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Multicenter Evaluation of the Verigene Clostridium difficile Nucleic Acid Assay 1
Running title: Verigene C. difficile Assay 2
Karen C. Carroll1, 2 #, Blake W. Buchan3, Sokha Tan2, Paul D. Stamper1, Katherine M. Riebe3, 3
Preeti Pancholi4, Cheryl Kelly4, Arundhati Rao5, Robert Fader5, 4
Robert Cavagnolo6, Wendy Watson6, Richard V. Goering7, Ernest A. Trevino, Alice S. 5
Weissfeld, Ph.D8, Nathan A. Ledeboer3 6
1 Department of Pathology, the Johns Hopkins University School of Medicine, 2 Medical 7
Microbiology Laboratory, the Johns Hopkins Hospital, Baltimore, MD; 3Medical College of 8
Wisconsin, Dynacare Laboratories, Milwaukee WI; 4Ohio State University, Columbus OH; 9
5Scott and White Hospital, Temple TX; 6Medfusion, Lewisville, TX; 7Creighton University 10
Medical Center, Omaha, NE, 8Microbiology Specialists, Houston, TX 11
12
# Corresponding Author: 13
Karen C. Carroll, M.D. 14
Professor Pathology 15
The Johns Hopkins University School of Medicine 16
Meyer B1-193 17
600 N. Wolfe St. 18
Baltimore MD 21287 19
Kcarrol7@jhmi.edu 20
410-955-5077 21
FAX 410-614-8087 22
JCM Accepts, published online ahead of print on 2 October 2013J. Clin. Microbiol. doi:10.1128/JCM.01690-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.
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Abstract 24
25
The Verigene Clostridium difficile Nucleic Acid Test (Verigene CDF Test) (Nanosphere, 26
Northbrook, IL) is a multiplex qualitative PCR assay that utilizes a nanoparticle-based array 27
hybridization method to detect C. difficile tcdA and tcdB in fecal specimens. In addition, the 28
assay detects binary toxin gene sequences and the single base pair deletion at nucleotide 117 (ǻ 29
117) in tcdC to provide a presumptive identification of the epidemic strain ribotype 027. This 30
study compared the Verigene CDF Test with anaerobic direct and enriched toxigenic culture on 31
stool specimens from symptomatic patients among five geographically diverse laboratories 32
within the USA. The Verigene CDF test was performed according to the manufacturer’s 33
instructions, and the reference methods performed by a central laboratory included direct culture 34
onto cycloserine cefoxitin fructose agar (CCFA) and enriched culture using cycloserine cefoxitin 35
mannitol broth with taurocholate and lysozyme. Recovered isolates were identified as C. 36
difficile using gas liquid chromatography and were tested for toxin using a cell culture 37
cytotoxicity neutralization assay. Strains belonging to ribotype 027 were determined by PCR 38
ribotyping and bidirectional sequencing for ǻ 117 in tcdC. A total of 1,875 specimens were 39
evaluable. Of these, 275 specimens (14.7%) were culture positive by either direct or enriched 40
culture methods. Compared to direct culture alone, the overall sensitivity, specificity, positive 41
predictive value, and negative predictive value for the Verigene CDF test were: 98.7%, 87.5%, 42
42%, and 99.9%, respectively. Compared to combined direct and enriched culture results, the 43
sensitivity, specificity, positive predictive value, and negative predictive values of the Verigene 44
CDF test were: 90.9%, 92.5%, 67.6%, and 98.3%, respectively. Of the 250 concordantly culture 45
positive specimens, 59 (23.6%) were flagged as “hypervirulent”; 53 were confirmed as ribotype 46
027 and all 59 possessed ǻ 117 in tcdC. Time to results was approximately 2.5 h per specimen. 47
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The Verigene CDF Test is a novel nucleic acid microarray that reliably detects both C. difficile 48
toxins A and B in unformed stool specimens and appears to adequately identify ribotype 027 49
isolates. 50
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Introduction 52
The optimum testing method for the diagnosis of Clostridium difficile (C. difficile) is still 53
a matter of debate. A variety of testing methods are available and include enzyme 54
immunoassays (EIAs) for detection of toxins A and B, cell culture cytotoxicity neutralization 55
assays (CCNAs), anaerobic toxigenic culture, detection of glutamate dehydrogenase and nucleic 56
acid amplification tests (NAATs). Although EIAs provide same day results, are relatively 57
inexpensive, and easy to perform, there is general agreement in the USA and in some European 58
countries that EIAs should not be performed as stand-alone tests due to their lack of sensitivity 59
(1, 2). CCNAs and anaerobic toxigenic culture do not provide timely results and have never been 60
