Inclusive and specific detection of toxinogenic Clostridium difficile by SIBA , a novel isothermal amplification technology
®
Pirjo Matero1, Kaisu Rantakokko-Jalava2, Anni Soikkeli2, Riikka Kärkkäinen1, Jenna Flinck1, Tuula Siljander1, Tiina Tomperi1, Sanna Kukkamaa1, Päivi Rikkinen-Holm1, Kirsi Pousi1, Tuomas Ojalehto1, Sanna Filén1 and Minna Mäki1
Orion Corporation, Orion Diagnostica Oy, P.O. Box 83, 02101 Espoo, Finland
2
Clinical Microbiology Laboratory, Turku University Central Hospital, Turku, Finland
1
Introduction and purpose
Clostridium difficile is a Gram positive spore-forming anaerobic bacillus that is the leading cause of healthcare-associated
diarrhoea. C. difficile has several toxinotypes that represent variability in the PaLoc gene region encoding two main virulence
factors, toxins TcdA and TcdBa. Currently, 38 toxinotypes (0-XXXIII) that differ in their toxin production pattern have been
characterised.
Here we present a rapid assay for detection of toxinogenic C. difficile. The assay, which is being developed for the Orion
GenRead® System, is based on a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification
(SIBA®), and detects a region in the C. difficile tcdB gene. The inclusivity of the assay is demonstrated with 37 different
toxinotypes, and the accuracy of the assay is shown with 125 patient samples and 10 Quality Control for Molecular Diagnostics
(QCMD) 2013 C. difficile samples.
Methods
The SIBA-based C. difficile assay consists of a freeze-dried reaction mixture, containing an internal amplification control (IAC)
and specific fluorophore and quencher pairs on separate detection probes (one for the IAC and one for the C. difficile target).
A rapid lysis- and filter-based procedure aimed at purifying C. difficile from faeces was used as the sample preparation method.
The analysis was performed on the Orion GenRead Instrument at 40 / 41°C.
37 C. difficile toxinotypes were collected at the Institute of Public Health Maribor, Slovenia and the extracted DNA was
used for the SIBA-based C. difficile assay. The obtained results were compared to those of RFLP-PCR-based toxinotyping and
toxigenic culture.
125 retrospective faecal samples of patients suspected of having C. difficile infection (CDI) were collected at Turku University
Central Hospital, Finland and analysed with the SIBA-based C. difficile assay. The results were compared to those of freshly
analysed Loop mediated isothermal amplification (LAMP, illumigene® C. difficile, Meridian Bioscience), PCR (GenomEra™ C.
difficile, Abacus Diagnostica), TcdA & TcdB toxins (VIDAS® C. difficile Toxin A & B, bioMérieux) or Glutamate dehydrogenase
(GDH, VIDAS® C. difficile GDH, bioMérieux) based methods.
Also, 10 QCMD 2013 C. difficile proficiency panel samples were analysed.
Results
All of the 35 TcdA and TcdB toxin-producing C. difficile toxinotypes were reported positive by the SIBA-based assay, while
the two out of the 37 analysed toxinotypes that were negative for TcdA and TcdB production were reported negative. 27
toxinotypes were characterised positive for both TcdA and TcdB toxin production and 6 toxinotypes only for the TcdB toxin
(Table 1).
Comparison of the SIBA-based assay to the LAMP-based illumigene® C. difficile assay showed an overall percent agreement
of 96.7%, whereas when compared to the PCR-based GenomEra™ C. difficile assay the agreement was 96.8%. The agreement
was 93.6% and 92.8% when compared to the TcdA & TcdB toxin and GDH detection based VIDAS® C. difficile assays, respectively.
Invalid results of the comparative methods were excluded from the calculations (Table 2).
Analysis of the QCMD 2013 C. difficile proficiency panel illustrated good concordance with the background information
of the sample. All replicates of the core samples were correctly detected (Table 3).
Conclusions
Rapid and accurate C. difficile detection may contribute to the establishment of faster, evidence-based patient management.
The SIBA-based C. difficile assay with an easy-to-use workflow, including freeze-dried reagents and a simple sample purification
method, has great potential as a powerful diagnostic tool, providing inclusive and specific detection of C. difficile.
