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Methods for Detection of STEC in Humans

An Overview

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E. coli

Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 73))

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Abstract

Timely and accurate diagnosis of Shiga toxigenic Escherichia coli (STEC) disease in humans is extremely important from both a public health and a clinical management perspective. In the outbreak setting, rapid diagnosis of cases and immediate notification of health authorities is essential for effective epidemiological intervention. Early diagnosis also creates a window of opportunity for therapeutic intervention. Agents capable of adsorbing and neutralizing free Shiga toxin (Stx) in the gut lumen have been described (1,2), and these are likely to be most effective when adminstered early in the course of disease, before serious systemic sequelae develop. Also, the clinical presentation of STEC disease can sometimes be confused with other bowel conditions; thus, early definitive diagnosis may prevent unnecessary invasive and expensive surgical and investigative procedures or administation of antibiotic therapy, which may be contraindicated (3). However, detection of STEC is fraught with difficulty, particularly for strains belonging to serogroups other than O157. In the early stages of infection, there may be very high numbers of STEC in feces (the STEC may constitute >90% of aerobic flora), but as disease progresses, the numbers may drop dramatically. In cases of hemolytic uraemic syndrome (HUS), the typical clinical signs may become apparent as much as 2 wk after the onset of gastrointestinal symptoms, by which time the numbers of the causative STEC may be very low indeed. Also, in some cases, diarrhea is no longer present and only a rectal swab may be available at the time of admission to the hospital, limiting the amount of specimen available for analysis. For these reasons, STEC detection methods need to be very sensitive and require minimal specimen volumes.

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References

  1. Armstrong G. D., Rowe P. C., Goodyer P., Orrbine E., Klassen T. P., Wells G., et al. (1995) A phase I study of chemically synthesized verotoxin (Shiga-like toxin) Pk-trisaccharide receptors attached to chromosorb for preventing hemolytic-uremic syndrome. J. Infect. Dis. 171, 1042–1045.

    CAS  PubMed  Google Scholar 

  2. Paton A. W., Morona R., and Paton J. C. (2000) A new biological agent for treatment of Shiga toxigenic Escherichia coli infections and dysentery in humans. Nat. Med. 6, 265–270.

    Article  CAS  PubMed  Google Scholar 

  3. Tarr P. I. (1995) Escherichia coli O157:H7 clinical, diagnostic, and epidemiological aspects of human infection. Clin. Infect. Dis. 20, 1–8.

    CAS  PubMed  Google Scholar 

  4. Karmali M. A. (1989) Infection by verocytotoxin-producing Escherichia coli. Clin. Microbiol. Rev. 2, 15–38.

    CAS  PubMed  Google Scholar 

  5. Paton J. C. and Paton A. W. (1998) Pathogenesis and diagnosis of Shiga toxinproducing Escherichia coli infections. Clin. Microbiol. Rev. 11, 450–479.

    CAS  PubMed  Google Scholar 

  6. Strockbine N. A., Wells J. G., Bopp C. A., and Barrett T.J. (1998) Overview of detection and subtyping methods, in Escherichia coli O157:H7 and Other Shiga Toxin-Producing E. coli Strains (Kaper J. B. and O’Brien A. D. eds.), American Society for Microbiology Washington, DC, pp. 331–356.

    Google Scholar 

  7. Karch H., Bielaszewska M., Bitzan M., and Schmidt H. (1999) Epidemiology and diagnosis of Shiga toxin-producing Escherichia coli infections. Diagn. Microbiol. Infect. Dis. 34, 229–243.

    Article  CAS  PubMed  Google Scholar 

  8. Karmali M. A., Petric M., Lim C., Cheung R., and Arbus G. S. (1985) Sensitive method for detecting low numbers of verotoxin-producing Escherichia coli in mixed cultures by use of colony sweeps and polymyxin extraction of verotoxin. J. Clin. Microbiol. 22, 614–619.

