Comparison of FilmArray® and qRT-PCR for the detection of Zaire ebolavirus from contrived and 1

clinical specimens 2

Running Title: Detection of Ebolavirus 3

Keywords: Ebola virus disease, Zaire ebolavirus, ZEBOV, FilmArray®, qRT-PCR 4

5

Timothy R. Southern*1,2, Lori D. Racsa*3, César G. Albariño4, Paul D. Fey1, Steven H. Hinrichs1, Caitlin 6

N. Murphy1,2, Vicki L. Herrera2, Anthony R. Sambol2, Charles E. Hill3, Emily L. Ryan3, Colleen S. 7

Kraft3,5,Shelley Campbell4, Tara K. Sealy4, Amy Schuh4, James C. Ritchie3, G. Marshall Lyon III5, 8

Aneesh K. Mehta5, Jay B. Varkey5, Bruce S. Ribner5, Kent P. Brantly6, Ute Ströher4 Peter C. Iwen1,2, 9

Eileen M. Burd3,5, # 10

*Both authors contributed equally to this work. 11

12

1Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 13

2Nebraska Public Health Laboratory, Omaha, NE 14

3Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 15

4Centers for Disease Control and Prevention, Atlanta, GA 16

5Department of Medicine, Division of Infectious Diseases, Emory University School of Medicine, 17

Atlanta, GA 18

6Samaritan’s Purse, Boone, NC 19

20

#Corresponding author: 21

Eileen M. Burd, Ph.D. 22

1364 Clifton Road NE 23

Atlanta, GA 30322 24

Phone number: 404-712-7297 25

Fax number: 404-712-4632 26

JCM Accepted Manuscript Posted Online 8 July 2015J. Clin. Microbiol. doi:10.1128/JCM.01317-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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eburd@emory.edu 27

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ABSTRACT 28 29

Rapid, reliable and easy to use diagnostic assays for detection of Zaire ebolavirus (ZEBOV) are 30

urgently needed. The goal of this study was to examine the percentage agreement among Emergency Use 31

Authorization (EUA) tests for the detection of ZEBOV nucleic acid including the BioFire FilmArray® 32

BioThreat (BT) panel, the FilmArray BT E-panel and the NP2 and VP40 quantitative real-time reverse 33

transcriptase polymerase chain reaction (qRT-PCR) assays from the Centers for Disease Control and 34

Prevention (CDC). Specimens used in this study included whole blood spiked with inactivated ZEBOV 35

at known titers and whole blood, plasma and urine clinical specimens from persons diagnosed with Ebola 36

virus disease (EVD). The percentage agreement for FilmArray and qRT-PCR assays using contrived 37

whole blood specimens was 100% (6/6) for each ZEBOV dilution from 4x107-4x102 virus/ml as well as 38

the no-virus negative control. The limit of detection for FilmArray and qRT-PCR based on duplicate 39

positive results was determined to be 4x102 inactivated ZEBOV/ml. Percentage agreement between 40

FilmArray and qRT-PCR using clinical specimens from patients with EVD was 85% (23/27) when testing 41

whole blood, 90%(18/20) when testing whole blood by FilmArray and matched plasma by qRT-PCR and 42

85% (11/13) when testing urine. Of 60 specimens, eight discordant results were noted with ZEBOV 43

nucleic acid detected only by FilmArray in four specimens and only by qRT-PCR in the remaining four. 44

These findings demonstrate that the rapid and easy to use FilmArray panels are effective tests for 45

evaluating patients with EVD. 46

47

FUNDING STATEMENT 48

None of the authors received external grant support for this study. PDF has previously received 49

grant support from BioFire to support clinical validation studies of the FilmArray respiratory and 50

gastrointestinal panels. 51

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The findings and conclusions in this report are those of the authors and do not necessarily 53

represent the official position of the Centers for Disease Control and Prevention. 54

55

INTRODUCTION 56

Ebolavirus is an enveloped, single-stranded ribonucleic acid (RNA) virus that is the cause of 57

Ebola virus disease (EVD). EVD is characterized by fever, emesis, diarrhea and a hemorrhagic disorder 58

that can include maculopapular rash, petechiae, ecchymoses and mucosal hemorrhage (1). Zaire 59

ebolavirus (ZEBOV) is the cause of the 2014-2015 outbreak of EVD in West Africa, which to-date has 60

resulted in 27,352 total cases, 15,052 laboratory-confirmed cases and 11,178 deaths (2,3). 61

