INCLUSIVITY

Presented at the Association for Molecular Pathology (AMP) Annual Meeting, November 2017

Poster ID13

CONTACT INFORMATION

Corike Toxopeus, PhD.Corike.Nuibe@BioFireDefense.com

Cynthia Phillips, PhD.Cynthia.Phillips@BioFireDefense.com

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Corike Toxopeus1, Jared R. Helm1, Wendy A. Smith1, Natalie Batty1, Olivia Davidson1, Brandon Marble1, Lex Border1, Alex Kelley1, Bryan T. Gnade2, Stefan Fernandez2, Cheryce Smith1, Erika Pollard1, Jubal Stewart1, Juan-David Rouillard1, Cynthia L. Phillips1

1. BioFire Defense, LLC, Salt Lake City, UT2. U.S. Army Medical Materiel Development Activity (USAMMDA), Fort Detrick, MD

The FilmArray® Global Fever Panel:

Goal of Quick Diagnosis of Infectious Diseases Presenting with Acute Febrile Illness

INTRODUCTION / BACKGROUNDThe FilmArray Global Fever Panel is a multiplexed investigational molecular diagnostics tool that tests whole blood specimens for the presence of 17 different pathogens that cause Acute Febrile Illness (AFI) and is being developed in collaboration with the Department of Defense1 and the NIAID2. The Global Fever Panel requires minimal handling of blood specimens, only about 200 µL specimen volume, and test results are available within one hour. Quick diagnosis of the cause of AFI in a patient allows for early intervention using the appropriate treatment. This has several benefits including prevention of disease progression to a stage that would require more expensive intervention (e.g. hospitalization, additional medication), prevention of any potential negative consequences from incorrect medication (e.g. use of aspirin in the case of hemorrhagic fever), and preventing the spread of disease.

The organisms detected by the Global Fever Panel and associated BioSafety Level (BSL) for handling are listed in Table 1.

Table 1. Organisms Detected by the Global Fever Panel and Associated BSL Levels.BSL Level Bacteria Viruses Protozoa

BSL2Leptospira spp.Salmonella enterica serovar ParatyphiSalmonella enterica serovar Typhi

Dengue virusZika virus

Leishmania spp.Plasmodium spp.

BSL3Bacillus anthracisFrancisella tularensisYersinia pestis

Chikungunya virusWest Nile virusYellow fever virus

N/A

BSL4 N/ACrimean-Congo hemorrhagic virusEbola virusLassa virusMarburg virus

N/A

FILMARRAY SYSTEM

The FilmArray is a lab-in-a-pouch medium-scale fluid manipulation system performed in a self-contained, disposable, thin-film plastic pouch. The FilmArray platform processes a single sample, from nucleic acid purification to result, in a fully automated fashion.

Testing requires minimal pre-processing of specimens. The sample is loaded into the FilmArray pouch using a filter-injection vial. The user enters the sample and pouch type (using a barcode reader) into the software and initiates a run.

The FilmArray pouch has a fitment containing all needed freeze-dried reagents and plungers that plunge liquids to the film portion of the pouch. This portion consists of stations for cell lysis (blister C), magnetic-bead based

nucleic acid purification (D & E), reverse transcription and first-stage multiplex PCR (F & G), and an array of 102, second-stage nested PCR reactions (I).

PCR primers are dried into the wells of the array and each primer set amplifies a unique product of the first-stage multiplex PCR. The second-stage PCR product is detected by melt analysis using a fluorescent-double-stranded DNA

binding dye, LCGreen® Plus.

MATERIALS AND METHODS

CONCLUSIONSThe initial evaluation of the sensitivity and specificity of the FilmArray Global Fever Panel demonstrated performance equivalent to previous FilmArray panels. More thorough non-clinical evaluations of the Global Fever Panel, and an in-depth clinical evaluation are planned next. The goal for this panel is to provide a quick diagnosis of infectious diseases presenting with acute febrile illness.

Inclusivity of the Global Fever Panel assays was evaluated by challenging the system with different strains/isolates/serovars/species of on-panel organisms at near LoD levels in whole blood samples. Initially, three replicates were tested at 3×LoD. If all replicates tested positive (detection rate of 3/3), the tested isolate was considered inclusive. If there was one undetected result, two additional replicates were tested, and if both had a Detected result (detection rate of 4/5), the tested isolate was considered inclusive. If either criteria were not met, testing at higher concentrations was performed, and in some cases further investigation was warranted.

