Presented at the American Society of Tropical Medicine and Hygiene (ASTMH) Annual Meeting, November 20 - 24, 2019

Poster C-1803See all of BioFire’s scientific posters by scanning the QR code to access the Scientific Poster Page

Brian W. Jones, David Rabiger, Mark A. Gurling, Madeline Veloz, Olivia Jackson, Marissa Burton, Natalie Batty, Pascal Belgique, Cynthia D. Andjelic, Cynthia L. Phillips

BioFire Defense, LLC, Salt Lake City, Utah, USA

Clinical Evaluation of the BioFire® FilmArray® Global Fever Panel

ABSTRACT

Acute Febrile Illness (AFI) is caused by a diverse set of pathogens. Standard testing for the causal agent is often difficult, slow, costly, and complicated by the overlapping differential diagnoses for each potential pathogen. Testing for multiple pathogens simultaneously would simplify and accelerate diagnosis. The FilmArray Global Fever (GF) Panel, developed by BioFire Defense in collaboration with the U.S. Department of Defensea and NIAIDb, uses an automated, multiplex PCR system to evaluate whole blood samples for 19 pathogens simultaneously in under an hour. Targets of the GF Panel are Chikungunya virus, CCHF virus, dengue virus (serotypes 1-4), Ebolavirus, Lassa virus, Marburgvirus, West Nile virus, Yellow fever virus, Zika virus, Bacillus anthracis, Francisella tularensis, Leptospira spp., Salmonella enterica serovar Typhi and Paratyphi A, Yersinia pestis, Leishmania donovani complex, Plasmodium spp., P. falciparum, and P. ovale/vivaxc. BioFire Defense is conducting a prospective clinical study to evaluate the sensitivity and specificity of the GF Panel when used to test blood collected from subjects with fever or who have recently had fever. This multi-center study is being conducted at locations around the world. Comparator testing consists of in-house developed PCR assays followed by bidirectional sequencing. Here we report the positive percent agreement and negative percent agreement of the GF Panel versus comparator testing, the rate of positive detections, and the fraction of specimens with more than one detected infection. Our results show that the FilmArray GF Panel could aid in rapid and actionable AFI diagnosis.

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

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

c. This test has not been reviewed by FDA and has not been approved for diagnostic use.

INTRODUCTION

The FilmArray Global Fever (GF) Panel uses an automated, multiplex PCR system to evaluate whole blood samples for 19 pathogenic bacteria, viruses, and parasites in a single test. Using prospectively collected whole blood specimens, BioFire Defense is conducting a pivotal clinical study to evaluate the specificity and sensitivity of the FilmArray GF Panel.

SUBJECT ENROLLMENT

Prospectively collected whole blood specimens have been collected in ten geographically distinct locations (Figure 1) between March 2018 and September 2019. Clinical whole blood samples were collected from 1630 consented male and female subjects representing all age groups (Table 1). Enrollment and testing is ongoing. This preliminary report includes results in sensitivity and specificity calculations only when FilmArray and comparator testing data are both available for the given assay.

FIGURE 1. STUDY SITES

BioFireDefense

GWUWashU

UNAH,Honduras

NAMRU-6,Peru

NHRC/NAMRU-3,Ghana

USAMRD-A,Kenya

IDI,Uganda AFRIMS,

Thailand

NAMRU-2,Cambodia

KCMC-Duke,Tanzania

AFRIMS – Armed Forces Research Institute of Medical SciencesGWU – George Washington UniversityIDI – Infectious Diseases Institute KCMC – Kilimanjaro Christian Medical CenterNAMRU – Naval Medical Research Unit

NHRC – Navrongo Health Research Centre UNAH – Universidad Nacional Autónoma de HondurasUSAMRD-A – U.S. Army Medical Research Division/Unit - AfricaWashU – Washington University in St. Louis *updated status of all sites can be viewed anytime at www.clinicaltrials.gov

TABLE 1. SUBJECT DEMOGRAPHICS

Total 01 02 05 07 08 09 11 12 13 14

Sex Female 851 58 79 126 113 107 78 87 134 65 4

Male 779 74 48 74 66 145 89 71 163 46 3

Age

<5 165 42 23 0 0a 26 0b 67 3 4 0a

5 to 21 616 22 48 128 14 128 56 69 101 50 0

22 to 50 599 41 34 60 106 64 73 21 147 50 3

>50 250 27 22 12 59 34 38 1 46 7 4

Totals 1630 132 127 200 179 252 167 158 297 111 7

a Site was not enrolling subjects <18 yrs oldb Site was not enrolling subjects <7 yrs old

