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Serological identification of Escherichia coli 0157:H7 infection in haemolytic uraemic syndrome
138
Serological identification of Escherichia coli O157:H7 infection in haemolytic uraemic syndrome
To test the value of serological tests as an adjunct to bacteriological methods and toxin testing in haemolytic uraemic syndrome (HUS), 60 patients with the disorder were examined for evidence of faecal Escherichia coli producing verocytotoxin (VTEC), particularly of serogroup 0157. They were also tested for serum antibodies reacting with the lipopolysaccharide of E coli 0157 by means of an enzyme-linked immunosorbent assay (ELISA) and immunoblotting; for faecal VTEC by means of DNA probes hybridising with the genes encoding verocytotoxins VT1 and VT2; and for "free" faecal VT. Strains of E coli serotype O157:H7 were isolated from 9 patients, and faecal VT2 was detected in 3 of them. Strains of E coli of serotypes O5:H-, O55:H10, O105ac:H18, and O163:H19 were isolated from 4 patients, but faecal VT was not detected. Faecal VT2 was present in 1 patient from whom VTEC were not isolated. Antibodies to the lipopolysaccharide of E coli 0157 were detected in serum samples from 44 patients. The 9 patients with faecal O157:H7 all had high titres of these antibodies, but serum samples from 16 healthy control children were negative. Serological testing of patients with HUS for antibodies to the lipopolysaccharide of E coli O157 provides evidence of infection with E coli O157 when faecal bacteria or VT cannot be detected. Introduction
Haemolytic uraemic syndromes (HUS) are characterised by microangiopathic anaemia and renal failure, arising predominantly in infants and young children. Two subtypes are recognised: the first is common in children and is associated with a diarrhoeal prodrome (D + ), whereas the second is rare in childhood and is not associated with diarrhoea (D -). Various agents have been associated with HUS,2 but since 1983 strains of Escherichia coli producing verocytotoxin (VTEC) have emerged as an important cause of this disease.3 VTEC strains of several serotypes have been isolated from patients with HUS, but the majority of such isolates belong to serotype 0157 :H7.4 VTEC produce two types of verocytotoxin (VT1 and VT2),’ which resemble Shiga toxin in structure and mode of action; however, unlike Shiga toxin the genes for both VTand VT2 are phage encoded.6,7 Detection of VTEC or VT in patients with HUS provides valuable information on the epidemiology of this disease. Gene probes for VT1and VT2 have been used for both detection and characterisation of faecal VTEC,4,6,7 and VT has been detected in culture filtrates and faecal extracts,
Vero cell monolayers.8 In addition, strains of E coli of serotype 0157:H7 metabolise sorbitol only slowly, and the characteristic growth as colourless colonies on sorbitol MacConkey agar9 is a rapid means to distinguish this particular VTEC from other faecal coliforms. The probability of isolating VTEC from patients with HUS depends very much on the time between the onset of symptoms and the examination of faecal specimens. 10 In our collaborative study with the British Association for Paediatric Nephrologists4,10 evidence of VTEC infection was found in only 31 % of HUS cases. Since VTEC and VT can be detected in patients’ stools for only a short time," failure to obtain evidence of VTEC infection hinders the accurate epidemiology of HUS cases, and other methods for obtaining evidence of infection by VTEC have been sought. We have shown that patients infected with E coli 0157:H7 produce serum antibodies to the lipopolysaccharide of this organism12 and that an enzymelinked immunosorbent assay (ELISA), used in conjunction with immunoblotting, may provide evidence of infection when faecal E coli 0 157 or VT cannot be detected. 13 We now report a more extensive study in which 60 patients with HUS were examined for VTEC, faecal VT, and serum antibodies to E coli 0157 lipopolysaccharide to assess fully the value of serology as an adjunct to established bacteriological methods.
