- Email: [email protected]
HAEMORRHAGIC COLITIS AND VERO-CYTOTOXIN-PRODUCING ESCHERICHIA COLI IN ENGLAND AND WALES
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Case D had classical type 1 GSD with complete absence of glucose-6-phophatase itself. Although he had exhibited all the symptoms of classical type la GSD since at least the age of 7 years, this condition had remained undiagnosed until his final admission with primary liver cell carcinoma. Hepatocellular carcinoma often develops in patients with classical type 1 GSD,32 although the reason for this association is unclear. The establishment of the diagnosis of GSD cannot be made with any certainty unless enzymes are assayed in fresh (unfrozen) tissue samples.2O In classical type la GSD, the clinical findings and biochemical screening procedures that have been worked out for childrenll.33 can also indicate GSD in adults, and justify liver biopsy to establish the nature of the underlying cause. In contrast the much milder clinical symptoms of our cases A-C had not previously suggested GSD and, without liver biopsy, they would have remained undiagnosed. The fact that we have diagnosed four such patients suggests that defects which make the hepatic glucose-6-phosphatase system function at a reduced level are more common than was thought. Adult type 1 GSD should therefore be considered in patients with unresolved hypoglycaemic symptoms and/or unresolved
hepatomegaly. We thank Miss L. Stewart for technical assistance, Ms M. Hughes for typing, and Dr D. Walsh for arranging the lactate measurements. This work was supported by grants from the Scottish Hospitals Endowment Research Trust, the Scottish Home and Health Departments, and the British Diabetic Association
to
A.B.
Correspondence should be addressed to A. B., Department of Medicine, Ninewells Hospital and Medical School, Dundee DD1 9SY. REFERENCES 1. Cori GT, Cori CF. Glucose-6-phosphatase of the liver m glycogen storage disease. J Biol Chem 1952; 199: 661-67. 2. Howell RR, Ashton DM, Wyngaarden JB. Glucose-6-phosphatase deficiency glycogen storage disease. Pediatrics 1962; 29: 553-65. 3. Howell RR, Williams JC. The glycogen storage diseases. In: Stanbury JA, Wyngaarden JB, Frederickson DS, Goldstein JL, Brown MS, eds. The metabolic basis of inherited disease. New York: McGraw Hill, 1983: 141-66. 4. Cornblath M, Schwartz R. Disorders of glycogen metabolism. In: Schaffer AJ, Markowitz M, eds. Disorders of carbohydrate metabolism in infancy. Philadelphia: W. R. Saunders & Co., 1976: 231-93. 5. Arion WJ, Wallin BK, Lange AJ, Ballas LM. On the involvement of a glucose-6-phosphate transport system in the function of microsomal glucose-6phosphatase. Mol Cell Biochem 1975; 6: 75-83. 6. Arion WJ, Ballas LM, Lange AJ, Wallin BK. Microsomal membrane permeability and the hepatic glucose-6-phosphatase system. J Biol Chem 1976; 251: 4901-07. 7. Arion WJ, Lange AJ, Walls HE, Ballas LM. Evidence of the participation of independent translocases for phosphate and glucose-6-phospate in the microsomal glucose-6-phosphatase system. J Biol Chem 1980; 225: 10396-406. 8. Lange AJ, Arion WJ, Beaudet AL. Type 1B glycogen storage disease is caued by a defect in the glucose-6-phosphate translocase of the microsomal glucose-6Chem 1980; 225: 8381-84. J phosphatase system. Biol 9. Nordlie RC, Sukalski K, Munoz JM, Baldwin JJ. Type IC, a novel glycogenosis. J Biol Chem 1983; 258: 9739-44. 10. Schaub J, Heyne K. Glycogen storage disease type 1B. Eur J Pediatr 1983; 140: 283-88. 11. Huijing F, Van de Kamer JH. A screening method for liver glycogen disease. Arch Dis Child 1969; 44: 311-17. 12. Arion WJ, Wallin BK. Kinetics of the glucose-6-phosphate-glucose exchange activity and glucose inhibition of glucose-6-phosphatase of intact and disrupted rat liver microsomes. J Biol Chem 1973; 248: 2373-79. 13. Bickerstaff GF, Burchell B. Studies on the purification of glucose-6-phosphatase from rabbit liver microsomal fraction. Biochem Soc Trans 1980; 8: 389-90. 14. Burchell A, Burchell B. Stabilisation of partially purified glucose-6-phosphatase by fluoride: Is enzyme inactivation caused by dephosphory lation. FEBS Lett 1980; 113: 180-84. 15. Arion WJ, Walls HE. The importance of membrane integrity in kinetic characerizations of the microsomal glucose-6-phosphatase system. J Biol Chem 1982; 257: 11217-20. 16. Arion WJ, Lange AJ, Walls HE. Microsomal membrane integrity and the interactions of phlorizin with the glucose-6-phosphatase system. J Biol Chem 1980; 225: 10387-95. 17. Peterson GL. A simplification of the protein assay method of Lowry et al which is more generally applicable. Anal Biochem 1977; 83: 346-56. 18. Van Handel E. Estimation of glycogen in small amounts of tissue. Anal Biochem 1965; 11: 256-65. 19. Huijing F. A rapid enzyme method for glycogen estimation in very small tissue samples. Clin Chim Acta 1970; 30: 567-72.
HAEMORRHAGIC COLITIS AND VEROCYTOTOXIN-PRODUCING ESCHERICHIA COLI IN ENGLAND AND WALES H. R. SMITH R. J. GROSS
B. ROWE N. K. FRY
S. M. SCOTLAND Division of Enteric Pathogens, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT
Vero-cytotoxin-producing strains of Escherichia coli (VTEC) were identified by the use of DNA probes in 39% of faecal samples from patients with haemorrhagic colitis in England and Wales. The patients with VTEC were distributed widely and their ages ranged from 2·5 to 86 years (mean 41). 3 patients died, including a child of 2·5 years. 30 of the 32 VTEC strains belonged to serogroup 0157. Plating on sorbitol agar for non-fermenters followed by agglutination with a specific 0157 antiserum was a useful screening method for 0157 Summary
VT + strains. However, it was not as sensitive as the DNA probe technique and did not detect VTEC of other serogroups. Introduction
(HC) is characterised by grossly bloody diarrhoea, usually in the absence of pyrexia but sometimes preceded by watery diarrhoea and often accompanied by abdominal cramps. Outbreaks and sporadic cases of HC have been reported in the United States, Canada, and Japan in the past 4 years.l-6 Two of the HAEMORRHAGIC colitis
outbreaks in the United States were associated with undercooked hamburger meat1,7 and one in Canada with milk.6 The epidemiological studies in North America show that the disease occurs over a wide geographical area and affects all age-groups. Konowalchuk et al8 described a cytotoxin active on Vero cells and produced by certain strains of Escherichia coli; it was termed Vero cytotoxin (VT). VT is clearly distinguishable, biologically and immunologically, from the heat-labile and heat-stable enterotoxins of E coli and has been reported to be closely related to Shiga toxin, produced by strains of Shigella dysenteriae type 1.9 At least two distinct Vero cytotoxins, VT1 and VT2, can be identified in toxin
20.
Spencer Peet J, Norman ME, Lake BD, McNamara J, Patrick AD. Hepatic glycogen 1971; 157: 95-114. storage disease. Quart Med J
21. Sokal
JE, Lowe CU, Sarcione EJ, et al. Studies of glycogen metabolism in liver glycogen storage disease. J Clin Invest 1961; 40: 364-74. 22. Narisawa K, Otoma H, Igarashi Y. Glycogen storage disease Type 1b. Pediatrics 1983; 17: 545-49.
