FilmArray Meningitis/Encephalitis Panel

Christine C. Ginocchio, PhD, MT (ASCP) VP, Scientific and Medical Affairs, BioFire Diagnostics

VP, Global Microbiology, bioMerieux

Professor of Medicine, Hofstra North Shore-LIJ School of Medicine, NY

2

Delayed Treatment: Increased

Morbidity and Mortality

The causative agents of meningitis or encephalitis should be rapidly identified to guide

appropriate patient management, but are many times impossible to identify based on

clinical indications alone due to overlapping symptoms1,2

Delays in appropriate therapy can be associated with adverse outcomes1

Early diagnosis facilitates timely and appropriate therapeutic interventions and

can minimize the risks of adverse outcomes and mortality2

Viral

Brain damage, including

behavioral and personality

changes and memory and

speech problems

Focal neurological signs

Seizures

Bacterial

Permanent brain and nerve

damage

Behavioral changes

Cognitive disabilities

Lack of muscle control

Seizures

Fungal

Increased intracranial

pressure

Hydrocephalus

IRIS

Blindness, sometimes with

optic atrophy

3

Traditional Diagnostics

CSF

Examination5

Cell count

Protein

Glucose

CSF

Culture5,6

Pathogen-specific

media

Bacterial culture

Viral culture

Fungal culture

Rapid Latex

Agglutination Test8

India

Ink Stain5 Gram Stain7

CSF=cerebrospinal fluid; PCR=polymerase chain reaction. 1. Bamberger DM. Am Fam Physician. 2010;82:1491-1498. 2. Tunkel AR et al. Clin Infect Dis. 2008;47:303-327. 3. NINDS. Meningitis and Encephalitis Fact Sheet. www.ninds.nih.gov/disorders/encephalitis_meningitis/detail_encephalitis_ meningitis.htm. Updated November 24, 2014. Accessed January 6, 2015. 4. College of American Pathologists. Microbiology Checklist. April 21, 2014. 5. Bahr NC et al. Biomark Med. 2014;9:1085-1103. 6. Markoulatos P et al. J Clin Microbiol. 2001;39:4426-4432. 7. Kumal S. Textbook of Microbiology. Jaypee Brothers Medical Publishers (P) Ltd. First Edition. 2012. 8. Duval BD et al. Am J Trop Med Hyg. 2014;90:1043-1046.

Traditional

PCR5 Crystal

violet

Iodine

Alcohol

Safranin

Positive Negative

<1 hr 72 hr-14 d <1 hr < 1 hr 2-4 hr 1-24 hr

2-5 d

4

Challenges in Diagnosing Meningitis

and Encephalitis Infections Meningitis and encephalitis often present with similar symptoms, sometimes as

flu-like symptoms1

The causative agents underlying the disease may not be distinguishable based

on clinical symptoms alone2

Challenges associated with currently available testing methods1,3:

Time-consuming

Technically

complex/requires

specific expertise

Lack sensitivity and

specificity

Small volume of CSF

obtained

Physician must choose

which tests to select

based on symptoms and

available CSF volume

Need to order multiple

tests specific for

suspected organisms Accuracy may be

affected by antibiotic

administration

CSF=cerebrospinal fluid. 1. NINDS. Meningitis and Encephalitis Fact Sheet. www.ninds.nih.gov/disorders/encephalitis_meningitis/detail_

encephalitis_meningitis.htm. Updated November 24, 2014. Accessed January 6, 2015. 2. Bamberger DM. Am Fam Physician. 2010;82:1491-1498. 3. Bahr NC et al. Biomark Med. 2014;9:1085-1103.