by means of
Subjects and methods Blood samples were obtained from 60 patients with HUS (53 part of the previous study1O). There were 57 D + cases and 3 D - cases; the latter had a relapsing illness, and 2 children were known to have a family history of a similar disorder. Blood and faecal samples were obtained in the acute phase, and serum samples taken during convalescence were available for analysis from 22 children. Serum samples from 16 healthy children were used as controls. Faecal samples were examined for coliforms9n’ and for VTEC by means of DNA probes for VT1 and VT2.’ Faecal VT was sought and titrated by means of Vero cell monolayers.11,’s E coli strain E32511 (0157:H -) was isolated from a patient with HUS,12 and stored on Dorset egg slopes at room temperature. For the preparation of lipopolysaccharide, bacteria were grown in trypticase soy broth (BBL Microbiology Systems, Cockeysville, Maryland, USA).12 Lipopolysaccharide was prepared by the hot-phenol procedure." For sodium dodecyl sulphate were
polyacrylamide gel electrophoresis 5 pg purified lipopolysaccharide was applied to a 4-5% stacking gel and a 12-5% separation gelY Electrophoresis was done with a constant current of 50 mA for 3 25 h, and the gels were either stained with silver17-19 or used for ADDRESSES Division of Enteric Pathogens, Central Public Health Laboratory, London (H Chart, PhD, H R Smith, PhD, S M Scotland, PhD, B Rowe, MB) and Department of Nephrology, Children’s Hospital, Birmingham, UK (D V. Milford, MD, C M Taylor, MD). Correspondence to Dr H. Chart, Division of Enteric Pathogens, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK
139
of an alkaline-phosphate-conjugated goat antiserum to total human immunoglobulin (Sigma, diluted to the manufacturer’s specification in phosphate-buffered saline/tween) and pnitrophenol phosphate (1mg/ml, Sigma) in diethanolamine buffer. The intensity of the resultant colour was determined by reading the means
optical density at 405 nm.
Results
immunoblotting. The lipopolysaccharide profiles were transferred
9 children had faecal E coli 0157:H7 and 3 of these also had faecal VT2, with titres of 2500, 2500, and 50 000, respectively. 4 children were infected with VTEC of serotypes 05:H -, O55:H10, 0105ac:H18, and O163:H19, respectively, but faecal VT was not detected in these patients. Faecal VT2 was detected in the stool of 1 patient from whom VTEC were not isolated. DNA probes for VT1 and VT2 gave positive hybridisation results only with faecal samples from which VTEC were also isolated. In the ELISA for antibodies to E coli 0157 lipopolysaccharide, 42 samples had values greater than 0-7 and 10 values below 0-4, including all three D - cases. In our previous studies,12,13 a cutoff value of about 0- 5 separated immunoblot-positive and immunoblot-negative serum samples. The 8 samples with intermediate ELISA values (0-4-0-7) were tested by immunoblotting. 2 of the 8 (ELISA values 0-53 and 0-67) contained serum antibodies reactive with 0157 lipopolysaccharide by immunoblotting (eg, fig 1, lane 1); the other 6 samples (ELISA values 0.4-0’64) did not react with this 0-antigen (fig 1, lane 2). Serum samples taken from 2 children about 1 month after the onset of disease gave ELISA values of 1-01 and 0-53 and both gave positive immunoblots. All of 26 samples with ELISA values greater than 0-7 randomly selected for immunoblotting gave positive antibody reactions. The child with faecal E coli 0163:H19 also had serum antibodies to E coli 0157 by immunoblot and ELISA (1’06). ELISA values for the 16 controls ranged from 0-09 to 0-39 with a mean of 0-24 (SD
to nitrocellulose sheets and reacted with antiserum (30
0’11).
Fig 1-Reaction of patients’ serum with E coli 0157 lipopolysaccharide by immunoblotting. Protein molecular weight standards are given in kD. Lane1 = positive, lane 2 negative reaction. =
IgM
µl/lane)12,13
antibodies binding to lipopolysaccharide antigens were detected by means of iodine-125-labelled immunoglobuliniz,l3 raised against human IgM (Sigma Chemical Co, Poole, Dorset). About 5 Ilg immunoglobulin, containing 101 cpm, was added to each lane and antibody-antigen reactions were detected by
autoradiography. ELI SAs were carried out as previously described.12,13 Plates were coated with 06 µg lipopolysaccharide and reacted with serum samples diluted (x 1000) in phosphate-buffered saline containing 0-5% ’Tween-20’. Antibody-antigen complexes were detected by
Fig 2-Serum antibody reactions with E coli 0157 lipopolysaccharide in initial and subsequent serum samples.
convalescence from 22 used to determine the duration of circulating patients E antibodies to coli 0157 lipopolysaccharide. The antibody titres in 4 patients increased over 16-20 days, whereas in the other 18 patients serum antibodies showed a gradual fall with time (fig 2). In 1 patient, antibodies could be detected up to 74 days after the initial blood sample.
