiorgn dysfunction, shock, peripherl ischemi, nd limb loss nd hs mortlity rte of pproximtely 5-10% [43]. Pseudomons eruginos is grm-negtive opportunistic pthogen. It rrely cuses disese in helthy individuls but cn cuse sepsis in ptients with burn wounds, mlignncies or immunodeficiency, or in preterm infnts [44-45]. P. eruginos is leding cuse of nosocomil infections nd is responsible for 10% of ll hospitl cquired infections [46]. Mortlity rtes due to P. eruginos bcteremi re greter thn 20%, nd my be s high s 50% for intensive cre unit ptients nd burn victims [44-45]. P. eruginos is susceptible to limited number of ntibiotics (ntipseudomonl penicillins nd cephlosporins, crbpenems, fluorquinolones nd ciprofloxcin) [44], nd multi-drug resistnt (MDR) P. eruginos infection is becoming n incresing problem in hospitls [46]. The crbpenemse, KPC, hs recently been identified in isoltes of P. eruginos [47]. Yest Cndid lbicns, Cndid glbrt, Cndid krusei, Cndid prpsilosis, Cndid tropiclis. Cndid species re yests tht re ubiquitous in the environment nd s members of the norml humn microbiot, especilly in the digestive trct nd on mucous membrnes. These fungi re importnt custive gents of opportunistic nosocomil infections rnging from superficil (e.g., orl thrush) to systemic (e.g., septicemi) [48]. Cndid spp. re the 4 th most common cuse of nosocomil bloodstrem infections, hving been detected in pproximtely 9% of ll cses nd over 10% of cses from the ICU in lrge U.S. surveillnce study [4]. The mortlity rte for Cndid bloodstrem infection is pproximtely 40% nd they often occur in combintion with bcteri or second Cndid sp. [49]. The five most common species cusing bloodstrem infections re C. lbicns, C. glbrt, C. prpsilosis, C. tropiclis, nd C. krusei. Species distribution hs chnged in the pst three decdes so tht non-c. lbicns hve become more frequent thn C. lbicns. This shift is significnt due to non-c. lbicns species, especilly C. glbrt nd C. krusei, hving incresed rtes of resistnce to fluconzole, the drug most often used to tret Cndid bloodstrem infections [50]. Less common Cndid species my be misidentified s one of the five common species using phenotypic lbortory testing. In prticulr, C. dubliniensis cn be misidentified s C. lbicns; nd C. orthopsilosis nd C. metpsilosis (previously clssified s Group II nd Group III C. prpsilosis, respectively) cn be misidentified s C. prpsilosis [51]. Antimicrobil Resistnce Genes meca Methicillin resistnce. Methicillin-resistnt (MR) stphylococci re concern in both hospitl-cquired nd community-cquired infections. Few options exist for tretment of these infections, s the bcteri re resistnt to both nturl nd semi-synthetic β-lctm ntibiotics (e.g., oxcillin/methicillin) [10]. The primry mechnism of methicillin resistnce in stphylococci is through cquisition of the meca gene tht encodes penicillin binding protein (PBP2) tht hs low ffinity for β-lctms. meca is prt of gene complex crried on chromosomlly-integrted mobile genetic element clled the stphylococcl cssette chromosome mec (SCCmec). Ten mjor SCCmec types (I - X) hve been chrcterized [52]. In 2011, n SCCmec type XI cssette crrying divergent meca homologue (mecalga251/mecc), which lso confers methicillin resistnce, ws identified [53]. Although cogulse-negtive stphylococci (CoNS) hve higher rtes of methicillin resistnce (MR-CoNS) thn S. ureus (MRSA), CoNS is less virulent thn S. ureus nd is primrily limited to infections in the immunocompromised or individuls with indwelling devices [54]. Other mechnisms, such s penicillin binding protein muttions nd hyperproduction of stphylococcl β-lctmse, cn fcilitte reduced methicillin susceptibility in S. ureus in the bsence of meca [55]. vna/b Vncomycin resistnce. The prevlence of vncomycin-resistnt enterococcus (VRE) hs incresed rpidly, with VRE ccounting for 60% of E. fecium nd 2% of E. feclis isolted from the bloodstrem [3-4]. Infection with VRE increses the risk of deth to 75%, compred with 45% for infection with susceptible strin [3]. Eight gene clusters BioFire Dignostics, LLC FilmArry BCID Pnel CE-IVD Instruction Booklet 10

