PCR Detection of Clostridium difficile Triose Phosphate Isomerase (tpi), Toxin A (tcdA), Toxin B (tcdB), Binary Toxin (cdtA, cdtB), and tcdC Genes in Vhembe District, South Africa

Amidou Samie AIDS Virus Research Laboratory and Laboratory for Molecular Microbiology, Department of Microbiology, University of Venda, Thohoyandou, South Africa; College of Agriculture and Environmental Sciences, School of Agriculture and Life Sciences, University of South Africa, Pretoria, South Africa; Centre for Global Health, Division of Infectious Diseases and International Health University of Virginia, Charlottesville, Virginia

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Chikwelu L. Obi AIDS Virus Research Laboratory and Laboratory for Molecular Microbiology, Department of Microbiology, University of Venda, Thohoyandou, South Africa; College of Agriculture and Environmental Sciences, School of Agriculture and Life Sciences, University of South Africa, Pretoria, South Africa; Centre for Global Health, Division of Infectious Diseases and International Health University of Virginia, Charlottesville, Virginia

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Jason Franasiak AIDS Virus Research Laboratory and Laboratory for Molecular Microbiology, Department of Microbiology, University of Venda, Thohoyandou, South Africa; College of Agriculture and Environmental Sciences, School of Agriculture and Life Sciences, University of South Africa, Pretoria, South Africa; Centre for Global Health, Division of Infectious Diseases and International Health University of Virginia, Charlottesville, Virginia

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Laurie Archbald-Pannone AIDS Virus Research Laboratory and Laboratory for Molecular Microbiology, Department of Microbiology, University of Venda, Thohoyandou, South Africa; College of Agriculture and Environmental Sciences, School of Agriculture and Life Sciences, University of South Africa, Pretoria, South Africa; Centre for Global Health, Division of Infectious Diseases and International Health University of Virginia, Charlottesville, Virginia

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Pascal O. Bessong AIDS Virus Research Laboratory and Laboratory for Molecular Microbiology, Department of Microbiology, University of Venda, Thohoyandou, South Africa; College of Agriculture and Environmental Sciences, School of Agriculture and Life Sciences, University of South Africa, Pretoria, South Africa; Centre for Global Health, Division of Infectious Diseases and International Health University of Virginia, Charlottesville, Virginia

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Cirle Alcantara-Warren AIDS Virus Research Laboratory and Laboratory for Molecular Microbiology, Department of Microbiology, University of Venda, Thohoyandou, South Africa; College of Agriculture and Environmental Sciences, School of Agriculture and Life Sciences, University of South Africa, Pretoria, South Africa; Centre for Global Health, Division of Infectious Diseases and International Health University of Virginia, Charlottesville, Virginia

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Richard L. Guerrant AIDS Virus Research Laboratory and Laboratory for Molecular Microbiology, Department of Microbiology, University of Venda, Thohoyandou, South Africa; College of Agriculture and Environmental Sciences, School of Agriculture and Life Sciences, University of South Africa, Pretoria, South Africa; Centre for Global Health, Division of Infectious Diseases and International Health University of Virginia, Charlottesville, Virginia

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Specific polymerase chain reaction (PCR) protocols were used to determine the prevalence of toxigenic Clostridium difficile in Vhembe, South Africa. Of 322 stool samples collected, toxigenic C. difficile was found in 23 (7.1%) cases and was significantly associated with diarrhea 20 (11.4%) compared with 3 (2%) in non-diarrheal samples (χ2 = 426, P = 0.001), intestinal inflammation in 18 (12.1%) compared with 5 (2.9%) in lactoferrin-negative samples (χ2 = 10.194, P = 0.001), and occult blood in 19 (16%) compared with 4 (2%) in occult blood–negative samples (χ2 = 22.157, P < 0.001). Toxigenic C. difficile was more common among individuals > 50 years of age (20%), followed by those between 30 and 39 years of age (19%) and was not associated with HIV infections (χ2 = 0.289, P = 0.591). Co-infection with other pathogens was common. Multivariate analysis indicated that toxigenic C. difficile was associated with E. bieneusi (P = 0.028), C. parvum (P = 0.007), and Enteroaggregative Escherichia coli (EAEC) (P = 0.007) in diarrheal samples. This study confirms the usefulness of PCR methodologies in the detection of toxigenic C. difficile and suggests that C. difficile is responsible for a small, but underappreciated, proportion of diarrheal cases in the region, and further study is warranted in this area.

