Kridsada Sirisabhabhorn1,2, Wanna Chaijaroenkul2, and Kesara Na-Bangchang2* Abstract: Toxin A (TcdA) and B (TcdB) are virulence factors of Clostridium difficilethat cause diarrhea and pseudomembranous colitis. The global prevalence of C. difficileinfection (CDI)has been increasing due to resistance of the bacteria to the standard treatment with vancomycin. The aim of the study was to investigate the prevalence rates ofC. difficileproducing TcdAand TcdBin fecal specimens collected in 2014 (n=646) and 2015 (n=649) from CDI patients at ThammasatChalermprakietHospital, Pathumtani, Thailand. TcdAand TcdBin all samples were detected using immunochromatographicdipstick test. The number (%) of bacteria producing TcdA, TcdB, and a mixture of TcdAand TcdBfound in 2014 were 11 (1.7%), 14 (2.2%), and 22 (3.41%) samples, respectively. The corresponding numbers (%) for samples collected in 2015 were 5 (0.7%), 27 (4.2%), and 17 (2.6%), respectively. The prevalence of fecal specimens with TcdBpositive was significantly increased in 2015 compared with 2014. This observation may be a warning sign for the progress of C. difficileresistant strains. Close monitoring of their prevalence rates in the hospital including sensitivity to chemotherapeutics is essential. Key words: C. difficile, TcdA, TcdB, prepvalence, resistance

1. Toxin A (TcdA) and B (TcdB) are virulence factors of Clostridium difficile that cause diarrhea and pseudomembranous colitis. The
global prevalence of C. difficile infection (CDI) has been increasing due to resistance of the bacteria to the standard treatment with
vancomycin. The aim of the study was to investigate the prevalence rates of C. difficile producing TcdA and TcdB in fecal specimens
collected in 2014 (n=646) and 2015 (n=649) from CDI patients at Thammasat Chalermprakiet Hospital, Pathumtani, Thailand. TcdA and
TcdB in all samples were detected using immunochromatographic dipstick test. The number (%) of bacteria producing TcdA, TcdB, and a
mixture of TcdA and TcdB found in 2014 were 11 (1.7%), 14 (2.2%), and 22 (3.41%) samples, respectively. The corresponding numbers (%)
for samples collected in 2015 were 5 (0.7%), 27 (4.2%), and 17 (2.6%), respectively. The prevalence of fecal specimens with TcdB positive
was significantly increased in 2015 compared with 2014. This observation may be a warning sign for the progress of C. difficile resistant
strains. Close monitoring of their prevalence rates in the hospital including sensitivity to chemotherapeutics is essential.
Increase in the prevalence of Clostridium difficile producing toxin B
in fecal specimens collected from patients
at Thammasat Chalermprakiet Hospital, Pathumtani, Thailand
Kridsada Sirisabhabhorn1,2, Wanna Chaijaroenkul2, and Kesara Na-Bangchang2*
1Medical Technology Laboratory, Thammasat University Hospital
2Center of Excellence in Pharmacology and Molecular Biology of Malaria and Cholangiocarcinoma, Chulabhorn International College of
Medicine, Thammasat University, Pathumtani, Thailand
* Corresponding Author
Abstract
Introduction
Clostridium difficile is gram positive anaerobic bacteria that produces two types of toxins, i.e., toxin A (TdcA) and toxin B (TdcB).
Both cause gastrointestinal irritation [1]. The virulence of TcdB is about 100 to 1,000 times of that of TcdA [2]. Immunochromatography is
currently used as a routine laboratory test for differential identification of these two toxins. Patients who are fecal positive for either
bacterial toxin may have serious complications that result in antimicrobial resistance, prolongation of hospital admission, as well as death.
Reporting to Hospital Pharmacy Department is required in case of positive result of either TcdA or TcdB. The incidence of C. difficile
resistance has been increasing dramatically throughout the world [3]. Establishment of surviellance database and continuous monitoring of
drug resistance is essential.
Key words: C. difficile, TcdA, TcdB, prepvalence, resistance
Objective
To investigate the prevalence rates of C. difficile producing TcdA and TcdB in fecal specimens in CDI patients who attended the
Thammasat Charlerm Prakiet Hospital (Thammasat University Hospital), Pathumtani, Thailand in 2014 and 2015.
Results
The current study provides preliminary data of the prevalence rates of C. difficile
producing toxin A (TcdA) and B (TcdB) in CDI patients at Thammasat Chalermprakiet
Hospital in 2014 and 2015. Results suggest a significant increase in the prevalence of C.
difficile producing TcdB of about 2-fold in 2015 compared with 2014. On the other hand, the
prevalence of either C. difficile producing TcdA or a mixture of both TcdA and TcdB are more
or less stable. Additionally, five samples collected from each year were found to be recurrent
cases. All were TcdB producing strain, while some were also TcdA producing strain. This
observation may be a warning sign for the outbreak of virulent strains of C. difficile and the
consequence of antimicrobial drug resistance. A recent study demonstrated that the gene
from toxigenic producing C. difficile strain can be transferred to non-toxigenic strain, and thus
non-toxigenic strain is converted to toxigenic strain [4]. It is possible that C. difficile
producing TcdB strain can also induce C. difficile producing TcdA strain. Further study in a
larger sample size is required to confirm this observation.
Discussion & Conclusion
References
Acknowledgements
Positive cases
of
C. diffile
toxins
Year p-value
2014
(n = 646)
2015
(n = 649)
TcdA TcdB TcdA + TcdB TcdA TcdB TcdA + TcdB
n 11 14 22 5 27 17
<0.001*%
(n /N)
1.7
(11/646)
2.2*
(14/646)
3.41
(22/646)
0.7
(5/649)
4.2*
(27/649)
2.6
(17/649)
Note: n = number of positive case each types in that year, N = All cases in that year
* = statistically significant difference (χ2- test)
Immunochromatography for typing of
C. difficile toxin
Methods
C. difficile Toxin Typing
Patient’s fecal specimen in a sterile container
Mixed with buffer
Centrifuged at 7,000 rpm ., 2 min.
Transferred supernatant (4 drops) to well kit
Interpretation
Data Analysis
Prevalence analysis of prevalence of
TcdA & TcdB in samples
2014
Chi-square test (SPSS® ), α = 0.05
2015
Results interpretation
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Rev. 2008; 32(3): 541–555.
2. Just I., Gerhard R. Large Clostridial cytotoxins. Rev. Physiol. Biochem. Pharmacol. 2004; 152: 23–47.
3. Kuijper E.J., Coignard B., Tüll P., ESCMID Study Group for Clostridium difficile EU Member States; European Centre
for Disease Prevention and Control. Emergence of Clostridium difficile-associated disease in North America and
Europe. Clin Microbiol Infect. 2006; 12(6): 2-18.
4. Brouwer, M. S. M., Roberts A.P., Hussain H., Williams R.J., Allan E., Mullany P. et al. Horizontal gene transfer converts
nontoxigenic Clostridium difficile strains into toxin producers. Nat. Commun. 2013; 4:2601: 1-6.
We would like thank Mr. Palakorn Puttaruk, Director of Medical Technology Laboratory, Thammasat University Hospital and Director of Thammasat
University Hospital for the study specimens.
TcdB positive band
TcdA positive band
Control band