JCM Accepts, published online ahead of print on 17 December 2014 J. Clin. Microbiol. doi:10.1128/JCM.03297-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Evaluation of the Cepheid Xpert® C. difficile/Epi and Meridian Bioscience
2
illumigene® C. difficile for detecting Clostridium difficile ribotype 033 strains
3 4 5
Grace Androga1, Alan McGovern1, Briony Elliott1, Barbara J. Chang1, Timothy
6
Perkins1, Niki Foster1, Thomas V. Riley1, 2, #
7 8 9 10
1
School of Pathology and Laboratory Medicine, The University of Western Australia,
Perth, Australia.
11 12 13
2
14
Medical Centre, Nedlands 6009 Western Australia.
Department of Microbiology, PathWest Laboratory Medicine Queen Elizabeth II
15 16 17
Running Title: Detecting C. difficile ribotype 033
18 19 20
Corresponding Author: # Professor Thomas V. Riley,
21
Microbiology & Immunology
22
School of Pathology & Laboratory Medicine
23
The University of Western Australia
24
Queen Elizabeth II Medical Centre
25
Nedlands 6009
26
Western Australia
27
E-mail: [email protected]
28 29 30
Keywords: Clostridium difficile, Binary toxin, RT033, CDT, diagnostics, A-B+
31 32 33
Word count: Abstract 55, Text 1357
34 1
35
Abstract
36
Clostridium difficile PCR ribotype (RT) 033 is found in the gastrointestinal tracts of
37
production animals and, occasionally, humans. The illumigene® C. difficile (Meridian
38
Bioscience, Inc.) failed to detect any of 52 C. difficile RT033 isolates while all strains
39
signaled positive for the binary toxin genes but were reported as negative for
40
C. difficile by the Xpert® C. difficile/Epi (Cepheid).
41 42
Text
43
Clostridium difficile is the most common cause of infection associated with healthcare
44
in the United States (1). The diagnosis of C. difficile infection (CDI) remains a vexing
45
problem in hospitals and laboratories. Most current methods of laboratory diagnosis
46
involve detecting tcdA and/or tcdB, two genes located in the C. difficile pathogenicity
47
locus (PaLoc) that encode the two main virulence factors of the bacterium, the large
48
clostridial toxins A (TcdA) and B (TcdB). Detection of a third, binary toxin (CDT),
49
an actin-specific ADP-ribosyltransferase also produced by some strains of C. difficile
50
and not encoded in the PaLoc, has been largely overlooked (2). This has been
51
attributed to the difficulty of detecting CDT by non-molecular means as well as the
52
fact that it is usually produced by strains that also produce toxins A and B (A+B+) (3).
53
An exception is the real-time PCR-based Xpert® C. difficile/Epi system (Cepheid,
54
Sunnyvale CA). This U.S. Food and Drug Administration (FDA) approved assay
55
detects CDT gene sequences but only as a way of identifying putative RT027/NAP1
56
C. difficile strains in fecal specimens. The assay also detects tcdB and the single base
57
pair deletion at nucleotide position 117 of tcdC found in epidemic fluoroquinolone-
58
resistant RT027/NAP1 strains (http://www.cepheid.com/us/cepheid-solutions/clinical-
59
ivd-tests/healthcare-associated-infections/xpert-c-difficile-epi
60
November 2014]). The illumigene® C. difficile assay (Meridian Bioscience, Inc.) is
61
also FDA approved but instead targets a highly conserved region at the 5’ end of tcdA
62
and uses loop-mediated isothermal DNA amplification (LAMP) technology
63
(http://www.meridianbioscience.com/diagnostic-products/c-difficile/illumigene-
64
molecular-diagnostic-system/illumigene-c-difficile.aspx [Accessed 17th November
65
2014]).