Table 4 summarizes the results for the inclusivity evaluation. For some organisms no additional isolates besides the primary isolate were available for testing, these organisms have ‘1/1’ listed in the ‘# Isolates Detected/Tested’ column, and ‘1×’ in the ‘× Est. LoD’ column.

Table 4. Global Fever Panel Results for the Mini-Inclusivity Evaluation.

FilmArray Global Fever Panel Analyte

# Isolates Detected/

TestedTested Isolate

Concentration Detected

(copies/mL)× Est. LoD

Test Results (Detected/

Tested)

BACTERIA

Bacillus anthracis 4/4

Ames 6.4E+02 1× 3/3Ames35 (genomic) 9.0E+01 3× 3/3Sterne (-pXO2) 9.0E+01 3× 3/3UM23-1 (genomic) 9.0E+01 3× 3/3

Francisella tularensis 1/1 Schu S4 Ag 1.2E+04 1× 3/3

Leptospira spp. 4/5

interrogans 3.9E+02 1× 3/3kirschneri (bogvere) 1.2E+03 3× 3/3noguchi 1.2E+03 3× 3/3borgpetersenii 3.9E+04 100x 3/3weilii serotype celledoni Ia 3.9E+04 100x 2/5

Salmonella enterica serovar Paratyphi A 2/2

ATCC® 9150™ 1.2E+01 1× 3/3I.N. Asheshov Collection 3.6E+01 3× 3/3

Salmonella enterica serovar Typhi 4/4

Subsp. Enterica Ty2 1.2E+01 1× 3/3NCTC 8385 [Ty 2] 3.6E+01 3× 5/5X-28 3.6E+01 3× 3/3CDC 2862-79 [H901] 3.6E+01 3× 3/3

Yersinia pestis 1/1 CO92 1.5E+02 1× 3/3PROTOZOA

Leishmania spp. 4/4

donovani 1.1E+01 1× 3/3donovani, 1S 3.3E+01 3× 3/3braziliensis Vianna 1:2.6*105 N/A 3/3infantum Nicolle 1.1E+02 10× 3/3

Plasmodium 7/7

falciparum, Pursat 1.0E+02 (spp.)1.0E+03 (falc.) 1× 3/3

vivax, 11 Strain Chesson 7.7E+01 1× 3/3brasilianum 2.7E+02 3× 3/3b

cynomolgi 2.7E+02 3× 3/3b,c

fieldi 2.7E+02 3× 3/3b,c

inui 2.7E+02 3× 3/3b

falciparum Tanzania 3.0E+03 3× 3/3b,d

falciparum SenTh021.09 3.0E+03 3× 3/3b,d

falciparum St. Lucia 3.0E+03 3× 3/3b,d

ESTIMATE OF LIMIT OF DETECTION (LoD) CROSS-REACTIVITY

A. Fitment with freeze-dried reagentsB. Plungers- deliver reagents to blistersC. Sample lysis and bead collectionD. Wash stationE. Magnetic bead collection blister

F. Elution StationG. Multiplex Outer PCR blisterH. Dilution blisterI. Inner Nested PCR array

• Whole blood (EDTA) was obtained from BioreclamationIVT.

• Organism stocks were obtained from BEI, ATCC, and Zeptometrix for most organisms.

• Live organism was used when possible in the on-site BSL2 facility.

• Nucleic acid concentration of organism stocks was quantified using qPCR kits.

STUDIESThree initial pre-clinical studies were performed with organisms to evaluate performance of the Global Fever Panel:

1) Estimate of LoD2) Cross-reactivity (intra- and extra-panel organisms)3) Inclusivity

References and AcknowledgementsThe FilmArray Global Fever Panel is being developed in collaboration with the Department of Defense1 and NIAID2

1. MCS-JPEO and USAMMDA Contract No. W911QY-13-D-0080, under the NGDS program

2. NIAID Contract No. HHSN272201600002C, “Advanced Development of Multiplex Diagnostic Platforms for Infectious Diseases (Global Fever Panel)”

The Global Fever Panel pouch has two internal controls in order to verify proper pouch functioning:

1. RNA Process ControlThe RNA Process Control assay targets a sequence of a spliced mRNA specific for Schizosaccharomyces pombe. The S. pombe is freeze-dried into the sample injection port of each pouch. When the sample is injected, the S. pombe is rehydrated and enters the pouch with the sample. S. pombe nucleic acid is extracted, purified, and tested along with the nucleic acid from the test sample. A positive result for the RNA Process Control indicates that all processes (nucleic acid extraction, reverse transcription, two polymerase chain reactions (PCR1 and PCR2), and melt analysis) are completed successfully.