standardized. Laboratories are beginning to adopt nucleic acid amplification tests (NAATs) 61
alone or as a component of multi-test algorithms that include screening with glutamate 62
dehydrogenase (GDH) (3-5). Such strategies are endorsed by various professional societies in 63
their C. difficile testing and treatment guidelines (1, 6). 64
Nucleic acid amplification tests (NAATs) have evolved as improved fecal extraction 65
protocols and real-time polymerase chain reaction (PCR) methods have overcome inhibition 66
accompanied with improved assay performance (4, 7). Currently there are nine FDA-cleared 67
NAATs available in the USA with several more in development (8-14). The majority of these 68
are real-time PCR assays that detect the toxin B gene (tcdB) and include the BD-GeneOhm Cdiff 69
assay (BD-GeneOhm, San Diego CA), the GeneXpert Cdiff and Cdiff Epi tests, (Cepheid, Inc. 70
Sunnyvale, CA), the Prodesse ProGastro CD assay (Hologic Gen-Probe, Inc. San Diego, CA) 71
and the Simplexa Universal Direct assay (Focus Technologies, Inc. Cypress, CA). The 72
GeneXpert Epi assay is a multiplex PCR method that not only detects tcdB, but also detects 73
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binary toxin genes and the single base pair deletion at nucleotide 117 (ǻ 117) in tcdC to provide 74
presumptive identification of the epidemic strain 027/NAP1/BI. 75
Several tests are based upon other novel chemistries including loop-mediated isothermal 76
amplification such as the illumigene assay (Meridian Bioscience, Inc., Cincinnati, OH) (11, 12), 77
helicase dependent amplification in the Portrait C. difficile assay (Great Basin, Inc., Salt Lake 78
City, UT) (13) and AmpliVue C. difficile assay (Quidel Molecular, Inc., San Diego CA), and 79
array technologies, Verigene Cdiff assay (Nanosphere, Inc. Northbrook IL). Several of these 80
assays detect conserved regions of the toxin A gene (tcdA) either alone (illumigene) [11, 12] or 81
in combination with other genetic targets (Verigene CDF test). One manufacturer, Luminex, Inc. 82
Austin, TX has included C. difficile in a multiplex assay that detects other enteric pathogens 83
(xTAG Gastrointestinal Pathogen Panel). 84
The Verigene CDF Test is a multiplex qualitative assay that utilizes PCR-amplified DNA 85
in a nanoparticle-based microarray to detect C. difficile tcdA and tcdB in fecal specimens. In 86
addition, the assay detects binary toxin gene sequences and the single base pair deletion at 87
nucleotide 117 in tcdC to provide presumptive identification of the epidemic strain 027/NAP1/BI 88
(henceforth referred to as ribotype 027). This study evaluated and established the analytical 89
performance of the Verigene CDF Test using the Verigene System compared to anaerobic 90
toxigenic culture, ribotyping and bidirectional sequencing as the reference methods. 91
(This research was presented in part at the 113th General Meeting of the American Society for 92
Microbiology, Denver, CO. and in part at the 23rd European Congress of Clinical Microbiology 93
and Infectious Diseases, Berlin, Germany). 94
Materials and Methods 95
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Participating Centers and Overall Study Design: This was a prospective study from February 96
28, 2012 until June 27, 2012 to assess the analytical performance of the Verigene CDF Test 97
among five geographically diverse clinical microbiology laboratories. These included: The Johns 98
Hopkins Hospital Microbiology Laboratory, Baltimore, MD; The Medical College of Wisconsin 99
Milwaukee, WI; Ohio State University, Columbus, OH; Scott and White Hospital, Temple, TX; 100
and Medfusion Laboratories, Inc. Lewisville, TX. Each site obtained appropriate institutional 101
review board approval or waiver consistent with local human subjects research requirements. 102
All sites performed Verigene CDF testing on leftover de-identified stool samples submitted to 103
the clinical laboratory specifically for C. difficile testing according to the institution’s routine 104
practices. In addition, all sites reserved an aliquot of the stool for shipping to a central reference 105
laboratory, Microbiology Specialists, Inc. (Houston, TX) for performance of anaerobic toxigenic 106
culture methods (see reference methods section). Toxin-producing isolates were sent to 107
independent laboratories for PCR ribotyping and bidirectional sequencing (see PCR Ribotyping 108
and Bi-directional sequencing section). 109
Specimen collection and handling: A minimum of 2 mL of liquid stool or a marble-size of soft 110