[Accessed
17th
66 67
C. difficile RT033 belongs to toxinotype XIa/b and has a truncated PaLoc but an
68
intact CDT locus (4, 5). It is an emerging strain of clinical and veterinary importance 2
69
that has been detected in cattle, veal calves, piglets, horses and soil from various
70
geographical locations world-wide (6-8). Its exact role in human CDI has yet to be
71
determined, however, enterotoxic effects have been observed in rabbit ileal loop tests
72
with A-B-CDT+ strains of C. difficile, indicating that CDT could be a virulence factor
73
in these strains (9). In addition, CDT has been linked to an increase in the prevalence
74
of community acquired-CDI (CA-CDI), a high rate of recurrent CDI, and the need for
75
hospital admission due to re-infection (10, 11). A-B-CDT+ strains of C. difficile such
76
as RT033 are not detected by most current diagnostic methods and hence may be
77
missed by clinical laboratories.
78 79
Meridian Bioscience Inc. claims that the tcdA target sequence of their illumigene®
80
C. difficile assay allows for detection of the PaLoc of toxigenic C. difficile regardless
81
of the expression phenotype and that the assay was designed to detect all known A+B+
82
and A-B+ toxinotypes (http://www.meridianbioscience.com/diagnostic-products/c-
83
difficile/illumigene-molecular-diagnostic-system/illumigene-c-difficile.aspx
84
[Accessed 17th November 2014]). This means that PaLoc-positive, phenotypically
85
A-B-CDT+ C. difficile strains such as RT033 may be detected by the illumigene® C.
86
difficile assay as well as signal CDT-positive by the Xpert® C. difficile/Epi assay. In
87
this report, we test C. difficile RT033 isolates for their ability to signal in the
88
illumigene® C. difficile and the Xpert® C. difficile/Epi assays.
89 90
RT033 isolated from human (n = 15), porcine (n = 16) and bovine (n = 21) sources
91
were used in this study (Table 1). The animal isolates were collected as part of
92
Australian livestock prevalence studies (6, 8). The clinical isolates were mostly from
93
pathology laboratories around Australia. Dr. Frederick Barbut (National Reference
94
Laboratory for C. difficile, France) kindly contributed 6 of the clinical RT033 isolates
95
(12). For comparison, 11 non-RT 033 isolates of various origins and 5 reference
96
strains were also tested (Table 1).
97 98
The identity of C. difficile isolates was confirmed by their distinct colony
99
morphology, yellow-green fluorescence under 360nm UV light, and horse-dung odour
100
when grown on blood agar for 48 h under anaerobic conditions (A35 Anaerobic
101
Workstation, Don Whitley Scientific, Shipley, West Yorkshire). Toxin gene profiling
3
102
and PCR ribotyping of all isolates was performed using previously described methods
103
(6). The illumigene and Xpert testing was performed according to the manufacturers’
104
instructions except that 0.5 McFarland saline suspensions of C. difficile from 48 h
105
blood agar cultures were used instead of the recommended stool samples.
106 107
All 52 human, bovine and porcine C. difficile RT033 isolates failed to signal with the
108
illumigene assay, as did the two A-B- reference strains (RT033 and RT010) and four
109
non-RT033 test ribotypes (the only A-B- ribotype [RT239] and 3 of 4 A-B+ ribotypes).
110
The four RT017 strains were the only A-B+ isolates tested that signaled in the
111
illumigene assay. The Xpert assay detected the CDT analyte in all 52 RT033 strains,
112
all CDT+ non-RT033 test strains and both CDT+ reference strains. Only the tcdB-
113
positive strains were identified as toxigenic C. difficile and only the RT027 reference
114
strain was identified as a presumptive 027/NAP1 (Table 1).
115 116
RT033 characteristically contains a truncated PaLoc region that only retains the
117
putative translocation domain fragment (A2) and parts of the repetitive domain
118
fragment (A3) of the tcdA gene (Fig 1); hence neither large clostridial toxin is
119
produced (4, 5). The illumigene® C. difficile assay was unable to detect the remnant
120
fragments of the tcdA gene in any of the RT033 isolates tested, consistent with a
121
deletion in the 5’ end region (4, 5). The illumigene® C. difficile assay was designed to
122
target a ‘conserved’ region at the 5’ end of tcdA present in all known A+B+ and A-B+
123
toxinotypes (13). This was supported by Couturier et al who reported detection of the
124
tcdA target sequence in A-B+ strains belonging to toxinotypes VIII and X using the
125
illumigene® C. difficile assay (13) and in the present study for RT017, toxinotype VIII
126
strains which have a truncated tcdA gene retaining only the A1 fragment of the gene
127
(4). Other A-B+ ribotypes tested (RT237, RT280 and RT281) that were known to
128
contain a truncated tcdA region were not detected by the illumigene® C. difficile
129
assay (14, 15). Therefore, in addition to PaLoc-negative C. difficile, the target region
130
of the illumigene® C. difficile assay is not conserved in the PaLocs of RT033, RT237,
131
RT280 and RT281 strains.