23, Illingworth B. Glycogen storage diseases. J Clin Nutrition 1961; 9: 683-90. 24. Hers HG. Glycogen storage disease. Adv Metab Disorders 1964; 1: 1-44. 25. Stamm WE, Webb DI. Partial deficiency of hepatic glucose-6-phosphatase in an adult patient. Arch Intern Med 1975; 135: 1107-09. 26. Chen Y-T, Comblath M, Sidbury JB. Cornstarch therapy in Type 1 glycogen storage disease. N Engl J Med 1984; 310: 171-75. 27. Burchell A, Cain DI. Rat hepatic microsomal glucose-6-phosphatase protein levels are increased in streptozotocin-induced diabetics. Diabetologia 1985; 28: 852-56. 28. Greenbaum AL, Gumaa KA, McLean P. The distribution of hepatic metabolites and the control of the pathways of carbohydrate metabolism in animals of different dietary and hormonal status. Arch Biochem Biophys 1971; 143: 617-63. 29. Nordlie RC. Fine tuning of blood glucose concentrations. Trends Biochem Sci 1985; 10: 70-75. 30. Okado S, Seino Y, Kodama H, et al. Insulin and glucagon secretion in hepatic glycogenosis. Acta Paediatr Scand 1979; 68: 735-38. 31. Wolf BA, Colca JR, Cowans PG, et al. Glucose-6-phosphate regulates Ca steady state in endoplasmic reticulum of islets. A possible link in glucose-induced Secretion. J Biol Chem 1986; 261: 16284-87. 32. Howell RR, Stevenson RE, Ben-Menachem Y, et al. Hepatic adenomata with type 1 glycogen storage disease. JAMA 1976; 236: 1481-84. 33. Femandes J, Koster JF, Crose WFA, Sargedrager N. Hepatic phosphorylase deficiency: Its differentiation from other glycogenoses. Arch Dis Child 1974; 49: 186-91.
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neutralisation experiments.10 Vero-cytotoxin-producing strains of E coli (VTEC) have been associated with the cases of haemorrhagic colitis described above, the commonest serotype of these VTEC strains being 0157 .H7 .1--4.6.7 VTEC have also been isolated from cases of haemolytic uraemic syndrome (HUS), for which bloody diarrhoea can be a
prodromal symptom.ll.12 VT production is bacteriophage determined
in
some
E
coli strainS13.14 and VT genes have been cloned in E coli K12 from phages isolated from wild-type strains.15,16 In our laboratory genes encoding VTl were cloned from a strain of serotype 026.H11, and VT2 genes were cloned from a non-motile (H - ) strain of serogroup 0157. Probes for the detection of VTEC were developed from these cloned genes and DNA hybridisation experiments showed that the VT1 and VT2
probe sequences did not cross-hybridise under conditions.16.17 stringent An outbreak of haemorrhagic colitis occurred in widely separated areas in East Anglia in the summer of 1985.18 A VT + E coli of serotype 0157.H7 was the epidemic strain and was found in 15 of 20 patients with haemorrhagic colitis in the Norwich area.19 During the outbreak we found that faecal specimens from 58% of 43 patients possessed E coli 0157.H7 which produced VT1 and VT2. A prospective study was thus conducted in England and Wales to evaluate the role of VTEC in haemorrhagic colitis. Since October, 1985, faecal samples from cases of HC have been examined for the presence of VTEC with a DNA probe technique. It has been reported that, with one exception, VTEC strains of serogroup 0157 do not ferment sorbit0120 whereas 81 % of E coli strains are sorbitol fermenters.21 This has led to the development of a MacConkey-sorbitol agar to screen for E coli of serogroup 0157. We have compared the DNA probe method with the MacConkey-sorbitol agar technique in the investigation of cases of haemorrhagic colitis in England and Wales for 1 year. Methods For this study haemorrhagic colitis was defmed as an acute episode of diarrhoea associated with frank blood in the stools and from which Salmonella, Shigella, or Campylobacter was not isolated. Recent travel abroad or the use of antibiotics before the specimen was taken were criteria for exclusion from this study. Only faecal samples which had been taken within 14 days of onset of symptoms were included. A request for samples was made in Communicable Disease Report, circulated to all public health and hospital laboratories in England and Wales. The samples were received from 48 laboratories during the year October, 1985, to October, 1986, and the clinical details were provided on a standard questionnaire. Faecal specimens were suspended in an equal volume of phosphate buffered saline and 01ml samples of ten-fold dilutions were spread on MacConkey agar plates. Plates with several hundred well-separated colonies were replicated on to nylon membranes (’Hybond-N’, Amersham) placed on agar plates. After incubation at 37°C for at least 5 h, the membranes were prepared for hybridisation.16 The DNA probe for the VT1 genes was derived
from E coli strain H19, of serotype 026.Hll, and that for VT2 from E coli strain E32511, of serotype ’0 157.H -. A HindI fragment of 0-75 kb was used as the VT1 probe, and the VT2 probe consisted of a 0 85 kb AvaI-Pstl fragment.16.17 Hybridisation conditions and detection of probe-positive colonies were as reported by Willshaw et al.16 Up to five probe-positive colonies from each sample were identified biochemically24 and serotyped by the method of Gross and Rowe.25 They were also examined for cytotoxin production, by the use of Vero cells, as described previously.26 Faecal samples were plated directly on MacConkey-sorbitol agar (Oxoid MacConkey agar no 3 + 1 % sorbitol). Where possible, five sorbitol non-fermenting colonies were picked and tested by slide agglutination with a specific E coli 0157 antiserum prepared in this
laboratory. All 0157 positive colonies were identified biochemically serotyped.24.25 They were tested for cytotoxin production and hybridisation with the VT probes. All VT + E coli strains were tested for resistance27 to amikacin, ampicillin, ceftazidime, cephaloridine, colomycin, cephalexin, chloramphenicol, furazolidone, gentamicin, kanamycin, mecillinam, nalidixic acid, netilmicin, streptomycin, spectinomycin, sulphathiazole, tetracyclines, tobramycin, and trimethoprim. and
Results Clinical and Epidemiological Features of Haemorrhagic Colitis in England and Wales Between October, 1985, and October, 1986, 89 cases of HC were admitted to this study. Over 80% of the cases occurred between April and October, which suggests a seasonality. The cases, whether associated with VTEC or not, were distributed widely throughout the country and were not noticeably related to urban or rural areas. CLINICAL FEATURES OF PATIENTS WITH HAEMORRHAGIC COLITIS
The main clinical features were similar whether VTEC isolated or not (see accompanying table). There were 3 deaths and from all these 3 patients VTEC were isolated. 2 of the patients who died were aged over 80 years and 1 of these died of renal failure after the development of HUS. The 3rd fatal case was a child aged 2-5years who died only 3 days after the onset of the bloody diarrhoea. Another patient, aged 74 years, had HUS. were
Laboratory Studies Coliforms were isolated from 83 of these 89 patients and in 33 of these 83 samples E coli colonies were obtained that hybridised with one or both of the specific VT probes. Except for colonies from one sample all the probe-positive colonies produced cytotoxin in Vero cell tests. The hybridisation results showed considerable differences in the proportion of probe-positive colonies. In the 32 samples in which VTEC were detected 8 had < 1 % probe-positive colonies, 4 contained between 1 and 10%, and in 20 faecal samples over 10 % of the coliforms hybridised. 30 of the 32 E coli strains that produced VT belonged to serogroup 0157. One of the remaining strains was 091.H— and the other strain did not belong to 0 serogroups 1 to 170 but possessed the H8 flagellar antigen. 26 of the VTEC 0157 strains possessed the H7 flagellar antigen and 4 were non-motile. From the hybridisation results the 0157 strains fell intothree classes; one strain hybridised with the VT1probe only, 16 with the VT2 probe only, and 13 with both probes. The 091.H- strain hybridised with both VTand VT2 probes while the O?H8 isolate was positive with the VT2 probe alone. One faecal sample contained bacteria that hybridised with the VT1 probe only but were negative in
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for cytotoxin production; the VT - strain belonged to serotype O?H25. 3 of the 30 VT + strains of serogroup 0157 were resistant to antimicrobial drugs. One strain was resistant to ampicillin (2000 g/m1), streptomycin (500 g/m1), and sulphathiazole ( > 2000 g/m1), another was resistant to streptomycin (1000 tg/ml) and sulphathiazole ( > 2000 g/ml), and the third was resistant to streptomycin alone (250 g/m1). The remaining VT + E coli were sensitive to all antimicrobials tested. Vero cell
tests
difficult to detect a small number of sorbitol non-fermenters plates with a large number of sorbitol fermenters. Another limitation of the use of sorbitol agar is that VTEC strains may belong to serogroups other than 0157 and ferment sorbitol, including the 091.H- and O?H8 strains identified in our survey. In a survey of faecal samples from HUS cases we have detected VT + E coli belonging to 10 serogroups other than 0157 and all these strains fermented sorbitol (ref 29 and unpublished data). It should be recognised that MacConkey-sorbitol agar has limitations as a screening technique for VTEC. During the study period we examined faecal specimens from a group of 9 travellers who had recently returned from a hotel in Ibiza. 4 had diarrhoea or bloody diarrhoea. DNA probes showed VTEC in 3 out of 4 cases and 3 out of 5 who were symptomless. The VT + strains were all 0157.H7 and produced VT2 only. Although in our present study subjects with a history of recent travel abroad were excluded, this incident indicates-that VTEC should be considered a cause of travellers’ diarrhoea. We conclude that VTEC are an important cause of haemorrhagic colitis in England and Wales, as is the case in North America. This preliminary study needs to be extended to identify the sources and pathways of infection, and a prospective case-control study is indicated.
