evaluation of the biofire filmarray biothreat e-test (v2. 5) for rapid
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Evaluation of the Biofire FilmArray Biothreat E-test (v2.5) for rapid identification of Ebola virus 1
disease in heat-treated blood samples obtained in Sierra Leone and United Kingdom 2
Running Title: FilmArray E-test against Sierra Leone and UK Ebola samples 3
Simon A Weller1#, Daniel Bailey2, Steven Matthews3, Sarah Lumley2, Angela Sweed2, Derren Ready4, 4
Gary Eltringham5, Jade Richards6, Richard Vipond2 Roman Lukaszewski1, Phillippa M Payne1, Emma 5
Aarons2, Andrew J Simpson2, Emma J Hutley3, and Tim Brooks2 6
Affiliations 7
1Defence Science and Technology Laboratory, Ministry of Defence, Porton Down, Salisbury, UK 8 9 2Rare and Imported Pathogens Laboratory, Public Health England, Porton Down, Salisbury, UK 10 11 3Centre of Defence Pathology, Royal Centre of Defence Medicine, Vincent Drive, Edgbaston, 12 Birmingham, UK 13 14 4Public Health Laboratory London, 80 Newark Street, London, UK 15 16 5Newcastle Molecular Laboratory, Public Health England, Royal Victoria Infirmary, Newcastle Upon 17
Tyne, UK 18 19 6MEHT Microbiology, NHS Trust, Broomfield Hospital, Chelmsford, UK 20 21
#Corresponding author: Email [email protected]; Tel +44 (0) 1980 617404. 22
© Crown copyright (2015), Dstl. This material is licensed under the terms of the Open Government 23
Licence except where otherwise stated. To view this licence, visit 24
http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3 or write to the 25
Information Policy Team, The National Archives, Kew, London TW9 4DU, or email: 26
28
JCM Accepted Manuscript Posted Online 4 November 2015J. Clin. Microbiol. doi:10.1128/JCM.02287-15© Crown copyright 2015.
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Abstract 29
Rapid Ebola virus (EBOV) detection is crucial for appropriate patient management and care. The 30
performance of the FilmArray Biothreat E-test (v2.5), against whole blood samples, was evaluated 31
in Sierra Leone and the United Kingdom (UK), and compared with results generated by a real-time 32
Ebola Zaire PCR reference method. In diagnostic laboratories samples were tested on availability, 33
included successive samples from individual patients, and were heat treated to facilitate EBOV 34
inactivation prior to PCR. In Sierra Leone (n=60; 44 patients), the Biothreat E-test had a sensitivity of 35
84% (confidence interval, CI: 64-95%) and specificity of 89% (CI: 73-97%); and in the UK (n=108; 70 36
patients), a sensitivity of 75%, (CI: 19-99%) and specificity of 100% (CI: 97-100%), when compared 37
to the reference real-time PCR. Statistical analysis (Fisher’s Exact test) indicated there was no 38
significant difference between methods at the 99% confidence level in either country. In 9 39
discrepant results (5 real-time PCR positives and E-test negatives; 4 real-time PCR negatives and E-40
test positives), the majority (8) were obtained from samples with an observed, or probable, low 41
viral load. The FilmArray E-test (v2.5) therefore provides an attractive option for laboratories (either 42
in austere field settings or in countries with an advanced technological infrastructure), which do not 43
routinely offer an EBOV diagnostic capability. 44
45
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Introduction 47
An Ebola virus (EBOV) outbreak has been ongoing in West Africa since December 2013 and was 48
declared as a Public Health Emergency of International Concern (PHEIC), by the World Health 49
Organisation (WHO) in August 2014 (1). Since the outbreak began the designated United Kingdom 50
(UK) EBOV testing laboratory, the Rare and Imported Pathogens Laboratory, Public Health England 51
(PHE) has been testing 10-15 samples per week for EBOV, an approximate 10 fold increase over the 52
normal testing frequency. These samples are primarily taken from UK citizens with a recent travel 53
history to West Africa and the vast majority are not infected with EBOV. 54
The preferred sample for detecting EBOV, an enveloped negative-sense single strand RNA virus, is 55
EDTA-blood, serum or plasma with the primary diagnostic technology being real-time PCR (2). EBOV 56
is designated in the UK by the Advisory Committee on Dangerous Pathogens (ACDP) as a Hazard 57
Group 4 (HG4) pathogen that must be handled under containment level (CL)-4 standards, (Biosafety 58
Level, or BSL-4 in other countries). As such stringent laboratory infrastructure and containment 59