132 133
The Xpert® C. difficile/Epi assay successfully detected CDT gene sequences in all
134
RT033 isolates as expected. However, it was necessary to view the raw data obtained
4
135
by the assay to identify a CDT-positive result because the assay does not recognize a
136
test as positive for toxigenic C. difficile based on this result alone. The CDT target is
137
only used to give a presumptive positive report of C. difficile RT027/NAP1 strains
138
and this is only when both tcdB and the tcdC Δ117 deletion are detected also.
139
Furthermore, the assay does not distinguish between the two CDT genes cdtA and
140
cdtB (2). Variability and truncations within the CDT locus have been reported,
141
however, the 5’ end region of cdtA and the 3’ end region of cdtB are usually highly
142
conserved (2).
143 144
A limitation of this study was the use of pure cultures of C. difficile. Both assays have
145
only been validated for stool specimens, however, this should not have reduced the
146
sensitivity or specificity of the assays in the present study. As a common ribotype in
147
veal calves and piglets in some parts of the world, the presence of RT033 should be a
148
concern for the veterinary industry (6, 16). While the role of C. difficile in idiopathic
149
diarrhea in calves is still unclear, C. difficile is the most frequent cause of piglet
150
neonatal diarrhea in the USA (15). RT033 has also been isolated from symptomatic
151
human patients suggesting possible zoonotic transmission (12). However, our study
152
highlights a problem when attempting to undertake surveillance for RT033 C. difficile
153
strains using current molecular methods, making it difficult to assess the true scale
154
and significance of such strains in CDI in both humans and production animals. The
155
Xpert® C. difficile/Epi assay detects CDT gene sequences and hence could play a role
156
in surveillance programs. There are other commercial diagnostic PCR kits, such as the
157
EasyScreen™ C. difficile Reflex Kit and the Verigene® C. difficile, capable of
158
detecting CDT gene sequences but these were not evaluated in this study. Ultimately,
159
the development of a multiplex PCR assay that targets all four C. difficile toxin genes
160
(tcdA, tcdB, cdtA, cdtB) is essential in maximizing C. difficile detection in clinical
161
laboratories.
162
5
163
Acknowledgements
164
We would like to thank Cepheid Australia and Meridian (Bioline Australia) for the
165
provision of materials for the study.
166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195
6
196
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197 198
1. Magill SS, Edwards JR, Bamberg W, Beldavs ZG, Dumyati G, Kainer MA,
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Lynfield R, Maloney M, McAllister-Hollod L, Naddle J, Ray SM, Thompson
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DL, Wilson LE, Fridkin SK. 2014. Multistate point-prevalence survey of health
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care-associated infections. N. Engl. J. Med. 370:1198-1208.
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2. Stare BG, Delmée M, Rupnik M. 2007. Variant forms of the binary toxin CDT
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locus and tcdC gene in Clostridium difficile strains. J. Med. Microbiol. 56: 329-
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335.
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3. Geric B, Johnson S, Gerding DN, Grabnar M, Rupnik M. 2003. Frequency of
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binary toxin genes among Clostridium difficile strains that do not produce large
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clostridial toxins. J. Clin. Microbiol. 41:5227-5232.
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4. Rupnik M. 2008. Heterogeneity of large clostridial toxins: importance of
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Clostridium difficile toxinotypes. FEMS Microbiol. Rev. 32: 541-555.
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5. Rupnik M, Brazier JS, Duerden BI, Grabnar M, Stubbs SL. 2001.
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Comparison of toxinotyping and PCR ribotyping of Clostridium difficile strains
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and description of novel toxinotypes. Microbiology. 147: 439-447.
