Diagnosis of Clostridium difficile infection by toxigenic culture and PCR assay

  • Elnaze Zare Mirzaei Infectious Diseases & Tropical Medicine Research Center, Babol University of Medical Sciences, Babol, Iran and Department of Microbiology, Faculty of Medicine, Babol University of Medical Sciences, Babol, Iran
  • Mahdi Rajabnia Department of Microbiology, Faculty of Medicine, Babol University of Medical Sciences, Babol, Iran
  • Farzin Sadeghi Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran
  • Elaheh Ferdosi-Shahandashti Department of Microbiology, Faculty of Medicine, Babol University of Medical Sciences, Babol, Iran
  • Mahmoud Sadeghi-Haddad-Zavareh Infectious Diseases & Tropical Medicine Research Center, Babol University of Medical Sciences, Babol, Iran
  • Soraya Khafri Infertility and Reproductive Health Research Center, Babol University of Medical Sciences, Babol, Iran
  • Abolfazl Davoodabadi Infectious Diseases & Tropical Medicine Research Center, Babol University of Medical Sciences, Babol, Iran and Department of Microbiology, Faculty of Medicine, Babol University of Medical Sciences, Babol, Iran
Keywords: Antibiotic associated diarrhea, Clostridium difficile, PCR, Toxigenic culture


Background and Objectives: Clostridium difficile is responsible for 15-25% of nosocomial antibiotic associated diarrhea (AAD) cases and all cases of pseudomembranous colitis. C. difficile has two major virulence factors, toxin A (enterotoxin) and toxin B (cytotoxin). The aim of this study was to determine the frequency of C. difficile strains in patients with diarrhea in Babol' hospitals with toxigenic culture and PCR assay. Materials and Methods: One hundred stool specimens were taken from diarrheal patients in hospitals of the city of Babol. All patients had a history of antibiotic use. The samples were cultured on CCFA medium. In the next stage, toxigenic culture was performed for isolated C. difficile strains. Then, PCR assay was used to identify gdh, tcdA and tcdB genes among isolated C. difficile strains. Results: From the 100 stool samples, eight (8%) samples were positive in C. difficile culture. In toxigenic culture, two (2%) of these strains had cytopathic effects on Vero cells. All eight strains had the gdh gene. This gene is specific for C. difficile. Two strains that had cytopathic effects on toxigenic culture were positive for toxin genes. Conclusion: The frequency of toxigenic strains in different parts of the world is variable, and needs to be continually investigated. In the present study, the PCR method had a good correlation with toxigenic culture. Thus, it can replace the laborious and costly cell culture method.


De Boer E, Zwartkruis-Nahuis A, Heuvelink AE, Harmanus C, Kuijper EJ. Prevalence of Clostridium difficile in retailed meat in the Netherlands. Int J Food Microbiol 2011;144(3):561-564.

Dubberke ER, Wertheimer AI. Review of current literature on the economic burden of Clostridium difficile infection. ‎Infect Control Hosp Epide 2009;30(1):57-66.

Landelle C, Verachten M, Legrand P, Girou E, Barbut F, Buisson CB. Contamination of healthcare workers' hands with Clostridium difficile spores after caring for patients with C. difficile infection. ‎Infect Control Hosp Epide 2014;35(1):10-15.

Bowman KA, Broussard EK, Surawicz CM. Fecal microbiota transplantation: current clinical efficacy and future prospects. Clin Exp Gastroenterol 2015;8:285.

Davies KA, Longshaw CM, Davis GL, Bouza E, Barbut F, Barna Z, et al. Underdiagnosis of Clostridium difficile across Europe: the European, multicentre, prospective, biannual, point-prevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID). Lancet Infect Dis 2014;14(12):1208-1219.

Deneve C, Janoir C, Poilane I, Fantinato C, Collignon A. New trends in Clostridium difficile virulence and pathogenesis. International journal of antimicrobial agents 2009;33:S24-S28.

Bauer MP, van Dissel JT. Alternative strategies for Clostridium difficile infection. Int J Antimicrob Agents 2009;33:S51-S56.