Serum
samples taken during
were
Discussion
Bacteriological methods and toxin testing provided evidence of VTEC involvement in 14 of the 60 children investigated. The 9 children from whom E coli 0157:H7 were cultured had antibody values much higher than the 0,5 cutoff ELISA value12,13 and than the values for control serum samples. Since 35 of the other 51 children also had antibodies to 0 157 lipopolysaccharide, our data suggest that a total of 44 (73%) had been infected with E coli 0157. It is interesting that the D - cases had ELISA values comparable with those of the controls and were negative on immunoblot testing. Screening for antibodies by ELISA alone identified patients with high antibody values and was a simple means of obtaining evidence of infection with E coli 0157. However, our studies suggest that serum samples with ELISA values between 0-4 and 0-7 should also be tested by immunoblotting. 1 child excreted faecal E coli 0163:H19 and yet had antibodies to E coli 0157, so might have been infected with strains of both serogroups concomitantly.
140
Isolation of VTEC from cases of HUS provides definitive evidence of the probable cause of disease; however, our serological tests detected evidence of infection by E coli 0157 in 73% of children with HUS, compared with only 23% by bacteriology and toxin testing alone. VTEC and VT can be detected in stools for only a short time after onset of diarrhoea. Our serological tests can provide evidence of infection for several weeks after the onset of disease. Serology therefore forms a valuable adjunct to established bacteriological techniques. Ongoing studies on serological tests for antibodies to the lipopolysaccharides of other VTEC may provide information on the role of non-0157 VTEC in these infections. We thank members of the British Paediatric Association who supplied serum samples for this study.
REFERENCES C, Gautier E, Steck A, Siebebmann AE, Oechslin R. Hamolytisch-uramische syndrome: bilaterale nierenrindennekrosen bei akutenerworbenen hamolytiscen anamien. Schweiz Med
1. Von Gasser
Wochenschr 1955; 85: 905-09. M, Barratt JM. Haemolytic uraemic syndrome. Arch Dis Child 1984; 59: 397-400. 3. Karmali MA, Petric M, Lim C, Fleming PC, Arbus GS, Lior H. The association between idiopathic hemolytic uremic syndrome and infection by Verotoxin-producing Escherichia coli. J Infect Dis 1985; 151: 775-82. 4. Kleanthous H, Smith HR, Scotland SM, et al. Haemolytic uraemic syndrome in the British Isles, 1985-1988; association with Vero cytotoxin-producing Eschenchia coli Part 2, microbiological aspects. Arch Dis Child 1990; 65: 722-27. 5. Scotland SM, Smith HR, Rowe B. Two distinct toxins active on Vero cells from Eschenchia coli O157. Lancet 1985; ii: 885-86. 6. Smith HR, Day NP, Scotland SM, Gross RJ, Rowe B. Phagedetermined production of verocytotoxin in strains of Eschenchia coli O157. Lancet 1984; i: 1242-43. 7. Willshaw GA, Smith HR, Scotland SM, Field AM, Rowe B. Heterogeneity of Escherichia coli phages encoding Vero cytotoxins: comparison of cloned sequences determining VT1 and VT2 and development of specific gene probes. J Gen Microbiol 1987; 133: 1309-17. 8. Konowalchuk J, Speirs JI, Stavric S. Vero response to a cytotoxin of Escherichia coli. Infect Immun 1978; 18: 775-79. 9. Kleanthous H, Fry NK, Smith HR, Gross RJ, Rowe B. The use of sorbitol MacConkey agar in conjunction with a specific antiserum for the detection of Vero cytotoxin-producing strains of Escherichia coli 0157. Epidemiol Infect 1988; 101: 327-35. 10. Milford DV, Taylor CM, Gutteridge B, Hall S, Rowe B, Kleanthous H. Haemolytic uraemic syndrome in the British Isles, 1985-1988; association with Vero cytotoxin-producing Escherichia coli part 1, clinical and epidemiological aspects. Arch Dis Child 1990; 65: 716-21. 