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6. Knight DR, Thean S, Putsathit P, Fenwick S, Riley TV. 2013. Cross-sectional
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study reveals high prevalence of Clostridium difficile non-PCR ribotype 078
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strains in Australian veal calves at slaughter. Appl. Environ. Microbiol. 79:2630-
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2635.
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7. Janezic S, Zidaric V, Pardon B, Indra A, Kokotovic B, Blanco JL, Seyboldt
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C, Diaz CR, Poxton IR, Perreten V, Drigo I, Jiraskova A, Ocepek M, Weese
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JS, Songer JG, Wilcox MH, Rupnik M. 2014. International Clostridium difficile
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animal strain collection and large diversity of animal associated strains. BMC
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Microbiol. 14:173.
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8. Knight DR, Squire MM, Riley TV. 2014. Clostridium difficile in Australian
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neonatal pigs; nationwide surveillance study shows high prevalence and
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heterogeneity of PCR ribotypes. Appl. Environ. Microbiol.
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doi:10.1128/AEM.03032-14.
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9. Geric B, Carman RJ, Rupnik M, Genheimer CW, Sambol SP, Lyerly DM,
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Gerding DN, Johnson S. 2006. Binary toxin-producing, large clostridial toxin-
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negative Clostridium difficile strains are enterotoxic but do not cause disease in
229
hamsters. J. Infect. Dis. 193:1143-1140. 7
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10. Stewart DB, Berg A, Hegarty J. 2013. Predicting recurrence of C. difficile colitis
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using bacterial virulence factors: binary toxin is the key. J. Gastrointest. Surg. 17:
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118-124.
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11. Barbut F, Decré D, Lalande V, Burghoffer B, Noussair L, Gigandon A,
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Espinasse F, Raskine L, Robert J, Mangeol A, Branger C, Petit JC. 2005.
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Clinical features of Clostridium difficile-associated diarrhea due to binary toxin
236
(actin-specific ADP-ribosyltransferase)-producing strains. J. Med. Microbiol. 54:
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181-185.
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12. Eckert C, Emirian A, Le Monnier a, Cathala L, De Montclos H, Goret J,
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Berger P, Petit A, De Chevigny A, Jean-Pierre H, Nebbad B, Camiade S,
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Meckenstock R, Lalande V, Merchandin H, Barbut F. 2014. Prevalence and
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pathogenicity of binary toxin-positive Clostridium difficile strains that do not
242
produce toxins A and B. New Microbes and New Infections. doi:
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10.1016/j.nmni.2014.10.003.
244
13. Couturier B, Schlaberg R, Konzk C, Nicholes J, Law C, She RC. 2013. tcdA
245
as a diagnostic target in a loop-mediated amplification assay for detecting
246
toxigenic Clostridium difficile. J. Clin. Lab. Anal. 27:171-176.
247
14. Elliot B, Squire MM, Thean S, Chang BJ, Brazier JS, Rupnik M, Riley TV.
248
2011. New types of toxin A-negative, toxin B-positive strains among clinical
249
isolates of Clostridium difficile in Australia. J. Med. Microbiol. 60: 1108-1111.
250
15. Squire MM, Carter GP, Mackin KE, Chakravorty A, Norén T, Elliot B,
251
Lyras D, Riley TV. 2013. Novel molecular type of Clostridium difficile in
252
neonatal pigs, Western Australia. Emerg. Infect. Dis. 19: 790-792.
253
16. Schneeberg A, Neubauer H, Schmoock G, Grossmann E, Seyboldt C. 2013.
254
Presence of Clostridium difficile PCR ribotype clusters related to 033, 078 and
255
045 in diarrhoeic calves in Germany. J. Med. Microbiol. 62: 1190-1198.
256 257 258 259 260 261 262
8
263
FIG 1.
264
A1
265 266
(A)
tcdD
tcdE
tcdB
267 268 269
A2
Δ
tcdC
tcdA A2
(B)
A3
A3 Δ
270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295
9
296
FIG 1. Comparison of a complete PaLoc (A) with the virulence genes (tcdA, tcdB)
297
and accessory genes (tcdD, tcdE, tcdC) and the truncated RT033 PaLoc (B)
298
containing parts of tcdA (fragments A2 and A3) and tcdC. tcdD, positive regulator
299
gene; tcdE, holin encoding protein; tcdC, negative regulator; tcdA, toxin A gene;
300
tcdB, toxin B gene (modified from 4, 5).