Early detection of ZEBOV is critical for management of persons with EVD and for outbreak 62

control. A significant challenge in areas without Ebola, such as the United States, is the rapid assessment 63

of individuals with travel history to West Africa presenting with symptoms of EVD. Currently, the 64

standard protocol for EVD testing involves collection of whole blood or plasma followed by testing using 65

quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) assays. Although 66

highly sensitive and specific, qRT-PCR assays for ZEBOV are complex, limiting their use to state public 67

health laboratories and the CDC (4, 5). Depending on the location of the patient being tested and the 68

laboratory performing the testing, turn-around time for qRT-PCR results could be measured in days 69

whereas initial testing performed at or near the point-of-care using a rapid test could be completed within 70

hours. Rapid, reliable and easy to use tests for the detection of ZEBOV are needed not only for testing in 71

endemic areas but for screening of health care workers, international travelers and other potentially 72

exposed individuals. 73

The first United States nationals infected in Africa were returned to our facilities for treatment in 74

the Serious Communicable Diseases Unit (SCDU) at Emory University (EU) and the Nebraska 75

Biocontainment Unit (NBU) at Nebraska Medicine, the affiliated academic medical hospital to the 76

University of Nebraska Medical Center (UNMC) between 2 August and 28 October, 2014 (6,7, 8). At 77

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that time, the available assays for the detection of ZEBOV were the research use only FilmArray 78

BioThreat (BT) panel (BioFire Defense, Salt Lake City, Utah) and the ZEBOV nucleoprotein (NP2) and 79

matrix protein (VP40) gene assays from the Centers for Disease Control and Prevention, Atlanta, 80

Georgia. The NP2 and VP40 qRT-PCR assays and the FilmArray BT-E (a modified version of the BT 81

panel) assays were granted Emergency Use Authorization (EUA) in October 2014, during the course of 82

treatment of our patients. The FilmArray BT assay detects a panel of biothreat agents but the ZEBOV 83

primers detecting the L-gene are identical in both the FilmArray BT and the FilmArray BT-E (EUA) 84

tests. The FilmArray BT-E assay also includes a freeze dried protease to add with the blood and loading 85

buffer and has an additional primer in the second stage PCR that perfectly matches the current circulating 86

strain. This study provides a comparative analysis of the FilmArray BT-E-panel and the NP2 and VP40 87

qRT-PCR assays for the detection of ZEBOV using contrived whole blood specimens. This study also 88

provides a prospective analysis of the FilmArray BT panel and qRT-PCR using whole blood, plasma and 89

urine specimens from 6 persons with EVD who were treated in our facilities. 90

91

MATERIALS AND METHODS 92

FilmArray. BioFire FilmArray (BioFire LLC, Salt Lake City, Utah) is an automated, nested PCR that 93

allows for the extraction and detection of nucleic acid targets in a closed system. The BT panel and BT 94

E-panel (BioFire Defense, Salt Lake City, Utah) are used on the FilmArray system to detect ZEBOV from 95

whole blood or urine specimens (9). Briefly, each panel was rehydrated with the provided Rehydration 96

Solution and whole blood (100 µl) or urine (200 µl) was mixed with the provided Sample Buffer. The 97

resulting solution was injected into the panel and the panel was loaded into the FilmArray instrument. 98

One sample is tested at a time with a result of detected or not detected provided in approximately one 99

hour. FilmArray testing was performed in a satellite laboratory within the SCDU at EU, at the Nebraska 100

Public Health Laboratory (NPHL) biosafety level 3 (BSL3) facility which is located on the UNMC 101

campus, and at the CDC in the Viral Special Pathogens Branch (VSPB) laboratory. Specimens were 102

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processed for FilmArray testing in a class II biological safety cabinet by individuals wearing enhanced 103

personal protective equipment as defined by the biosafety committees at each institution. 104

qRT-PCR. Testing was also performed by the VSPB at the CDC using the two qRT-PCR assays for the 105

detection NP2 and VP40 genes(10,11). While only whole blood was evaluated with FilmArray, plasma 106

and whole blood were tested by qRT-PCR at various times. Results are generated in about three hours and 107

up to 96 specimens can be run simultaneously. Total RNA was extracted from whole blood, plasma or 108

urine specimens using the BeadRetriever system (Invitrogen, Grand Island, New York) and the MagMax 109