on
’
Use
of MacConkey-sorbitol Agar During this study we examined faecal samples from 51 of the patients by direct plating on MacConkey-sorbitol agar. 28 of the 51 faecal samples contained bacteria that did not ferment sorbitol, and in 11 of these some of the colonies agglutinated with the 0157 antiserum. Further tests confirmed that the colonies that agglutinated with the 0157 antiserum were VTEC. The 51 samples described above were also tested with the VT probes and in 17 cases probe-positive colonies were identified. The 11 specimens in which 0157 sorbitol non-fermenting colonies were identified were also positive in probe tests; the percentage of probe-positive colonies in these 11 specimens ranged from 13 to 100%. Thus there were 6 cases in which VTEC were detected by probes but not by plating on MacConkeysorbitol agar followed by testing with a specific 0157 antiserum. 4 of these 6 samples contained 0157 VTEC which were sorbitol non-fermenters; the percentage of probe-positive colonies in these 4 samples ranged from to 8%. The VTEC in the other 2 samples were <1% 091.H- and O?H8, and these fermented sorbitol.
Discussion This study of haemorrhagic colitis in England and Wales from October, 1985, to October, 1986, showed that in 32 (39%) of the 83 samples that grew coliforms Vero cytotoxinproducing E coli could be identified by hybridisation with specific VT probes. All but 2 of the VTEC belonged to serogroup 0157; the remaining strains were serotypes 091.H and O?H8. The spread of a single strain, or clone, of E coli 0157 does not seem likely since the VT+ 0157 strains could be distinguished from each other by the types of VT produced. The strains of 0157 from 13 cases of HC produced both VTland VT2 but in other cases the strains produced only VT1 or VT2, which suggests that both cytotoxins may be virulence factors. VTEC in faecal samples may be detected by a number of different techniques.7,20,28 We have used DNA probes, which permits examination of a large number of individual colonies, and demonstrated that in 12 of 32 samples there were less than 10% VTEC. Detection of faecal VT has been used in studies of VTEC disease,11,28,29 including HUS. In one HUS study29 faecal VT was detected in 3 samples when no VTEC were identified by probes. Therefore, it is preferable when possible to test for the presence of faecal VT as well as to identify VTEC by the use of DNA probes. MacConkey-sorbitol agar is a useful screening method for VTEC of serogroup 0157 in HC but it requires a specific antiserum for serogroup 0157. In our survey the sensitivity of this MacConkey-sorbitol agar method, compared with DNA probes, was 73% but the specificity was only 39% since sorbitol non-fermenters that were not E coli 0 157 were detected in 17 of 28 samples. In this survey, 4 samples that contained VT + E coli 0157 were negative in tests using MacConkey-sorbitol agar and the 0157 antiserum. It can be ,
We thank
colleagues who sent specimens and information for this study. REFERENCES
Riley LW, Remis RS, Helgerson SD, et al. Hemorrhagic colitis associated with a rare Escherichia coli serotype. N Engl J Med 1983; 308: 681-85. 2. Johnson WM, Lior H, Bezanson GS. Cytotoxic Echerichia coli O157:H7 associated with haemorrhagic colitis m Canada. Lancet 1983; i: 76. 3. Pai CH, Gordon R, Sims HV, Bryan LE. Sporadic cases of hemorrhagic colitis associated with Escherichia coli O157:H7. Clinical, epidemiologic, and bacteriologic features. Ann Intern Med 1984; 101: 738-42. 4. Remis RS, MacDonald KL, Riley LW, et al. Sporadic cases of hemorrhagic colitis associated with Escherichia coli 0157:H7. Ann Intern Med 1984; 101: 624-26. 5. Itoh T, Kai A, Saito K, et al. Epidemiological and laboratory investigation of an outbreak of acute enteritis associated with cytotoxin-producing Escherichia coli O145:H-. Ann Rep Tokyo Metr Res Lab PH 1985; 36: 16-22. 6. Borczyk AA, Karmali MA, Lior H, Duncan LMC. Bovine reservoir for verotoxinproducing Escherichia colt O157:H7. Lancet 1987; i: 98. 7. Wells JG, Davis BR, Wachsmuth IK, et al. Laboratory investigation of haemorrhagic colitis outbreaks associated with a rare Escherichia coli serotype. J Clin Microbiol 1983; 18: 512-20. 8. Konowalchuk J, Spiers JI, Stavric S. Vero response to a cytotoxin of Escherichia coli. Infect Immun 1977; 18: 775-79. 9. O’Brien AD, Lively IA, Chen ME, Rothman SW, Formal SB. Escherichia coli O157:H7 strains associated with haemorrhagic colitis in the United States produce a Shigella dysenteriae 1 (Shiga) like cytotoxin. Lancet 1983; i: 702. 10. Scotland SM, Smith HR, Rowe B. Two distinct toxins active on Vero cells from Escherichia coli O157. Lancet 1985; ii: 885-86. 11. Karmali MA, Steele BT, Petric M. Lim C. Sporadic cases of haemolyticuraemic syndrome associated with faecal cytotoxin and cytotoxin-producing Escherichia coli in stools. Lancet 1983; i: 619-20. 12. Taylor CM, White RHR, Winterbom MH, Rowe B. Haemolytic-uraemic syndrome. clinical experience of an outbreak in the East Midlands. Br Med J 1986; 292: 1513-16. 13. Scotland SM, Smith HR, Willshaw GA, Rowe B. Vero cytotoxin production in strain of Escherichia coli is determined by genes carried on bacteriophage. Lancet 1983; ii: 216. 14. Smith HW, Green P, Parsell Z. Vero cell toxins in Escherichia coli and related bacteria: transfer by phage and conjugation and toxic action in laboratory animals, chickens and pigs. J Gen Microbiol 1983; 129: 3121-37. 15. Newland JW, Strockbine NA, Miller SF, O’Brien AD, Holmes RK. Cloning of Shiga-like toxin structural genes from a toxin converting phage of Escherichia coli. Science 1985; 230: 179-81. 16. Willshaw GA, Smith HR, Scotland SM, Rowe B. Cloning of genes determining the production of Vero cytotoxin by Escherichia coli. J Gen Microbiol 1985; 131: 3047-53. 17. Willshaw GA, Smith HR, Scotland SM, Field AM, Rowe B. Heterogeneity of Eschenchia coli phages encoding Vero cytotoxins: comparison of cloned sequences determining VT1 and VT2 and development of specific gene probes. J Gen Microbiol (in press). 18. Communicable Disease Report 1985; 85/28. 19. Allen J, Coe O. Escherichia coli O157:H7 haemorrhagic colitis associated with salad vegetables. Med Lab Sci 1986; 43: S52. 20. Farmer JJ, Davis BR. H7 antiserum-sorbitol fermentation medium a single tube screening medium for detecting Escherichia colt 0157:H7 associated with haemorrhagic colitis. J Clin Microbiol 1985; 22: 620-25.