ssocited with vncomycin resistnce hve been identified to dte (vna, vnb, vnc, vnd, vne, vng, vnl, nd vnm), with vna nd vnb being the most common in clinicl isoltes [56-57]. Both the vna nd vnb gene clusters re borne on mobile genetic elements (trnsposons) nd cn be locted either on the chromosome or crried on plsmid. Enterococci crrying vna or vnb re resistnt to high levels of vncomycin. Isoltes crrying vna re lso resistnt to high levels of teicoplnin [57]. The next most commonly detected Enterococci, E. gllinrium nd E. csseliflvus, disply intrinsic low-level resistnce (intermedite resistnce, vnc phenotype) to vncomycin in the bsence of vna/b [58]. KPC (bl KPC) Crbpenem resistnce. Crbpenem-resistnt Enterobctericee (CRE) re incresingly importnt pthogens in the hospitl setting. Limited tretment options exist for CRE nd they re ssocited with high mortlity rtes. Those most t risk include ptients receiving long courses of ntibiotics nd those with indwelling devices (e.g. ventiltors, urinry ctheters, or intrvenous ctheters). The most common mechnism of crbpenemse-resistnce in CRE in the United Sttes is tht conferred by Klebsiell pneumonie crbpenemses (KPCs) genes (blkpc), fmily of crbpenemse enzymes first described in 2001 [59]. Since their emergence, KPCs hve become endemic in severl countries. Twelve vrints hve been identified to dte, KPC-2 through KPC-13, which differ by one to three mino cids [60-61]. KPCs re frequently crried on mobile genetic elements with the potentil to spred between orgnisms. KPCs hve been identified in mny Enterobctericee, the most common being K. pneumonie. In ddition to CRE, non- Enterobctericee such s Pseudomons eruginos nd Acinetobcter bumnnii my lso hrbor KPCs [62]. However, KPCs re not the most common mechnisms of crbpenem resistnce in these two orgnisms. P. eruginos nd A. bumnnii isoltes frequently crry other β-lctmses tht confer crbpenem resistnce (e.g. VIM, IMP, SIM, OXAs) or hve porin downregultion leding to crbpenem resistnce [63-64]. Detection of KPCs using phenotypic susceptibility testing (e.g., MIC brekpoints or Modified Hodge Test) is very difficult, not only becuse other mechnisms of crbpenem-resistnce exist, but lso becuse KPC ctivity is regulted by multiple mechnisms tht my not be ccurtely ssessed in vitro resulting in incorrect susceptibility reporting [65-66]. Alterntively, moleculr methods (e.g., PCR) re incresingly being used to specificlly identify KPC genes in clinicl isoltes [67]. Principle of the Procedure The FilmArry BCID pouch is closed system disposble tht houses ll the chemistry required to isolte, mplify nd detect nucleic cid from multiple bloodstrem pthogens within single blood culture smple. The rigid plstic component (fitment) of the FilmArry BCID pouch contins regents in freeze-dried form. The flexible plstic portion of the pouch is divided into discrete segments (blisters) where the required chemicl processes re crried out. The user of the FilmArry BCID Pnel lods the smple into the FilmArry BCID pouch, plces the pouch into the FilmArry instrument, nd strts the run. All other opertions re utomted. The following is n overview of the testing procedure: 1. Remove the pouch from its vcuum-seled pckge. Since solutions re drwn into the pouch by vcuum, it is importnt to keep pouches in their protective pckging until the time of use. 2. Plce the pouch into the FilmArry Pouch Loding Sttion. The FilmArry Pouch Loding Sttion hs been designed to prevent error by providing instructions nd visul cues in the form of color-coded rrows to ensure tht the pouch is properly loded. 3. Lod Hydrtion Solution into the pouch using the Pouch Hydrtion Syringe. The syringe is fitted with blunt stinless steel cnnul, which is used to deliver the solution into the pouch. Loding the pouch with Hydrtion Solution rehydrtes the freeze-dried regents contined in the pouch fitment. 4. Remove blood culture medi from the bottle (using 28-guge syringe to prevent clogging from resin) nd dd it to the Smple Buffer vil. Mix with Trnsfer Pipette. The Smple Buffer contins regents tht promote binding of nucleic cids to mgnetic beds for isoltion. BioFire Dignostics, LLC FilmArry BCID Pnel CE-IVD Instruction Booklet 11

