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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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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
52
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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
259
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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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