1.
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Reviews of Books Advances in Viral Oncology Vol 6, Experimental Approaches to Multifactorial Interactions in Tumor Development ; vol 7, Analysis of Multistep Scenarios in the Natural History of Human or Animal Cancer. Edited by George Klein. New York: Raven. 1987. Pp 174 and 217.$72.50 and $79.50. ISBN 0-881671908 and 0-881671916.
MOST
compilations appearing at regular intervals under
such convenient titles as Recent Advances or Current Trends are heterogeneous in subject matter, style, and quality. The best of them attract authoritative contributions and are eagerly awaited as publication dates approach, though most readers expect to find in any given issue only one or two articles of specific interest to themselves. These two volumes bear little relation to that stereotype. Published simultaneously (presumably to compensate for a gap in the series since 1985) they represent highly rewarding cover-to-cover reading for anyone interested in the molecular basis of malignancy. The link with "viral oncology", of course, is the historical fact that oncogenes were first discovered in RNA tumour viruses, but the emphasis has moved rapidly (and, one suspects, irreversibly) to the malignant cell and alterations in its genome, whether mediated by virus or not. Firm editing shows in the logical assembly of review articles spanning both volumes. A modicum of repetition is retained and
actually serves to emphasise the links between chapters. Thus the different rearrangements giving. rise to a deregulated c-myc gene are covered three times, once in the context of chromosome translocation involving 8q24 in human B cell tumours (Croce, Erikson, Tsujimoto, and Nowell), again in relation to the development of mouse plasmacytomas (Potter), and thirdly in the course of a lively and informative debate between Lenoir and Bornkamm, on the one side, and Klein on the other, about the place of the c-myc rearrangements in the natural history of Burkitt’s lymphoma. At each mention, a different perspective is given on the same topic. The same is true, for example, of the role of the polyoma T antigens in oncogenicity which is covered from two different viewpoints by Ruley ("oncogene complementation") and by Cuzin and Meneguzzi
("stepwise transformation"). In sum, the two volumes probably represent the most comprehensive yet succinct collection currently available of second-generation reviews on the subject of oncogenes; "second generation" because the authors are now past the point of recording phenomena for their own sake and are much more concerned with interpretation of mechanisms, causes, and effects. The two chapters just cited deal with complementation, or positive cooperation, between oncogenes while three more (by Standbridge, by Bassin and Noda, and by Sachs) cover cellular control mechanisms that
21. Ewing WH. Edwards and Ewing’s identification of Enterobacteriaceae. 4th ed. New York: Elsevier, 1986. 22. Harris AA, Kaplan RL, Goodman LJ, et al. Results of a screening method used m a 12-month stool survey for Escherichia coli O157:H7. J Infect Dis 1985, 152: 775-77. 23. March SB, Ratnam S. Sorbitol-MacConkey medium for detection of Escherichia coh associated with haemorrhagic colitis. J Clin Microbiol 1986; 23: 869-72 24. Cowan ST, ed. Cowan and Steel’s manual for the identification of medical bacteria. Cambridge: Cambridge University Press, 1974. 25. Gross RJ, Rowe B. Serotypmg of Escherichia coli. In: Sussman M, ed. The virulence of Escherichia coli. Reviews and methods. London: Academic Press, 1985: 345-63. 26. Scotland SM, Day NP, Rowe B. Production of a cytotoxin affecting Vero cells by
may counteract either the expression of oncogenes or the consequences of that expression. Other contributions discuss the various ways in which cellular oncogenes may become activated and/or amplified and the role of oncogenes
in tumour progression and metastasis. There is not a single redundant article-indeed, scarcely a single redundant sentence-in the whole collection. The balance between discussion of established experimental findings and speculation about future directions for research is exactly right. The editor and contributors are to be congratulated on a publication that is timely, authoritative, and readable. MRC Clinical and Population Western General Hospital,
Edinburgh
Cytogenetics Unit, C. M. STEEL
Therapeutic Controversies in the Rheumatic Diseases Edited by Robert F. Willkens and Stephen L. Dahl. Orlando: Grune & Stratton. 1986. Pp 399.$47.50. ISBN 0 8089 1834 6.
IN their preface to this volume the editors argue that the collected essays will not replace standard texts but will supplement them by providing informal dialogue on contentious therapeutic problems. Many of the 22 chapters by 20 American contributors are arranged in pairs, with one rheumatologist arguing for a particular drug strategy and a second arguing against it. Despite the editors’ anxieties, there is enough material to provide a comprehensive, if somewhat simplified, primer on drug management of arthritis in the USA. Accounts of the use of standard drugs are adequately referenced and written in innovative style--eg, the chapter on analgesics includes a section on the psychotropic modulation of pain. The choice of drugs reflects FDA guidelines rather than the British tendency to explore drugs already in the pharmacopoeia for other purposes. In the treatment of rheumatoid arthritis, auranofin and cyclosporin are reviewed whereas sulphasalazine, dapsone, and phenytoin are not. Dimethyl sulphoxide and the political ramifications of its use are described in detail, and a coloured chart identifies all antirheumatic tablets available in the USA. The discussion chapters appealed to me most, with intriguing insight into styles of American care. Old chestnuts-when to treat symptomless hyperuricaemia; analgesics or anti-inflammatory agents for osteoarthritis; the case for and against steroids in rheumatoid arthritis-are soundly reviewed. The argument for combination cytotoxic therapy in a non-fatal disease spans eleven pages; singleagent cytotoxic therapy has to be content with four. Against this aggressive therapeutic background, the arguments for and against the availability of OTC (over the counter) ibuprofen sold to the public at barely therapeutic doses provide a striking contrast. Pharmacists now describe this as a BTC (behind the counter) drug: they face the risk of litigation on two fronts if they decide to keep it in a back
of Escherichia coli belonging to traditional enteropathogenic serogroups. FEMS Microbiol Lett 1980; 7: 15-17. 27 Anderson ES, Threlfall EJ. The characterisation of plasmids in the Enterobacteria. strains
J Hyg 1974; 72: 471-87. 28. Karmali MA, Petric M, Lim C, Cheung R, Arbus GS. 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 1985; 22: 614-19. 29. Scotland SM, Rowe B, Smith HR, Willshaw GA, Gross RJ. Vero cytotoxinproducing strains of Escherichia coli from children with haemolytic uraemic syndrome and their detection by specific DNA probes. J Med Microbiol (in press).