    CAS  PubMed  Google Scholar 

  9. Ashkenazi S. and Cleary T. G. (1990) A method for detecting Shiga toxin and Shiga-like toxin-I in pure and mixed culture. J. Med. Microbiol. 32, 255–261.

    Article  CAS  PubMed  Google Scholar 

  10. Law D., Ganguli L. A., Donohue-Rolfe A., and Acheson D. W. (1992) Detection by ELISA of low numbers of Shiga-like toxin-producing Escherichia coli in mixed cultures after growth in the presence of mitomycin C. J. Med. Microbiol. 36, 198–202.

    Article  CAS  PubMed  Google Scholar 

  11. Kehl K. S., Havens P., Behnke C. E., and Acheson D. W. K. (1997) Evaluation of the Premier EHEC assay for detection of Shiga toxin-producing Escherichia coli. J. Clin. Microbiol. 35, 2051–2054.

    CAS  PubMed  Google Scholar 

  12. Mackenzie A. M. R., Lebel P., Orrbine E., Rowe P. C., Hyde L., Chan F., et al., and the Synsorb PK Study Investigators (1998) Sensitivities and specificities of Premier E. coli O157 and Premier EHEC enzyme immunoassays for diagnosis of infection with verotoxin (Shiga-like toxin)-producing Escherichia coli. J. Clin. Microbiol. 36, 1608–1611.

    CAS  PubMed  Google Scholar 

  13. Beutin L., Zimmermann S., and Gleier K. (1996) Rapid detection and isolation of Shiga-like toxin (Verocytotoxin)-producing Escherichia coli by direct testing of individual enterohemolytic colonies from washed sheep blood agar plates in the VTEC-RPLA assay. J. Clin. Microbiol. 34, 2812–2814.

    CAS  PubMed  Google Scholar 

  14. Karmali M. A., Petric M., and Bielaszewska M. (1999) Evaluation of a microplate latex agglutination method (Verotox-F assay) for detecting and characterizing verotoxins (Shiga toxins) in Escherichia coli. J. Clin. Microbiol. 37, 396–399.

    CAS  PubMed  Google Scholar 

  15. Thomas A., Smith H. R., Willshaw G. A., and Rowe B. (1991) Non-radioactively labelled polynucleotide and oligonucleotide DNA probes, for selectively detecting Escherichia coli strains producing Vero cytotoxins VT1, VT2 and VT2 variant. Mol. Cell. Probes 5, 129–135.

    Article  CAS  PubMed  Google Scholar 

  16. Begum D., Strockbine N. A., Sowers E. G., and Jackson M. P. (1993) Evaluation of a technique for identification of Shiga-like toxin-producing Escherichia coli by using polymerase chain reaction and digoxigenin-labeled probes. J. Clin. Microbiol. 31, 3153–3156.

    CAS  PubMed  Google Scholar 

  17. Brian M. J., Frosolono M., Murray B. E., Miranda A., Lopez E. L., Gomez H. F., and Cleary T. G. (1992) Polymerase chain reaction for diagnosis of enterohemorrhagic Escherichia coli infection and hemolytic-uremic syndrome. J. Clin. Microbiol. 30, 1801–1806.

    CAS  PubMed  Google Scholar 

  18. Gannon V. P. J., King R. K., Kim J. Y., and Thomas E. J. (1992) Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction. Appl. Environ. Microbiol. 58, 3809–3815.

    CAS  PubMed  Google Scholar 

  19. Pollard D. R., Johnson W. M., Lior H., Tyler S. D., and Rozee K. R. (1990) Rapid and specific detection of verotoxin genes in Escherichia coli by the polymerase chain reaction. J. Clin. Microbiol. 28, 540–545.

    CAS  PubMed  Google Scholar 

  20. Karch H. and Meyer T. (1989) Single primer pair for amplifying segments of distinct Shiga-like toxin genes by polymerase chain reaction. J. Clin. Microbiol. 27, 2751–2757.