301
10
TABLE 1 Comparison of Meridian illumigene C. difficile and Cepheid Xpert® C. difficile/Epi assays for detection of C. difficile RT033 Ribotype (strain)
No. tested
Toxin profile
Toxinotype
Test strains RT033
52
A-B-CDT+
Xla, Xlb
RT017
4
A-B+CDT-
VIII
RT237 RT239 RT280 RT281 Reference strains RT087 (VPI 10463) RT033 (IS 58) RT012 (CD630) RT027 (R 20291) RT010 (CD062)
4 1 1
Xpert result
illumigene result
Human (15) Bovine (21) Porcine (16) Human
NEG
POS
NEG
Toxigenic C. difficile NEG; Presumptive RT027 NEG
NEG
POS
NEG
NEG
POS
POS
POS
NEG
NEG
POS
NEG
Toxigenic C. difficile POS; Presumptive RT027 NEG Toxigenic C. difficile POS; Presumptive RT027 NEG Toxigenic C. difficile NEG; Presumptive RT027 NEG Toxigenic C. difficile POS; Presumptive RT027 NEG Toxigenic C. difficile POS; Presumptive RT027 NEG
XXXI
-
-
+
NA
Human (2) Porcine (2) Human
-
+
+
XXX
Human
POS
POS
NEG
-
+
+
XXX
Human
POS
POS
NEG
A B CDT A B CDT
A B CDT
A+B+CDT-
1
tcdC
+
1
1
Xpert CDT
+
A B CDT
1
tcdB
-
1
1
Source (No. of isolates)
0
Human
POS
NEG
NEG
-
-
+
Xla
Human
NEG
POS
NEG
+
+
-
0
Human
POS
NEG
NEG
+
+
+
III
Human
POS
POS
POS
-
-
-
NA
Human
NEG
NEG
NEG
A B CDT
A B CDT A B CDT A B CDT
POS, Positive; NEG, Negative; NA, not applicable (no pathogenicity locus present)
Toxigenic C. difficile POS; Presumptive RT027 NEG Toxigenic C. difficile NEG; Presumptive RT027 NEG Toxigenic C. difficile POS; Presumptive RT027 NEG Toxigenic C. difficile POS; Presumptive RT027 POS Toxigenic C. difficile NEG; Presumptive RT027 NEG
NEG NEG NEG NEG
POS NEG POS POS NEG
1
Evaluation of the Cepheid Xpert® C. difficile/Epi and Meridian Bioscience
2
illumigene® C. difficile for detecting Clostridium difficile ribotype 033 strains
3 4 5
Grace Androga1, Alan McGovern1, Briony Elliott1, Barbara J. Chang1, Timothy
6
Perkins1, Niki Foster1, Thomas V. Riley1, 2, #
7 8 9 10
1
School of Pathology and Laboratory Medicine, The University of Western Australia,
Perth, Australia.
11 12 13
2
14
Medical Centre, Nedlands 6009 Western Australia.
Department of Microbiology, PathWest Laboratory Medicine Queen Elizabeth II
15 16 17
Running Title: Detecting C. difficile ribotype 033
18 19 20
Corresponding Author: # Professor Thomas V. Riley,
21
Microbiology & Immunology
22
School of Pathology & Laboratory Medicine
23
The University of Western Australia
24
Queen Elizabeth II Medical Centre
25
Nedlands 6009
26
Western Australia
27
E-mail: [email protected]
28 29 30
Keywords: Clostridium difficile, Binary toxin, RT033, CDT, diagnostics, A-B+
31 32 33
Word count: Abstract 55, Text 1357
34 1
35
Abstract
36
Clostridium difficile PCR ribotype (RT) 033 is found in the gastrointestinal tracts of
37
production animals and, occasionally, humans. The illumigene® C. difficile (Meridian
38
Bioscience, Inc.) failed to detect any of 52 C. difficile RT033 isolates while all strains