procedures are required to handle viable EBOV material, and only a few laboratories in Europe and 60
elsewhere are equipped to do so (3), though UK guidelines do allow for primary clinical analyses of 61
samples from individuals with possible EBOV infection to be carried out under lower containment 62
(4). 63
The FilmArray PCR platform (Biofire Diagnostics, UT), integrates sample processing, nucleic acid 64
extraction and purification, and a multiple PCR analysis into a single-use, disposable pouch (5). The 65
FilmArray was developed to fulfil a low operative burden, Point-of-Care (PoC), diagnostic screening 66
capability within a healthcare setting. Results are available around an hour after crude sample is 67
added to the pouch. Evaluations of the FilmArray Biothreat Panel pouch (a multivalent pouch which 68
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contains an EBOV assay), in Sierra Leone (6) and the US (7) have demonstrated its utility in the 69
identification and treatment of Ebola positive patients. 70
In 2014 a Biothreat E-test (v2.5) pouch (hereafter referred as the Biothreat E-test) was released (8). 71
This pouch detects EBOV only and has been optimised for detection of the current strain (Makona) 72
in clinical sample types. This pouch was awarded an Emergency Use Authorisation (EUA) by the U.S. 73
Food and Drug Administration (FDA) for the presumptive detection of EBOV in whole blood or urine 74
specimens (9), and a conditional, temporary, derogation from the medical devices regulations 2002: 75
regulation 39(2) by the UK regulatory body, Medicines and Healthcare Products Regulatory Agency, 76
permitting supply and use of a non-CE marked device. 77
In this study we evaluate the performance of the Biothreat E-test against whole blood samples from 78
patients presenting in both Sierra Leone and United Kingdom. In both countries a range of samples 79
(including successive samples from individual patients), were tested by each method to generate 80
data on the sensitivity (i.e. positives from varying stages of infection with high and low viral loads), 81
and specificity (i.e. negative samples), of the Biothreat E-test. Samples were also heat treated to 82
address concerns associated with handling and processing of specimens potentially containing a 83
HG4 pathogen at lower containment. Therefore the test conditions were outside of the parameters 84
stated by the manufacturer. The overall aim was to provide further confidence, over that supplied 85
by the manufacturers validation data and Emergency Use orders, that the Biothreat E-test could be 86
used to provide a safe, reliable, and sensitive diagnostic for EBOV detection in whole blood samples 87
by laboratories (both in austere field settings or in countries with an advanced technological 88
infrastructure), which do not routinely offer an EBOV diagnostic capability. 89
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Materials and Methods 91
Sierra Leone and UK studies 92
This study was conducted at the PHE and UK Ministry of Defence (MOD) laboratory at the Kerry 93
Town Ebola Treatment Centre in the Western Urban Area of Sierra Leone, and PHE laboratories in 94
the UK. This evaluation was performed as a field evaluation in the context of an ongoing 95
emergency. Consequently operational and logistical requirements both in the UK and Sierra Leone 96
considerably limited the scope of comparative testing that could be employed during the evaluation 97
period. 98
Whole blood samples (taken by venepuncture and collected into EDTA tubes), submitted for Ebola 99
testing, were tested on receipt by a method (RNA extraction from plasma; real-time PCR), validated 100
for routine use in PHE laboratories in Sierra Leone and the UK and then stored at 4 oC as whole 101
bloods. These samples were then tested by Biothreat E-test within 0-6 days of the diagnostic real-102
time PCR. Samples were tested on availability rather than through the application of specific 103
selection criteria and included successive samples from the same patients. 104
The UK study was conducted under Royal College of Pathology guidelines (10) for the in service 105
development of diagnostic capability (i.e. performance assessment), and as such ethical approval 106
was not sought from individual organisational ethics committees. The Sierra Leone Ethics and 107
Scientific Review Committee was consulted but, after considering the parameters of the study and 108