5

Meningitis/Encephalitis Panel

Bacteria

Escherichia coli K1

Haemophilus influenzae

Listeria monocytogenes

Neisseria meningitidis

Streptococcus agalactiae

Streptococcus pneumoniae

Fungi

Cryptococcus neoformans/gattii

Viruses

Cytomegalovirus (CMV)

Enterovirus

Herpes simplex virus 1 (HSV-1)

Herpes simplex virus 2 (HSV-2)

Human herpesvirus 6 (HHV-6)

Human parechovirus

Varicella zoster virus (VZV)

6

E. coli

GBS

L. monocytogenes

H. influenzae

HSV-1, 2

EV, PeV

S. pneumoniae

N. meningitidis

H. influenzae (NT)

EV, PeV

HHV-6

EBV, CMV

Arboviruses

N. meningitidis

S. pneumoniae

H. influenzae (NT)

EV, PeV

HSV-1, 2

EBV, CMV

Arboviruses

S. pneumoniae

N. meningitidis

H. influenzae (NT)

Gram neg (K. kingae)

HSV-1, 2, VZV (zoster)

EV

Arboviruses

Neuroborelliosis

All above +

L. monocytogenes

CMV, EBV

HIV

HHV-6

Polyomaviruses (BK)

Aspergillosis

Nocardiosis

Cryptococcosis

Toxoplasmosis

Neurosyphillis

Gram negatives

Gram positives

+++++++++

Comprehensive Diagnostics for the

Most Common Pathogens

7

BioFire FilmArray® System

8

BioFire FilmArray® Pouch

Sample extraction and

purification

1st stage multiplex

PCR

2nd stage PCR

9

8. The FilmArray performs a melt to confirm the presence or

absence of assay-specific temperature signatures of the second

stage PCR product for each well in the array

2. Nucleic acids bound by magnetic beads move from the lysis

chamber to the purification chamber. A wash buffer removes

cellular and pathogen debris

3. An elution buffer removes purified nucleic acids from the

magnetic beads

4. Nucleic acids move to the first-stage PCR chamber. Reverse

transcriptase converts target RNA to DNA, followed by a high-order

multiplex PCR

5. Products from the first-stage PCR are diluted to remove any

remaining PCR primers

6. First-stage PCR products are added to fresh master mix and are

aliquoted into each well of the array

7. Each well is pre-spotted with a single pair of second-stage PCR

primers, resulting in specific amplification of target DNA only. A

fluorescent double-stranded DNA binding dye monitors each reaction

1. Sample moves into lysis chamber. Cells and pathogens are lysed

by bead beating, releasing nucleic acids

65:00

65 minutes

run-time

64:00 63:00 62:00 61:00 60:00 59:00 58:00 57:00 56:00 55:00 54:00 53:00 52:00 51:00 50:00 49:00 48:00 47:00 46:00 45:00 44:00 43:00 42:00 41:00 40:00 39:00 38:00 37:00 36:00 35:00 34:00 33:00 32:00 31:00 30:00 29:00 28:00 27:00 26:00 25:00 24:00 23:00 22:00 21:00 20:00 19:00 18:00 17:00 16:00 15:00 14:00 13:00 12:00 11:00 10:00 09:00 08:00 07:00 06:00 05:00 04:00 03:00 02:00 01:00 00:00 65:00

Sample Extraction, Amplification,

and Detection: All Inclusive

15-0159-00/MK

10

Automated Results Analysis

102 individual 2nd stage PCR wells

Each well contains one reaction

2/3 wells must be positive

Melt curves generated for each well: must be within specific ranges

Process Control Freeze-dried Schizosaccharomyces pombe organism

Re-suspended with specimen

•PCR II Control

DNA template spotted on the array

FilmArray® Results Qualification

Staphylococcus aureus

15-0159-00/MK

11

Intended Use

Qualitative test for bacteria, viruses and

yeast

CSF (200 ml) obtained via lumbar

puncture from individuals with signs

and/or symptoms of meningitis and/or

encephalitis.