Case D had classical type 1 GSD with complete absence of glucose-6-phophatase itself. Although he had exhibited all the symptoms of classical type la GSD since at least the age of 7 years, this condition had remained undiagnosed until his final admission with primary liver cell carcinoma. Hepatocellular carcinoma often develops in patients with classical type 1 GSD,32 although the reason for this association is unclear. The establishment of the diagnosis of GSD cannot be made with any certainty unless enzymes are assayed in fresh (unfrozen) tissue samples.2O In classical type la GSD, the clinical findings and biochemical screening procedures that have been worked out for childrenll.33 can also indicate GSD in adults, and justify liver biopsy to establish the nature of the underlying cause. In contrast the much milder clinical symptoms of our cases A-C had not previously suggested GSD and, without liver biopsy, they would have remained undiagnosed. The fact that we have diagnosed four such patients suggests that defects which make the hepatic glucose-6-phosphatase system function at a reduced level are more common than was thought. Adult type 1 GSD should therefore be considered in patients with unresolved hypoglycaemic symptoms and/or unresolved
hepatomegaly. We thank Miss L. Stewart for technical assistance, Ms M. Hughes for typing, and Dr D. Walsh for arranging the lactate measurements. This work was supported by grants from the Scottish Hospitals Endowment Research Trust, the Scottish Home and Health Departments, and the British Diabetic Association
to
A.B.
Correspondence should be addressed to A. B., Department of Medicine, Ninewells Hospital and Medical School, Dundee DD1 9SY. REFERENCES 1. Cori GT, Cori CF. Glucose-6-phosphatase of the liver m glycogen storage disease. J Biol Chem 1952; 199: 661-67. 2. Howell RR, Ashton DM, Wyngaarden JB. Glucose-6-phosphatase deficiency glycogen storage disease. Pediatrics 1962; 29: 553-65. 3. Howell RR, Williams JC. The glycogen storage diseases. In: Stanbury JA, Wyngaarden JB, Frederickson DS, Goldstein JL, Brown MS, eds. The metabolic basis of inherited disease. New York: McGraw Hill, 1983: 141-66. 4. Cornblath M, Schwartz R. Disorders of glycogen metabolism. In: Schaffer AJ, Markowitz M, eds. Disorders of carbohydrate metabolism in infancy. Philadelphia: W. R. Saunders & Co., 1976: 231-93. 5. Arion WJ, Wallin BK, Lange AJ, Ballas LM. On the involvement of a glucose-6-phosphate transport system in the function of microsomal glucose-6phosphatase. Mol Cell Biochem 1975; 6: 75-83. 6. Arion WJ, Ballas LM, Lange AJ, Wallin BK. Microsomal membrane permeability and the hepatic glucose-6-phosphatase system. J Biol Chem 1976; 251: 4901-07. 7. Arion WJ, Lange AJ, Walls HE, Ballas LM. Evidence of the participation of independent translocases for phosphate and glucose-6-phospate in the microsomal glucose-6-phosphatase system. J Biol Chem 1980; 225: 10396-406. 8. Lange AJ, Arion WJ, Beaudet AL. Type 1B glycogen storage disease is caued by a defect in the glucose-6-phosphate translocase of the microsomal glucose-6Chem 1980; 225: 8381-84. J phosphatase system. Biol 9. Nordlie RC, Sukalski K, Munoz JM, Baldwin JJ. Type IC, a novel glycogenosis. J Biol Chem 1983; 258: 9739-44. 10. Schaub J, Heyne K. Glycogen storage disease type 1B. Eur J Pediatr 1983; 140: 283-88. 11. Huijing F, Van de Kamer JH. A screening method for liver glycogen disease. Arch Dis Child 1969; 44: 311-17. 12. Arion WJ, Wallin BK. Kinetics of the glucose-6-phosphate-glucose exchange activity and glucose inhibition of glucose-6-phosphatase of intact and disrupted rat liver microsomes. J Biol Chem 1973; 248: 2373-79. 13. Bickerstaff GF, Burchell B. Studies on the purification of glucose-6-phosphatase from rabbit liver microsomal fraction. Biochem Soc Trans 1980; 8: 389-90. 14. Burchell A, Burchell B. Stabilisation of partially purified glucose-6-phosphatase by fluoride: Is enzyme inactivation caused by dephosphory lation. FEBS Lett 1980; 113: 180-84. 15. Arion WJ, Walls HE. The importance of membrane integrity in kinetic characerizations of the microsomal glucose-6-phosphatase system. J Biol Chem 1982; 257: 11217-20. 16. Arion WJ, Lange AJ, Walls HE. Microsomal membrane integrity and the interactions of phlorizin with the glucose-6-phosphatase system. J Biol Chem 1980; 225: 10387-95. 17. Peterson GL. A simplification of the protein assay method of Lowry et al which is more generally applicable. Anal Biochem 1977; 83: 346-56. 18. Van Handel E. Estimation of glycogen in small amounts of tissue. Anal Biochem 1965; 11: 256-65. 19. Huijing F. A rapid enzyme method for glycogen estimation in very small tissue samples. Clin Chim Acta 1970; 30: 567-72.
HAEMORRHAGIC COLITIS AND VEROCYTOTOXIN-PRODUCING ESCHERICHIA COLI IN ENGLAND AND WALES H. R. SMITH R. J. GROSS
B. ROWE N. K. FRY
S. M. SCOTLAND Division of Enteric Pathogens, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT
Vero-cytotoxin-producing strains of Escherichia coli (VTEC) were identified by the use of DNA probes in 39% of faecal samples from patients with haemorrhagic colitis in England and Wales. The patients with VTEC were distributed widely and their ages ranged from 2·5 to 86 years (mean 41). 3 patients died, including a child of 2·5 years. 30 of the 32 VTEC strains belonged to serogroup 0157. Plating on sorbitol agar for non-fermenters followed by agglutination with a specific 0157 antiserum was a useful screening method for 0157 Summary
VT + strains. However, it was not as sensitive as the DNA probe technique and did not detect VTEC of other serogroups. Introduction
(HC) is characterised by grossly bloody diarrhoea, usually in the absence of pyrexia but sometimes preceded by watery diarrhoea and often accompanied by abdominal cramps. Outbreaks and sporadic cases of HC have been reported in the United States, Canada, and Japan in the past 4 years.l-6 Two of the HAEMORRHAGIC colitis
outbreaks in the United States were associated with undercooked hamburger meat1,7 and one in Canada with milk.6 The epidemiological studies in North America show that the disease occurs over a wide geographical area and affects all age-groups. Konowalchuk et al8 described a cytotoxin active on Vero cells and produced by certain strains of Escherichia coli; it was termed Vero cytotoxin (VT). VT is clearly distinguishable, biologically and immunologically, from the heat-labile and heat-stable enterotoxins of E coli and has been reported to be closely related to Shiga toxin, produced by strains of Shigella dysenteriae type 1.9 At least two distinct Vero cytotoxins, VT1 and VT2, can be identified in toxin
20.
Spencer Peet J, Norman ME, Lake BD, McNamara J, Patrick AD. Hepatic glycogen 1971; 157: 95-114. storage disease. Quart Med J
21. Sokal
JE, Lowe CU, Sarcione EJ, et al. Studies of glycogen metabolism in liver glycogen storage disease. J Clin Invest 1961; 40: 364-74. 22. Narisawa K, Otoma H, Igarashi Y. Glycogen storage disease Type 1b. Pediatrics 1983; 17: 545-49.