Pathogen RNA/DNA Isolation kit (Applied Biosystems, Grand Island, New York). The NP2 qRT-PCR 110

assay was then performed on the extracted nucleic acid and a cycle threshold (Ct) value was reported 111

back to each institution for use in patient care. Cycle threshold values of ≤40 were interpreted as positive. 112

Specimens with no specific amplification or with amplification curves that did not cross the baseline 113

threshold were interpreted as negative. Specimens that yielded positive results with a Ct value >38 in the 114

NP2 assay were further confirmed using the VP40 assay and/or an additional serology assay at the CDC 115

VSPB laboratory (the EUA provides for an equivocal interpretation for specimens with Ct values of 38-116

40 and suggests that additional analysis may be required). 117

Specimens. Contrived specimens were prepared at the VSPB using inactivated ZEBOV at known titers 118

to examine concordance between the FilmArray BT E-panel, NP2 qRT-PCR and VP40 qRT-PCR for the 119

detection of ZEBOV nucleic acid. ZEBOV (specimen number: 812592) at a titer of 4x107 TCID50/ml was 120

inactivated by gamma irradiation. Ten-fold serial dilutions of the inactivated virus from 4x107-4x101 121

TCID50/ml and a “no virus” negative control were prepared in whole blood. Contrived specimens at each 122

virus dilution, and the negative control, were tested in duplicate using the FilmArray BT E-panel and the 123

NP2 and VP40 qRT-PCR assays. 124

Clinical specimens from individuals with EVD who were treated at EU and UNMC were 125

evaluated using the FilmArray BT panel and qRT-PCR. Whole blood (n=27), plasma (n=20), and urine 126

(n=13) were collected and tested at various times throughout the course of patient management. Twenty-127

seven matched whole blood and 13 matched urine specimens were tested using the FilmArray BT panel 128

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and qRT-PCR. An additional 20 whole blood specimens were tested using the FilmArray BT panel with 129

qRT-PCR testing on matched plasma specimens for a total of 60 pairwise tests on clinical specimens from 130

patients being treated for EVD.. FilmArray BT panel testing was performed at the clinical sites using 131

whole blood and urine. Matched clinical specimens were tested by qRT-PCR at the VSPB once per 132

sample. Only the qRT-PCR results were used for patient management at this time. The interval between 133

FilmArray testing on site and testing at the VSPB ranged from 24 hours before to 5 days after qRT-PCR 134

results were received. 135

Calculations. Percentage agreement was calculated for results from FilmArray and qRT-PCR assays. 136

Ethics Statement. Approval was obtained from the institutional review boards at Emory University and 137

the University of Nebraska. 138

RESULTS 139

Ten-fold serial dilutions of inactivated ZEBOV from 4x107-4x101 TCID50/ml in whole blood were 140

tested in duplicate using the FilmArray BT-E panel, NP2 qRT-PCR and VP40 qRT-PCR at the VSPB 141

(Table 1). ZEBOV nucleic acid was detected in contrived whole blood specimens at virus concentrations 142

from 4x107-4x102 TCID50/ml resulting in 100% agreement between the 3 ZEBOV tests (6/6 at each titer; 143

36/36 overall). The VP40 qRT-PCR detected ZEBOV nucleic acid in 1 of 2 tests at the 4x101 virus 144

dilution while ZEBOV nucleic acid was not detected by either FilmArray or the NP2 qRT-PCR and 145

resulted in an 83.3% (5/6) agreement for that dilution. ZEBOV nucleic acid was not detected in whole 146

blood that did not contain inactivated virus resulting in a 100% (6/6) agreement between the three assays. 147

Limit of detection (LOD), or the lowest virus dilution at which each assay detected ZEBOV nucleic acid 148

in duplicate, was determined to be 4x102 TCID50/ml for FilmArray and both qRT-PCR assays. 149

Sixty specimens (47 whole blood and 13 urine) from individuals with EVD were tested using 150

FilmArray (S1 Table). Testing of matched whole blood, plasma and urine specimens was performed by 151

qRT-PCR at the CDC. Twenty-seven whole blood specimens were tested using both the FilmArray and 152

qRT-PCR. An additional 20 whole blood specimens were tested using FilmArray with corresponding 153

plasma tested by qRT-PCR. Thirteen urine specimens were also tested using both assays. 154

