Vol. 30, No. 5
JOURNAL OF CLINICAL MICROBIOLOGY, May 1992, p. 1174-1178 0095-1137/92/051174-05$02.00/0 Copyright C) 1992, American Society for Microbiology
Indirect Hemagglutination Assay for Diagnosis of Escherichia coli 0157 Infection in Patients with Hemolytic-Uremic Syndrome MARTIN BITZAN't AND HELGE KARCH2* Department of Pediatrics, University Hospital of Hamburg-Eppendorf, Hamburg,1 Institut for Hygiene and Microbiology, University of Wurzburg, Wurzburg, Germany Received 12 November 1991/Accepted 29 January 1992
An indirect hemagglutination assay consisting of sheep erythrocytes coated with lipopolysaccharide (LPS) from Shiga-like toxin-producing Escherichia coli 0157 was used for the serological diagnosis of E. coli 0157 infections in children with classical (enteropathic) hemolytic-uremic syndrome (HUS). One week after the onset of diarrhea (acute phase of the disease), the E. coli 0157 antibody titer was > 1:4,096 in 22 of 27 patients with HUS, compared with 4 of 249 controls, the majority of whom had 0157 antibody titers of between 1:4 and 1:256. This antibody response was observed in HUS patients with stool cultures positive and negative for E. coli 0157. Selective absorption with homologous LPS and heterologous LPS showed that the antibody response was specific for E. coli 0157. Because of its simplicity and ease of interpretation, the indirect hemagglutination assay described in this paper is recommended for the serological diagnosis of E. coli 0157 infections in patients with HUS.
samples were collected within 4 to 9 days (median, 7 days) after the onset of diarrhea. In addition, serum samples were collected between 1986 and 1990 from 200 hospitalized children (median age, 3.8 years) who had no diarrhea, gastrointestinal disorders, or renal diseases (negative controls). Since E. coli 0157 may be transmitted from person to person (11, 13), serum and fecal samples were collected from six family members of two HUS patients for investigation. In addition, serological testing for 0157 LPS antibodies was performed with serum samples from children involved in an outbreak of HUS in upper Bavaria (9). All fecal samples were cultured for enteropathogenic bacteria as described previously (9), and all sera were assayed for antibody titers. Preparation of LPSs from enteropathogenic bacteria. LPSs were prepared from E. coli 0157:H7 strains 933 (Centers for Disease Control, Atlanta, Ga.) and HUS-CL40 (M. A. Karmali, Toronto, Ontario, Canada); clinical isolates of E. coli of serotypes 0111:H-, 091:H-, 022:H8, and 02:H5; Yersinia enterocolitica 09 strain Ye 96; and strain 145/91 of Salmonella enteritidis 09,12:g,m: -. LPSs were extracted with hot aqueous phenol, purified by treatment with proteinase K (Sigma Chemicals, Deisenhofen, Germany), and subjected to further aqueous phenol extraction and repeated dialysis. The purity of the LPS preparations was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by immunoblotting as described previously (8). The LPS preparations were treated with sodium hydroxide (NaOH) before they were used for coating sheep erythrocytes (SRBC) and for the absorption of patient sera. The alkali treatment was done as described by Schlecht and Westphal (15), with minor modifications. In brief, 5.0 mg of each LPS preparation was suspended in 2.0 ml of distilled water, to which 0.4 ml of 1 N NaOH was added. The mixture was incubated at 56°C for 60 min, neutralized with 1 N acetic acid, and brought to 5.0 ml with distilled water. The NaOHtreated LPS preparations either were directly used to sensitize SRBC or were lyophilized to absorb patient sera. Preparation of LPS-sensitized SRBC. A 1.2-ml quantity of
In recent years, enterohemorrhagic Escherichia coli 0157:H7 and 0157:H- have emerged as pathogens of significant clinical importance to public health. They produce Shiga-like toxins (SLTs; verotoxins) and are responsible for diarrhea, hemorrhagic colitis, and occasionally classical hemolytic-uremic syndrome (HUS), which is characterized by a sudden onset of thrombocytopenia, hemolysis, and renal failure (5, 10, 12). HUS typically appears about 1 week after the onset of diarrhea, at which time E. coli 0157 is commonly absent from the stools of these patients (2, 4, 16). However, HUS patients do mount an antibody response to this organism, and two studies (2, 4) have reported elevated antibody titers to the lipopolysaccharide (LPS) of E. coli 0157 in over 70% of their cohorts. In this paper, we describe an indirect hemagglutination assay (IHA) for E. coli 0157 LPS antibody titers which can be used to improve the diagnosis of E. coli 0157 infections in patients with HUS.
