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NEW DIAGNOSTIC TEST FOR ENTEROPATHOGENIC ESCHERICHIA COLI
1337
"Novopen Group", and found that over half had seen occasional bubbles in the insulin vial. The problem is known to the manufacturers and other clinics, but it has not been associated with serious metabolic decompensation. We now recommend that before injection patients express one "click" (2 units) of insulin to remove any bubble. matter at a meeting of our
Department of Medicine, Arrowe Park Hospital, Wirral, Merseyside *
Present address:
KEVIN HARDY* GEOFFREY GILL
Department of Medicine, Broadgreen Hospital, Liverpool L14 3LB.
NEW DIAGNOSTIC TEST FOR ENTEROPATHOGENIC ESCHERICHIA COLI
Escherichia coli (EPEC) remain an of infant gastroenteritis in many parts of the developing world.’ EPEC are defmed as diarrhoea-causing E coli belonging to serogroups epidemiologically incriminated as pathogens but whose pathogenic mechanisms have not been shown to be related to heat-labile or heat-stable enterotoxins or to shigella-like invasiveness.2 Diagnostic tests for EPEC are timeconsuming, expensive, and not 100% sensitive. A DNA probe recognising EPEC adherence factor has also been used, but this too does not recognise all EPEC strains.3 EPEC adhere to the intestinal mucosa and produce a characteristic "attaching and effacing" lesion in the brush border microvillous membrane. The lesion is characterised by destruction of microvilli and intimate attachment of bacteria to cup-like projections of the apical enterocyte membrane, with which dense condensations of microfilaments are associated.4-ó These appearances are seen when EPEC adhere in vivo or to tissue culture cells in vitro.’ Up to now this lesion has only been detectable by electron microscopy. As a simple test for this membrane lesion we have made use of a fluorescein-labelled phallotoxin (FITC-phalloidin, Sigma), which specifically binds to filamentous actin, to identify the dense condensations of microfilaments produced when EPEC adhere to tissue culture cells. Ten EPEC strains known from electron microscopy to produce the characteristic lesion6 and ten tissue culture cell adherent non-EPEC strains were examined, and fluorescence and phase contrast images of identical fields were
SiR,—Enteropathogenic
important
cause
compared. Cells infected with all ten EPEC strains exhibited intense spots of fluorescence corresponding exactly with the position of each adherent bacterium (figure, A and B). Cells infected with non-EPEC strains did not exhibit this striking pattern and were indistinguishable from uninfected control cells (figure, C and D). The difference was striking and no false positives were seen. The test is so sensitive that even individual attaching and effacing bacteria can be identified. Indeed, EPEC strains previously described as tissue culture (HEp-2) cell non-adherenf have been found weakly adherent in this test. The test should also discriminate between EPEC and enterohaemorrhagic E coli, which produce an identical lesion to EPEC but in the large bowel;8 EHEC do not adhere to the tissue culture cell lines used for EPEC adhesion studies. This simple and inexpensive diagnostic test for EPEC may be particularly useful in settings where tissue culture cell adhesion assays are done but where serotyping, enterotoxin testing, and DNA probe testing are not routinely available. Institute of Child Health, University of Birmingham, Birmingham B16 SET; and Department of Genetics, University of Leicester
S. KNUTTON T. BALDWIN P. H. WILLIAMS A. S. MCNEISH
1. Toledo MRF, Alvariza MCB, Murahovschi J, Ramos SRTS, Trabulsi LR. Enteropathogenic Escherichia coli serotypes and endemic diarrhea in infants. Infect Immun 1983, 39: 586-89. 2. Edelman R, Levine MM Summary of a workshop on enteropathogenic Escherichia coli. J Infect Dis 1983; 147: 1108-18. 3. Nataro JP, Baldini MM, Kaper JB, Black RE, Bravo N, Levine MM. Detection of an adhesion factor of enteropathogenic Escherichia coli with a DNA probe.J Infect Dis
1985; 152: 560-65 4. Ulshen MH, Rollo JL. Pathogenesis of Escherichia coli gastroenteritis in mananother mechanism N Engl JMed 1980, 302: 99-101. 5 Rothbaum R, McAdams AJ, Gianella R, Partin JC. A clinicopathologic study of enterocyte adherent Escherichia coli a cause of protracted diarrhea in infants. Gastroenterology 1982; 83: 441-54 6. Knutton S, Lloyd DR, McNeish AS Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa. Infect Immun 1987, 55: 69-77. 7 Knutton S, Baldini MM, Kaper JB, McNeish AS. Role of plasmid-encoded adherence factors in adhesion of enteropathogenic Escherichia coli to HEp-2 cells. Infect Immun 1987; 55: 78-85 8. Tzipori S, Wachsmuth IK, Chapman C, et al The pathogenesis ofhemorrhagic colitis caused by Escherichia coli 0157.H7 in gnotobiotic piglets. J Infect Dis 1986; 154: 712-16.
Fluorescence (A, C) and phase contrast micrographs (B, D) of identical fields showing HEp-2 cells infected with EPEC and non-EPEC strains for 3 h and stained with FITC-phalloidin. Intense spots of fluorescence are seen at sites corresponding to each adherent bacterium with EPEC (A, B) but not with non-EPEC strains (C, D).