SENSITIVITY AND SPECIFICITY

Specimens were evaluated with the FilmArray Global Fever Panel – IUO (Investigational Use Only). Clinical sites extracted nucleic acid (DNA and RNA) from whole blood specimens using the MagNA Pure Compact system (Roche Diagnostics) and shipped frozen aliquots of this nucleic acid to BioFire Defense for comparator testing. In general, comparator testing consisted of two PCR assays for each analyte followed by bi-directional sequencing. The Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) were defined as a specimen having agreement between the FilmArray GF Panel and the comparator assays (Table 2).

TABLE 2. CALCULATION OF SENSITIVITY AND SPECIFICITY

Comparator Result

Positive Negative FilmArray Performance Two-sided 95%CI

Film

Arr

ay R

esul

t

Posi

tive

TP FP Sensitivity (PPA)=100 x (TP / TP + FN)

Exact Binomial Confidence Interval of Sensitivity Proportion

Neg

ativ

e

FN TN Specificity (NPA)= 100 x (TN / TN + FP)

Exact Binomial Confidence Interval of Specificity Proportion

Total TP + FN TN + FP

FP, False Positive; FN, False Negative; TP, True Positive; TN, True Negative

SYSTEM PERFORMANCE AND CONTROLS

FilmArray pouches contain freeze-dried reagents packaged under negative vacuum pressure (Figure 2). The pouches are hydrated immediately before use. Each pouch contains two internal controls that confirm proper function of the pouch run and the FilmArray software monitors the progress of the automated run. Table 3 shows the performance of all pouches run as part of the clinical study.

The External Control Material (ECM) kit is being developed for customer use with the GF Panel. During the clinical evaluation, one valid ECM run must be performed at each site at the start of each day of specimen testing. The ECM kit contains positive and negative control injection vials. During the clinical study, positive and negative ECMs are run on alternating days. The positive ECM contains a mix of nucleic acid sequences that are detected by the FilmArray GF Panel assays. A valid ECM run is one that exactly matches the expected results for each analyte in the mix (i.e., positive or negative). ECM performance is shown in Table 4. The ECM was reformulated during the study; data shown is only from the final version of the ECM.

TABLE 3. GF PANEL POUCH PERFORMANCE

Total Pouches

Used

% Failed of Total

Control FailureInternal

Incomplete Run

Instrument Error

Software Error

Aborted by User

Loading Failure

Hydration Failure

Total 3447 1.97% 39 4 9 0 1 0 15

TABLE 4. ECM PERFORMANCE

ECM Passed/Run

Positive 126/128a,b

Negative 122/124a,c

TOTAL 248/252

FIGURE 2. FILMARRAY POUCH

Of the 1179 samples tested by FilmArray and comparator, 282 were positive for at least one analyte (23.9% positivity). The GF Panel showed >90% sensitivity for eight analytes and >99% specificity for eighteen analytes (Table 5). In all FP or FN cases, the PCR amplification curves of the FilmArray or comparator testing suggest that pathogen was at or near the Limit of Detection. (For detailed analytical studies of the GF Panel, see poster C-1783.)

TABLE 5. FILMARRAY GF PANEL PERFORMANCE SUMMARY

AnalytePPA NPA

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

Viruses

Chikunguna virus 22/22 100% 85.1-100% 1142/1144 -99.80% 99.4-100%

Crimean-Congo hemorrhagic fever virus 1/1 100% 20.7-100% 1127/1127 -100% 99.7-100%