MATERIALS AND METHODS Serum and fecal specimens. The clinical criteria for classical HUS have been previously described (2, 10). Serum and fecal samples were collected from 27 children with HUS (median age, 3.5 years) during the acute phase (1 week after the onset of diarrhea) and during the postacute phase (2 to 3, 4 to 6, and >6 weeks after the onset of diarrhea) of the disease. Sera from 17 of the 27 children were collected between 1986 and 1989 as part of a prospective study of SLT-producing E. coli in northern Germany. Sera from the remaining 10 were obtained during 1990 from various parts of Germany. All of the HUS children had diarrhea. In 20 of the 27 children this diarrhoea was hemorrhagic. Control serum and fecal samples were obtained from 49 children (median age, 4 years) who had diarrheal disease but who did not develop HUS (diarrheal controls). The serum *
Corresponding author.
t Present address: Department of Microbiology, The Hospital for
Sick Children, University of Toronto, Toronto, Ontario, Canada.
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each NaOH-treated LPS preparation was added dropwise to 100 ml of 0.5% SRBC solution (vol/vol) in 0.9% saline. The mixture was incubated at 37°C for 30 min with gentle agitation, after which the SRBC were washed three times with 0.9% saline to remove unabsorbed LPS. The sensitized SRBC were suspended at a concentration of 0.6% (vol/vol) in 0.9% saline for the IHA. Control SRBC were prepared identically, except for the addition of LPS. Preparation of LPS-absorbed sera. The NaOH-treated LPS preparations were also used to absorb patient sera as follows. One milligram of each NaOH-treated LPS preparation was added to 0.5 ml of heat-inactivated (56°C for 20 min), undiluted, individual patient serum, and the mixture was incubated at 37°C for 30 min, cooled, and kept at 4°C for 1 day. The precipitate was removed by centrifugation at 1,000 x g for 20 min. The absorbed serum was kept for antibody titer measurements. LPSs prepared from E. coli of serogroups 02, 022, 091, 0111, and 0157 and from S. entenitidis were used for the absorption experiments. IHA. For the IHA, 30 RI of heat-inactivated patient serum diluted fourfold in the range of 1:2 to 1:32,768 was mixed in 96-well curved microtiter plates (Nunclon Delta, Roskilde, Denmark) with 30 ,pl of LPS-sensitized SRBC, yielding a final serum dilution of 1:4 to 1:65,536. The plates were incubated at room temperature for 3 h. The assay was carried out with absorbed and unabsorbed sera from HUS patients and controls. The highest dilution giving a clear agglutination pattern was considered the end point. Direct bacterial microagglutination assays. Y. enterocolitica and Brucella abortus have been reported to share common antigenic epitopes with 0157 LPS (14). To assess the possible relevance of cross-reacting antibodies, we performed bacterial microagglutination assays as described previously (1). Y. enterocolitica 09 strain Ye 96 and E. coli 0157:H7 strain 933 were grown in brain heart infusion broth and heated for 2 h at 100°C to prepare the respective 0 antigens. B. abortus suspensions were obtained from the Bundesgesundheitsamt, Berlin, Germany, and S. enteritidis 09,12:g,m:- suspensions were obtained from BehringWerke, Marburg, Germany. The assays were standardized with rabbit immune sera to the respective antigens. Rabbit antisera prepared against S. enteritidis 09,12: g,m: -, E. coli 0157:H7, and other 0 groups of E. coli were kindly provided by S. Aleksic (Hamburg, Germany) or were obtained from Behring-Werke. Antisera to B. abortus were purchased from the Bundesgesundheitsamt, and antisera to Y. enterocolitica 09 were purchased from the Robert Koch