11. Scotland SM, Rowe B, Smith HR, Willshaw GA, Gross RJ. Verocytotoxin-producing strains of Escherichia coli from children with haemolytic uraemic syndrome and their detection by specific DNA probes. J Med Microbiol 1988; 25: 237-43. 12. Chart H, Scotland SM, Rowe B. Serum antibodies to Escherichia coli serotype O157:H7 in patients with hemolytic uremic syndrome. J Clin Microbiol 1989; 27: 285-90. 13. Chart H, Scotland SM, Smith HR, Rowe B. Antibodies to Escherichia coli 0157 in patients with haemorrhagic colitis and haemolytic uraemic syndrome. J Clin Pathol 1989; 42: 973-76. 14. Gross RJ, Rowe B. Serotyping of Escherichia coli. In: Sussman M, ed. The virulence of Escherichia coli. London: Society for General Microbiology, special publication no 13, 1985: 345-63. 15. Scotland SM, Willshaw GA, Smith HR, Rowe B. Properties of strains of Escherichia coli belonging to serogroup 0157 with special reference to production of Vero cytotoxins VT1 and VT2. Epidemiol Infect 1987; 99: 613-24. 16. Westphal O, Jann K. Bacterial lipopolysaccharide: extraction with phenol-water and further applications of the procedure. Meth Carbohydr Chem 1965; 5: 83-91. 17. Wray W, Boulikas T, Wray VP, Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem 1981; 118: 197-203. 18. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-85. 19. Tsai C-M, Frasch CE. A sensitive silver stain for detecting lipopolysaccharide in polyacrylamide gels. Anal Biochem 1982; 119: 2. Levin
115-19.
SHORT REPORTS
Lymphoedema/hypercalcaemia syndrome mediated by
parathyroid-hormone-related protein 24-year-old woman presented with a cryptogenic, hypercalcaemic syndrome with metastatic pulmonary calcification and leg and massive breast enlargement and bilateral chylous pleural effusions compatible with a generalised lymphatic abnormality. The patient’s serum parathyroid hormone-related protein concentration was very high at a time when she was significantly hypercalcaemic, implying that the hypercalcaemia was mediated by this protein. A
Metastatic pulmonary calcification has been described in
hypercalcaemic syndromes, including primary hyperparathyroidism, malignant disease, chronic renal failure, and sarcoidosis. We describe here a patient with steroid-suppressible hypercalcaemia and metastatic pulmonary calcification in whom the cause of the hypercalcaemia was at first uncertain. Serial measurement of serum parathyroid-hormone-related protein (PTHRP) pointed strongly to this peptide as the explanation for the hypercalcaemia. The development of bilateral chylous pleural effusions and gross breast lymphoedema suggested an acquired lymphatic abnormality not previously described in hypercalcaemia. This case reinforces the view1 that PTHRP may mediate non-malignant hypercalcaemia.
most
common
The patient first presented at age 18 with systemic lupus erythematosus, which responded to non-steroidal antiinflammatory agents. She re-presented 4 years later with a 2-month history of nausea, polyuria, and polydipsia. She had hypercalcaemia (3-75 mmol/1) with normal serum phosphate and a serum PTH level below 08 pmol/1 (normal 09-54). A chest X-ray was normal. There was no history of vitamin D abuse or unusual diet. She was given rehydration and oral corticosteroids, and within 10 days the serum calcium was normal. One month later the symptoms recurred with breathlessness on exertion. She had bilateral pleural effusions and her right breast had increased in size. Her serum calcium was 4 22 mmol/1. Rehydration and corticosteroids resolved the hypercalcaemia within a week. For a year she remained normocalcaemic on prednisolone 10 mg daily. The pleural effusions required intermittent aspiration. Pleurodesis was unsuccessful. Moderate hypertension (diastolic blood pressure 110-115 mm Hg) necessitated atenolol and nifedipine.