Not centrifuged, xanthrochromic and

bloody acceptable

Not intended for testing of specimens

collected from indwelling CNS medical

devices (shunts)

“Aid in diagnosis” – must be used with

other clinical, lab and epidemiological

data

Culture is necessary for organism

recovery, typing, and antimicrobial

susceptibility testing of bacterial agents

FilmArray and FilmArray

2.0 Systems

12

Performance Studies

Analytical Validation

Limit of Detection (LoD)

Reactivity

Precision/Reproducibility

Clinical Validation

Prospective clinical trial

Archived specimen testing

Contrived specimen testing

13

ANALYTICAL STUDIES

14

LoD is 100 – 1,000 CFU/mL or cells/mL for all bacteria & yeast

ME Panel Test Result Species/Isolate Tested

LoD

Concentration BACTERIA

E. coli K1 E. coli K1, O18ac:K1:H7

1×103 CFU/mL ATCC 700973

H. influenzae H. influenzae, type b, biotype I

1×103 CFU/mL ATCC 10211

L. monocytogenes L. monocytogenes, type 4b

1×103 CFU/mL ATCC 13932

N. meningitidis N. meningitidis, type W135

100 CFU/mL ATCC 43744

S. agalactiae S. agalactiae, group B

1×103 CFU/mL ATCC 13813

S. pneumoniae S. pneumoniae, serotype 1 100 cells/mL*

YEAST

C. neoformans/gattii

C. neoformans

ATCC 20882 100 CFU/mL

C. gattii

ATCC MYA-4877 100 CFU/mL

*cells/mL is based on OD600 reading – no CFU/mL determined

Limit of Detection (LoD)

95% Detection Level

15

Limit of Detection (LoD)

95% Detection Level

ME Panel Test Result Species/Isolate Tested LoD Concentration

viruses

CMV CMV, strain AD-169 100 TCID50/mL

Zeptometrix 0810003CF (4.30×103 copies/mL)

EV

(Species A-D)

Coxsackievirus A6, species A, strain Gdula 50 TCID50/mL

ATCC VR-1801

Coxsackievirus A9, species B 5 TCID50/mL

Zeptometrix 0810017CF

Coxsackievirus A17, species C, strain G-12 5 TCID50/mL

ATCC VR-1023

EV 70, species D, strain J670/71 50 TCID50/mL

ATCC VR-836

HSV-1 HSV-1, strain MacIntyre 250 TCID50/mL

Zeptometrix 0810005CF (1.51×103 copies/mL)

HSV-2 HSV-2, strain MS 50 TCID50/mL

Zeptometrix 0810006CF (1.29×103 copies/mL)

HHV-6 HHV-6A, strain U1102, NCPV 0003121v

1×104 copies/mL HHV-6B, strain HST, NCPV 0006111v

HPeV HPeV, type 3

500 TCID50/mL Zeptometrix 0810147CF

VZV VZV, strain Ellen 0.10 TCID50/mL

Zeptometrix 0810171CF (1.66×103 copies/mL)

LoD is 5-500 TCID50/mL or 100 – 1,000 copies/mL for all viruses

16

Summary of ME Analytical Reactivity

(Inclusivity)

FilmArray ME

Panel

Test Result

# Tested and

Detected

Concentration

Detected Isolates Tested and Detected

Bacteria

E. coli K1 5 ≤ 3,000 CFU/mL E. coli strains of the K1 serotype only

H. influenzae 9 ≤ 3,000 CFU/mL Non-typeable and typeable (types a-f) strains of H. influenzae

L. monocytogenes 6 ≤ 3,000 CFU/mL Types 1/2a, 1/2b, and 4b of L. monocytogenesa

N. meningitidis 7 ≤ 300 CFU/mL Encapsulated N. meningitis (serotypes W135, A, B, C, D, Y and

DNA from a strains with a variant ctrA gene)

S. agalactiae 5 ≤ 3,000 CFU/mL Multiple serotypes or isolates of S. agalactiae (Group B

Streptococcus)

S. pneumoniae 6 ≤ 300 cells/mL Multiple serotypes of S. pneumoniae

Viruses

CMV 5 ≤ 300 TCID50/mL

(≤ 1×104 copies/mL) Multiple strains of Cytomegalovirus (CMV).