stool specimen was required for testing. Each study site de-identified the fresh prospectively-111
collected specimen submitted to the laboratory for clinical testing by assigning each a unique 112
specimen identification number. Duplicate specimens from the same patient were not enrolled. 113
The collection time and date, patient age, and consistency of the specimen were recorded for 114
each specimen. Enrolled specimens were thoroughly mixed in the original container for 5 sec. 115
and then > 2 mL of specimen were pipetted into a 5 mL conical tube (tube A) for culture and 116
molecular testing within 48 h of collection. Specimens were kept at 2-8ºC until tested. A 0.5 117
mL aliquot of liquid stool or a pea-size soft stool specimen in tube A was transferred to 118
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Anaerobe Tissue Transport Medium (ATTM) at the time of Verigene testing, (Anaerobe 119
Systems, Inc. Morgan Hill, CA). The ATTM was stored at room temperature and shipped each 120
weekday in an insulated shipping container to Microbiology Specialists (MSI), Inc. for anaerobic 121
toxigenic culture. 122
Reference Culture Methods: Upon arrival to MSI, the stool was inoculated onto pre-reduced 123
CCFA (cycloserine cefoxitin fructose agar) [CCFA-D] and CCMB-TAL broth (cycloserine 124
cefoxitin mannitol broth with taurocholate and lysozyme) (Anaerobic Systems). The CCFA and 125
CCMB-TAL broth were incubated anaerobically for 48 h. All TAL broths were then subcultured 126
to CCFA and CCFA-HT (CCFA with horse blood and taurocholate, CCFA-E). These plates 127
were subsequently incubated under anaerobic conditions for 48 h. On CCFA, colonies of C. 128
difficile exhibit a yellow, ground glass appearance and chartreuse fluorescence under a Wood’s 129
lamp. On CCFA-HT, colonies of C. difficile exhibit a grey, ground glass appearance. A Gram 130
stain was performed on suspicious colonies; large gram-positive rods were further tested for 131
aerotolerance and susceptibility to vancomycin. 132
Any vancomycin-susceptible, strictly anaerobic, large gram-positive rods were placed in 133
chopped meat carbohydrate (CMC) broth for 48 h. Part of the broth was then extracted for gas 134
liquid chromatography (GLC); the presence of isovaleric, isocaproic and isobutyric acids in the 135
volatile product represented a definitive identification for C. difficile. Another portion of the 136
CMC was centrifuged, and the supernatant was then removed and used to determine the presence 137
of toxin by inoculation onto human fetal foreskin cells. Toxin was neutralized with specific 138
antitoxin to C. difficile (Diagnostic Hybrids, Athens, OH) to confirm the presence of toxigenic C. 139
difficile (10). 140
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For the purposes of this study, if C. difficile was isolated from the CCFA-D plate and the 141
isolate was positive by the cell cytotoxicity assay, the specimen was classified as “toxigenic C. 142
difficile positive” and the CCFA-E plate was not further analyzed. If no C. difficile was isolated 143
from the CCFA-D plate or if the isolate was negative by the cell culture cytotoxicity assay, the 144
CCFA-E plate was further analyzed. If CCFA-E was positive for C. difficile and the isolate was 145
positive for cell cytotoxicity assay, the specimen was classified as “toxigenic C. difficile 146
positive”. The specimen was reported as “negative” if CCFA-E was negative for C. difficile or 147
the isolate was tested negative by the cell cytotoxicity assay. 148
Description of the Verigene CDF Assay: 149
The Verigene System is a bench-top sample-to result platform consisting of two modules: 150
the Verigene Processor SP and the Verigene Reader. The Processor SP automates specimen 151
nucleic acid extraction, PCR amplification, and amplicon hybridization. The Processor SP 152
utilizes four single-use consumables per test: an extraction tray, amplification tray, test cartridge, 153
and pipette tip holder assembly. The user tests a specimen by loading the single-use consumables 154
into the Processor SP, pipetting the test specimen into the sample well of the extraction tray, and 155
initiating the test protocol on the Reader by scanning the test cartridge barcode and specimen 156
information. Upon test completion, the user inserts the microarray slide from the test cartridge 157
into the Reader for optical analysis and generation of test results. 158
For C. difficile testing on the Verigene System, 100 ȝl of the prepared stool sample is 159