Author Notes

Reprint requests: A. Samie, Department of Microbiology, University of Venda, Private Bag X5050, Thohoyandou 0950, Limpopo Province, South Africa, E-mail: samieamidou@yahoo.com.
  • 1

    Cleary RK, 1998. Clostridium difficile-associated diarrhea and colitis: clinical manifestations, diagnosis, and treatment. Dis Colon Rectum 41 :1435–1449.

  • 2

    Wilcox MH, 1996. Cleaning up Clostridium difficile infection. Lancet 348 :767–768.

  • 3

    Surawicz CM, McFarland LV, 1999. Pseudomembranous colitis: causes and cures. Digestion 60 :91–100.

  • 4

    Karlström O, Fryklund B, Tullus K, Burman LG, 1998. A prospective nationwide study of Clostridium difficile-associated diarrhea in Sweden. The Swedish C. difficile Study Group. Clin Infect Dis 26 :141–145.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 5

    Kuijper EJ, Coignard B, Tull P, the ESCMID Study Group for Clostridium difficile (ESGCD)*, EU Member States and the European Centre for Disease Prevention and Control (ECDC), 2006. Emergence of Clostridium difficile-associated disease in North America and Europe. Clin Microbiol Infect 6 :2–18.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 6

    Reichardt C, Chaberny IF, Kola A, Mattner F, Vonberg RP, Gastmeier P, 2007. Dramatic increase of diarrhea associated with Clostridium difficile in Germany: has the new strain PCR-ribotype 027 reached us? Dtsch Med Wochenschr 132 :223–228.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 7

    Lyerly DM, Lockwood DE, Richardson SH, Wilkins TD, 1982. Biological activities of toxins A and B of Clostridium difficile. Infect Immun 35 :1147–1150.

  • 8

    Borriello SP, 1998. Pathogenesis of Clostridium difficile infection. J Antimicrob Chemother 41 :13–19.

  • 9

    Riegler M, Sedivy R, Pothoulakis C, Hamilton G, Zacherl J, Bischof G, Cosentini E, Feil W, Schiessel R, LaMont JT, Wenzl E, 1995. Clostridium difficile toxin B is more potent than toxin A in damaging human colonic epithelium in vitro. J Clin Invest 95 :2004–2011.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 10

    Barbut F, Decre D, Lalande V, Burghoffer B, Noussair L, Gigandon A, Espinasse F, Raskine L, Robert J, Mangeol A, Branger C, Petit JC, 2005. Clinical features of Clostridium difficile-associated diarrhea due to binary toxin (actin-specific ADP-ribosyltransferase)-producing strains. J Med Microbiol 54 :181–185.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 11

    Johnson S, Kent SA, O’Leary KJ, Merrigan MM, Sambol SP, Peterson LR, Gerding DN, 2001. Fatal pseudomembranous colitis associated with a variant Clostridium difficile strain not detected by toxin A immunoassay. Ann Intern Med 135 :434–438.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 12

    Moncrief JS, Zheng L, Neville LM, Lyerly DM, 2000. Genetic characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates by PCR. J Clin Microbiol 38 :3072–3075.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 13

    Spigaglia P, Mastrantonio P, 2002. Molecular analysis of the pathogenicity locus and polymorphism in the putative negative regulator of toxin production (TcdC) among Clostridium difficile clinical isolates. J Clin Microbiol 40 :3470–3475.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 14

    Geric B, Carman RJ, Rupnik M, Genheimer CW, Sambol SP, Lyerly DM, Gerding DN, Johnson S, 2006. Binary toxin-producing, large clostridial toxin-negative Clostridium difficile strains are enterotoxic but do not cause disease in hamsters. J Infect Dis 193 :1143–1150.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 15

    Poilane I, Karjalainen T, Barc MC, Bourlioux P, Collignon A, 1998. Protease activity of Clostridium difficile strains. Can J Microbiol 44 :157–161.