    CAS  PubMed  Google Scholar 

  21. Paton A. W., Paton J. C., Goldwater P. N., and Manning P. A. (1993) Direct detection of Escherichia coli Shiga-like toxin genes in primary fecal cultures by polymerase chain reaction. J. Clin. Microbiol. 31, 3063–3067.

    CAS  PubMed  Google Scholar 

  22. Lin Z., Kurazono H., Yamasaki S., and Takeda Y. (1993) Detection of various variant verotoxin genes in Escherichia coli by polymerase chain reaction. Microbiol. Immunol. 37, 543–548.

    CAS  PubMed  Google Scholar 

  23. Russmann H., Schmidt H., Heesemann J., Caprioli A., and Karch H. (1994) Variants of Shiga-like toxin II constitute a major toxin component in Escherichia coli O157 strains from patients with haemolytic uraemic syndrome. J. Med. Microbiol. 40, 338–343.

    Article  CAS  PubMed  Google Scholar 

  24. Tyler S. D., Johnson W. M., Lior H., Wang G., and Rozee K. R. (1991) Identification of verotoxin type 2 variant B subunit genes in Escherichia coli by the polymerase chain reaction and restriction fragment length polymorphism analysis. J. Clin. Microbiol. 29, 1339–1343.

    CAS  PubMed  Google Scholar 

  25. Chen S., Xu R., Yee A., Wu K. Y., Wang C. N., Read S., et al. (1998) An automated fluorescent PCR method for detection of shiga toxin-producing Escherichia coli in foods. Appl. Environ. Microbiol. 64, 4210–4216.

    CAS  PubMed  Google Scholar 

  26. Sharma V. K., Dean-Nystrom E. A., and Casey T. A. (1999) Semi-automated fluorogenic PCR assays (TaqMan) for rapid detection of Escherichia coli O157:H7 and other Shiga toxigenic E. coli. Mol. Cell. Probes 13, 291–302.

    Article  CAS  Google Scholar 

  27. Gannon V.P.J., Rashed M., King R.K., and Thomas E.J.G. (1993) Detection and characterization of the eae gene of Shiga-like toxin-producing Escherichia coli using polymerase chain reaction. J. Clin. Microbiol. 31, 1268–1274.

    CAS  PubMed  Google Scholar 

  28. Schmidt H., Beutin L., and Karch H. (1995) Molecular analysis of the plasmidencoded hemolysin of Escherichia coli O157:H7 strain EDL 933. Infect. Immun. 63, 1055–1061.

    CAS  PubMed  Google Scholar 

  29. Barrett T. J., Kaper J. B., Jerse A. E., and Wachsmuth I. K. (1992) Virulence factors in Shiga-like toxin-producing Escherichia coli isolated from humans and cattle. J. Infect. Dis. 165, 979–980.

    CAS  PubMed  Google Scholar 

  30. Schmidt H. and Karch H. (1996) Enterohemolytic phenotypes and genotypes of Shiga toxin-producing Escherichia coli O111 strains from patients with diarrhea and hemolytic-uremic syndrome. J. Clin. Microbiol. 34, 2364–2367.

    CAS  PubMed  Google Scholar 

  31. Louie M., De-Azavedo J., Clarke R., Borczyk A., Lior H., Richter M., et al. (1994) Sequence heterogeneity of the eae gene and detection of verotoxin-producing Escherichia coli using serotype-specific primers. Epidemiol. Infect. 112, 449–461.

    Article  CAS  PubMed  Google Scholar 

  32. Paton A. W. and Paton J. C. (1998) Detection and characterization of Shiga toxigenic Escherichia coli using multiplex PCR assays for stx1, stx2, eaeA, Enterohemorrhagic E. coli hlyA, rfbO111 and rfbO157. J. Clin. Microbiol. 36, 598–602.