EV 18 ≤ 50 TCID50/mL Representative isolates from all species (A-D) and several

serotypes of human Enterovirus, Coxsackievirus, and Echovirusb

HSV-1 5 ≤ 750 TCID50/mL

(≤ 4.5×103 copies/mL) Multiple strains of Herpes simplex virus 1 (HSV-1)

HSV-2 5 ≤ 150 TCID50/mL

(≤ 4×103 copies/mL) Multiple strains of Herpes simplex virus 2 (HSV-2)

HHV-6 4 ≤ 3×104 copies/mL A and B variants of Human herpesvirus 6 (HHV-6)

HPeV 6 500 - 5,000 TCID50/mL Serotypes 1-6 of Human parechovirus (HPeV)c

VZV 5 ≤ 0.3 TCID50/mL

(≤ 5×103 copies/mL) Multiple strains of Varicella zoster virus (VZV)

Yeast

C. neoformans/gattii 10

(5 per species) ≤ 300 CFU/mL

Multiple strains, serotypes, and genotypes of Cryptococcus

neoformans and Cryptococcus gattii

17

Analytical Specificity

(Exclusivity/Cross-Reactivity)

>100 off-panel isolates tested at high concentration Minor cross-reactivity identified:

Enterovirus (EV2) assay will cross-react with various rhinoviruses

H. influenzae assay (H. influenzae 1) will cross-react with H. haemolyticus

C. neoformans/gattii assay will cross-react with C. amylolentus

Rhinovirus and H. haemolyticus are rarely isolated from CSF – but can be in respiratory tract – avoid sample contamination

C. amylolentus is not isolated from humans

18

Precision/Reproducibility –

Study Design

Every analyte tested @ 3 levels

Moderate Positive (3× LoD)

- Expect positive results in >95% of samples

Low Positive (1× LoD)

- Expect positive results in ≥95% of samples

Negative

- Expect negative results in all samples

Testing occurs:

At 3 test sites

Multiple runs per day, on multiple (5) days

On multiple instruments/ 2.0 systems

By multiple operators (2 per day per site)

With multiple pouch/reagent lots

19

Precision/Reproducibility

Results

Expected qualitative results (Detected/Not Detected) reported at all sites, with all instruments/systems, etc.

Test Result

(Organism/Isol

ate Tested)

Concentratio

n Tested

Expecte

d Result

Agreement with Expected Result

FilmArray FilmArray 2.0

Site A Site B Site C

All Sites

(95%

Confidence

Interval)

System A System B System

C

All Systems

(95%

Confidence

Interval)

BACTERIA

E. coli K1

(ATCC 700973)

Moderate

Positive

3× LoD

3×103

CFU/mL

Detected 30/30

100%

30/30

100%

30/30

100%

90/90

100%

(96.0%-

100%)

30/30

100%

30/30

100%

30/30

100%

90/90

100%

(96.0%-100%)

Low Positive

1× LoD

1×103

CFU/mL

Detected 30/30

100%

30/30

100%

30/30

100%

90/90

100%

(96.0%-

100%)

29/30

96.7%

29/30

96.7%

29/30

96.7%

87/90

96.7%

(90.6%-99.3%)

Negative

(No analyte)

Not

Detected

60/60

100%

60/60

100%

60/60

100%

180/180

100%

(98.0%-

100%)

60/60

100%

60/60

100%

60/60

100%

180/180

100%

(98.0%-100%)