2. PCR2 ControlThe PCR2 Control assay detects a synthetic DNA amplicon that is dried into triplicate wells of the second-stage PCR (PCR2) array along with its corresponding primers. A positive result indicates that the array filled properly and that PCR2 and the melt analysis were successful.

Serial 10-fold dilutions were tested in triplicate and the lowest concentration of an organism to have a Detected result for all 3 replicates was considered the Estimated LoD. Whole blood was multi-spiked with up to 5 organisms.

Table 2 shows the estimated LoD values obtained for each organism expressed in nucleic acid copies/mL and in values based on provided stock concentration when provided.

Table 2. Global Fever Panel Estimated LoD for Organisms

Organism Species/Strain

Estimated LoD ConcentrationLive or

InactivatedCopies/mL1Based on

Concentration Provided on the CofA

BACTERIABacillus anthracis Ames vegetative 6.4E+02 3.0E+01 CFU/mL InactivatedFrancisella tularensis Schu S4 Ag 1.2E+04 N/A Inactivated

Leptospira spp.interrogans 3.9E+02 N/A Livewoolfii2 1:106 N/A Live

Salmonella entericaserovar Typhi 1.2E+01 N/A Liveserovar Paratyphi A 1.2E+01 N/A Live

Yersinia pestis CO92 1.5E+02 3.0E+01 CFU/mL InactivatedVIRUSES

Chikungunya virus R80422 5.5E+02 1.1E+00 TCID50/mL InactivatedCCHF IbAr10200 6.4E+00 N/A Inactivated

Dengue virus

DENV-1 Hawaii 2.7E+01 N/A LiveDENV-2 New Guinea C 3.6E+01 N/A LiveDENV-3 H87 1.6E+03 N/A LiveDENV-4 H241 7.6E-01 N/A Live

Ebolavirus

Bundibugyo 1.4E+04 N/A InactivatedTai Forest 8.3E+01 N/A InactivatedReston 2.8E+03 N/A InactivatedSudan 1.1E+04 N/A InactivatedZaire Mayinga 1.1E+03 N/A Inactivated

Lassa Virus Josiah 5.6E+03 N/A Inactivated

Marburg MarburgvirusRavn 2.6E+01 N/A InactivatedCi67 5.0E+01 N/A Inactivated

West Nile virus B-956 Uganda 3.2E+03 N/A InactivatedYellow Fever virus strain 17D 1.2E+02 1.1E+01 TCID50/mL InactivatedZika virus PRVABC59 1.3E+02 4.7E+02 TCID50/mL Live

PROTOZOALeishmania spp. donovani 1.0E+01 2.2E+01 Cells/mL Live

Plasmodiumfalciparum Pursat Cambodia 2011,

IPC 48841.0E+023

1.0E+034 N/A Live

vivax 11 (Strain Chesson) 7.7E+01 N/A Liveovale TBD5 TBD N/A Live

1 Quantified using commercially available qPCR kits.2 No qPCR kit available3 Plasmodium spp. assay4 Plasmodium falciparum assay5 No organism source had been identified at time of testing.

Samples were prepared by diluting organism stocks with sterile saline to a final concentration of 10% of the original stock concentration. Exclusivity of the Global Fever Panel was verified if the expected Detected or Not Detected result was produced for each organism tested. Only one sample was tested for each organism; both intra-panel (A) and extra-panel (B) organisms were tested.

A. Intra-PanelAll intra-panel organisms were detected by the appropriate assay only and no cross-reactivity was observed during intra-panel testing of the Global Fever Panel.

B. Extra-PanelTable 3 lists the selection of extra-panel organisms evaluated at high concentrations for potential cross-reactivity with the FilmArray Global Fever Panel. Three Plasmodium species were detected and are bolded; this result was expected and these organisms may crossover into humans. No other off-panel organism was detected by the Global Fever Panel.