References
Maja Rupnik. Heterogeneity of large clostridial
toxins: importance of Clostridium difficile
toxinotypes. FEMS Microbiol Rev 32 (2008):
541–555
a
Table 2. Method comparison results
Table 1. Toxinotype testing results
Toxinotype
Type strain
Toxin
production
Reaction in SIBA
C. difficile assay
0
VPI 10463
A+ B+ CDT-
+
I
EX 623
A+ B+ CDT-
+
II
AC 008
A+ B+ CDT-
+
IIIa
SE 844
A+ B+ CDT+
+
IIIb
R 12087
A+ B+ CDT+
+
IIIc
CH6230
A+ B+ CDT+
+
IV
55767
A+ B+ CDT+
+
V
SE 881
A+ B+ CDT+
+
VI
51377
A+
B+
CDT+
+
VII
57267
A+ B+ CDT+
+
VIII
1470
A- B+ CDT-
+
IX
51680
A+ B+ CDT+
+
X
8864
A- B+ CDT+
+
XIa
IS 58
A- B- CDT+
-
XIb
R 11402
A- B- CDT+
-
A+
CDT-
+
B+
XII
IS 25
XIII
R 9367
A+ B+ CDT-
+
XIV
R 10870
A+ B+ CDT+
+
XV
R 9385
A+ B+ CDT+
+
XVI
SUC36
A- B+ CDT+
+
XVII
J9965
A- B+ CDT+
+
XVIII
K095
A+ B+ CDT-
+
XIX
TR13
A+
B+
CDT-
+
XX
TR14
A+ B+ CDT-
+
XXI
CH6223
A+ B+ CDT-
+
A+
CDT+
+
B+
XXII
CD07-468
XXIII
8785
A+ B+ CDT+
+
XXIV
597B
A+ B+ CDT+
+
XXV
7325
A+
CDT+
+
XXVI
7459
A+ B+ CDT-
+
XXVII
KK2443/2006
A+ B+ CDT-
+
XXVIII
CD08-070
A+ B+ CDT+
+
XXIX
CD07-140
A+ B+ CDT-
+
A-
B+
B+
CDT+
+
SIBA C. difficile assay*
illumigene® C. difficile
GenomEra™ C. difficile
VIDAS® C. difficile Toxin A & B
VIDAS® C. difficile GDH
Positive
Negative
Total
Positive
28
4
32
Negative
0
90
90
Invalid
2
1
3
Positive
Negative
Total
Positive
30
4
34
Negative
0
91
91
Invalid
-
-
Positive
Negative
Total
Positive
24
2
26
Negative
6
93
99
Invalid
-
-
Positive
Negative
Total
Positive
30
9
39
Negative
0
86
86
Invalid
-
-
n = 125
n = 125
n = 125
n = 125
* in development phase
Table 3. Results of the QCMD 2013 Clostridium difficile proficiency panel
% correct result of tested samples
Sample ID
Sample content
Sample
conc.,
CFU/ml
CD13-02
C. difficile 017
8.0x106
CD13-05
C. difficile 027
4.6x106
CD13-06
C. difficile 017
8.0x105
Sample
status
Frequently
detected
Frequently
detected
Frequently
detected
Frequently
detected
Frequently
detected
Sample type
SIBA
C. difficile
Real time
PCR,
commercial*
Real time
PCR,
in-house*
LAMP*
Core
100
97.6
100
100
Core
100
100.0
100
100
Core
100
96.3
100
100
Core
100
93.9
100
100
Core
100
93.9
100
100
XXX
ES 130
XXXI
WA 151
A- B+ CDT+
+
CD13-01
C. difficile 027
4.6x105
XXXII
173070
nd
+
CD13-09
C. difficile 027
4.6x105
XXXIII
2402
nd
+
CD13-03
Cd negative
–
Negative
Core
100
97.6
90.9
100
CD13-10
C. difficile 017
8.0x104
Detected
Educational
100
78.0
100
75
CD13-04
C. difficile 027
4.6x104
Detected
Educational
25
75.6
100
100
CD13-07
C. difficile 027
4.6x103
Detected
Educational
0
61.0
100
25
CD13-08
C. sordellii
2.1x105
Negative
Educational
100
98.8
100
100
Toxinotype type strain DNA samples: Professor Maja Rupnik, Institute of Public Health
Maribor, Centre for Microbiology, Maribor, Slovenia.
nd = not determined, + = positive reaction, - = negative reaction.
* Results provided by QCMD
SIBA® and Orion GenRead® are registered trademarks of Orion Diagnostica Oy.
24th European Congress of Clinical Microbiology and Infectious Diseases, Barcelona, Spain, May 10 –13, 2014
987-02GB, 05/2015