5. Lod the smple/buffer mixture into the pouch using the Smple Loding Syringe. When the smple mixture is loded, process control contined in the fitment of the pouch is introduced into the smple. The process control monitors ll of the criticl processes tht occur in the pouch. 6. Trnsfer the pouch to the instrument nd initite run. The FilmArry softwre provides on-screen nimtions illustrting the steps needed to strt the run. 7. View results on the report t the completion of the run. The following is n overview of the opertions nd processes tht occur during FilmArry run: 1. Nucleic Acid Purifiction - Nucleic cid purifiction occurs in the first three blisters of the pouch. The smple is lysed by gittion (bed beting) nd the liberted nucleic cid is cptured, wshed nd eluted using mgnetic bed technology. These steps require bout ten minutes nd the bed-beter pprtus cn be herd s highpitched whine during the first minute of opertion. 2. 1 st Stge Multiplex PCR - The purified nucleic cid solution is combined with preheted mster mix to initite thermocycling for multiplex PCR. The effect of 1 st stge PCR is to enrich for the trget nucleic cids present in the smple. 3. 2 nd Stge PCR - The products of first stge PCR re diluted nd mixed with fresh PCR regents contining double strnded DNA binding dye (LCGreen Plus, BioFire Dignostics, LLC). This solution is distributed over the 2 nd stge PCR rry. The individul wells of the rry contin primers for different ssys (ech present in triplicte) tht trget specific nucleic cid sequences from ech of the pthogens detected, s well s control templte mteril. These primers re nested or internl to the specific products of the 1 st stge multiplex rection, which enhnces both the sensitivity nd specificity of the rections. 4. DNA Melting Anlysis After 2 nd stge PCR, the temperture is slowly incresed nd fluorescence in ech well of the rry is monitored nd nlyzed to generte melt curve. The temperture t which specific PCR product melts (melting temperture or Tm) is consistent nd predictble nd the FilmArry softwre utomticlly evlutes the dt from replicte wells for ech ssy to report results. For description of dt interprettion nd reporting see the Interprettion of Results section of this booklet. The FilmArry softwre controls the opertion of the instrument, collects nd nlyzes dt, nd utomticlly genertes test report t the end of the run. The entire process tkes bout n hour. Additionl detils cn be found in the FilmArry Opertor s Mnul. MATERIALS PROVIDED Ech kit contins sufficient regents to test 30 smples (30 pouch kit) or 6 smples (6 pouch kit): Individully pckged FilmArry BCID Pnel pouches Single-use (0.5 ml) Smple Buffer vils (red lid) Single-use (1.5 ml) Hydrtion Solution vils (blue lid) Individully pckged Trnsfer Pipettes Individully pckged Smple Loding Syringes with ttched cnnul (red cp) BioFire Dignostics, LLC FilmArry BCID Pnel CE-IVD Instruction Booklet 12

Individully pckged Pouch Hydrtion Syringes with ttched cnnul (blue cp) MATERIALS REQUIRED BUT NOT PROVIDED FilmArry system including: FilmArry or FilmArry 2.0 instrument nd softwre FilmArry Pouch Loding Sttion Syringes with 28-guge needle cpble of mesuring 0.1 ml (100 µl) smple volume BD 1cc Insulin Syringe #329410, or equivlent WARNINGS AND PRECAUTIONS Generl Precutions 1. For in vitro dignostic use only. 2. This device is restricted to sle by or on the order of physicin, or to clinicl lbortory; its use is restricted to, by, or on the order of physicin. 3. A trined helthcre professionl should crefully interpret the results from the FilmArry BCID Pnel in conjunction with ptient s signs nd symptoms nd results from other dignostic tests. 4. FilmArry BCID Pnel pouches re only for use with FilmArry systems. 5. Clinicl performnce chrcteristics of the FilmArry BCID Pnel hve only been determined with positive blood culture smples using the BD BACTEC Plus Aerobic/F Medium tht demonstrted the presence of orgnisms by Grm stin evlution. Other blood culture bottle types were evluted nlyticlly only (see Interference Section). 6. FilmArry pouches re stored under vcuum in individully-wrpped cnisters. To preserve the integrity of the pouch vcuum for proper opertion, be sure tht FilmArry instrument will be vilble nd opertionl before unwrpping ny pouches for loding. 7. Alwys check the expirtion dte on the pouch nd do not use pouch fter its expirtion dte. Sfety Precutions 1. Wer pproprite Personl Protective Equipment (PPE), including (but not limited to) disposble powder-free gloves nd lb cots. Protect skin, eyes, nd mucus membrnes. Chnge gloves often when hndling regents or smples. 2. Hndle ll smples nd wste mterils s if they were cpble of trnsmitting infectious gents. Observe sfety guidelines such s those outlined in CDC/NIH Biosfety in Microbiologicl nd Biomedicl Lbortories [68], the CLSI Document M29 Protection of Lbortory Workers from Occuptionlly Acquired Infections [69], or other pproprite guidelines. BioFire Dignostics, LLC FilmArry BCID Pnel CE-IVD Instruction Booklet 13