23, Illingworth B. Glycogen storage diseases. J Clin Nutrition 1961; 9: 683-90. 24. Hers HG. Glycogen storage disease. Adv Metab Disorders 1964; 1: 1-44. 25. Stamm WE, Webb DI. Partial deficiency of hepatic glucose-6-phosphatase in an adult patient. Arch Intern Med 1975; 135: 1107-09. 26. Chen Y-T, Comblath M, Sidbury JB. Cornstarch therapy in Type 1 glycogen storage disease. N Engl J Med 1984; 310: 171-75. 27. Burchell A, Cain DI. Rat hepatic microsomal glucose-6-phosphatase protein levels are increased in streptozotocin-induced diabetics. Diabetologia 1985; 28: 852-56. 28. Greenbaum AL, Gumaa KA, McLean P. The distribution of hepatic metabolites and the control of the pathways of carbohydrate metabolism in animals of different dietary and hormonal status. Arch Biochem Biophys 1971; 143: 617-63. 29. Nordlie RC. Fine tuning of blood glucose concentrations. Trends Biochem Sci 1985; 10: 70-75. 30. Okado S, Seino Y, Kodama H, et al. Insulin and glucagon secretion in hepatic glycogenosis. Acta Paediatr Scand 1979; 68: 735-38. 31. Wolf BA, Colca JR, Cowans PG, et al. Glucose-6-phosphate regulates Ca steady state in endoplasmic reticulum of islets. A possible link in glucose-induced Secretion. J Biol Chem 1986; 261: 16284-87. 32. Howell RR, Stevenson RE, Ben-Menachem Y, et al. Hepatic adenomata with type 1 glycogen storage disease. JAMA 1976; 236: 1481-84. 33. Femandes J, Koster JF, Crose WFA, Sargedrager N. Hepatic phosphorylase deficiency: Its differentiation from other glycogenoses. Arch Dis Child 1974; 49: 186-91.
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neutralisation experiments.10 Vero-cytotoxin-producing strains of E coli (VTEC) have been associated with the cases of haemorrhagic colitis described above, the commonest serotype of these VTEC strains being 0157 .H7 .1--4.6.7 VTEC have also been isolated from cases of haemolytic uraemic syndrome (HUS), for which bloody diarrhoea can be a
prodromal symptom.ll.12 VT production is bacteriophage determined
in
some
E
coli strainS13.14 and VT genes have been cloned in E coli K12 from phages isolated from wild-type strains.15,16 In our laboratory genes encoding VTl were cloned from a strain of serotype 026.H11, and VT2 genes were cloned from a non-motile (H - ) strain of serogroup 0157. Probes for the detection of VTEC were developed from these cloned genes and DNA hybridisation experiments showed that the VT1 and VT2
probe sequences did not cross-hybridise under conditions.16.17 stringent An outbreak of haemorrhagic colitis occurred in widely separated areas in East Anglia in the summer of 1985.18 A VT + E coli of serotype 0157.H7 was the epidemic strain and was found in 15 of 20 patients with haemorrhagic colitis in the Norwich area.19 During the outbreak we found that faecal specimens from 58% of 43 patients possessed E coli 0157.H7 which produced VT1 and VT2. A prospective study was thus conducted in England and Wales to evaluate the role of VTEC in haemorrhagic colitis. Since October, 1985, faecal samples from cases of HC have been examined for the presence of VTEC with a DNA probe technique. It has been reported that, with one exception, VTEC strains of serogroup 0157 do not ferment sorbit0120 whereas 81 % of E coli strains are sorbitol fermenters.21 This has led to the development of a MacConkey-sorbitol agar to screen for E coli of serogroup 0157. We have compared the DNA probe method with the MacConkey-sorbitol agar technique in the investigation of cases of haemorrhagic colitis in England and Wales for 1 year. Methods For this study haemorrhagic colitis was defmed as an acute episode of diarrhoea associated with frank blood in the stools and from which Salmonella, Shigella, or Campylobacter was not isolated. Recent travel abroad or the use of antibiotics before the specimen was taken were criteria for exclusion from this study. Only faecal samples which had been taken within 14 days of onset of symptoms were included. A request for samples was made in Communicable Disease Report, circulated to all public health and hospital laboratories in England and Wales. The samples were received from 48 laboratories during the year October, 1985, to October, 1986, and the clinical details were provided on a standard questionnaire. Faecal specimens were suspended in an equal volume of phosphate buffered saline and 01ml samples of ten-fold dilutions were spread on MacConkey agar plates. Plates with several hundred well-separated colonies were replicated on to nylon membranes (’Hybond-N’, Amersham) placed on agar plates. After incubation at 37°C for at least 5 h, the membranes were prepared for hybridisation.16 The DNA probe for the VT1 genes was derived
from E coli strain H19, of serotype 026.Hll, and that for VT2 from E coli strain E32511, of serotype ’0 157.H -. A HindI fragment of 0-75 kb was used as the VT1 probe, and the VT2 probe consisted of a 0 85 kb AvaI-Pstl fragment.16.17 Hybridisation conditions and detection of probe-positive colonies were as reported by Willshaw et al.16 Up to five probe-positive colonies from each sample were identified biochemically24 and serotyped by the method of Gross and Rowe.25 They were also examined for cytotoxin production, by the use of Vero cells, as described previously.26 Faecal samples were plated directly on MacConkey-sorbitol agar (Oxoid MacConkey agar no 3 + 1 % sorbitol). Where possible, five sorbitol non-fermenting colonies were picked and tested by slide agglutination with a specific E coli 0157 antiserum prepared in this
laboratory. All 0157 positive colonies were identified biochemically serotyped.24.25 They were tested for cytotoxin production and hybridisation with the VT probes. All VT + E coli strains were tested for resistance27 to amikacin, ampicillin, ceftazidime, cephaloridine, colomycin, cephalexin, chloramphenicol, furazolidone, gentamicin, kanamycin, mecillinam, nalidixic acid, netilmicin, streptomycin, spectinomycin, sulphathiazole, tetracyclines, tobramycin, and trimethoprim. and
Results Clinical and Epidemiological Features of Haemorrhagic Colitis in England and Wales Between October, 1985, and October, 1986, 89 cases of HC were admitted to this study. Over 80% of the cases occurred between April and October, which suggests a seasonality. The cases, whether associated with VTEC or not, were distributed widely throughout the country and were not noticeably related to urban or rural areas. CLINICAL FEATURES OF PATIENTS WITH HAEMORRHAGIC COLITIS
The main clinical features were similar whether VTEC isolated or not (see accompanying table). There were 3 deaths and from all these 3 patients VTEC were isolated. 2 of the patients who died were aged over 80 years and 1 of these died of renal failure after the development of HUS. The 3rd fatal case was a child aged 2-5years who died only 3 days after the onset of the bloody diarrhoea. Another patient, aged 74 years, had HUS. were
Laboratory Studies Coliforms were isolated from 83 of these 89 patients and in 33 of these 83 samples E coli colonies were obtained that hybridised with one or both of the specific VT probes. Except for colonies from one sample all the probe-positive colonies produced cytotoxin in Vero cell tests. The hybridisation results showed considerable differences in the proportion of probe-positive colonies. In the 32 samples in which VTEC were detected 8 had < 1 % probe-positive colonies, 4 contained between 1 and 10%, and in 20 faecal samples over 10 % of the coliforms hybridised. 30 of the 32 E coli strains that produced VT belonged to serogroup 0157. One of the remaining strains was 091.H— and the other strain did not belong to 0 serogroups 1 to 170 but possessed the H8 flagellar antigen. 26 of the VTEC 0157 strains possessed the H7 flagellar antigen and 4 were non-motile. From the hybridisation results the 0157 strains fell intothree classes; one strain hybridised with the VT1probe only, 16 with the VT2 probe only, and 13 with both probes. The 091.H- strain hybridised with both VTand VT2 probes while the O?H8 isolate was positive with the VT2 probe alone. One faecal sample contained bacteria that hybridised with the VT1 probe only but were negative in
1064
for cytotoxin production; the VT - strain belonged to serotype O?H25. 3 of the 30 VT + strains of serogroup 0157 were resistant to antimicrobial drugs. One strain was resistant to ampicillin (2000 g/m1), streptomycin (500 g/m1), and sulphathiazole ( > 2000 g/m1), another was resistant to streptomycin (1000 tg/ml) and sulphathiazole ( > 2000 g/ml), and the third was resistant to streptomycin alone (250 g/m1). The remaining VT + E coli were sensitive to all antimicrobials tested. Vero cell
tests
difficult to detect a small number of sorbitol non-fermenters plates with a large number of sorbitol fermenters. Another limitation of the use of sorbitol agar is that VTEC strains may belong to serogroups other than 0157 and ferment sorbitol, including the 091.H- and O?H8 strains identified in our survey. In a survey of faecal samples from HUS cases we have detected VT + E coli belonging to 10 serogroups other than 0157 and all these strains fermented sorbitol (ref 29 and unpublished data). It should be recognised that MacConkey-sorbitol agar has limitations as a screening technique for VTEC. During the study period we examined faecal specimens from a group of 9 travellers who had recently returned from a hotel in Ibiza. 4 had diarrhoea or bloody diarrhoea. DNA probes showed VTEC in 3 out of 4 cases and 3 out of 5 who were symptomless. The VT + strains were all 0157.H7 and produced VT2 only. Although in our present study subjects with a history of recent travel abroad were excluded, this incident indicates-that VTEC should be considered a cause of travellers’ diarrhoea. We conclude that VTEC are an important cause of haemorrhagic colitis in England and Wales, as is the case in North America. This preliminary study needs to be extended to identify the sources and pathways of infection, and a prospective case-control study is indicated.