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Of the 60 specimens tested, ZEBOV nucleic acid was detected in 40 specimens using both 155

FilmArray and qRT-PCR while 12 specimens were negative by both assays (Table 2). Eight discrepant 156

results were noted with ZEBOV nucleic acid detected by FilmArray but negative by qRT-PCR in four 157

cases and positive by qRT-PCR but not detected by FilmArray in four cases. The specimens that yielded 158

discrepant results included four whole blood, two plasma (paired with whole blood), and two urine (S1 159

Table). Overall percentage agreement between FilmArray and qRT-PCR was 87% (52/60). Percentage 160

agreement was 85% (23/27) for whole blood specimens tested by both FilmArray and qRT-PCR, 90% 161

(18/20) when whole blood was tested using FilmArray and matched plasma specimens were tested by 162

qRT-PCR and 85% (11/13) for urine specimens (Table 3). 163

The two plasma specimens tested ZEBOV negative by qRT-PCR but for which the corresponding 164

whole blood tested positive by FilmArray. To explore this further, a series of six specimens from one of 165

the patients at Emory was collected every 24 hours for six days and tested on the day of collection using 166

both whole blood and plasma obtained from the same collection tube. ZEBOV was detected in whole 167

blood by both the FilmArray and qRT-PCR in all six specimens, however, detectable virus was found in 168

only two of the corresponding plasma specimens on FilmArray, suggesting a trend toward the virus 169

clearing from plasma before clearing from whole blood (Table 4). 170

DISCUSSION 171

Rapid, reliable and easy to use assays for the detection of ZEBOV from clinical specimens are 172

needed in response to the unprecedented outbreak in West Africa and the emergence of infected 173

individuals beyond outbreak zones. This study evaluated FilmArray and two qRT-PCR assays for 174

detection of ZEBOV from whole blood specimens spiked with inactivated virus at known titers, and 175

whole blood, plasma and urine clinical specimens from 6 individuals with EVD who were treated in the 176

United States. 177

Parallel testing of contrived whole blood specimens at virus dilutions of 4x107-4x102 TCID50/ml 178

revealed 100% agreement between the three diagnostic assays evaluated in this study. The only 179

disagreement noted was a single positive result following VP40 qRT-PCR testing of the 4x101 TCID50/ml 180

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dilution that resulted in an 83.3% (5/6) agreement. Detection of the VP40 qRT-PCR target in the one 181

sample at a ten-fold lower dilution compared to FilmArray and NP2 qRT-PCR results may suggest 182

somewhat greater sensitivity for VP40 qRT-PCR. 183

Parallel testing of contrived whole blood specimens with inactivated virus also provided insight 184

into the limit of detection which, given the concentrations tested, was determined to be 4x102 TCID50/ml 185

for all three assays. Inactivation is known to affect nucleic acid integrity and to shift the detection limit, 186

usually about 10-100 fold higher, compared to that obtained with viable virus (10, 11)). Studies 187

performed by BioFire Defense, LLC on the BT-E panel indicated an LOD of 6x105 plaque forming units 188

(PFU) per ml of whole blood using inactivated ZEBOV (9). BioFire Defense, LLC also reported 189

successful BT-E panel detection of synthetic ZEBOV L-gene RNA template in Tris-EDTA buffer at 190

1x100-1x105 genome equivalents (8). Studies at the CDC documented a limit of detection for both the NP 191

and VP40 qRT-PCR assays of 30 TCID50/reaction (~ 5,400 TCID50/ml) using inactivated ZEBOV in both 192

whole blood and urine.(10,11) The apparent differences likely have more to do with variations in the 193

harshness of the protocols used to inactivate the virus in the analyte preparations than the ability of the 194

test systems to detect ZEBOV. Our studies did not evaluate specificity of the assays, but the EUA 195

documentation includes results of extensive studies that were done with the help of the United States 196