    CAS  PubMed  Google Scholar 

  33. Paton A. W. and Paton J. C. (1999) Direct detection of Shiga toxigenic Escherichia coli strains belonging to serogroups O111, O157, and O113 by multiplex PCR. J. Clin. Microbiol. 37, 3362–3365.

    CAS  PubMed  Google Scholar 

  34. Gannon V. P. J., D’Souza S., Graham T., King R. K., Rahn K., and Read S. (1997) Use of the flagellar H7 gene as a target in multiplex PCR assays and improved specificity and identification of enterohemorrhagic Escherichia coli strains. J. Clin. Microbiol. 35, 656–662.

    CAS  PubMed  Google Scholar 

  35. Cebula T. A., Payne W. L., and Feng P. (1995) Simultaneous identification of strains of Escherichia coli serotype O157:H7 and their Shiga-like toxin type by mismatch amplification mutation assay-multiplex PCR. J. Clin. Microbiol. 33, 248–250. [Published erratum appears in J. Clin. Microbiol. 33, 1048.]

    CAS  PubMed  Google Scholar 

  36. March S. B. and Ratnam S. (1986) Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis. J. Clin. Microbiol. 23, 869–872.

    CAS  PubMed  Google Scholar 

  37. Chapman P. A., Siddons C. A., Zadik P. M., and Jewes L. (1991) An improved selective medium for the isolation of Escherichia coli O157. J. Med. Microbiol. 35, 107–110.

    Article  CAS  PubMed  Google Scholar 

  38. Zadik P. M., Chapman P. A., and Siddons C. A. (1993) Use of tellurite for the selection of verocytotoxigenic Escherichia coli O157. J. Med. Microbiol. 39, 155–158.

    Article  CAS  PubMed  Google Scholar 

  39. Gunzer F., Bohm H., Russmann H., Bitzan M., Aleksic S., and Karch H. (1992) Molecular detection of sorbitol-fermenting Escherichia coli O157 in patients with hemolytic-uremic syndrome. J. Clin. Microbiol. 30, 1807–1810.

    CAS  PubMed  Google Scholar 

  40. Bielaszewska M., Janda J., Blahova K., Karch H., Karmali M. A., Preston M. A., et al. (1997) Isolation of sorbitol-fermenting (SF) verocytotoxin-producing Escherichia coli O157:H-in the Czech Republic. 13rd International Symposium and Workshop on Shiga Toxin (Verotoxin)-producing Escherichia coli Infections, Baltimore, MD.

    Google Scholar 

  41. Ratnam S., March S. B., Ahmed R., Bezanson G. S., and Kasatiya S. (1988) Characterization of Escherichia coli serotype O157:H7. J. Clin. Microbiol. 26, 2006–2012.

    CAS  PubMed  Google Scholar 

  42. Thompson J. S., Hodge D. S., and Borczyk A. A. (1990) Rapid biochemical test to identify verocytotoxin-positive strains of Escherichia coli serotype O157. J. Clin. Microbiol. 28, 2165–2168.

    CAS  PubMed  Google Scholar 

  43. Novicki T.J., Daly J.A., Mottice S.L., and Carroll K.C. (2000) Comparison of sorbitol MacConkey agar and a two-step method which utilizes enzyme-linked immunosorbent assay toxin testing and a chromogenic agar to detect and isolate enterohemorrhagic Escherichia coli. J. Clin. Microbiol. 38, 547–551.

    CAS  PubMed  Google Scholar 

  44. Park C.H., Hixon D.L., Morrison W.L., and Cook C.B. (1994) Rapid diagnosis of enterohemorrhagic Escherichia coli O157:H7 directly from fecal specimens using immunofluorescence stain. Am. J. Clin. Pathol. 101, 91–94.

    CAS  PubMed  Google Scholar 

  45. Park C. H., Vandel N. M., and Hixon D. L. (1996) Rapid immunoassay for detection of Escherichia coli O157 directly from stool specimens. J. Clin. Microbiol. 34, 988–990.