  • 16

    Eveillard M, Fourel V, Barc MC, Kerneis S, Coconnier MH, Karjalainen T, Bourlioux P, Servin AL, 1993. Identification and characterization of adhesive factors of Clostridium difficile involved in adhesion to human colonic enterocyte-like Caco-2 and mucus-secreting HT 29 cells in culture. Mol Microbiol 7 :371–381.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 17

    Karjalainen T, Barc MC, Collignon A, Trolle S, Boureau H, Cotte-Laffitte J, Bourlioux P, 1994. Cloning of a genetic determinant from Clostridium difficile involved in adherence to tissue culture cells and mucus. Infect Immun 62 :4347–4355.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 18

    Bartlett JG, 2006. Narrative review: the new epidemic of Clostridium difficile-associated enteric disease. Ann Intern Med 145 :758–764.

  • 19

    Johal SS, Hammond J, Solomon K, James PD, Mahida YR, 2004. Clostridium difficile associated diarrhea in hospitalised patients: onset in the community and hospital and role of flexible sigmoidoscopy. Gut 53 :673–677.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 20

    Rupnik M, Avesani V, Janc M, von Eichel-Streiber C, Delmee M, 1998. A novel toxinotyping scheme and correlation of toxino-types with serogroups of Clostridium difficile isolates. J Clin Microbiol 36 :2240–2247.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 21

    Rahmati A, Gal M, Northey G, Brazier JS, 2005. Subtyping of Clostridium difficile polymerase chain reaction (PCR) ribotype 001 by repetitive extragenic palindromic PCR genomic fingerprinting. J Hosp Infect 60 :56–60.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 22

    Silletti RP, Lee G, Ailey E, 1996. Role of stool screening tests in diagnosis of inflammatory bacterial enteritis and in selection of specimens likely to yield invasive enteric pathogens. J Clin Microbiol 34 :1161–1165.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 23

    Ruiz-Pelaez JG, Mattar S, 1999. Accuracy of fecal lactoferrin and other stool tests for diagnosis of invasive diarrhea at a Colombian pediatric hospital. Pediatr Infect Dis J 18 :342–346.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 24

    Vaishnavi C, Thapa BR, Thennarasu K, Singh K, 2002. Faecal lactoferrin assay as an adjunct to Clostridium difficile diarrhea. Indian J Pathol Microbiol 45 :69–73.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 25

    Samie A, Obi CL, Barrett LJ, Powell SM, Guerrant RL, 2006. Prevalence of Campylobacter species, Helicobacter pylori and Arcobacter species in stool samples from the Venda region, Limpopo, South Africa: studies using molecular diagnostic methods. J Infect 54 :558–566.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 26

    Lemee L, Dhalluin A, Testelin S, Mattrat MA, Maillard K, Lemeland JF, Pons JL, 2004. Multiplex PCR targeting tpi (triose phosphate isomerase), tcdA (Toxin A), and tcdB (Toxin B) genes for toxigenic culture of Clostridium difficile. J Clin Microbiol 42 :5710–5714.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 27

    Terhes G, Urbán E, Sóki J, Hamid KA, Nagy E, 2004. Community-acquired Clostridium difficile caused by binary toxin, toxin A, toxin B gene-positive isolates in Hungary. J Clin Microbiol 42 :4316–4318.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 28

    Cohen SH, Tang YJ, Silva J Jr, 2000. Analysis of the pathogenicity locus in Clostridium difficile strains. J Infect Dis 181 :659–663.

  • 29

    Stubbs S, Rupnik M, Gibert M, Brazier J, Duerden B, Popoff M, 2000. Production of actin-specific ADP-ribosyltransferase (binary toxin) by strains of Clostridium difficile. FEMS Microbiol Lett 186 :307–312.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 30

    Nataro JP, Mai V, Johnson J, Blackwelder WC, Heimer R, Tirrell S, Edberg SC, Braden CR, Glenn Morris J Jr, Hirshon JM, 2006. Diarrheagenic Escherichia coli infection in Baltimore, Maryland, and New Haven, Connecticut. Clin Infect Dis 43 :402–407.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 31

    Kyne L, Merry C, O’Connell B, Keane C, O’Neill D, 1998. Community-acquired Clostridium difficile infection. J Infect 36 :287–288.