Army Medical Research Institute of Infectious Diseases (USAMRIID) and the United States Department 197

of Defense which confirmed the high analytical specificity of all three tests (9,10,11). Further, clinical 198

specificity was documented in a study in Sierra Leone in which the FilmArray assay was used for 199

research purposes to test patients and healthcare workers who were referred for diagnostic testing (12). In 200

that study, 83 individuals were tested. Six of the individuals tested positive with 5 of the 6 confirmed as 201

positive by CDC mobile laboratories using the NP2 and VP 40 tests in Sierra Leone. The remaining 202

patient had a throat swab that tested positive by FilmArray but the patient died with typical EVD 203

symptoms before a specimen for confirmatory testing could be obtained. Notably, one asymptomatic 204

healthcare worker tested positive, became symptomatic the following day and also tested positive by the 205

CDC assay on a blood specimen taken 4 days later. There were 19 asymptomatic individuals who were 206

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exposed to confirmed EVD patients who tested negative by FilmArray and did not meet the suspected 207

EVD case definition so were not tested further. Whole blood specimens from 57 symptomatic patients 208

tested negative by FilmArray and were confirmed as negative by the CDC mobile laboratory. In follow-209

up, none of these patients developed EVD. One symptomatic patient with a urine specimen that tested 210

negative by FilmArray was diagnosed with EVD by a blood specimen tested three days later by the CDC 211

mobile lab. This study showed high specificity and perfect correlation between the FilmArray and CDC 212

assays when used as a diagnostic test with whole blood as the specimen type. 213

Testing of clinical specimens in the current study using FilmArray and qRT-PCR resulted in an 214

overall percentage agreement of 87% (52/60) with a percentage agreement of 85% (23/27) for whole 215

blood specimens and 85% (11/13) for urine specimens. Eight discrepancies between FilmArray and qRT-216

PCR were noted with four from whole blood, two from plasma and two from urine, which occurred when 217

EVD was resolving and viral load was waning, as indicated by patient improvement and qRT-PCR results 218

with high or negative Ct values. Agreement between the FilmArray and NP2 qRT-PCR assays, regardless 219

of specimen type, was 100% (30/30) when testing the clinical specimens obtained initially, during the 220

early stages of disease and through early recovery in our patients. These specimens all had Ct values of 221

37 or lower. In the later stages of recovery, there was some variability between the assays. Of the 14 222

specimens with qRT-PCR Ct values of 38 or higher, 4 were not detected by FilmArray. Conversely, of 223

the 16 specimens that tested negative by qRT-PCR, 4 had detectable virus by FilmArray. 224

Specimen type, sample volume, handling, and storage are important to consider when interpreting 225

results of FilmArray and qRT-PCR testing for ZEBOV. Plasma and whole blood were tested by qRT-226

PCR while only whole blood was evaluated with FilmArray. In this study, two discrepancies were noted 227

when plasma specimens tested by qRT-PCR were negative for ZEBOV while corresponding whole blood 228

specimens were positive using FilmArray. Further evaluation of six additional specimens corroborated 229

the results and showed that this discordance occurred when viral loads were waning as evidenced by Ct 230

values of 36 or higher. This observation gives some insight into viral kinetics and might suggest that 231

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whole blood is the more appropriate specimen for ZEBOV detection since corresponding plasma 232

specimens may not contain virus at detectable levels. This is perhaps not unexpected since monocytes are 233

infected early in EVD, and the plasma compartment clearing first has been shown with other RNA viruses 234

(13, 14)). Sample volume is also an important consideration for molecular detection of ZEBOV. We 235

initially performed FilmArray testing with 100 μL of whole blood but the EUA FilmArray BT-E 236

Instructions for use indicate that 200 μL of whole blood should be used (9). We tested two whole blood 237

specimens using FilmArray to compare input volumes of 100µl and 200µl. Positive FilmArray results 238

were obtained with both input volumes. The corresponding Ct values from the NP assay were 33 and 37. 239

Specimens with higher Ct values were not tested. Four whole blood specimens that tested negative using 240

FilmArray were also negative by qRT-PCR. Finally, specimen handling and storage is an important 241

consideration for molecular detection of ZEBOV. Whole blood specimens should be collected in plastic 242

collection tubes with EDTA, sodium polyanethol sulfonate, or citrate as preservative and tested promptly 243

following collection. If necessary, specimens may be stored short-term at 4°C or frozen to prevent 244

degradation of the specimen and viral nucleic acid. If shipping is required, the specimen should be 245

packaged and shipped at 2-8°C with cold packs according to the Department of Transportation 246

recommendations for a Category A infectious substance using an approved courier. (15). 247