Dengue virus 29/33 88% 72.7-95.2% 1080/1080 -100% 99.6-100%

Ebola virus 0/0 - - 968/968 -100% 99.6-100%

Lassa virus 0/0 - - 1088/1088 -100% 99.6-100%

Marburg virus 0/0 - - 1048/1048 -100% 99.6-100%

West Nile virus 1/1 100% 20.7-100% 1087/1087 -100% 99.6-100%

Yellow fever virus 0/0 - - NAa - -

Zika Virus 0/0 - - 1078/1078 -100% 99.6-100%

Bacteria

Bacillus anthracis 0/0 - - 1127/1127 -100% 99.7-100%

Francisella tularensis 0/0 - - 1127/1127 -100% 99.7-100%

Leptospira spp. 5/5 100% 56.6-100% 1158/1159 -99.90% 99.5-100%

Salmonella enterica serovar Paratyphi A 0/0 - - 1087/1087 -100% 99.6-100%

Salmonella enterica serovar Typhi 1/1 100% 20.7-100% 1126/1126 -100% 99.7-100%

Yersinia pestis 0/0 - - 1127/1127 -100% 99.7-100%

Protozoa

Leishmania spp. 0/0 - - 1048/1048 -100% 99.6-100%

Plasmodium falciparum 142/153 93% 87.6-95.9% 990/993 -99.70% 99.1-99.9%

Plasmodium spp. 213/216 99% 96-99.5% 907/915 -99.10% 98.3-99.6%

Plasmodium vivax/ovale 70/74 95% 86.9-97.9% 1069/1069 -100% 99.6-100%

One advantage of the FilmArray syndromic testing approach is the ability to identify more than one pathogen in a single test. Seven samples were positive by FilmArray for more than one analyte (Table 6). Dual infection data has not yet been confirmed by comparator testing.

TABLE 6. DETECTION OF DUAL INFECTIONSa

Co-Detected Analytes No. Observed

Dengue virus and Chikungunya virus 2

Dengue virus and Zika virus 1

Dengue virus and Plasmodium spp./P. falciparum 1

Dengue virus and Plasmodium spp./P. vivax/ovale 1

Leptospira spp. and Plasmodium spp. 1

Leptospira spp. and Plasmodium spp./P. vivax/ovale 1

Plasmodium spp./P. falciparum and Plasmodium spp./P. vivax/ovale 22aDual infection data has not yet been confirmed by comparator testing

SUMMARY

Of the 19 analytes detected by GF Panel, 18 have been evaluated by GF Panel and comparator. Comparator testing is ongoing and will be completed for all analytes for all specimens. The NPA for all select agents was 100% and for all non-select agents was over 99%. Positive comparator results were obtained for nine analytes and seven met the desired performance goal of at least 95% PPA. Overall, these results indicate that the FilmArray GF Panel is highly sensitive and specific when testing clinical specimens.

Data presented are from assays that are Investigational Use Only (IUO) and have not been cleared or approved for diagnostic use.

CONTACT INFORMATION

Brian W. Jones, PhD.Brian.Jones@BioFireDefense.com

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

During a run, the FilmArray system:• Lyses the sample by agitation (bead beating).• Extracts and purifies all nucleic acids from the sample using magnetic bead technology.• Performs nested multiplex PCR by:

o First performing a single, large volume, highly multiplexed first-stage PCR reaction (PCR1).

o Second performing multiple, nested singleplex second-stage PCR reactions (PCR2) to amplify sequences within the PCR1 products.

• Uses endpoint melting curve data to detect and generate a result for each target on the array.

a. Site ran a positive and a negative ECM on the same day and accidentally swapped the pouches when loading into the instruments.

b. Positive ECM failure due to several as-says going undetected; all subsequent positive ECMs at this site passed.

c. Negative ECM failure due to detection of Plasmodium spp.; all subsequent negative ECMs at this site passed.

a Comparator testing for this assay is incomplete

ACKNOWLEDGMENTSThe authors thank personnel at BioFire who designed assays: Wendy Smith, Amy Carmichael, and Jeffery Nicholes. The authors also thank the principal investigators and their dedicated study teams: Matthew Rubach (KCMC-Duke); Abraham Oduro, Victor Asoala, and Michael Kaburise (NHRC); Andrew Letizia, David Wolfe, and Anne Fox (NAMRU-3); Stacey House (WashU); Chris Myers (OID); Crystyan Siles and Isabel Bazán (NAMRU-6); Ivette Lorenzana and Concepción Zuniga (UNAH); Mohammed Lamorde and Yukari Manabe (IDI/JHU); John Waitumbi (USAMRD-A); Jose Garcia, Kimberly Edgel, and Dennis Faix (NAMRU-2); Aileen Chang and Chris Mores (GWU); and Stefan Fernandez (AFRIMS).