loaded into the sample well of the extraction tray. The test specimen is prepared from a soft or 160
liquid stool sample by swabbing the stool with the provided flocked swab and transferring the 161
evenly coated swab to the provided stool prep buffer tube, which is subsequently vortexed for 15 162
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seconds and centrifuged for 30 seconds. Following test initiation on the Processor SP, sample 163
bacteria are lysed and target DNA is extracted using a magnetic bead-based extraction. 164
Extraction is followed by multiplex PCR-based amplification, which generates target specific 165
amplicons. Finally, amplicon hybridization to target specific capture DNA occurs in a 166
microarray setting using mediator and gold nanoparticle probe hybridization to the captured 167
amplicons. Silver enhancement of the bound gold nanoparticle probes at the capture sites results 168
in gold-silver aggregates that are assessed optically on the Reader to produce the final test result. 169
Controls 170
A negative control is included in the array to detect whether the melt temperature of the 171
hybridization chamber is too low due to an instrument malfunction. The Verigene Reader 172
automatically checks the target oligonucleotide signal relative to the negative control 173
oligonucleotide signal. If the target signal is not adequately higher than the negative control 174
signal, a “no call” result will be generated. 175
An artificial DNA construct serves as a hybridization control and is referred to as 176
Processing Control 1 (PC1). This control material along with the primers, enzymes and 177
amplification buffers are included within the amplification tray. If the process control is not valid 178
a “no call” result will be obtained and the test should be repeated. Bacillus subtilis serves as a 179
specimen preparation and amplification control and is referred to as Processing Control 2 (PC2). 180
This control is automatically added by the Processor SP to each specimen prior to the extraction 181
step. If the process control is not valid, a “no call” result will be obtained and the test should be 182
repeated. Additional positive controls are immobilized on the test slide and are used to 183
determine that hybridization was performed correctly. The Verigene CDF Test algorithm 184
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requires that these controls be valid before decisions regarding the presence or absence of any 185
other target on the panel can be determined. If these controls are not detected a “no call” result 186
will be obtained and the test is repeated. 187
Verigene CDF Test Performance: For liquid samples, 150 µL of the specimen from Tube A 188
was transferred to a small screw capped microcentrifuge tube containing stool lysis buffer. For 189
soft stool specimens, a sterile swab was fully immersed in the specimen and once evenly coated, 190
it was transferred to the stool lysis buffer tube and the swab was broken at the preformed score 191
breakpoint. In both cases the screw cap was gently tightened by hand. All specimen transfers 192
were performed in such a manner as to ensure a minimum risk of contamination. Gloves were 193
changed between pipetting and transfer of specimens and only one specimen was open and 194
processed at a time. All sample buffer tubes containing specimen were vortexed for 15 sec. and 195
microcentrifuged for 30 sec., after which 100 µL of supernatant were inoculated into the 196
specimen well of the extraction tray. Testing was performed in a blinded manner on the de-197
identified aliquots according the manufacturer’s protocol and product package insert. 198
Three external positive controls were supplied to each test site for quality control 199
purposes. These external positive controls were run on an alternating basis once every 24 h 200
during the period of time that clinical testing was conducted (e.g. CDF Control 1 was run on Day 201
1 of testing, CDF Control 2 on Day 2, and so on) with the goal being an approximately equal 202
distribution of each CDF control run during the study at each site. Several test-specific negative 203
controls are immobilized on the Test Slide and are used to guide test decisions (see Description 204
of the Verigene Test). A single external negative control (which is a non-toxigenic C. difficile) 205
was run every 24 h during the period when clinical specimen testing was conducted. 206
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Results interpretation: The Verigene CDF assay provides a qualitative result for the presence 207