248

CONCLUSION 249

The unprecedented outbreak of ZEBOV in West Africa highlights the need for rapid, reliable and 250

easy to use tests for the detection of ZEBOV from clinical specimens. Data presented here suggest that 251

FilmArray BT panel and BT E-panel perform comparably to the CDC qRT-PCR assays for the rapid 252

detection of ZEBOV in blood and urine specimens from individuals suspected of having EVD. Reduced 253

manipulation of clinical specimens, ease of use and rapid turn-around time make this an appropriate 254

screening test for healthcare institutions and public health laboratories that lack qRT-PCR capability yet 255

need the ability to provide presumptive identification of ZEBOV from individuals suspected of having 256

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EVD. According to the EUA, all FilmArray BT-E results should be confirmed by state public health 257

laboratories or the CDC using EUA methods. 258

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TABLE 1. Results of FilmArray® BioThreat E-Panel and qRT-PCR testing using serial dilutions of inactivated Zaire ebolavirus at known titers in whole blood.a,b,c Virus NP2c VP40c FilmArray®d Titer (TCID50/ml) Test 1 Test 2 Test 1 Test 2 Test 1 Test 2 40,000,000 Positive (20) Positive (20) Positive (18) Positive (18) Detected Detected 4,000,000 Positive (23) Positive (23) Positive (22) Positive (22) Detected Detected 400,000 Positive (26) Positive (26) Positive (25) Positive (25) Detected Detected 40,000 Positive (30) Positive (30) Positive (28) Positive (28) Detected Detected 4,000 Positive (33) Positive (33) Positive (32) Positive (32) Detected Detected 400 Positive (37) Positive (35) Positive (34) Positive (34) Detected Detected 40 Negative Negative Negative Positive (37) Not Detected Not Detected 0 Negative Negative Negative Negative Not Detected Not Detected aZaire ebolavirus (strain: 812592) stock at a known titer of 4x108 TCID50/ml was inactivated by gamma irradiation prior to testing. bEmergency Use Authorization-approved NP2 and VP40 qRT-PCR assays and the FilmArray® Biothreat E-panel were performed in duplicate using ten-fold serial dilutions of inactivated virus in whole blood at the CDC. cqRT-PCR results are presented as positive and negative with cycle threshold values in parentheses. dFilmArray® results are presented as detected or not-detected.

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TABLE 2. Results of FilmArray® BioThreat Panel and qRT-PCR testing of clinical specimens throughout the course of disease from patients diagnosed with EVD.a,b NP2 qRT-PCR Result FA Result Positive (Ct ≤ 40) Negative Detected 40 4c Not Detected 4d 12 Abbreviation: FA, FilmArray®; EVD, ebola virus disease aTesting was performed using both the FilmArray® BioThreat panel and the CDC quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) assay. bIncludes whole blood, plasma and urine. c Includes two samples that were whole blood paired with plasma. dThe Ct values for these four samples were 38, 39, 39 and 39

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TABLE 3. Comparison of the FilmArray® BioThreat panel and the CDC NP2 qRT-PCR assay results for the detection of Zaire ebolavirus from whole blood, plasma and urine.a

Specimen # Specimens Result Percent NP2 qRT-PCR FilmArray® Evaluated RT+/FA+ RT-/FA- RT+/FA- RT-/FA+ Agreement

Whole Blood Whole Blood 27 19 4 3 1 85% Plasma Whole Blood 20 13 5 0 2 90% Urine Urine 13 8 3 1 1 85% Abbreviations: RT, qRT-PCR; FA, FilmArray® aTesting was performed using both the FilmArray® BioThreat panel and the CDC real-time reverse transcriptase polymerase chain reaction (qRT-PCR) NP2 assay.

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TABLE 4. Detectiona of low-level ZEBOV viremia using whole blood vs plasma in serial samples from a single patient Sample # FilmArray® result NP2 qRT-PCR result (CT value)

Whole Blood Plasma Whole Blood1 Detected Detected Positive (36)2 Detected Not Detected Positive (36)3 Detected Detected Positive (37)4 Detected Detected Positive (37)5 Detected Not Detected Positive (37)6 Detected Not Detected Positive (40)aTesting was performed using both the FilmArray® BioThreat panel and the CDC real-time reverse transcriptase polymerase chain reaction (qRT-PCR) NP2 assay. Numbers in parentheses indicate Ct values.

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