the UK guidance, determined that they did not need to provide approval. 109
Conventional real-time PCR in Sierra Leone and the UK 110
In the Sierra Leone laboratory, and within a flexible film isolator, EDTA-blood samples were 111
centrifuged to produce plasma. In a fresh 2 mL screw cap microtube, 80 µL of plasma was mixed 112
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with 320 µL of Buffer AVL (Qiagen), the tube surface was decontaminated by wiping with 5% 113
chlorine solution and the tube was left to stand for 10 mins. The tube was removed from the 114
isolator and the sample was heated to 60 oC for 15 mins, the required temperature to inactivate 115
EBOV in a blood sample mixed with Buffer AVL (11). RNA extraction was performed from the entire 116
heat-treated 400 µL plasma / Buffer AVL sample using the Qiagen EZ1 Virus Mini Kit v2.0 and 117
Qiagen EZ1 Advanced XL platform to generate a final eluted RNA extract volume of 60 µL. Whole 118
MS2 bacteriophage was incorporated during the extraction procedure as an internal and inhibition 119
control. 120
Real-time PCR comprised the Ebola Zaire-MGB PCR (12) multiplexed with a MS2 control PCR (13). A 121
total reaction volume of 25 µL comprised: 5 µL template; Ebola Zaire-MGB primers (0.9 µM F565 122
[TCTGACATGGATTACCACAAGATC] and 0.9 µM R6405 [GGATGACTCTTTGCCGAACAATC]); probe 123
(0.25 µM P597S [6FAM-AGGTCTGTCCGTTCAA- MGBNFQ]); MS2 PCR primers (0.08 µM MS2 F1 124
[TGGCACTACCCCTCTCCGTATTCACG]; 0.08 µM MS2 R1 [GTACGGGCGACCCCACGATGAC]); probe 125
(0.16 µM MS2 [VIC-CACATCGATAGATCAAGGTGCCTACAAGC-QSY]); 6.25 µL TaqMan Fast Virus 1-126
Step Master Mix (Thermo Fisher Scientific) and water. Controls (Positive; No template [H2O]; and 127
negative extraction) were performed with each run. Positive results were recorded as a Cq value, 128
the cycle during which fluorescence was first detected above threshold during the PCR. Tests 129
conducted in the UK were performed similarly though initial sample inactivation occurred under 130
BSL-3 standards. 131
FilmArray PCR in Sierra Leone and the UK 132
Within the isolator comparator whole blood samples (200 µL) were added to a 2 mL micro-tube 133
containing the contents of FilmArray Sample Buffer ampoule (800 µL) which had previously been 134
used to re-suspend the Protease vial also supplied with the Biothreat E-test pouch. The sample was 135
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mixed and tube surface decontaminated by wiping with 5% chlorine solution and left to stand for 136
10 mins. The tube was removed from the isolator and heated to 60 oC for 15 mins. The sample was 137
then added to a hydrated Biothreat E-test as per the manufacturer instructions and run on a 138
benchtop FilmArray. Three hundred µL of the blood / buffer suspension is drawn into a FilmArray 139
pouch (5) indicating around 60 µL of blood was processed and tested by the Biothreat E-test. The 140
FilmArray presents results in a qualitative Positive / Negative interpretation. No Cq data is available, 141
although melt curve peaks are viewable by the operator. Internal control PCRs (RNA extraction and 142
PCR), monitor the success of the FilmArray process. To maintain a comparison between results in 143
both countries UK FilmArray testing also included the pre-PCR heat treatment, though all work 144
occurred under BSL-3 standards. Data from both methods and countries was analysed using the ‘R’ 145
language and environment for statistical computing and graphics (14). 146
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Results 148
Sierra Leone study 149
PCR results from 60 samples, obtained from 44 patients, are summarised in Table 1. EBOV was 150
detected in 25 samples by conventional real-time PCR and 25 samples by Biothreat E-test. 151
Discrepant PCR results were returned from 8 samples: four real-time PCR positives and Biothreat E-152
test negatives; four real-time PCR negative and Biothreat E-test positives. In a further five samples 153
control PCRs in the Biothreat E-test pouch failed and each run was declared invalid by the FilmArray 154
software, including one which returned a EBOV real-time PCR positive. These five samples were 155
excluded from the statistical analysis. The respective distribution of Sierra Leone Biothreat E-test 156
PCR results against positive real-time Cq values are included in Table 2. 157
In Sierra Leone the Biothreat E-test had sensitivity of 84% (confidence interval, CI: 64-95%) and 158