(detected) or absence (not detected or not applicable) of tcdA, tcdB, binary toxin genes and the 208
ǻ117 deletion in tcdC. Table 1 demonstrates the possible outcomes for valid test results. Invalid 209
results may be generated by failure to image the array slide (No Call-No Grid) or from internal 210
control failures or because the Reader could not read the target specific signals due to variability. 211
Other sources of invalid results include pre-analytical errors. All invalid results were repeated 212
within 48 h of specimen collection from a refrigerated reserved aliquot of the original stool 213
sample. 214
PCR Ribotyping: Following the culture testing at MSI, the toxigenic C. difficile positive isolates 215
were sent for strain identification by PCR-Ribotyping to an external third-party site (Creighton 216
University Medical Center, Omaha, NE) as per the study protocol. In addition to conventional 217
agarose-gel electrophoresis, for added discrimination, PCR products were analyzed using the 218
Agilent 2100 Bioanalyzer (Agilent Technologies, Clara, CA). The strains were designated into 219
two PCR-Ribotyping categories, 027 and non-027. Strain type assignments were based on a 220
comparison of isolate profiles with known C. difficile reference strains from the C. difficile 221
collection of the Cardiff-European Centre for Disease Prevention and Control [ECDC] obtained 222
from Dr. Ed Kuijper, Leiden University Medical Center, Netherlands using the BioNumerics 223
software, version 6.6 (Applied Maths, Belgium). 224
Bi-directional sequencing: During the study, DNA from the Extraction step of the Verigene 225
CDF Test was saved and stored frozen at -20 °C until needed. If the PCR amplification for tcdC 226
failed for a DNA sample, the DNA extracted during the PCR-Ribotyping was 227
tested. Sequencing templates were prepared by PCR amplification of the stored DNA using 228
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sequencing primers. The same set of primers was also used for sequencing the amplified 229
material. In parallel, following central culture testing, DNA from the culture-confirmed C. 230
difficile positive isolates (that was extracted during the Verigene CDF Test) was sent to an 231
external third-party site (ACGT, Inc, Wheeling, IL) for tcdC bi-directional sequencing as per the 232
protocol. A 295 bp fragment of tcdC was sequenced. 233
Data analysis and statistical methods: Results of the Verigene CDF Test were compared to 234
direct culture on CCFA with isolates confirmed and analyzed independently by ribotyping and 235
bi-directional sequencing. In addition, the Verigene CDF Test was also compared to enriched 236
culture, with positives verified by ribotyping and bi-directional sequencing (see Reference 237
Culture Methods sections). 238
Results were entered into EXCEL (Microsoft Corporation, Redmond, Washington) and 239
the sensitivity, specificity, positive predictive and negative predictive values were calculated. 240
The exact binomial methods in SAS 9.1.3 (SAS Institute, Inc., Cary, North Carolina) were used 241
to calculate the lower and upper two-sided confidence limits. 242
RESULTS 243
Among the five study sites 1,877 specimens met the inclusion criteria. Seventy one 244
specimens (3.8%) required repeat testing: 46 specimens (2.4%) had an initial “No Call” result 245
due to assay internal control errors; 17 specimens (1.0%) had an initial “Indeterminate” call (No 246
Call-IND); and eight specimens (0.4%) had pre-analytical errors (four motor stalls, two tip 247
failures, one cracked slide and one cartridge not detected). The eight specimens that experienced 248
pre-analytical errors and the 46 “No Call” specimens all produced a valid call upon repeat testing 249
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as per the protocol. However, two of the “No Call” specimens required a second repeat test. 250
Repeat testing of the 17 No Call-IND specimens called all but two specimens. Therefore, two 251
specimens had a final “Indeterminate” call and were not included in the clinical data analysis of 252
evaluable results. Thus, 1,875 specimens were analyzed in this clinical evaluation. 253
Table 2 summarizes the overall Verigene CDF Test clinical performance. Of the 1,875 254
specimens that were analyzed in this trial, there were 275 specimens positive for C. difficile and 255
1600 negative specimens by either direct or enriched culture methods after all repeat testing. The 256
majority (98.8%) of positive specimens contained genes for both toxins A and B (data not 257