    CAS  PubMed  Google Scholar 

  46. Beutin L., Montenegro M. A., Orskov I., Orskov F., Prada J., Zimmermann S., et al. (1989) Close association of verotoxin (Shiga-like toxin) production with enterohemolysin production in strains of Escherichia coli. J. Clin. Microbiol. 27, 2559–2564.

    CAS  PubMed  Google Scholar 

  47. Hull A. E., Acheson D. W., Echeverria P., Donohue-Rolfe A., and Keusch G. T. (1993) Mitomycin immunoblot colony assay for detection of Shiga-like toxinproducing Escherichia coli in fecal samples: comparison with DNA probes. J. Clin. Microbiol. 31, 1167–1172.

    CAS  PubMed  Google Scholar 

  48. Chapman P. A. and Siddons C. A. (1996) A comparison of immunomagnetic separation and direct culture for the isolation of verocytotoxin-producing Escherichia coli O157 from cases of bloody diarrhoea, non-bloody diarrhoea and asymptomatic contacts. J. Med. Microbiol. 44, 267–271.

    Article  CAS  PubMed  Google Scholar 

  49. Karch H., Janetzki-Mittmann C., Aleksic S., and Datz M. (1996) Isolation of enterohemorrhagic Escherichia coli O157 strains from patients with hemolyticuremic syndrome by using immunomagnetic separation, DNA-based methods and direct culture. J. Clin. Microbiol. 34, 516–519.

    CAS  PubMed  Google Scholar 

  50. Paton A. W., Ratcliff R., Doyle R. M., Seymour-Murray J., Davos D., Lanser J. A., et al. (1996) Molecular microbiological investigation of an outbreak of hemolytic uremic syndrome caused by dry fermented sausage contaminated with Shiga-like toxin-producing Escherichia coli. J. Clin. Microbiol. 34, 1622–1627.

    CAS  PubMed  Google Scholar 

  51. Jenkins C., Chart H., Smith H.R., Hartland E.L., Batchelor M., Delahay R.M., et al. (2000) Antibody response of patients infected with verocytotoxin-producing Escherichia coli to protein antigens encoded on the LEE locus. J. Med. Microbiol. 49, 97–101.

    CAS  PubMed  Google Scholar 

  52. Barrett T. J., Green J. H., Griffin P. M., Pavia A. T., Ostroff S. M., and Wachsmuth I. K. (1991) Enzyme-linked immunosorbent assays for detecting antibodies to Shiga-like toxin I, Shiga-like toxin II, and Escherichia coli O157:H7 lipopolysaccharide in human serum. Curr. Microbiol. 23, 189–195.

    Article  CAS  Google Scholar 

  53. Chart H., Law D., Rowe B., and Acheson D. W. (1993) Patients with haemolytic uraemic syndrome caused by Escherichia coli O157: absence of antibodies to Vero cytotoxin 1 (VT1) or VT2. J. Clin. Pathol. 46, 1053,1054.

    Article  Google Scholar 

  54. Karmali M. A., Petric M., Winkler M., Bielaszewska M., Brunton J., van-de-Kar N., et al. (1994) Enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Escherichia coli Vero cytotoxin 1. J. Clin. Microbiol. 32, 1457–1463.

    CAS  PubMed  Google Scholar 

  55. Reymond D., Karmali M. A., Clarke I., Winkler M., and Petric M. (1997) Comparison of the western blot assay with the neutralizing-antibody and enzymelinked immunosorbent assays for measuring antibody to verocytotoxin 1. J. Clin. Microbiol. 35, 609–613.

    CAS  PubMed  Google Scholar 

  56. Bitzan M. and Karch H. (1992) Indirect hemagglutination assay for diagnosis of Escherichia coli O157 infection in patients with hemolytic-uremic syndrome. J. Clin. Microbiol. 30, 1174–1178.