On referral to this hospital, 15 months after the first of hypercalcaemia, the patient had worsening breathlessness on exertion. Her breasts were much enlarged and lymphoedematous and there were large bilateral pleural effusions. After left talc pleurodesis the oedema worsened and her postoperative course was complicated by hypoxia. Hypercapnic respiratory failure ensued (PaOz 5-6 kPa, PaC02 10-12 kPa). The left pleural effusion did not recur. High-dose diuretic therapy resulted in some resolution of the lymphoedema but no improvement in the breathlessness or blood gases.
episode
Serological identification of Escherichia coli O157:H7 infection in haemolytic uraemic syndrome
To test the value of serological tests as an adjunct to bacteriological methods and toxin testing in haemolytic uraemic syndrome (HUS), 60 patients with the disorder were examined for evidence of faecal Escherichia coli producing verocytotoxin (VTEC), particularly of serogroup 0157. They were also tested for serum antibodies reacting with the lipopolysaccharide of E coli 0157 by means of an enzyme-linked immunosorbent assay (ELISA) and immunoblotting; for faecal VTEC by means of DNA probes hybridising with the genes encoding verocytotoxins VT1 and VT2; and for "free" faecal VT. Strains of E coli serotype O157:H7 were isolated from 9 patients, and faecal VT2 was detected in 3 of them. Strains of E coli of serotypes O5:H-, O55:H10, O105ac:H18, and O163:H19 were isolated from 4 patients, but faecal VT was not detected. Faecal VT2 was present in 1 patient from whom VTEC were not isolated. Antibodies to the lipopolysaccharide of E coli 0157 were detected in serum samples from 44 patients. The 9 patients with faecal O157:H7 all had high titres of these antibodies, but serum samples from 16 healthy control children were negative. Serological testing of patients with HUS for antibodies to the lipopolysaccharide of E coli O157 provides evidence of infection with E coli O157 when faecal bacteria or VT cannot be detected. Introduction
Haemolytic uraemic syndromes (HUS) are characterised by microangiopathic anaemia and renal failure, arising predominantly in infants and young children. Two subtypes are recognised: the first is common in children and is associated with a diarrhoeal prodrome (D + ), whereas the second is rare in childhood and is not associated with diarrhoea (D -). Various agents have been associated with HUS,2 but since 1983 strains of Escherichia coli producing verocytotoxin (VTEC) have emerged as an important cause of this disease.3 VTEC strains of several serotypes have been isolated from patients with HUS, but the majority of such isolates belong to serotype 0157 :H7.4 VTEC produce two types of verocytotoxin (VT1 and VT2),’ which resemble Shiga toxin in structure and mode of action; however, unlike Shiga toxin the genes for both VTand VT2 are phage encoded.6,7 Detection of VTEC or VT in patients with HUS provides valuable information on the epidemiology of this disease. Gene probes for VT1and VT2 have been used for both detection and characterisation of faecal VTEC,4,6,7 and VT has been detected in culture filtrates and faecal extracts,
Vero cell monolayers.8 In addition, strains of E coli of serotype 0157:H7 metabolise sorbitol only slowly, and the characteristic growth as colourless colonies on sorbitol MacConkey agar9 is a rapid means to distinguish this particular VTEC from other faecal coliforms. The probability of isolating VTEC from patients with HUS depends very much on the time between the onset of symptoms and the examination of faecal specimens. 10 In our collaborative study with the British Association for Paediatric Nephrologists4,10 evidence of VTEC infection was found in only 31 % of HUS cases. Since VTEC and VT can be detected in patients’ stools for only a short time," failure to obtain evidence of VTEC infection hinders the accurate epidemiology of HUS cases, and other methods for obtaining evidence of infection by VTEC have been sought. We have shown that patients infected with E coli 0157:H7 produce serum antibodies to the lipopolysaccharide of this organism12 and that an enzymelinked immunosorbent assay (ELISA), used in conjunction with immunoblotting, may provide evidence of infection when faecal E coli 0 157 or VT cannot be detected. 13 We now report a more extensive study in which 60 patients with HUS were examined for VTEC, faecal VT, and serum antibodies to E coli 0157 lipopolysaccharide to assess fully the value of serology as an adjunct to established bacteriological methods.