shown). 258
The overall agreement between direct culture and the Verigene CDF Test for detection of 259
toxigenic C. difficile in stool was 88.4%. The overall sensitivity, specificity, positive predictive 260
values and negative predictive values of the Verigene CDF Test compared to direct culture on 261
CCFA are listed in Table 2. Overall agreement between the combined direct and enriched 262
culture results and the Verigene CDF assay was 92.3% (Table 2). 263
Table 3 lists the performance characteristics of the Verigene CDF Test compared to 264
combined culture methods stratified by investigational site. There were no major differences in 265
the performance of the Verigene assay by investigational site. 266
There were 72 non-toxigenic C. difficile isolates recovered by culture during the study. 267
Eleven of these were detected as toxigenic by the Verigene CDF test and were classified as false 268
positives. Nine of the eleven false positive results were confirmed positive for tcdC by bi-269
directional sequencing. Two of the 11 false positive results were negative for tcdC and tcdB by 270
bi-directional sequencing (data not shown). 271
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Table 4 shows the distribution by Verigene CDF Test results for strain characterization. 272
Of the 250 concordantly culture and Verigene positive specimens, 59 (23. 5%) were flagged as 273
“hypervirulent”; 53 were confirmed as ribotype 027 and all 59 possessed the ǻ 117 mutation in 274
tcdC. One isolate that was ribotype 027 and contained the ǻ117 tcdC mutation was incorrectly 275
identified as non-O27 by the Verigene CDF Test. Compared to ribotyping, the Verigene CDF 276
Test correctly assigned the ribotype in 89.7% of the cases and when compared to sequencing the 277
percent agreement was 100 %. 278
DISCUSSION 279
Clostridium difficile remains a formidable pathogen that continues to cause significant 280
morbidity and mortality among hospitalized patients (15). Between 2001 and 2006, the 281
incidence of C. difficile infection in the USA more than doubled and the incidence continues 282
to rise in North America and Europe (15). In part, this increase was due to the appearance of 283
“hypervirulent’ strains such as ribotype 027. In addition to being more transmissible and a 284
hyperproducer of toxins A and B, this ribotype is notably more drug resistant compared to 285
historical strains (15). Increased severity of illness has resulted in more colectomies, 286
prolonged hospitalizations, and an increase in the observed number of recurrences (16). 287
More significantly, mortality rates have also increased and in North America and Europe are 288
reported to be as high as 22-37% (15). The economic burden is substantial, as was shown in 289
a systematic review of healthcare costs associated with patients with primary and recurrent 290
CDI (17). In 2008 US dollars, the additional costs for treating relatively mild primary CDI 291
varied from $9,822 to $13,854 (17). In Europe, the incremental increase in costs ranges in 292
Euros from €4067 to €9276 (15). 293
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Over the last decade, the emergence of “hypervirulent strains” such as ribotype 027, 294
ribotype 078 and the newly described ribotypes 176 and 198 (18, 19), have spurred many 295
clinical laboratories to abandon insensitive EIAs for more sensitive methods such as NAATs. 296
The published performance characteristics among the nine FDA-cleared NAATs varies 297
depending upon the comparative method, but in general those reports that use toxigenic 298
culture as the reference method have demonstrated sensitivities ranging from 77% to 100% 299
and specificities that range from 90-100% (4,7,8,11-14, 20). The Verigene CDF test is one 300
of the newer assays to obtain FDA 510(k) clearance and is novel. The assay uses multiplex 301
PCR combined with silver enhancement of gold nanoparticle probe hybridization to capture 302
amplicons in a microarray format. The Verigene CDF test is the only FDA-cleared assay that 303
detects both toxins A and B and identifies ribotype 027 by identifying both the binary toxin 304
gene and the ǻ117 deletion of tcdC. In terms of performance characteristics, the overall 305
sensitivity and specificity of 98.7% and 87.5%, respectively compared to direct toxigenic 306
culture and 90.9% and 92.5%, respectively compared to enriched culture are comparable to 307
the published clinical performance of other NAATs (8-14). As has been noted for other 308