  • 32

    Johal SS, Hammond J, Solomon K, James PD, Mahida YR, 2004. Clostridium difficile associated diarrhoea in hospitalised patients: onset in the community and hospital and role of flexible sigmoidoscopy. Gut 53 :673–677.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 33

    Raveh D, Rabinowitz B, Breuer GS, Rudensky B, Yinnon AM, 2006. Risk factors for Clostridium difficile toxin-positive nosocomial diarrhea. Int J Antimicrob Agents 28 :231–237.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 34

    Pulvirenti JJ, Mehra T, Hafiz I, DeMarais P, Marsh D, Kocka F, Meyer PM, Fischer SA, Goodman L, Gerding DN, Weinstein RA, 2002. Epidemiology and outcome of Clostridium difficile infection and diarrhea in HIV infected inpatients. Diagn Microbiol Infect Dis 44 :325–330.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 35

    Hutin Y, Molina JM, Casin I, Daix V, Sednaoui P, Welker Y, Lagrange P, Decazes JM, Modai J, 1993. Risk factors for Clostridium difficile-associated diarrhea in HIV-infected patients. AIDS 7 :1441–1447.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 36

    Colarian J, 1988. Clostridium difficile colitis following antiviral therapy in the acquired immunodeficiency syndrome. Am J Med 84 :1081.

  • 37

    McDonald LC, Coignard B, Dubberke E, Song X, Horan T, Kutty PK, Ad Hoc Clostridium difficile Surveillance Working Group, 2007. Recommendations for surveillance of Clostridium difficile-associated disease. Infect Control Hosp Epidemiol 28 :140–145.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 38

    Taha S, Johansson O, Rivera Jonsson S, Heimer D, Krovacek K, 2007. Toxin production by and adhesive properties of Clostridium difficile isolated from humans and horses with antibiotic-associated diarrhea. Comp Immunol Microbiol Infect Dis 30 :163–174.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 39

    Songer JG, Anderson MA, 2006. Clostridium difficile: an important pathogen of food animals. Anaerobe 12 :1–4.

  • 40

    Arroyo LG, Staempfli H, Weese JS, 2007. Molecular analysis of Clostridium difficile isolates recovered from horses with diarrhea. Vet Microbiol 120 :179–183.

  • 41

    Rodriguez-Palacios A, Stampfli HR, Duffield T, Peregrine AS, Trotz-Williams LA, Arroyo LG, Brazier JS, Weese JS, 2006. Clostridium difficile PCR ribotypes in calves, Canada. Emerg Infect Dis 12 :1730–1736.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 42

    Simango C, 2006. Prevalence of Clostridium difficile in the environment in a rural community in Zimbabwe. Trans R Soc Trop Med Hyg 100 :1146–1150.

  • 43

    Wultanska D, Pituch H, Obuch-Woszczatynski P, Meisel-Mikolajczyk F, Luczak M, 2005. Profile of toxigenicity of Clostridium difficile strains isolated from paediatric patients with clinical diagnosis of antibiotic associated diarrhea (AAD). Med Dosw Mikrobiol 57 :377–382.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 44

    Pituch H, Obuch-Woszczatynski P, Luczak M, Meisel-Mikolajczyk F, 2003. Clostridium difficile and enterotoxigenic Bacteroides fragilis strains isolated from patients with antibiotic associated diarrhea. Anaerobe 9 :161–163.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 45

    Price MF, Dao-Tran T, Garey KW, Graham G, Gentry LO, Dhungana L, DuPont HL, 2007. Epidemiology and incidence of Clostridium difficile-associated diarrhea diagnosed upon admission to a university hospital. J Hosp Infect 65 :42–46.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 46

    Kazakova SV, Ware K, Baughman B, Bilukha O, Paradis A, Sears S, Thompson A, Jensen B, Wiggs L, Bessette J, Martin J, Clukey J, Gensheimer K, Killgore G, McDonald LC, 2006. A hospital outbreak of diarrhea due to an emerging epidemic strain of Clostridium difficile. Arch Intern Med 166 :2518–2524.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 47

    Curry SR, Marsh JW, Muto CA, O’Leary MM, Pasculle AW, Harrison LH, 2007. tcdC genotypes associated with severe TcdC truncation in an epidemic clone and other strains of Clostridium difficile. J Clin Microbiol 45 :215–221.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 48

    Rexach CE, Tang-Feldman YJ, Cantrell MC, Cohen SH, 2006. Epidemiologic surveillance of Clostridium difficile diarrhea in a freestanding pediatric hospital and a pediatric hospital at a university medical center. Diagn Microbiol Infect Dis 56 :109–114.

    • PubMed
    • Search Google Scholar
    • Export Citation
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