3. Follow your institution's sfety procedures for hndling biologicl smples. 4. Dispose of mterils used in this ssy, including regents, smples, nd used buffer vils, ccording to federl, stte, nd locl regultions. 5. Smple Buffer is ssigned the following clssifictions: Acute toxicity (Ctegory 4), Serious Eye dmge (Ctegory 1), nd Skin irrittion (Ctegory 2). Plese refer to the FilmArry Regent Kit Sfety Dt Sheet (SDS) for more informtion. 6. Smple Buffer will form hzrdous compounds nd fumes when mixed with blech or other disinfectnts. WARNING: Blech should never be dded to Smple Buffer or smple wste. Lbortory Precutions 1. Preventing orgnism contmintion Due to the sensitive nture of the FilmArry BCID Pnel, it is importnt to gurd ginst contmintion of the work re by following these guidelines: Positive blood culture smples contin high concentrtions of orgnisms nd require creful dherence to the smple processing steps described in this booklet. To void possible contmintion, smples should be processed in biosfety cbinet. If biosfety cbinet is not used, ded ir box (e.g., AirClen PCR worksttion), splsh shield (e.g., Bel-Art Sciencewre Splsh Shields), or fce shield should be used when prepring smples. A biosfety cbinet tht is used for performing bcteril culture should not be used for smple preprtion or pouch loding. Prior to processing smple, thoroughly clen both the work re nd the FilmArry Pouch Loding Sttion using suitble clener such s freshly prepred 10% blech or similr disinfectnt. To void residue buildup nd potentil PCR inhibition, wipe disinfected surfces with wter. Smples nd pouches should be hndled one-t--time. Chnge gloves nd clen the work re between ech smple. 2. Preventing mplicon contmintion A common concern with PCR-bsed ssys is flse positive results cused by contmintion of the work re with PCR mplicon. Becuse the FilmArry BCID Pnel pouch is closed system, the risk of mplicon contmintion is low provided tht pouches remin intct fter the test is completed. Adhere to the following guidelines to prevent mplicon contmintion: Discrd used pouches in n pproprite biohzrd continer immeditely fter the run hs completed. Avoid excessive hndling of pouches fter test runs. Avoid exposing pouches to shrp edges or nything tht might cuse puncture. WARNING: If liquid is observed on the exterior of pouch, the liquid nd pouch should be immeditely contined nd discrded in biohzrd continer. The instrument nd work spce must be decontminted s described in the FilmArry Opertor s Mnul. DO NOT PERFORM ADDITIONAL TESTING UNTIL THE AREA HAS BEEN DECONTAMINATED. BioFire Dignostics, LLC FilmArry BCID Pnel CE-IVD Instruction Booklet 14

3. Eliminting resin beds from the blood culture smples Some blood culture medi contin resin beds. The presence of resin beds in the FilmArry BCID Pnel test hs been shown to cuse pouch control filures nd ffect test performnce. Blood culture smples should be collected in mnner tht prevents resin beds from entering the smple nd the pouch. Use of 28-guge needle hs been shown to prevent resin beds from the BD BACTEC Plus Aerobic/F Medium from being drwn out of the bottle with the smple. USE OF SYRINGES WITH A LARGER NEEDLE BORE SIZE (SMALLER GAUGE) IS NOT RECOMMENDED. If the lbortory uses n lternte blood culture collection method (possibly to void the use of needles), the method should prevent resin beds from entering the FilmArry test. 4. Blood culture medi my contin non-vible orgnisms nd/or nucleic cids t levels tht cn be detected by the FilmArry BCID Pnel. The presence of non-vible orgnisms nd/or nucleic cids my led to flse positive test results. Typiclly, these flse positives will present with more thn one positive result becuse the BCID Pnel will lso detect the orgnism tht is growing in the culture bottle. Do not use blood culture medi tht contins chrcol (e.g., BcT/ALERT FA FAN Aerobic). There is n incresed risk of flse positive test results for Pseudomons eruginos nd Enterococcus when the FilmArry BCID Pnel is used to test biomérieux BcT/ALERT SN stndrd nerobic blood culture bottles. If the FilmArry