39
signaled positive for the binary toxin genes but were reported as negative for
40
C. difficile by the Xpert® C. difficile/Epi (Cepheid).
41 42
Text
43
Clostridium difficile is the most common cause of infection associated with healthcare
44
in the United States (1). The diagnosis of C. difficile infection (CDI) remains a vexing
45
problem in hospitals and laboratories. Most current methods of laboratory diagnosis
46
involve detecting tcdA and/or tcdB, two genes located in the C. difficile pathogenicity
47
locus (PaLoc) that encode the two main virulence factors of the bacterium, the large
48
clostridial toxins A (TcdA) and B (TcdB). Detection of a third, binary toxin (CDT),
49
an actin-specific ADP-ribosyltransferase also produced by some strains of C. difficile
50
and not encoded in the PaLoc, has been largely overlooked (2). This has been
51
attributed to the difficulty of detecting CDT by non-molecular means as well as the
52
fact that it is usually produced by strains that also produce toxins A and B (A+B+) (3).
53
An exception is the real-time PCR-based Xpert® C. difficile/Epi system (Cepheid,
54
Sunnyvale CA). This U.S. Food and Drug Administration (FDA) approved assay
55
detects CDT gene sequences but only as a way of identifying putative RT027/NAP1
56
C. difficile strains in fecal specimens. The assay also detects tcdB and the single base
57
pair deletion at nucleotide position 117 of tcdC found in epidemic fluoroquinolone-
58
resistant RT027/NAP1 strains (http://www.cepheid.com/us/cepheid-solutions/clinical-
59
ivd-tests/healthcare-associated-infections/xpert-c-difficile-epi
60
November 2014]). The illumigene® C. difficile assay (Meridian Bioscience, Inc.) is
61
also FDA approved but instead targets a highly conserved region at the 5’ end of tcdA
62
and uses loop-mediated isothermal DNA amplification (LAMP) technology
63
(http://www.meridianbioscience.com/diagnostic-products/c-difficile/illumigene-
64
molecular-diagnostic-system/illumigene-c-difficile.aspx [Accessed 17th November
65
2014]).
[Accessed
17th
66 67
C. difficile RT033 belongs to toxinotype XIa/b and has a truncated PaLoc but an
68
intact CDT locus (4, 5). It is an emerging strain of clinical and veterinary importance 2
69
that has been detected in cattle, veal calves, piglets, horses and soil from various
70
geographical locations world-wide (6-8). Its exact role in human CDI has yet to be
71
determined, however, enterotoxic effects have been observed in rabbit ileal loop tests
72
with A-B-CDT+ strains of C. difficile, indicating that CDT could be a virulence factor
73
in these strains (9). In addition, CDT has been linked to an increase in the prevalence
74
of community acquired-CDI (CA-CDI), a high rate of recurrent CDI, and the need for
75
hospital admission due to re-infection (10, 11). A-B-CDT+ strains of C. difficile such
76
as RT033 are not detected by most current diagnostic methods and hence may be
77
missed by clinical laboratories.
78 79
Meridian Bioscience Inc. claims that the tcdA target sequence of their illumigene®
80
C. difficile assay allows for detection of the PaLoc of toxigenic C. difficile regardless
81
of the expression phenotype and that the assay was designed to detect all known A+B+
82
and A-B+ toxinotypes (http://www.meridianbioscience.com/diagnostic-products/c-
83
difficile/illumigene-molecular-diagnostic-system/illumigene-c-difficile.aspx
84
[Accessed 17th November 2014]). This means that PaLoc-positive, phenotypically
85
A-B-CDT+ C. difficile strains such as RT033 may be detected by the illumigene® C.
86
difficile assay as well as signal CDT-positive by the Xpert® C. difficile/Epi assay. In
87
this report, we test C. difficile RT033 isolates for their ability to signal in the
88
illumigene® C. difficile and the Xpert® C. difficile/Epi assays.
89 90
RT033 isolated from human (n = 15), porcine (n = 16) and bovine (n = 21) sources
91
were used in this study (Table 1). The animal isolates were collected as part of
92
Australian livestock prevalence studies (6, 8). The clinical isolates were mostly from
93
pathology laboratories around Australia. Dr. Frederick Barbut (National Reference
94
Laboratory for C. difficile, France) kindly contributed 6 of the clinical RT033 isolates
95
(12). For comparison, 11 non-RT 033 isolates of various origins and 5 reference
96
strains were also tested (Table 1).