by means of
Subjects and methods Blood samples were obtained from 60 patients with HUS (53 part of the previous study1O). There were 57 D + cases and 3 D - cases; the latter had a relapsing illness, and 2 children were known to have a family history of a similar disorder. Blood and faecal samples were obtained in the acute phase, and serum samples taken during convalescence were available for analysis from 22 children. Serum samples from 16 healthy children were used as controls. Faecal samples were examined for coliforms9n’ and for VTEC by means of DNA probes for VT1 and VT2.’ Faecal VT was sought and titrated by means of Vero cell monolayers.11,’s E coli strain E32511 (0157:H -) was isolated from a patient with HUS,12 and stored on Dorset egg slopes at room temperature. For the preparation of lipopolysaccharide, bacteria were grown in trypticase soy broth (BBL Microbiology Systems, Cockeysville, Maryland, USA).12 Lipopolysaccharide was prepared by the hot-phenol procedure." For sodium dodecyl sulphate were
polyacrylamide gel electrophoresis 5 pg purified lipopolysaccharide was applied to a 4-5% stacking gel and a 12-5% separation gelY Electrophoresis was done with a constant current of 50 mA for 3 25 h, and the gels were either stained with silver17-19 or used for ADDRESSES Division of Enteric Pathogens, Central Public Health Laboratory, London (H Chart, PhD, H R Smith, PhD, S M Scotland, PhD, B Rowe, MB) and Department of Nephrology, Children’s Hospital, Birmingham, UK (D V. Milford, MD, C M Taylor, MD). Correspondence to Dr H. Chart, Division of Enteric Pathogens, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK
139
of an alkaline-phosphate-conjugated goat antiserum to total human immunoglobulin (Sigma, diluted to the manufacturer’s specification in phosphate-buffered saline/tween) and pnitrophenol phosphate (1mg/ml, Sigma) in diethanolamine buffer. The intensity of the resultant colour was determined by reading the means
optical density at 405 nm.
Results
immunoblotting. The lipopolysaccharide profiles were transferred
9 children had faecal E coli 0157:H7 and 3 of these also had faecal VT2, with titres of 2500, 2500, and 50 000, respectively. 4 children were infected with VTEC of serotypes 05:H -, O55:H10, 0105ac:H18, and O163:H19, respectively, but faecal VT was not detected in these patients. Faecal VT2 was detected in the stool of 1 patient from whom VTEC were not isolated. DNA probes for VT1 and VT2 gave positive hybridisation results only with faecal samples from which VTEC were also isolated. In the ELISA for antibodies to E coli 0157 lipopolysaccharide, 42 samples had values greater than 0-7 and 10 values below 0-4, including all three D - cases. In our previous studies,12,13 a cutoff value of about 0- 5 separated immunoblot-positive and immunoblot-negative serum samples. The 8 samples with intermediate ELISA values (0-4-0-7) were tested by immunoblotting. 2 of the 8 (ELISA values 0-53 and 0-67) contained serum antibodies reactive with 0157 lipopolysaccharide by immunoblotting (eg, fig 1, lane 1); the other 6 samples (ELISA values 0.4-0’64) did not react with this 0-antigen (fig 1, lane 2). Serum samples taken from 2 children about 1 month after the onset of disease gave ELISA values of 1-01 and 0-53 and both gave positive immunoblots. All of 26 samples with ELISA values greater than 0-7 randomly selected for immunoblotting gave positive antibody reactions. The child with faecal E coli 0163:H19 also had serum antibodies to E coli 0157 by immunoblot and ELISA (1’06). ELISA values for the 16 controls ranged from 0-09 to 0-39 with a mean of 0-24 (SD
to nitrocellulose sheets and reacted with antiserum (30
0’11).
Fig 1-Reaction of patients’ serum with E coli 0157 lipopolysaccharide by immunoblotting. Protein molecular weight standards are given in kD. Lane1 = positive, lane 2 negative reaction. =
IgM
µl/lane)12,13
antibodies binding to lipopolysaccharide antigens were detected by means of iodine-125-labelled immunoglobuliniz,l3 raised against human IgM (Sigma Chemical Co, Poole, Dorset). About 5 Ilg immunoglobulin, containing 101 cpm, was added to each lane and antibody-antigen reactions were detected by
autoradiography. ELI SAs were carried out as previously described.12,13 Plates were coated with 06 µg lipopolysaccharide and reacted with serum samples diluted (x 1000) in phosphate-buffered saline containing 0-5% ’Tween-20’. Antibody-antigen complexes were detected by
Fig 2-Serum antibody reactions with E coli 0157 lipopolysaccharide in initial and subsequent serum samples.
convalescence from 22 used to determine the duration of circulating patients E antibodies to coli 0157 lipopolysaccharide. The antibody titres in 4 patients increased over 16-20 days, whereas in the other 18 patients serum antibodies showed a gradual fall with time (fig 2). In 1 patient, antibodies could be detected up to 74 days after the initial blood sample.