specificity of 89% (CI: 73-97%) when compared to the reference real-time PCR method. Statistical 159
analysis (Fisher’s Exact test) indicated there was no significant difference between methods at the 160
99% confidence level. 161
United Kingdom study 162
PCR results from 108 samples, obtained from 70 patients, are summarised in Table 1. In total EBOV 163
was detected in four samples by either method with one discrepant result: real-time PCR positive 164
and Biothreat E-test negative. The respective distribution of UK Biothreat E-test PCR results against 165
positive real-time Cq values are included in Table 2. In the UK (n=108; 70 patients) the Biothreat E-166
test had a sensitivity of 75%, (CI: 19-99%) and specificity of 100% (CI: 97-100%) when compared to 167
the reference real-time PCR. Statistical analysis (Fisher’s Exact test) indicated there was no 168
significant difference between methods at the 99% confidence level. 169
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Discussion 170
The West African Ebola outbreak has led the international community to deploy a number of Ebola 171
diagnostic laboratories into the countries mainly affected. There has also been pressure to increase 172
the number of laboratories in the UK able to provide an EBOV diagnostic capability (for suspect 173
patients with a recent travel history to West Africa), thereby improving test turnaround times 174
irrespective of where in the UK patients present for medical attention. 175
In West Africa it has been impossible to rapidly create a BSL-4 laboratory infrastructure and to 176
ensure operative safety laboratories have used methods which rapidly inactivate EBOV prior to 177
testing. A separate study (11) has indicated that a combination of Buffer AVL (containing a 178
chaotropic guanidine salt) and heat is required to inactivate EBOV in blood samples - with individual 179
Buffer AVL or heat treatments not inactivating EBOV in samples. In Sierra Leone the EZ1 RNA 180
extraction and SmartCycler PCR platforms were operated outside of biological containment. 181
Therefore, using a flexible film isolator, all plasma samples were prepared and inactivated with a 182
combination of Buffer AVL and heat prior to RNA extraction and real-time PCR. 183
This heat-treatment step was also included for whole blood samples mixed with FilmArray Sample 184
Buffer and is outside the parameters stated by the manufacturer and the FDA EUA. We have not 185
experimentally evaluated the EBOV viricidal efficacy of FilmArray Sample Buffer (which like Buffer 186
AVL also contains a guanidine salt), with or without an additional heat treatment. Without any 187
experimental evidence we included the heat treatment as the previous study (11) indicated that a 188
dual treatment (guanidine based buffer and heat), was required to ensure EBOV inactivation. This 189
provided confidence to allow operation of the Sierra Leone FilmArray outside of containment. 190
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Overall there was good concordance between the results from Biothreat E-test and real-time EBOV 191
PCR assays, particularly when used against high load (low Cq) samples (Table 2). The Sierra Leone 192
sample set, with a range of positive samples, confirmed the diagnostic sensitivity of the Biothreat E-193
test assay whilst the UK sample set, with a large number of true negatives, helped confirm the 194
specificity of the Biothreat E-test assay. 195
In total nine samples provided discrepant results, all of which came from patients who were 196
confirmed to be infected with EBOV, based on real-time PCR results and clinical findings. The results 197
comprised 4 potential Biothreat E-test false-positives and 5 false-negatives when measuring the 198
performance of the FilmArray against the reference PCR method. Timeline patient sample data is 199
summarised in Figure 1 to provide contextual information for each discrepancy. 200
Of the potential false-positive results one was from a patient sample (SL Patient 6) where repeat, 201
and next-day, samples generated real-time PCR Cq values above 36. Another (SL Patient 5), was 202
returned two days after a previous sample generated a real-time Cq result of 31.5. Two other 203
samples (SL Patients 2 and 7), tested positive by Biothreat E-test 10 and 14 days, respectively, from 204
initial samples which provided strong EBOV real-time PCR positives. Although it cannot be 205
definitively proven that these last three results are not Biothreat E-test false positive reactions a 206