97 98
The identity of C. difficile isolates was confirmed by their distinct colony
99
morphology, yellow-green fluorescence under 360nm UV light, and horse-dung odour
100
when grown on blood agar for 48 h under anaerobic conditions (A35 Anaerobic
101
Workstation, Don Whitley Scientific, Shipley, West Yorkshire). Toxin gene profiling
3
102
and PCR ribotyping of all isolates was performed using previously described methods
103
(6). The illumigene and Xpert testing was performed according to the manufacturers’
104
instructions except that 0.5 McFarland saline suspensions of C. difficile from 48 h
105
blood agar cultures were used instead of the recommended stool samples.
106 107
All 52 human, bovine and porcine C. difficile RT033 isolates failed to signal with the
108
illumigene assay, as did the two A-B- reference strains (RT033 and RT010) and four
109
non-RT033 test ribotypes (the only A-B- ribotype [RT239] and 3 of 4 A-B+ ribotypes).
110
The four RT017 strains were the only A-B+ isolates tested that signaled in the
111
illumigene assay. The Xpert assay detected the CDT analyte in all 52 RT033 strains,
112
all CDT+ non-RT033 test strains and both CDT+ reference strains. Only the tcdB-
113
positive strains were identified as toxigenic C. difficile and only the RT027 reference
114
strain was identified as a presumptive 027/NAP1 (Table 1).
115 116
RT033 characteristically contains a truncated PaLoc region that only retains the
117
putative translocation domain fragment (A2) and parts of the repetitive domain
118
fragment (A3) of the tcdA gene (Fig 1); hence neither large clostridial toxin is
119
produced (4, 5). The illumigene® C. difficile assay was unable to detect the remnant
120
fragments of the tcdA gene in any of the RT033 isolates tested, consistent with a
121
deletion in the 5’ end region (4, 5). The illumigene® C. difficile assay was designed to
122
target a ‘conserved’ region at the 5’ end of tcdA present in all known A+B+ and A-B+
123
toxinotypes (13). This was supported by Couturier et al who reported detection of the
124
tcdA target sequence in A-B+ strains belonging to toxinotypes VIII and X using the
125
illumigene® C. difficile assay (13) and in the present study for RT017, toxinotype VIII
126
strains which have a truncated tcdA gene retaining only the A1 fragment of the gene
127
(4). Other A-B+ ribotypes tested (RT237, RT280 and RT281) that were known to
128
contain a truncated tcdA region were not detected by the illumigene® C. difficile
129
assay (14, 15). Therefore, in addition to PaLoc-negative C. difficile, the target region
130
of the illumigene® C. difficile assay is not conserved in the PaLocs of RT033, RT237,
131
RT280 and RT281 strains.
132 133
The Xpert® C. difficile/Epi assay successfully detected CDT gene sequences in all
134
RT033 isolates as expected. However, it was necessary to view the raw data obtained
4
135
by the assay to identify a CDT-positive result because the assay does not recognize a
136
test as positive for toxigenic C. difficile based on this result alone. The CDT target is
137
only used to give a presumptive positive report of C. difficile RT027/NAP1 strains
138
and this is only when both tcdB and the tcdC Δ117 deletion are detected also.
139
Furthermore, the assay does not distinguish between the two CDT genes cdtA and
140
cdtB (2). Variability and truncations within the CDT locus have been reported,
141
however, the 5’ end region of cdtA and the 3’ end region of cdtB are usually highly
142
conserved (2).