20

CLINICAL PERFORMANCE PROSPECTIVE, ARCHIVED, CONTRIVED TESTING

21

Prospective Study

Overview

In hospital laboratories

Prospectively-collected patient specimens

Residual specimens leftover from clinician order

for CSF bacterial culture

At least 1500 tests to show performance in

intended use setting

22

Comparator Methods

CSF Bacterial Culture Escherichia coli K1

Haemophilus influenza

Listeria monocytogenes

Neisseria meningitidis

Streptococcus agalactiae

Streptococcus pneumoniae

PCR & Sequencing* Cytomegalovirus

Epstein-Barr virus

Herpes simplex virus1

Herpes simplex virus 2

Human herpesvirus 6

Varciella zoster virus

Human parechovirus

Human enterovirus

Cryptococcus neoformans/gattii

* 2 PCR assays + sequencing

23

Data Collection

Basic Demographics

Age range (<2 mo, 2 – 23 mo, 2 – 17 yrs, 18 – 34 yrs, 35 – 64

yrs, >65 yrs)

Sex

Status (ED, inpatient, outpatient)

Results of bacterial culture

Results of any other CSF testing ordered

CSF chemistry (protein, glucose, WBC + differential)

For positives:

Abx issued within 24 hrs prior to LP

Immune status

Clinician final diagnosis

24

Enrollment Summary

1560 prospectively-collected, residual specimens

Of which 545 (35%) had

been previously frozen*

Feb – Sept 2014

11 U.S. Study Sites

All age groups included

(41% under 18)

Mostly hospitalized (59%) or ED (34%)

*IUO pouches available June 2014

25

System Performance

98.9% of specimens were successfully

tested on the initial test

11 instrument / software errors (~0.7%)

6 pouch control failures (~0.4%)

26

19.40%

11.90% 7.60% 6.70%

4.40% 4.60%

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

100.0%

< 2 mo.(n=299)

2-23 mo.(n=143)

2-17 years(n=197)

18-34 years(n=224)

35-64 years(n=522)

65+ years(n=175)

Overall Positivity = 8.7%

Positivity by Age Group

27

Prevalence by FilmArray

February to September 2014 - United States

FilmArray ME Panel

Result

Overall

(n=1560)

< 2 mo.

(n=299)

2-23 mo.

(n=143)

2-17 years

(n=197)

18-34 years

(n=224)

35-64 years

(n=522)

65+ years

(n=175)

Bacteria

E. coli K1 3 (0.2%) 0 (0%) 1 (0.7%) 0 (0%) 0 (0%) 2 (0.4%) 0 (0%)

H. influenzae 2 (0.1%) 0 (0%) 1 (0.7%) 0 (0%) 0 (0%) 1 (0.2%) 0 (0%)

L. monocytogenes 0 (0.0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%)

N. meningitidis 0 (0.0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%)

S. agalactiae 1 (0.1%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (0.6%)

S. pneumoniae 16 (1.0%) 2 (0.7%) 2 (1.4%) 2 (1%) 3 (1.3%) 4 (0.8%) 3 (1.7%)

Viruses

CMV 6 (0.4%) 4 (1.3%) 0 (0%) 1 (0.5%) 1 (0.4%) 0 (0%) 0 (0%)

EV 51 (3.3%) 31 (10.4%) 5 (3.5%) 11 (5.6%) 4 (1.8%) 0 (0%) 0 (0%)

HSV-1 4 (0.3%) 0 (0%) 2 (1.4%) 0 (0%) 0 (0%) 2 (0.4%) 0 (0%)

HSV-2 12 (0.8%) 0 (0%) 0 (0%) 0 (0%) 1 (0.4%) 8 (1.5%) 3 (1.7%)

HHV-6 22 (1.4%) 9 (3%) 7 (4.9%) 2 (1%) 3 (1.3%) 1 (0.2%) 0 (0%)

HPeV 12 (0.8%) 12 (4%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%)

VZV 7 (0.4%) 0 (0%) 0 (0%) 0 (0%) 3 (1.3%) 3 (0.6%) 1 (0.6%)