previous study (7), when testing patient samples, also generated real-time PCR negatives (from 207
plasmas) but FilmArray PCR positives (from whole bloods). These were subsequently confirmed to 208
be from patients whose viral load was waning (Cq > 36). Analytical sensitivity studies in the same 209
paper produced positive (Cq = 37) and negative real-time PCR results at the EBOV titre of 4 × 101 210
TCID50∙mL-1. Therefore the four, discrepant, Biothreat E-test positive results discussed above are 211
consistent with the presence of residual, low, EBOV concentrations in blood, as has also been 212
observed in the other study (7). 213
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To support this hypothesis the four potential FilmArray false-negative results (from SL Patients 1 214
and 4, UK Patient 1), were generated from samples where corresponding real-time PCR values were 215
high (>36) again indicating these samples contained low amounts of target nucleic acid but in this 216
case a potential Biothreat E-test false negative occurred. Of note, the UK discrepant result (real-217
time Cq = 37.53), was generated in a sample where a repeat sample (taken at the same time but 218
into a different EDTA blood tube), tested positive by FilmArray PCR (comparator Cq = 36.7). 219
The remaining discrepant result (a potential Biothreat E-test false-negative), was returned from a 220
patient (SL Patient 3), with a higher viral load (Cq = 29.1), than the other discrepant samples. It is 221
unclear why the Filmarray failed to detect EBOV in this sample, though it is unlikely to be due to 222
variation in the Biothreat E-test target sequence as subsequent samples from the same patient 223
were positive. This is potentially an inhibition effect with whole blood, the FilmArray sample type, 224
containing more PCR inhibitors (15), than the plasma sample type tested by real-time PCR. Indeed 225
plasma generated from the same sample and tested by Biothreat E-test did provide an EBOV 226
positive (data not shown). Although FilmArray control PCRs on the discrepant sample passed, the 227
performance of these assays is measured by the system using only qualitative melt-curve data (5), 228
and therefore an increase in control PCR Cq value, which might indicate a general inhibition effect 229
and therefore explain the EBOV negative, would not be detected by the system. Individual PCRs are 230
known to be differentially affected by the same inhibitor (16), supporting the hypothesis of a 231
control PCR pass but EBOV PCR failure. 232
In general although other potential variables within our test protocol (storage of samples prior to 233
FilmArray testing; differential sample volumes processed by each method; differential viral loads in 234
blood and plasma; laboratory error), could have affected the number and nature of the discrepant 235
results, the majority appear to correlate with samples with observed, or probable, low 236
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concentrations of EBOV. At low concentrations nucleic acids are distributed stochastically (17), and 237
stochastic effects occur commonly in PCR tests, including FilmArray studies (18), when the copy 238
load in the sample is low. This gives rise to apparent discrepancies, even when the same assay is 239
used in duplicate. This requires careful interpretation and explanation by the laboratory to 240
clinicians, in the context of each case and their likely phase in the evolution of the disease. 241
While not formally assessed, a number of operational considerations were noted during the 242
operation of the Biothreat E-test. We did not find evidence of cross contamination between runs, 243
even when high viral load positive and negative samples were run in succession. Several pouches 244
also failed to hydrate properly (both countries), though this event is readily noticed during set-up 245
(and does not result in the loss of sample), and the manufacturer has recently released a new 246
version of the pouch loading station in mitigation. In addition the FilmArray, designed as a low-247
throughput screening capability and only able to test one sample per run, may not be readily 248
suitable as the primary diagnostic in an Ebola outbreak situation where high numbers of samples 249
need to be rapidly tested. Integrated PCR platforms able to test multiple EBOV samples 250
concurrently (19), or other rapid diagnostic technologies already evaluated in West Africa (20), may 251
