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 Bin 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

IntroductionClostridium 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

ObjectiveTo 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. difficileproducing toxin A (TcdA) and B (TcdB) in CDI patients at Thammasat ChalermprakietHospital 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 thusnon-toxigenic strain is converted to toxigenic strain [4]. It is possible that C. difficileproducing 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 casesof

C. diffiletoxins

Year p-value2014

(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

1. Rupnik M. Heterogeneity of large clostridial toxins: importance of Clostridium difficile toxinotypes. FEMS Microbiol 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 convertsnontoxigenic 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 bandTcdA positive band

Control band