Yeast

C. neoformans/gattii 5 (0.3%) 1 (0.3%) 0 (0%) 0 (0%) 1 (0.4%) 2 (0.4%) 1 (0.6%)

28

Analyte Sensitivity Specificity

TP/(TP + FN) % 95%CI TN/(TN + FP) % 95%CI

Bacteria

E. coli K1 2/2 100 34.2-100 1557/1558 99.9 99.6-100

H. influenzae 1/1 100 - 1558/1559 99.9 99.6-100

L. monocytogenes 0/0 - - 1560/1560 100 99.8-100

N. meningitidis 0/0 - - 1560/1560 100 99.8-100

S. agalactiae 0/1 0.0 - 1558/1559 99.9 99.6-100

S. pneumoniae 4/4 100 51.0-100 1544/1556 99.2 98.7-99.6

Analyte Positive Percent Agreement Negative Percent Agreement

TP/(TP + FN) % 95%CI TN/(TN + FP) % 95%CI

Viruses

CMV 3/3 100 43.9-100 1554/1557 99.8 99.4-99.9

EV 44/46 95.7 85.5-98.8 1507/1514 99.5 99.0-99.8

HSV-1 2/2 100 34.2-100 1556/1558 99.9 99.5-100

HSV-2 10/10 100 72.2-100 1548/1550 99.9 99.5-100

HHV-6 18/21 85.7 65.4-95.0 1532/1536 99.7 99.3-99.9

HPeV 9/9 100 70.1-100 1548/1551 99.8 99.4-99.9

VZV 4/4 100 51.0-100 1553/1556 99.8 99.4-99.9

Yeast

C. neoformans/gattii 1/1 100 - 1555/1559 99.7 99.3-99.9

Summary of Prospective Data

29

Concern for False Positives

Laboratory Precautions

Due to the sensitive nature of the FilmArray ME Panel, it is

important to guard against contamination.

Limitations in Product Literature S. pneumoniae and H. influenzae can be shed from the

respiratory tract of healthy individuals.

HSV-1 may also be shed from individuals with active or recurrent

cold sores.

Particular attention should be given to the Laboratory

Precautions noted under the Warnings and Precautions section.

Caution should also be exercised during specimen collection and

testing to prevent contamination leading to false positive results.

Results from this test must be correlated with the clinical history,

epidemiological data, and other data available to the clinician

evaluating the patient.

30

Cryptococcal Antigen Test

PCR negative/CrAg positive

Low level CrAg titers may persist for extended periods of

time following appropriate therapy and the resolution of

infection

- May be indicative of cryptococcal immune reconstitution

inflammatory syndrome (IRIS) with history of cryptococcal

meningitis and sterile cultures

Medical chart review indicated that each subject with

positive CrAg and negative FilmArray was on antifungal

therapy for treatment of cryptococcal meningitis or

cryptococcosis at the time of specimen collection and/or

had prior history of Cryptococcus infection.

- likely due to antigen persistence rather than the presence of live

organism

31

Co-Detections

Distinct Co-Detection Combinations

Analyte 1 Analyte 2

CMV S. pneumoniae

EV HPeV

HSV-1 HHV-6

HSV-2 S. agalactiae

VZV S. pneumoniae

136 Positive Specimens

141 Positive Results

5 dual detections

Recommend retesting

specimens showing

multiple detections, to

rule out potential

contamination

32

Considerations for

Herpesvirus Detections

HHV-6 or CMV can exist in latent form that is reactivated during infection due to other pathogens, including agents not detected by the FilmArray ME panel that may cause meningitis/encephalitis (e.g., Mycobacterium tuberculosis or HIV).

When detected by the FilmArray ME, HHV-6 or CMV should be considered as the likely cause of meningitis/encephalitis only in appropriate clinical settings and following expert consultation

Viral shedding into the CSF often occurs in cases of zoster (shingles; caused by reactivation of VZV). VZV may not be the cause of CNS disease in some cases.