Table 3. Global Fever Panel Exclusivity Testing Results for Extra-Panel Organisms

Bacteria Viruses Protozoa

Borrelia burgdorferi Guanarito virus Babesia microti

Clostridium botulism Hendra virus

Plasmodium

inui1

Leptospira

group Ialstonii Hepatitis A virus berghei1

kmetyi Influenza A virus simiovale1,2

group II licerasiae Machupo virusTrypanosoma

brucei

group III

biflexa Measles virus cruzi

meyeri Rift Valley fever virus

wolbachii

Salmonella enterica subsp. enterica

serovar Typhimurium

Outbreak 2004LT2isolate 4

serovar Newport

S11975SL317SL254

1 Detected by Plasmodium spp. assay; these organisms may cross-over into humans and detection was to be expected.2 Detected by Plasmodium vivax/ovale assay; this organism may cross-over into humans and detection was to be expected.

FilmArray Global Fever Panel Analyte

# Isolates Detected/

TestedTested Isolate

Concentration Detected

(copies/mL)× Est. LoD

Test Results (Detected/

Tested)

VIRUSESChikungunya virus 1/1 R80422 5.5E+02 1× 3/3CCHF 1/1 IbAr10200 6.4E+00 1× 3/3

Dengue virus

DENV-1 6/6

Hawaii 2.7E+01 1× 3/3VN/BID-V1792/2007 8.1E+01 3× 3/3276RK1 2.7E+03 100× 3/3strain 12150 2.7E+03 100× 3/3strain BC89/94 2.7E+03 100× 3/3228690 2.7E+03 100× 3/3

DENV-21 5/5

New guinea C 3.6E+01 1× 3/3VN/BID-V1002/2006 (2-1) 1.1E+03 30× 3/3DakArA1247 (2-1) 1.1E+03 30× 3/3BC102/94 (2-1) 1.1E+03 30× 3/3strain 429557 (2-2) 1.1E+03 30× 3/3

DENV-3 3/3H87 1.6E+03 1× 3/3VN/BID-V1329/2006 4.8E+03 3× 3/3strain C0360/94 4.8E+03 3× 3/3

DENV-4 5/5

H241 7.6E-01 1× 3/3BC258/97 2.3E+00 3× 4/5strain 703 7.6E+00 10× 3/3BC13/97 7.6E+01 100x 3/3BC287/97 7.6E+01 100x 3/3

Ebolavirus

1/1 each species

Bundibugyo 1.4E+04 1× 3/3Tai Forest 8.3E+01 1× 3/3Reston 2.8E+03 1× 3/3Sudan 1.1E+04 1× 3/3

2/2 Zaire Mayinga strain 1.1E+03 1× 3/3Makona strain 3.3E+03 3× 3/3

Lassa virus 1/1 Josiah 5.6E+03 1× 3/3

Marburg Marburgvirus 3/3

RAVN 2.6E+01 1× 3/3Ci67 5.0E+01 1× 3/3Musoke 1.5E+02 3× 3/3

West Nile virus1/1 B-956 Uganda (WNV2) 3.2E+03 1× 3/3

2/2 NY2001 (WNV 1)2 9.6E+02c 0.1× 3/31986 (WNV1)2 9.6E+02c 0.1× 3/3

Yellow fever virus 1/1 strain 17D 1.2E+02 1× 3/3

Zika virus 5/5

PRVABC59 1.3E+02 1× 3/3Ibh 30656 3.9E+02 3× 3/3MR 766 3.9E+02 3× 3/3H/PAN/2016/BEI-259634 3.9E+02 3× 3/3FLR 3.9E+02 3× 3/3a The Leptospira weilii isolate tested did not meet the inclusivity criteria and is being further investigated.

b Plasmodium spp. Detected c Plasmodium spp. and Plasmodium vivax/ovale Detectedd Plasmodium spp. and Plasmodium falciparum Detected

1 The estimated LoD was initially set at 3.6E+02 copies/mL due to late crossing point values. Inclusivity testing was based on this initial estimated LoD value.2 There are two assays for West Nile virus on the Global Fever Panel. LoD was set for a lineage 2 virus for one West Nile virus assay; inclusivity testing was

performed with two lineage 1 viruses that were detected at a very low crossing point by the other West Nile virus assay, and therefore an additional dilution was tested.