Russello G, Russo A, Sisto F, Scaltrito MM, Farina C. Laboratory diagnosis of Clostridium difficile associated diarrhoea and molecular characterization of clinical isolates. New Microbiologica 2012;35(3):307-316.

Hawkins MR, Drew RH, Lewis SS, Anderson DJ, Sexton DJ, Moehring RW, editors. 219Characteristics of Antimicrobial Stewardship (AS) Activities in Community Hospitals Upon Enrollment in the Duke Antimicrobial Stewardship Outreach Network (DASON). Open Forum Infect Dis; 2014: Oxford University Press.

Balamurugan R, Balaji V, Ramakrishna BS. Estimation of faecal carriage of Clostridium difficile in patients with ulcerative colitis using real time polymerase chain reaction. Indian J Med Res 2008;127(5):472.

Lemee L, Dhalluin A, Testelin S, Mattrat M-A, Maillard K, Lemeland J-F, et al. Multiplex PCR targeting tpi (triose phosphate isomerase), tcdA (Toxin A), and tcdB (Toxin B) genes for toxigenic culture of Clostridium difficile. J Clin Microbiol 2004;42(12):5710-5714.

Liu Y-L, Lu X-H, Tang YJ, Lam K, Silva J, Leung JW. Polymerase chain reaction for identification and typing of Clostridium difficile isolated from Chinese patients. Int J Infect Dis 1997;2(2):85-87.

Pancholi P, Kelly C, Raczkowski M, Balada-Llasat J. Detection of toxigenic Clostridium difficile: comparison of the cell culture neutralization, Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays. Journal of clinical microbiology 2012;50(4):1331-1335.

Kato H, Kato N, Watanabe K, Iwai N, Nakamura H, Yamamoto T, et al. Identification of toxin A-negative, toxin B-positive Clostridium difficile by PCR. J Clin Microbiol 1998;36(8):2178-2182.

Zheng L, Keller S, Lyerly D, Carman R, Genheimer C, Gleaves C, et al. Multicenter evaluation of a new screening test that detects Clostridium difficile in fecal specimens. J Clin Microbiol 2004;42(8):3837-3840.

Persson S, Torpdahl M, Olsen K. New multiplex PCR method for the detection of Clostridium difficile toxin A (tcdA) and toxin B (tcdB) and the binary toxin (cdtA/cdtB) genes applied to a Danish strain collection. Clinical microbiology and infection 2008;14(11):1057-1064.

Khanna S, Pardi DS. The growing incidence and severity of Clostridium difficile infection in inpatient and outpatient settings. Expert Rev Gastroenterol Hepatol 2010;4(4):409-416.

Worth L, Spelman T, Bull A, Brett J, Richards M. Epidemiology of Clostridium difficile infections in Australia: enhanced surveillance to evaluate time trends and severity of illness in Victoria, 2010–2014. J Hosp Infect 2016;93(3):280-285.

Davis BM, Yin J, Blomberg D, Fung IC-H. Impact of a prevention bundle on Clostridium difficile infection rates in a hospital in the Southeastern United States. Am J Infect Control 2016;44(12):1729-1731.

Geric B, Rupnik M, Gerding DN, Grabnar M, Johnson S. Distribution of Clostridium difficile variant toxinotypes and strains with binary toxin genes among clinical isolates in an American hospital. J Med Microbiol 2004;53(9):887-894.

Bourgault A-M, Lamothe F, Loo VG, Poirier L, group C-Cs. In vitro susceptibility of Clostridium difficile clinical isolates from a multi-institutional outbreak in Southern Quebec, Canada. Antimicrob Agents Chemother 2006;50(10):3473-3475.

Wozniak G, Trontelj P, Rupnik M. Genomic relatedness of Clostridium difficile strains from different toxinotypes and serogroups. Anaerobe 2000;6(4):261-267.

Sambol SP, Merrigan MM, Lyerly D, Gerding DN, Johnson S. Toxin gene analysis of a variant strain ofClostridium difficile that causes human clinical disease. Infect Immun 2000;68(10):5480-5487.