provide more appropriate EBOV diagnostics in this scenario. 252
This study has indicated the FilmArray Biothreat E-test v2.5 offers performance comparable with a 253
validated real-time PCR approach for Ebola diagnosis and therefore a positive result can be treated 254
as a presumptive diagnosis with confidence. Of equal importance is the confidence in a negative 255
result, as part of a screening process, as this will have considerable significance for both patient 256
care and wider public health response in both epidemic and non-epidemic countries. The slight 257
differences in results between both PCR methods likely reflect the stochastic differences at low 258
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levels of pathogen during recovery and sampling stages, rather than performance differences 259
between the different assays. 260
In summary we show that the FilmArray Biothreat E-test (v2.5) is sensitive and specific and, when 261
also considering the low logistic and operative burdens and speed to result, provides an attractive 262
option for a low-throughput laboratory, without either BSL-4 trained staff or infrastructure, wishing 263
to provide an EBOV diagnosis capability. Such a laboratory could either have an existing 264
containment infrastructure and therefore reduce the workload on a centralised reference 265
laboratory or, if using a method to rapidly inactivate EBOV prior to PCR, be a field laboratory in an 266
austere or remote environment. 267
Acknowledgements 268
This study was funded by the UK Ministry of Defence and Public Health England. The Sierra Leone 269
evaluation was performed at the Kerry Town Ebola Treatment Centre funded by the UK Department 270
for International Development. 271
We acknowledge and thank the volunteer and MOD staff in the laboratory in Kerrytown, SL for their 272
contribution to the study execution. Similarly we thank the staff in the UK for their role in 273
diagnostic processing of patient specimens. 274
© Crown copyright (2015), Dstl. This material is licensed under the terms of the Open Government Licence 275
except where otherwise stated. To view this licence, visit http://www.nationalarchives.gov.uk/doc/open-276
government-licence/version/3 or write to the Information Policy Team, The National Archives, Kew, London 277
TW9 4DU, or email: [email protected]. 278
279
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Leone, January to February 2015. Euro Surveill 2015;20(12):pii=21073. Available online: 351
http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=21073 352
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Table 1. Summary of EBOV real-time PCR and Biotheat E-test PCR results from patient samples tested 354
in this study 355
356 a Sensitivity and specificity of the E-test, measured against the reference real-time PCR method, 357 calculated using the R statistical language and environment (14). 95% confidence intervals in 358 parentheses. 359
360
361
362
EBOV PCR results from patient samples
Sierra Leone samples (n=60; 44 patients) UK samples (n=108; 70 patients)
real-time NP PCR real-time NP PCR
Positive Negative Positive Negative
E-test
Positive
21 4
E-test
Positive 3 0
Negative 4 31 Negative 1 104
E-test sensitivity 84% (64-95%)a 75% (19-99%)a
E-test specificity 89% (73-97%)a 100% (97-100%)a
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Table 2. Distribution of FilmArray Biothreat E-test (v2.5) PCR results against Cq values generated by 363
Ebola Zaire MGB (NP target) PCR from samples tested in Sierra Leone and UK 364
Cq range of Ebola Zaire-MGB
(NP) PCR positive results
Biothreat E-test (v2.5) result
Positive Negative
15-20 6 0
20-25 2 0
25-30 3 1
30-35 10 0
>35 3 3
n = 24 4
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Figure 1. Representation of nine patient samples in Sierra Leone (SL) and the United Kingdom (UK) 367
with discrepant Ebola Zaire-MGB and Biothreat E-test PCR results, with preceding and subsequent 368
test results. 369
370
371
a Cq = PCR cycle number during which fluorescence first detected in 40 cycle PCR; POS = Biothreat 372
E-test positive; NEG = Biothreat E-test negative; NT = not tested. Shading indicates sampling and 373
testing points by each method, Arrows indicate discrepant results. 374
b A subsequent real-time PCR test on the same sample was positive (Cq = 36.3) 375
c Two samples taken at the same time on day 8 (into different EDTA blood tubes). 376
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