on
’
Use
of MacConkey-sorbitol Agar During this study we examined faecal samples from 51 of the patients by direct plating on MacConkey-sorbitol agar. 28 of the 51 faecal samples contained bacteria that did not ferment sorbitol, and in 11 of these some of the colonies agglutinated with the 0157 antiserum. Further tests confirmed that the colonies that agglutinated with the 0157 antiserum were VTEC. The 51 samples described above were also tested with the VT probes and in 17 cases probe-positive colonies were identified. The 11 specimens in which 0157 sorbitol non-fermenting colonies were identified were also positive in probe tests; the percentage of probe-positive colonies in these 11 specimens ranged from 13 to 100%. Thus there were 6 cases in which VTEC were detected by probes but not by plating on MacConkeysorbitol agar followed by testing with a specific 0157 antiserum. 4 of these 6 samples contained 0157 VTEC which were sorbitol non-fermenters; the percentage of probe-positive colonies in these 4 samples ranged from to 8%. The VTEC in the other 2 samples were <1% 091.H- and O?H8, and these fermented sorbitol.
Discussion This study of haemorrhagic colitis in England and Wales from October, 1985, to October, 1986, showed that in 32 (39%) of the 83 samples that grew coliforms Vero cytotoxinproducing E coli could be identified by hybridisation with specific VT probes. All but 2 of the VTEC belonged to serogroup 0157; the remaining strains were serotypes 091.H and O?H8. The spread of a single strain, or clone, of E coli 0157 does not seem likely since the VT+ 0157 strains could be distinguished from each other by the types of VT produced. The strains of 0157 from 13 cases of HC produced both VTland VT2 but in other cases the strains produced only VT1 or VT2, which suggests that both cytotoxins may be virulence factors. VTEC in faecal samples may be detected by a number of different techniques.7,20,28 We have used DNA probes, which permits examination of a large number of individual colonies, and demonstrated that in 12 of 32 samples there were less than 10% VTEC. Detection of faecal VT has been used in studies of VTEC disease,11,28,29 including HUS. In one HUS study29 faecal VT was detected in 3 samples when no VTEC were identified by probes. Therefore, it is preferable when possible to test for the presence of faecal VT as well as to identify VTEC by the use of DNA probes. MacConkey-sorbitol agar is a useful screening method for VTEC of serogroup 0157 in HC but it requires a specific antiserum for serogroup 0157. In our survey the sensitivity of this MacConkey-sorbitol agar method, compared with DNA probes, was 73% but the specificity was only 39% since sorbitol non-fermenters that were not E coli 0 157 were detected in 17 of 28 samples. In this survey, 4 samples that contained VT + E coli 0157 were negative in tests using MacConkey-sorbitol agar and the 0157 antiserum. It can be ,
We thank
colleagues who sent specimens and information for this study. REFERENCES
Riley LW, Remis RS, Helgerson SD, et al. Hemorrhagic colitis associated with a rare Escherichia coli serotype. N Engl J Med 1983; 308: 681-85. 2. Johnson WM, Lior H, Bezanson GS. Cytotoxic Echerichia coli O157:H7 associated with haemorrhagic colitis m Canada. Lancet 1983; i: 76. 3. Pai CH, Gordon R, Sims HV, Bryan LE. Sporadic cases of hemorrhagic colitis associated with Escherichia coli O157:H7. Clinical, epidemiologic, and bacteriologic features. Ann Intern Med 1984; 101: 738-42. 4. Remis RS, MacDonald KL, Riley LW, et al. Sporadic cases of hemorrhagic colitis associated with Escherichia coli 0157:H7. Ann Intern Med 1984; 101: 624-26. 5. Itoh T, Kai A, Saito K, et al. Epidemiological and laboratory investigation of an outbreak of acute enteritis associated with cytotoxin-producing Escherichia coli O145:H-. Ann Rep Tokyo Metr Res Lab PH 1985; 36: 16-22. 6. Borczyk AA, Karmali MA, Lior H, Duncan LMC. Bovine reservoir for verotoxinproducing Escherichia colt O157:H7. Lancet 1987; i: 98. 7. Wells JG, Davis BR, Wachsmuth IK, et al. Laboratory investigation of haemorrhagic colitis outbreaks associated with a rare Escherichia coli serotype. J Clin Microbiol 1983; 18: 512-20. 8. Konowalchuk J, Spiers JI, Stavric S. Vero response to a cytotoxin of Escherichia coli. Infect Immun 1977; 18: 775-79. 9. O’Brien AD, Lively IA, Chen ME, Rothman SW, Formal SB. Escherichia coli O157:H7 strains associated with haemorrhagic colitis in the United States produce a Shigella dysenteriae 1 (Shiga) like cytotoxin. Lancet 1983; i: 702. 10. Scotland SM, Smith HR, Rowe B. Two distinct toxins active on Vero cells from Escherichia coli O157. Lancet 1985; ii: 885-86. 11. Karmali MA, Steele BT, Petric M. Lim C. Sporadic cases of haemolyticuraemic syndrome associated with faecal cytotoxin and cytotoxin-producing Escherichia coli in stools. Lancet 1983; i: 619-20. 12. Taylor CM, White RHR, Winterbom MH, Rowe B. Haemolytic-uraemic syndrome. clinical experience of an outbreak in the East Midlands. Br Med J 1986; 292: 1513-16. 13. Scotland SM, Smith HR, Willshaw GA, Rowe B. Vero cytotoxin production in strain of Escherichia coli is determined by genes carried on bacteriophage. Lancet 1983; ii: 216. 14. Smith HW, Green P, Parsell Z. Vero cell toxins in Escherichia coli and related bacteria: transfer by phage and conjugation and toxic action in laboratory animals, chickens and pigs. J Gen Microbiol 1983; 129: 3121-37. 15. Newland JW, Strockbine NA, Miller SF, O’Brien AD, Holmes RK. Cloning of Shiga-like toxin structural genes from a toxin converting phage of Escherichia coli. Science 1985; 230: 179-81. 16. Willshaw GA, Smith HR, Scotland SM, Rowe B. Cloning of genes determining the production of Vero cytotoxin by Escherichia coli. J Gen Microbiol 1985; 131: 3047-53. 17. Willshaw GA, Smith HR, Scotland SM, Field AM, Rowe B. Heterogeneity of Eschenchia coli phages encoding Vero cytotoxins: comparison of cloned sequences determining VT1 and VT2 and development of specific gene probes. J Gen Microbiol (in press). 18. Communicable Disease Report 1985; 85/28. 19. Allen J, Coe O. Escherichia coli O157:H7 haemorrhagic colitis associated with salad vegetables. Med Lab Sci 1986; 43: S52. 20. Farmer JJ, Davis BR. H7 antiserum-sorbitol fermentation medium a single tube screening medium for detecting Escherichia colt 0157:H7 associated with haemorrhagic colitis. J Clin Microbiol 1985; 22: 620-25.