Serum
samples taken during
were
Discussion
Bacteriological methods and toxin testing provided evidence of VTEC involvement in 14 of the 60 children investigated. The 9 children from whom E coli 0157:H7 were cultured had antibody values much higher than the 0,5 cutoff ELISA value12,13 and than the values for control serum samples. Since 35 of the other 51 children also had antibodies to 0 157 lipopolysaccharide, our data suggest that a total of 44 (73%) had been infected with E coli 0157. It is interesting that the D - cases had ELISA values comparable with those of the controls and were negative on immunoblot testing. Screening for antibodies by ELISA alone identified patients with high antibody values and was a simple means of obtaining evidence of infection with E coli 0157. However, our studies suggest that serum samples with ELISA values between 0-4 and 0-7 should also be tested by immunoblotting. 1 child excreted faecal E coli 0163:H19 and yet had antibodies to E coli 0157, so might have been infected with strains of both serogroups concomitantly.
140
Isolation of VTEC from cases of HUS provides definitive evidence of the probable cause of disease; however, our serological tests detected evidence of infection by E coli 0157 in 73% of children with HUS, compared with only 23% by bacteriology and toxin testing alone. VTEC and VT can be detected in stools for only a short time after onset of diarrhoea. Our serological tests can provide evidence of infection for several weeks after the onset of disease. Serology therefore forms a valuable adjunct to established bacteriological techniques. Ongoing studies on serological tests for antibodies to the lipopolysaccharides of other VTEC may provide information on the role of non-0157 VTEC in these infections. We thank members of the British Paediatric Association who supplied serum samples for this study.
REFERENCES C, Gautier E, Steck A, Siebebmann AE, Oechslin R. Hamolytisch-uramische syndrome: bilaterale nierenrindennekrosen bei akutenerworbenen hamolytiscen anamien. Schweiz Med
1. Von Gasser
Wochenschr 1955; 85: 905-09. M, Barratt JM. Haemolytic uraemic syndrome. Arch Dis Child 1984; 59: 397-400. 3. Karmali MA, Petric M, Lim C, Fleming PC, Arbus GS, Lior H. The association between idiopathic hemolytic uremic syndrome and infection by Verotoxin-producing Escherichia coli. J Infect Dis 1985; 151: 775-82. 4. Kleanthous H, Smith HR, Scotland SM, et al. Haemolytic uraemic syndrome in the British Isles, 1985-1988; association with Vero cytotoxin-producing Eschenchia coli Part 2, microbiological aspects. Arch Dis Child 1990; 65: 722-27. 5. Scotland SM, Smith HR, Rowe B. Two distinct toxins active on Vero cells from Eschenchia coli O157. Lancet 1985; ii: 885-86. 6. Smith HR, Day NP, Scotland SM, Gross RJ, Rowe B. Phagedetermined production of verocytotoxin in strains of Eschenchia coli O157. Lancet 1984; i: 1242-43. 7. Willshaw GA, Smith HR, Scotland SM, Field AM, Rowe B. Heterogeneity of Escherichia coli phages encoding Vero cytotoxins: comparison of cloned sequences determining VT1 and VT2 and development of specific gene probes. J Gen Microbiol 1987; 133: 1309-17. 8. Konowalchuk J, Speirs JI, Stavric S. Vero response to a cytotoxin of Escherichia coli. Infect Immun 1978; 18: 775-79. 9. Kleanthous H, Fry NK, Smith HR, Gross RJ, Rowe B. The use of sorbitol MacConkey agar in conjunction with a specific antiserum for the detection of Vero cytotoxin-producing strains of Escherichia coli 0157. Epidemiol Infect 1988; 101: 327-35. 10. Milford DV, Taylor CM, Gutteridge B, Hall S, Rowe B, Kleanthous H. Haemolytic uraemic syndrome in the British Isles, 1985-1988; association with Vero cytotoxin-producing Escherichia coli part 1, clinical and epidemiological aspects. Arch Dis Child 1990; 65: 716-21. 11. Scotland SM, Rowe B, Smith HR, Willshaw GA, Gross RJ. Verocytotoxin-producing strains of Escherichia coli from children with haemolytic uraemic syndrome and their detection by specific DNA probes. J Med Microbiol 1988; 25: 237-43. 12. Chart H, Scotland SM, Rowe B. Serum antibodies to Escherichia coli serotype O157:H7 in patients with hemolytic uremic syndrome. J Clin Microbiol 1989; 27: 285-90. 