NAATs, the NPV for the Verigene assay ranges from 98-99.9% and essentially excludes C 309
difficile as a cause of diarrheal disease, whereas the PPV is low ranging from 42-67% 310
compared to toxigenic culture methods. Some of the false positive samples were confirmed 311
by bi-directional sequencing as containing toxin-producing strains indicating possible mixed 312
infection with toxigenic and non-toxigenic isolates. A limitation of this study is that no 313
clinical data was collected and included in the assessment of a true positive or negative 314
specimen. The percentage of combined “No-Call”, “Invalid”, and pre-analytical problems of 315
3.9% necessitating repeat testing is also similar to what has been reported for the BD 316
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GeneOhm assay’s invalid rate of 0.5-7.3% (8, 12, 14), the Prodesse ProGastro CD invalid 317
rate of 2.7% (9, 14), and the illumigene rate of 0.8-4.4% (11, 12). This is slightly higher than 318
what is reported for the Quidel AmpliVue C. difficile assay (0.5%) [20]. 319
The usefulness of detecting ribotype 027 in any C. difficile assay remains controversial. 320
While some studies have demonstrated more severe disease and increased mortality associated 321
with ribotype 027 (16, 19), more recent studies have challenged this observation (21, 22). In the 322
latter studies, when other covariates were considered, the ribotype of the infecting organism was 323
not found to be a significant predictor of severe CDI (21). Currently, there are no 324
recommendations to alter infection control practices based upon ribotype. Likewise, there are 325
no formal recommendations to alter treatment based solely on the strain causing the infection. 326
Some strain types may correlate with an increased risk of recurrent disease (23). In the initial 327
fidaxomicin treatment study, the data showed that the risk of relapse may vary depending upon 328
the C. difficile strain type (24). For example, the rate of recurrence of non-027 CDI was 329
significantly lower in the fidaxomicin treated group compared to the vancomycin treated patients 330
(24). A more recent meta-analysis comparing fidaxomicin to vancomycin showed that 331
fidaxomicin reduced recurrence or death by 40% compared with vancomycin through day 40 332
(25). This observation did not hold true for ribotype 027 strains, where the rates of recurrence 333
with each agent were not different (24, 25). Considering the cost of this drug, a practical use for 334
real-time strain typing could be to include the ribotype information when triaging which patients 335
are best suited to receive fidaxomicin therapy. 336
One of the limitations to the Verigene system is the fact that it is a “one sample at a time” 337
method and the technologist has to manually remove the cartridge from the Processor into the 338
reader. While this allows for easy “on demand” testing, such a system is not amenable to large 339
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through-put laboratories. The instruments can be stacked and one Reader can accommodate up 340
to 32 Processors, but this requires space and may be operationally challenging. 341
In summary, the Verigene CDF assay is a novel PCR microarray test that is unique 342
among the current FDA-cleared platforms in its simultaneous detection of the genes that encode 343
toxins A and B, binary toxin and the ǻ 117 mutation in tcdC as surrogate markers for ribotype 344
027. In comparison to published performance characteristics of the other NAATs, the Verigene 345
test performs on the high end of the range of reported sensitivities. The unresolved specificity is 346
a bit lower as might be expected for a more sensitive assay. Finally the assay correlates well 347
with PCR-ribotyping and bidirectional sequencing in identifying ribotype 027 in clinical 348
samples. 349
ACKNOWLEDGEMENTS 350
The authors thank ACGT, Inc. under the direction of Semyon Rubinchik, Ph.D. for performing 351
the genetic analysis of the recovered isolates and Jon Marion and Niku Falandino from 352
Nanosphere, Inc. who assisted with the data analysis. In addition, all authors are grateful to the 353
staff of their clinical laboratories for their support of this study. This study was sponsored by 354
Nanosphere, Inc. NAL and BWB have served as consultants to Nanosphere. 355
REFERENCES 356
1. Cohen, SH, Gerding DN, Johnson S, Kelly CP, Loo VG, McDonald LC, Pepin J, 357
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Table 1: Interpretation of Valid Verigene CDF Test Results