BCID Pnel is used with these bottles, then positive results for Pseudomons eruginos nd Enterococcus should be confirmed by nother method prior to reporting the test results. In rre cses, the Grm stin result nd results of the FilmArry BCID Pnel my be discrepnt (for exmple, detection of grm-positive cocci by FilmArry BCID when grm-positive cocci were not observed in the Grm stin). In these cses, the results should be used in conjunction with other clinicl nd lbortory findings. Precution Relted to Public Helth Reporting in the United Sttes Locl, stte, nd federl regultions for notifiction of reportble disese re continully updted nd include number of orgnisms for surveillnce nd outbrek investigtions [70-71]. Additionlly, the Centers for Disese Control (CDC) recommends tht when pthogens from reportble diseses re detected by culture independent dignostic test (CIDT), the lbortory should fcilitte obtining the isolte or clinicl mterils for submission to the pproprite public helth lbortory to id in outbrek detection nd epidemiologicl investigtions. Lbortories re responsible for following their stte nd/or locl regultions nd should consult their locl nd/or stte public helth lbortories for isolte nd/or clinicl smple submission guidelines. REAGENT STORAGE, HANDLING AND STABILITY 1. Store the test kit, including regent pouches nd buffers, t room temperture (15 25 ºC). DO NOT REFRIGERATE. 2. Avoid storge of ny mterils ner heting or cooling vents or in direct sunlight. 3. Alwys check the expirtion dte nd do not use regents beyond the expirtion dte printed on the pouch or kit. 4. All kit components should be stored nd used together. Do not use components from one kit with those of nother kit. BioFire Dignostics, LLC FilmArry BCID Pnel CE-IVD Instruction Booklet 15

5. Do not remove pouches from their pckging until smple is redy to be tested. Once the pouch pckging hs been opened, the pouch should be loded s soon s possible (within pproximtely 30 minutes). 6. Once pouch hs been loded, the test run should be strted s soon s possible (within 60 minutes). SAMPLE REQUIREMENTS The FilmArry BCID Pnel is performed directly on positive blood culture smples tht demonstrte the presence of orgnisms s determined by Grm stin. Smple Volume 0.1 ml Smple Collection Smple should be collected from the blood culture bottle using syringe with 28-guge needle tht is cpble of mesuring 0.1 ml. Needles with lrger bore size (e.g., 18-guge) should not be used becuse they my llow resin beds from the blood culture medi to enter the smple. THE PRESENCE OF RESIN BEADS IN THE SAMPLE WILL AFFECT TEST PERFORMANCE AND SHOULD BE AVOIDED. If n lternte collection method is used, the lbortory should ensure tht the method dequtely excludes resin beds from the smple. Smple Age Blood culture smples should be processed nd tested s soon s possible fter being flgged s positive by the culture instrument. However, smples my be stored for up to 8 hours t room temperture or in the culture instrument prior to testing. PROCEDURE Refer to the FilmArry Blood Culture Identifiction Pnel Quick Guide, the FilmArry Trining Video or the FilmArry Opertor s Mnul for more detils nd pictoril representtions of these instructions. Gloves nd other Personl Protective Equipment (PPE) should be used when hndling pouches nd smples. Only one FilmArry BCID pouch should be prepred t time. Once smple is dded to the pouch, it should be promptly trnsferred to the instrument to strt the run. After the run is complete, the pouch should be discrded in biohzrd continer. Prepre Pouch 1. Thoroughly clen the work re nd the FilmArry Pouch Loding Sttion with freshly prepred 10% blech (or suitble disinfectnt) followed by wter rinse. 2. Remove the pouch from its vcuum-seled pckge by tering or cutting the notched outer pckging nd opening the protective luminum cnister. NOTE: If the vcuum sel of the pouch is not intct, the pouch my still be used. Attempt to hydrte the pouch using the steps in the Hydrte Pouch section. If hydrtion is successful, continue with the run. If hydrtion fils, discrd the pouch nd use new pouch to test the smple. 3. Slide the pouch into the FilmArry Pouch Loding Sttion so tht the red nd blue lbels on the pouch lign with the red nd blue rrows on the FilmArry Pouch Loding Sttion. BioFire Dignostics, LLC FilmArry BCID Pnel CE-IVD Instruction Booklet 16