13. Chart H, Scotland SM, Smith HR, Rowe B. Antibodies to Escherichia coli 0157 in patients with haemorrhagic colitis and haemolytic uraemic syndrome. J Clin Pathol 1989; 42: 973-76. 14. Gross RJ, Rowe B. Serotyping of Escherichia coli. In: Sussman M, ed. The virulence of Escherichia coli. London: Society for General Microbiology, special publication no 13, 1985: 345-63. 15. Scotland SM, Willshaw GA, Smith HR, Rowe B. Properties of strains of Escherichia coli belonging to serogroup 0157 with special reference to production of Vero cytotoxins VT1 and VT2. Epidemiol Infect 1987; 99: 613-24. 16. Westphal O, Jann K. Bacterial lipopolysaccharide: extraction with phenol-water and further applications of the procedure. Meth Carbohydr Chem 1965; 5: 83-91. 17. Wray W, Boulikas T, Wray VP, Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem 1981; 118: 197-203. 18. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-85. 19. Tsai C-M, Frasch CE. A sensitive silver stain for detecting lipopolysaccharide in polyacrylamide gels. Anal Biochem 1982; 119: 2. Levin
115-19.
SHORT REPORTS
Lymphoedema/hypercalcaemia syndrome mediated by
parathyroid-hormone-related protein 24-year-old woman presented with a cryptogenic, hypercalcaemic syndrome with metastatic pulmonary calcification and leg and massive breast enlargement and bilateral chylous pleural effusions compatible with a generalised lymphatic abnormality. The patient’s serum parathyroid hormone-related protein concentration was very high at a time when she was significantly hypercalcaemic, implying that the hypercalcaemia was mediated by this protein. A
Metastatic pulmonary calcification has been described in
hypercalcaemic syndromes, including primary hyperparathyroidism, malignant disease, chronic renal failure, and sarcoidosis. We describe here a patient with steroid-suppressible hypercalcaemia and metastatic pulmonary calcification in whom the cause of the hypercalcaemia was at first uncertain. Serial measurement of serum parathyroid-hormone-related protein (PTHRP) pointed strongly to this peptide as the explanation for the hypercalcaemia. The development of bilateral chylous pleural effusions and gross breast lymphoedema suggested an acquired lymphatic abnormality not previously described in hypercalcaemia. This case reinforces the view1 that PTHRP may mediate non-malignant hypercalcaemia.
most
common
The patient first presented at age 18 with systemic lupus erythematosus, which responded to non-steroidal antiinflammatory agents. She re-presented 4 years later with a 2-month history of nausea, polyuria, and polydipsia. She had hypercalcaemia (3-75 mmol/1) with normal serum phosphate and a serum PTH level below 08 pmol/1 (normal 09-54). A chest X-ray was normal. There was no history of vitamin D abuse or unusual diet. She was given rehydration and oral corticosteroids, and within 10 days the serum calcium was normal. One month later the symptoms recurred with breathlessness on exertion. She had bilateral pleural effusions and her right breast had increased in size. Her serum calcium was 4 22 mmol/1. Rehydration and corticosteroids resolved the hypercalcaemia within a week. For a year she remained normocalcaemic on prednisolone 10 mg daily. The pleural effusions required intermittent aspiration. Pleurodesis was unsuccessful. Moderate hypertension (diastolic blood pressure 110-115 mm Hg) necessitated atenolol and nifedipine.
On referral to this hospital, 15 months after the first of hypercalcaemia, the patient had worsening breathlessness on exertion. Her breasts were much enlarged and lymphoedematous and there were large bilateral pleural effusions. After left talc pleurodesis the oedema worsened and her postoperative course was complicated by hypoxia. Hypercapnic respiratory failure ensued (PaOz 5-6 kPa, PaC02 10-12 kPa). The left pleural effusion did not recur. High-dose diuretic therapy resulted in some resolution of the lymphoedema but no improvement in the breathlessness or blood gases.
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