143 144
A limitation of this study was the use of pure cultures of C. difficile. Both assays have
145
only been validated for stool specimens, however, this should not have reduced the
146
sensitivity or specificity of the assays in the present study. As a common ribotype in
147
veal calves and piglets in some parts of the world, the presence of RT033 should be a
148
concern for the veterinary industry (6, 16). While the role of C. difficile in idiopathic
149
diarrhea in calves is still unclear, C. difficile is the most frequent cause of piglet
150
neonatal diarrhea in the USA (15). RT033 has also been isolated from symptomatic
151
human patients suggesting possible zoonotic transmission (12). However, our study
152
highlights a problem when attempting to undertake surveillance for RT033 C. difficile
153
strains using current molecular methods, making it difficult to assess the true scale
154
and significance of such strains in CDI in both humans and production animals. The
155
Xpert® C. difficile/Epi assay detects CDT gene sequences and hence could play a role
156
in surveillance programs. There are other commercial diagnostic PCR kits, such as the
157
EasyScreen™ C. difficile Reflex Kit and the Verigene® C. difficile, capable of
158
detecting CDT gene sequences but these were not evaluated in this study. Ultimately,
159
the development of a multiplex PCR assay that targets all four C. difficile toxin genes
160
(tcdA, tcdB, cdtA, cdtB) is essential in maximizing C. difficile detection in clinical
161
laboratories.
162
5
163
Acknowledgements
164
We would like to thank Cepheid Australia and Meridian (Bioline Australia) for the
165
provision of materials for the study.
166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195
6
196
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255
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256 257 258 259 260 261 262
8
263
FIG 1.
264
A1
265 266
(A)
tcdD
tcdE
tcdB
267 268 269
A2
Δ
tcdC
tcdA A2
(B)
A3
A3 Δ
270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295
9
296
FIG 1. Comparison of a complete PaLoc (A) with the virulence genes (tcdA, tcdB)
297
and accessory genes (tcdD, tcdE, tcdC) and the truncated RT033 PaLoc (B)
298
containing parts of tcdA (fragments A2 and A3) and tcdC. tcdD, positive regulator
299
gene; tcdE, holin encoding protein; tcdC, negative regulator; tcdA, toxin A gene;
300
tcdB, toxin B gene (modified from 4, 5).
301
10
TABLE 1 Comparison of Meridian illumigene C. difficile and Cepheid Xpert® C. difficile/Epi assays for detection of C. difficile RT033 Ribotype (strain)
No. tested
Toxin profile
Toxinotype
Test strains RT033
52
A-B-CDT+
Xla, Xlb
RT017
4
A-B+CDT-
VIII
RT237 RT239 RT280 RT281 Reference strains RT087 (VPI 10463) RT033 (IS 58) RT012 (CD630) RT027 (R 20291) RT010 (CD062)
4 1 1
Xpert result
illumigene result
Human (15) Bovine (21) Porcine (16) Human
NEG
POS
NEG
Toxigenic C. difficile NEG; Presumptive RT027 NEG
NEG
POS
NEG
NEG
POS
POS
POS
NEG
NEG
POS
NEG
Toxigenic C. difficile POS; Presumptive RT027 NEG Toxigenic C. difficile POS; Presumptive RT027 NEG Toxigenic C. difficile NEG; Presumptive RT027 NEG Toxigenic C. difficile POS; Presumptive RT027 NEG Toxigenic C. difficile POS; Presumptive RT027 NEG
XXXI
-
-
+
NA
Human (2) Porcine (2) Human
-
+
+
XXX
Human
POS
POS
NEG
-
+
+
XXX
Human
POS
POS
NEG
A B CDT A B CDT
A B CDT
A+B+CDT-
1
tcdC
+
1
1
Xpert CDT
+
A B CDT
1
tcdB
-
1
1
Source (No. of isolates)
0
Human
POS
NEG
NEG
-
-
+
Xla
Human
NEG
POS
NEG
+
+
-
0
Human
POS
NEG
NEG
+
+
+
III
Human
POS
POS
POS
-
-
-
NA
Human
NEG
NEG
NEG
A B CDT
A B CDT A B CDT A B CDT
POS, Positive; NEG, Negative; NA, not applicable (no pathogenicity locus present)
Toxigenic C. difficile POS; Presumptive RT027 NEG Toxigenic C. difficile NEG; Presumptive RT027 NEG Toxigenic C. difficile POS; Presumptive RT027 NEG Toxigenic C. difficile POS; Presumptive RT027 POS Toxigenic C. difficile NEG; Presumptive RT027 NEG
NEG NEG NEG NEG
POS NEG POS POS NEG