tcdA tcdB Binary tcdC Result
Not detected Not Detected N/A N/A Toxigenic Clostridium difficile Not Detected
Detected Detected Detected Wild type Toxigenic Clostridium difficile Detected
Detected Detected Detected Mutant Toxigenic and PCR ribotype 027 Clostridium difficile Detected
Detected Detected Not Detected Wild type Toxigenic Clostridium difficile Detected
Detected Not Detected Detected Wild type Toxigenic Clostridium difficile Detected
Detected Not Detected Detected Mutant Toxigenic and PCR ribotype 027 Clostridium difficile Detected
Detected Not Detected Not Detected Wild type Toxigenic Clostridium difficile Detected
Not detected Detected Detected Wild type Toxigenic Clostridium difficile Detected
Not detected Detected Detected Mutant Toxigenic and PCR ribotype 027 Clostridium difficile Detected
Not detected Detected Not Detected Wild type Toxigenic Clostridium difficile Detected
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Table 2: Overall Performance Characteristics of the Verigene CDF Test Compared to Direct
Toxigenic Culture and Direct plus Enriched Toxigenic Culture
Method Number
of Specimens
Sensitivity
(% CI)
Specificity
(% CI)
Accuracy
(% CI)
PPV
(% CI)
NPV
(% CI)
Direct
Culture
1875 156/158
98.7%
(95.5-99.9)
1502/1717
87.5%
(85.8-89.0)
1658/1875
88.4%
(86.9-89.8)
156/371
42.1%
(37.0-47.3)
1502/1504
99.9%
(99.5-100)
Direct plus
Enriched
Culture
1875 250/275
90.9%
(86.9-94.0)
1480/1600
92.5%
(91.1-93.7)
1730/1875
92.3%
(91.0-93.4)
250/370
67.6%
(62.5-72.3)
1480/1505
98.3%
(97.6-98.9)
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Table 3: Direct Culture Results and Direct Culture plus Enriched Culture Results Stratified by Laboratory Site
Direct Culture Direct plus Enriched Culture Site # Sensitivity
(% CI)
Specificity
(% CI)
Accuracy
(% CI)
PPV
(%CI)
NPV
( % CI)
Sensitivity
(% CI)
Specificity
(% CI)
Accuracy
(% CI)
PPV
(% CI)
NPV
(% CI)
1 557
55/55
100%
(93.5-100)
424/502
84.5%
(81.0-
87.5)
479/557
86.3%
(83.2-
89.1)
55/133
41.4%
(32.9-
50.2)
424/424
100%
(99.1-
100)
98/106
92.5%
(85.7-96.7)
416/451
92.2%
(89.4-94.5)
514/557
92.3%
(89.7-
94.4)
98/133
73.7%
(65.4-
80.9)
416/424
98.1%
(96.3-
99.2)
2 482
36/36
100%
(90.3-100)
379/446
85.0%
(81.3-
88.2)
415/482
86.1%
(82.7-
89.1)
36/101
35.6%
(26.4-
45.8)
381/381
100%
(99.0-
100)
58/61
95.1%
(86.3-99.0)
378/421
89.8%
(86.5-92.5)
436/482
90.5%
(87.5-
92.9)
58/101
57.4%
(47.2-
67.2)
378/381
99.2%
(97.7-
99.8)
3 299
25/25
100%
(86.3-100)
251/274
91.6%
(87.7-
94.6)
276/299
92.3%
(88.7-
95.1)
25/47
53.2%
(38.1-
67.9)
252/252
100%
(98.5-
100)
36/40
90.0%
(76.3-97.2)
248/259
95.8%
(92.5-97.9)
284/299
95.0%
(91.9-
97.2)
36/47
76.6%
(62.0-
87.7)
248/252
98.4%
(96.0-
99.6)
4 371
25/27
92.6%
(75.7-
99.1)
310/344
90.1%
(86.5-
93.1)
335/371
90.3%
(86.8-
93.1)
25/59
42.4%
(29.6-
55.9)
310/312
99.4%
(97.7-
99.9)
38/44
86.4%
(72.7-94.8)
306/327
93.6%
(90.4-96.0)
344/371
92.7%
(89.6-
95.2)
38/59
64.4%
(50.9-
76.5)
306/312
98.1%
(95.9-
99.3)
5 166
15/15
100%
(78.2-100)
135/151
89.4%
(83.4-
93.8)
150/166
90.4%
(84.8-
94.4)
15/31
48.4%
(30.2-
66.9)
135/135
100%
(97.3-
100)
20/24
83.3%
(62.6-95.3)
132/142
93.0%
(87.4-96.6)
152/166
91.6%
(86.3-
95.3)
20/30
66.7%
(47.2-
82.7)
132/136
97.1%
(92.6-
99.2)
Total 1875
156/158
98.7%
(95.5-
99.9)
1502/1717
87.5%
(85.8-
89.0)
1658/187
5
88.4%
(86.9-
89.8)
156/371
42.1%
(37.0-
47.3)
1502/15
04
99.9%
(99.5-
100)
250/275
90.9%
(86.9-94.0)
1480/1600
****
92.5%
(91.1-93.7)
1730/187
5
92.3%
(91.0-
93.4)
250/37
0
67.6%
(62.5-
72.3)
1480/150
5
98.3%
(97.6-
98.9)
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***Of the 1875 specimens evaluated, seven specimens were culture positive but were either not PCR ribotyped or sequenced or both but
are included in the performance characteristics above.
****Of the 120 specimens that were toxigenic C. difficile positive (+) by Verigene CDF testing but toxigenic C. difficile negative (-) by direct/
enriched toxigenic culture, 90 were positive by bi-directional sequencing for tcdC, 6 were positive by bi-directional sequencing for tcdB, and 24
were negative by bi-directional sequencing for both tcdC and tcdB.
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Table 4: Results for Presumptive Detection of 027 Strains by the Verigene CDF Test
Compared to PCR-Ribotyping and Sequencing
PCR-Ribotyping or Sequencing Results
Verigene CDF Test
027 Not 027
PC
R-R
iboty
pin
g
027 53 1
Not 027 5 188
Not
performed
1 3
Total 59 192
Bi-
Dir
ecti
on
al
Seq
uen
cin
g
027 59 1
Not 027 0 191
Not
performed
0 0
Total 59 192
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