"Novopen Group", and found that over half had seen occasional bubbles in the insulin vial. The problem is known to the manufacturers and other clinics, but it has not been associated with serious metabolic decompensation. We now recommend that before injection patients express one "click" (2 units) of insulin to remove any bubble. matter at a meeting of our
Department of Medicine, Arrowe Park Hospital, Wirral, Merseyside *
Present address:
KEVIN HARDY* GEOFFREY GILL
Department of Medicine, Broadgreen Hospital, Liverpool L14 3LB.
NEW DIAGNOSTIC TEST FOR ENTEROPATHOGENIC ESCHERICHIA COLI
Escherichia coli (EPEC) remain an of infant gastroenteritis in many parts of the developing world.’ EPEC are defmed as diarrhoea-causing E coli belonging to serogroups epidemiologically incriminated as pathogens but whose pathogenic mechanisms have not been shown to be related to heat-labile or heat-stable enterotoxins or to shigella-like invasiveness.2 Diagnostic tests for EPEC are timeconsuming, expensive, and not 100% sensitive. A DNA probe recognising EPEC adherence factor has also been used, but this too does not recognise all EPEC strains.3 EPEC adhere to the intestinal mucosa and produce a characteristic "attaching and effacing" lesion in the brush border microvillous membrane. The lesion is characterised by destruction of microvilli and intimate attachment of bacteria to cup-like projections of the apical enterocyte membrane, with which dense condensations of microfilaments are associated.4-ó These appearances are seen when EPEC adhere in vivo or to tissue culture cells in vitro.’ Up to now this lesion has only been detectable by electron microscopy. As a simple test for this membrane lesion we have made use of a fluorescein-labelled phallotoxin (FITC-phalloidin, Sigma), which specifically binds to filamentous actin, to identify the dense condensations of microfilaments produced when EPEC adhere to tissue culture cells. Ten EPEC strains known from electron microscopy to produce the characteristic lesion6 and ten tissue culture cell adherent non-EPEC strains were examined, and fluorescence and phase contrast images of identical fields were
SiR,—Enteropathogenic
important
cause
compared. Cells infected with all ten EPEC strains exhibited intense spots of fluorescence corresponding exactly with the position of each adherent bacterium (figure, A and B). Cells infected with non-EPEC strains did not exhibit this striking pattern and were indistinguishable from uninfected control cells (figure, C and D). The difference was striking and no false positives were seen. The test is so sensitive that even individual attaching and effacing bacteria can be identified. Indeed, EPEC strains previously described as tissue culture (HEp-2) cell non-adherenf have been found weakly adherent in this test. The test should also discriminate between EPEC and enterohaemorrhagic E coli, which produce an identical lesion to EPEC but in the large bowel;8 EHEC do not adhere to the tissue culture cell lines used for EPEC adhesion studies. This simple and inexpensive diagnostic test for EPEC may be particularly useful in settings where tissue culture cell adhesion assays are done but where serotyping, enterotoxin testing, and DNA probe testing are not routinely available. Institute of Child Health, University of Birmingham, Birmingham B16 SET; and Department of Genetics, University of Leicester
S. KNUTTON T. BALDWIN P. H. WILLIAMS A. S. MCNEISH
1. Toledo MRF, Alvariza MCB, Murahovschi J, Ramos SRTS, Trabulsi LR. Enteropathogenic Escherichia coli serotypes and endemic diarrhea in infants. Infect Immun 1983, 39: 586-89. 2. Edelman R, Levine MM Summary of a workshop on enteropathogenic Escherichia coli. J Infect Dis 1983; 147: 1108-18. 3. Nataro JP, Baldini MM, Kaper JB, Black RE, Bravo N, Levine MM. Detection of an adhesion factor of enteropathogenic Escherichia coli with a DNA probe.J Infect Dis
1985; 152: 560-65 4. Ulshen MH, Rollo JL. Pathogenesis of Escherichia coli gastroenteritis in mananother mechanism N Engl JMed 1980, 302: 99-101. 5 Rothbaum R, McAdams AJ, Gianella R, Partin JC. A clinicopathologic study of enterocyte adherent Escherichia coli a cause of protracted diarrhea in infants. Gastroenterology 1982; 83: 441-54 6. Knutton S, Lloyd DR, McNeish AS Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa. Infect Immun 1987, 55: 69-77. 7 Knutton S, Baldini MM, Kaper JB, McNeish AS. Role of plasmid-encoded adherence factors in adhesion of enteropathogenic Escherichia coli to HEp-2 cells. Infect Immun 1987; 55: 78-85 8. Tzipori S, Wachsmuth IK, Chapman C, et al The pathogenesis ofhemorrhagic colitis caused by Escherichia coli 0157.H7 in gnotobiotic piglets. J Infect Dis 1986; 154: 712-16.
Fluorescence (A, C) and phase contrast micrographs (B, D) of identical fields showing HEp-2 cells infected with EPEC and non-EPEC strains for 3 h and stained with FITC-phalloidin. Intense spots of fluorescence are seen at sites corresponding to each adherent bacterium with EPEC (A, B) but not with non-EPEC strains (C, D).