    CAS  PubMed  Google Scholar 

  57. Bitzan M., Moebius E., Ludwig K., Muller-Wiefel D. E., Heesemann J., and Karch H. (1991) High incidence of serum antibodies to Escherichia coli O157 lipopolysaccharide in children with hemolytic-uremic syndrome. J. Pediatr. 119, 380–385.

    Article  CAS  PubMed  Google Scholar 

  58. Chart H., Smith H. R., Scotland S. M., Rowe B., Milford D. V., and Taylor C. M. (1991) Serological identification of Escherichia coli infection in haemolytic uraemic syndrome. Lancet 337, 138–140.

    Article  CAS  PubMed  Google Scholar 

  59. Greatorex J. S. and Thorne G.M. (1994) Humoral immune responses to Shigalike toxins and Escherichia coli O157 lipopolysaccharide in hemolytic-uremic syndrome patients and healthy subjects. J. Clin. Microbiol. 32, 1172–1178.

    CAS  PubMed  Google Scholar 

  60. Yamada S., Kai A., and Kudoh Y. (1994) Serodiagnosis by passive hemagglutination test and verotoxin enzyme-linked immunosorbent assay of toxin-producing Escherichia coli infections in patients with hemolytic-uremic syndrome. J. Clin. Microbiol. 32, 955–959.

    CAS  PubMed  Google Scholar 

  61. Chart H. (1999) Evaluation of a latex agglutination kit for the detection of human antibodies to the lipopolysaccharide of Escherichia coli O157, following infection with verocytotoxin-producing E. coli O157. Lett. Appl. Microbiol. 29, 434–436.

    Article  CAS  PubMed  Google Scholar 

  62. Bielaszewska M., Janda J., Blahova K., Feber J., Potuznik V., and Souckova A. (1996) Verocytotoxin-producing Escherichia coli in children with hemolytic uremic syndrome in the Czech Republic. Clin. Nephrol. 46, 42–44.

    CAS  PubMed  Google Scholar 

  63. Chart H. and Rowe B. (1990) Serological identification of infection by Vero cytotoxin producing Escherichia coli in patients with haemolytic uraemic syndrome. Serodiagn. Immunother. Infect. Dis. 4, 413–418.

    Article  Google Scholar 

  64. Caprioli A., Luzzi I., Rosmini F., Resti C., Edefonti A., Perfumo F., et al. (1994) Community-wide outbreak of hemolytic-uremic syndrome associated with non-O157 verocytotoxin-producing Escherichia coli. J. Infect. Dis. 169, 208–211.

    CAS  PubMed  Google Scholar 

  65. Paton A. W., Woodrow M. C., Doyle R. M., Lanser J. A. and Paton J. C. (1999) Molecular characterization of a Shiga-toxigenic Escherichia coli O113:H21 strain lacking eae responsible for a cluster of cases of hemolytic-uremic syndrome. J. Clin. Microbiol. 37, 3357–3361.

    CAS  PubMed  Google Scholar 

  66. Karch H., Meyer T., Russmann H., and Heesemann J. (1992) Frequent loss of Shiga-like toxin genes in clinical isolates of Escherichia coli upon subcultivation. Infect. Immun. 60, 3464–3467.

    CAS  PubMed  Google Scholar 

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  1. Department of Molecular Biosciences, University of Adelaide, Adelaide, Australia

    James C. Paton & Adrienne W. Paton

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  1. James C. Paton
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  1. Groupe d&'Immunité Innée et Signalisation, Institut Pasteur, Paris, France

    Dana Philpott

  2. Max-von-Pettenkofer-Institut, Bakteriologie, Munich, Germany

    Frank Ebel

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Paton, J.C., Paton, A.W. (2003). Methods for Detection of STEC in Humans. In: Philpott, D., Ebel, F. (eds) E. coli. Methods in Molecular Medicine™, vol 73. Humana Press. https://doi.org/10.1385/1-59259-316-X:09

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  • DOI: https://doi.org/10.1385/1-59259-316-X:09

  • Publisher Name: Humana Press

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