1.
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Reviews of Books Advances in Viral Oncology Vol 6, Experimental Approaches to Multifactorial Interactions in Tumor Development ; vol 7, Analysis of Multistep Scenarios in the Natural History of Human or Animal Cancer. Edited by George Klein. New York: Raven. 1987. Pp 174 and 217.$72.50 and $79.50. ISBN 0-881671908 and 0-881671916.
MOST
compilations appearing at regular intervals under
such convenient titles as Recent Advances or Current Trends are heterogeneous in subject matter, style, and quality. The best of them attract authoritative contributions and are eagerly awaited as publication dates approach, though most readers expect to find in any given issue only one or two articles of specific interest to themselves. These two volumes bear little relation to that stereotype. Published simultaneously (presumably to compensate for a gap in the series since 1985) they represent highly rewarding cover-to-cover reading for anyone interested in the molecular basis of malignancy. The link with "viral oncology", of course, is the historical fact that oncogenes were first discovered in RNA tumour viruses, but the emphasis has moved rapidly (and, one suspects, irreversibly) to the malignant cell and alterations in its genome, whether mediated by virus or not. Firm editing shows in the logical assembly of review articles spanning both volumes. A modicum of repetition is retained and
actually serves to emphasise the links between chapters. Thus the different rearrangements giving. rise to a deregulated c-myc gene are covered three times, once in the context of chromosome translocation involving 8q24 in human B cell tumours (Croce, Erikson, Tsujimoto, and Nowell), again in relation to the development of mouse plasmacytomas (Potter), and thirdly in the course of a lively and informative debate between Lenoir and Bornkamm, on the one side, and Klein on the other, about the place of the c-myc rearrangements in the natural history of Burkitt’s lymphoma. At each mention, a different perspective is given on the same topic. The same is true, for example, of the role of the polyoma T antigens in oncogenicity which is covered from two different viewpoints by Ruley ("oncogene complementation") and by Cuzin and Meneguzzi
("stepwise transformation"). In sum, the two volumes probably represent the most comprehensive yet succinct collection currently available of second-generation reviews on the subject of oncogenes; "second generation" because the authors are now past the point of recording phenomena for their own sake and are much more concerned with interpretation of mechanisms, causes, and effects. The two chapters just cited deal with complementation, or positive cooperation, between oncogenes while three more (by Standbridge, by Bassin and Noda, and by Sachs) cover cellular control mechanisms that
21. Ewing WH. Edwards and Ewing’s identification of Enterobacteriaceae. 4th ed. New York: Elsevier, 1986. 22. Harris AA, Kaplan RL, Goodman LJ, et al. Results of a screening method used m a 12-month stool survey for Escherichia coli O157:H7. J Infect Dis 1985, 152: 775-77. 23. March SB, Ratnam S. Sorbitol-MacConkey medium for detection of Escherichia coh associated with haemorrhagic colitis. J Clin Microbiol 1986; 23: 869-72 24. Cowan ST, ed. Cowan and Steel’s manual for the identification of medical bacteria. Cambridge: Cambridge University Press, 1974. 25. Gross RJ, Rowe B. Serotypmg of Escherichia coli. In: Sussman M, ed. The virulence of Escherichia coli. Reviews and methods. London: Academic Press, 1985: 345-63. 26. Scotland SM, Day NP, Rowe B. Production of a cytotoxin affecting Vero cells by
may counteract either the expression of oncogenes or the consequences of that expression. Other contributions discuss the various ways in which cellular oncogenes may become activated and/or amplified and the role of oncogenes
in tumour progression and metastasis. There is not a single redundant article-indeed, scarcely a single redundant sentence-in the whole collection. The balance between discussion of established experimental findings and speculation about future directions for research is exactly right. The editor and contributors are to be congratulated on a publication that is timely, authoritative, and readable. MRC Clinical and Population Western General Hospital,
Edinburgh
Cytogenetics Unit, C. M. STEEL
Therapeutic Controversies in the Rheumatic Diseases Edited by Robert F. Willkens and Stephen L. Dahl. Orlando: Grune & Stratton. 1986. Pp 399.$47.50. ISBN 0 8089 1834 6.
IN their preface to this volume the editors argue that the collected essays will not replace standard texts but will supplement them by providing informal dialogue on contentious therapeutic problems. Many of the 22 chapters by 20 American contributors are arranged in pairs, with one rheumatologist arguing for a particular drug strategy and a second arguing against it. Despite the editors’ anxieties, there is enough material to provide a comprehensive, if somewhat simplified, primer on drug management of arthritis in the USA. Accounts of the use of standard drugs are adequately referenced and written in innovative style--eg, the chapter on analgesics includes a section on the psychotropic modulation of pain. The choice of drugs reflects FDA guidelines rather than the British tendency to explore drugs already in the pharmacopoeia for other purposes. In the treatment of rheumatoid arthritis, auranofin and cyclosporin are reviewed whereas sulphasalazine, dapsone, and phenytoin are not. Dimethyl sulphoxide and the political ramifications of its use are described in detail, and a coloured chart identifies all antirheumatic tablets available in the USA. The discussion chapters appealed to me most, with intriguing insight into styles of American care. Old chestnuts-when to treat symptomless hyperuricaemia; analgesics or anti-inflammatory agents for osteoarthritis; the case for and against steroids in rheumatoid arthritis-are soundly reviewed. The argument for combination cytotoxic therapy in a non-fatal disease spans eleven pages; singleagent cytotoxic therapy has to be content with four. Against this aggressive therapeutic background, the arguments for and against the availability of OTC (over the counter) ibuprofen sold to the public at barely therapeutic doses provide a striking contrast. Pharmacists now describe this as a BTC (behind the counter) drug: they face the risk of litigation on two fronts if they decide to keep it in a back
of Escherichia coli belonging to traditional enteropathogenic serogroups. FEMS Microbiol Lett 1980; 7: 15-17. 27 Anderson ES, Threlfall EJ. The characterisation of plasmids in the Enterobacteria. strains
J Hyg 1974; 72: 471-87. 28. Karmali MA, Petric M, Lim C, Cheung R, Arbus GS. 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 1985; 22: 614-19. 29. Scotland SM, Rowe B, Smith HR, Willshaw GA, Gross RJ. Vero cytotoxinproducing strains of Escherichia coli from children with haemolytic uraemic syndrome and their detection by specific DNA probes. J Med Microbiol (in press).