33

Archived Study

Purpose:

Supplement prospective

study by testing known

positive sepcimens

Workflow:

Lab ID is confirmed by

molecular assay

Data Analysis:

Only confirmed

specimens are

considered for

performance

Analyte Received Confirmed (%)

E. coli K1 7 2 (29%) Haemophilus influenzae 3 3 (100%)

Listeria monocytogenes 1 1 (100%)

Neisseria meningitidis 8 7 (88%)

Streptococcus agalactiae 2 2 (100%)

Streptococcus pneumoniae 21 17 (81%)

CMV 11 8 (73%)

HSV-1 17 10 (59%)

HSV-2 26 18 (69%)

ARUP “HSV positive” 35 22 (63%)

HHV-6 22 16 (73%)

HPeV 6 3 (50%)

VZV 29 22 (76%)

Cryptococcus neoformans/gattii

22 19 (86%)

Total 210 150 (71%)

Previous Panel Archived Confirmation:

RP 86% confirmed and GI 85% confirmed

34

Archived Performance

Analyte Positive Percent Agreement Negative Percent Agreement

TP/(TP + FN) % 95% CI TN/(TN + FP) % 95% CI

Bacteria

E. coli K1 2/2 100 34.2-100 35/35 100 90.1-100

H. influenzae 3/3 100 43.9-100 39/39 100 91.0-100

L. monocytogenes 1/1 100 - 41/41 100 91.4-100

N. meningitidis 7/7 100 64.6-100 34/34 100 89.8-100

S. agalactiae 2/2 100 34.2-100 40/40 100 91.2-100

S. pneumoniae 17/17 100 81.6-100 21/21 100 84.5-100

Viruses

CMV 7/8 87.5 52.9-97.8 181/181 100 97.9-100

HSV-1 16/16 100 80.6-100 156/157 99.4 96.5-99.9

HSV-2 33/34 97.1 85.1-99.5 136/136 100 97.3-100

HHV-6 12/16 75.0 50.5-89.8 168/168 100 97.8-100

HPeV 2/3 66.7 20.8-93.9 187/187 100 98.0-100

VZV 22/22 100 85.1-100 162/164 98.8 95.7-99.7

Yeast

C. neoformans/gattii 19/19 100 83.2-100 171/171 100 97.8-100

35

Contrived Study

Residual CSF specimens negative for all ME

panel analytes (by FilmArray and comparator

methods) were co-spiked

235 Specimens - 35 Negative

- 50 specimens per analyte with at least six quantified isolates

evaluated - 25 at 2 × LoD

- 25 at four concentrations spanning the clinically relevant range (based on Cp values from

previous FilmArray ME Panel positive test results)

Specimens tested at six of the prospective study

clinical laboratories

36

Contrived Performance

a One E coli false negative was observed at 2 × LoD and one E coli false negative was observed at 0.2 × LoD. b Both S. agalactiae false negatives were observed at 0.2 × LoD. c Both CMV false negatives were observed at 0.2 × LoD.

Analyte

/PPA NPA

TP/(TP +

FN) % 95% CI TN/(TN + FP) % 95% CI

E. coli K1 47/49a 95.9 86.3-98.9 245/245 100 98.5-100

H. influenzae 50/50 100 92.9-100 243/244 99.5 97.7-99.9

L. monocytogenes 50/50 100 92.9-100 244/244 100 98.5-100

N. meningitidis 75/75 100 95.1-100 219/219 100 98.3-100

S. agalactiae 48/50b 96.0 86.5-98.9 244/244 100 98.5-100

CMV 47/49c 95.9 86.3-98.9 245/245 100 98.5-100

HHV-6 50/50 100 92.9-100 243/244 99.5 97.7-99.9

HPeV 50/50 100 92.9-100 244/244 100 98.5-100

37

Summary of Registration

Studies

FilmArray ME Panel is:

Sensitive

- LoD in 100 – 1,000 copies/mL

- Clinical study showed 95-100% PPA for most analytes

Specific

- Limited cross-reactivity

- Clinical study showed >99% NPA for all analytes

Reliable

- Highly reproducible test results

- Robust

- Few errors/control failures in intended use setting