Rupnik M, Grabnar M, Geric B. Binary toxin producing Clostridium difficile strains. Anaerobe 2003;9(6):289-294.

van den Berg RJ, Claas EC, Oyib DH, Klaassen CH, Dijkshoorn L, Brazier JS, et al. Characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates from outbreaks in different countries by amplified fragment length polymorphism and PCR ribotyping. J Clin Microbiol 2004;42(3):1035-1041.

Lotfian S, Douraghi M, Aliramezani A, Ghourchian S, Sarrafnejad A, Zeraati H. Detection of Clostridium difficile in Fecal Specimens: a Comparative Evaluation of Nucleic Acid Amplification Test and Toxigenic Culture. Clinical laboratory 2016;62(10):1887.

Sadeghifard N, Salari M, Ghassemi M, Shirazi M, Feizabadi M, Kazemi B, et al. Prevalence of Clostridium difficile-associated diarrhea in hospitalized patients with nosocomial diarrhea. Iran J Public Health 2005;34(4):67-72.

azizi o, aslani m, azimirad m, albouye l, mousavi f, m z. frequency of c.difficile toxigenic strains in tehran with PCR method. journal of kerman medical science university 2013;20(2).

Goudarzi M, Goudarzi H, Albouyeh M, Azimi Rad M, Zali MR, Aslan MM. Molecular typing of Clostridium difficile isolated from hospitalized patients by PCR ribotyping. Research in Medicine 2012;36(2):68-75.

Zaidi M, de León SP, Ortiz RM, de León SP, Calva JJ, Ruiz-Palacios G, et al. Hospital-acquired diarrhea in adults: a prospective case-controlled study in Mexico. Infection Control & Hospital Epidemiology 1991;12(6):349-355.

Gorenek L, Dizer U, Besirbellioglu B, Eyigun C, Hacibektasoglu A, Van Thiel D. The diagnosis and treatment of Clostridium difficile in antibiotic-associated diarrhea. Hepatogastroenterology 1999;46(25):343-348.

Garcia LB, Uzeda Md. Occurrence of clostridium difficile in fecal samples of children in Rio de Janeiro, RJ. Revista do Instituto de Medicina Tropical de São Paulo 1988;30(6):419-423.

Sachu A, Dinesh K, Siyad I, Kumar A, Vasudevan A, Karim S. A prospective cross sectional study of detection of Clostridium difficile toxin in patients with antibiotic associated diarrhoea. Iran J Microbiol 2018;10(1):1-6.

Segar L, Easow JM, Srirangaraj S, Hanifah M, Joseph NM, Seetha K. Prevalence of Clostridium difficile infection among the patients attending a tertiary care teaching hospital. Indian J Pathol Microbiol 2017;60(2):221.

Collins DA, Gasem MH, Habibie TH, Arinton I, Hendriyanto P, Hartana AP, et al. Prevalence and molecular epidemiology of Clostridium difficile infection in Indonesia. New Microbes New Infect 2017;18:34-37.

Arvand M, Ruscher C, Bettge-Weller G, Goltz M, Pfeifer Y. Prevalence and risk factors for colonization by Clostridium difficile and extended-spectrum β-lactamase-producing Enterobacteriaceae in rehabilitation clinics in Germany. J Hosp Infect 2017.

Kuijper E, Coignard B, Tüll P. Emergence of Clostridium difficile‐associated disease in North America and Europe. Clin Microbiol Infect 2006;12(s6):2-18.

Goudarzi M, Seyedjavadi SS, Goudarzi H, Mehdizadeh Aghdam E, Nazeri S. Clostridium difficile infection: epidemiology, pathogenesis, risk factors, and therapeutic options. Scientifica 2014;2014.

How to Cite
Zare Mirzaei E, Rajabnia M, Sadeghi F, Ferdosi-Shahandashti E, Sadeghi-Haddad-Zavareh M, Khafri S, Davoodabadi A. Diagnosis of Clostridium difficile infection by toxigenic culture and PCR assay. Iran J Microbiol. 10(5):287-293.
Original Article(s)