Institute, Berlin, Germany. Statistical analysis. The differences in antibody titers were evaluated statistically by the Wilcoxon rank test and the Fisher exact test as appropriate. RESULTS Recovery of enteropathogenic bacteria from stool cultures of HUS patients and controls. SLT-producing E. coli was isolated from stool samples from 9 of the 27 patients with HUS (E. coli 0157:H7, 1 patient; E. coli 0157:H-, 6 patients; E. coli 055:H6, 1 patient; and E. coli 0111:H-, 1 patient). Apart from the SLT-producing E. coli, no other enteropathogens were found. Stool samples from six family members of two patients with HUS (patients B and E; Table 1) were investigated for enteropathogens. The father of patient B and one of the three siblings of patient E excreted E. coli 0157 in stools, but both were asymptomatic.
IMMUNOSEROLOGY OF HUS
1175
Enteropathogenic bacteria were cultured from the stools of 29 of the 49 diarrheal controls (Campylobacter jejuni, 5 patients; Y enterocolitica 09, 5 patients; S. enteritidis, 10 patients; Salmonella typhimurium, 9 patients). None of these 49 controls developed HUS. E. coli 0157 LPS antibody titers in HUS patients and controls. None of the serum samples from HUS patients or controls agglutinated uncoated SRBC significantly. The IHA titer was between 1:4 and 1:16. Likewise, there was no spontaneous agglutination of LPS-coated SRBC with saline. E. coli 0157 LPS antibody titers in serum samples taken during the first week of illness (acute phase of HUS) were >1:4,096 in 22 of 27 (81.5%) patients with HUS, 3 of 49 (6.1%) patients with diarrheal illness but without HUS, and 1 of 200 (0.5%) patients with no diarrhea, gastrointestinal disorders, or renal disease (Table 2). Over 97% of control patients had E. coli 0157 LPS antibody titers below 1:1,024. HUS patients had significantly higher antibody titers to E. coli 0157 LPS than to E. coli 02, 022, 091, or 0111 LPS (Fig. 1 and Table 1). Moreover, antibody titers were in the same range, i.e., >1:4,096, in patients with positive and negative stool cultures. The two HUS patients who excreted E. coli 055:H6 and 0111:H- had E. coli 0157 LPS antibody titers of 1:64, and these titers did not change when serum samples were tested against homologous 055:H6 and 0111:H- LPSs. E. coli 0157 LPS antibody titers fell rapidly during the postacute phase of HUS (Fig. 1). Antibody titers of representative serum samples are given in Table 1. Specificity of E. coli 0157 LPS antibody titers. Sera from three patients with HUS were absorbed with E. coli 0157 LPS. The IHA titer fell from .1:65,336 to 51:4. Absorption of the same sera with LPS from E. coli of serogroup 02, 022, 091, or 0111 or with LPS from S. enteritidis did not reduce the 0157 LPS antibody titer. Prior to absorption, these sera had antibody titers of 1:10 to 1:20 to B. abortus and Y enterocolitica 09 and 1:40 to 1:160 to the 0-antigen preparation of E. coli 0157 in the direct bacterial microagglutination assay. These sera did not react with S. enteritidis somatic antigens. Sera from three control patients with diarrhea associated with S. enteritidis had high antibody titers to E. coli 0157 LPS. Absorption of these control sera with S. enteritidis LPS did not alter the E. coli 0157 LPS antibody titer in the IHA or direct microagglutination assay. However, in the direct microagglutination assay, S. enteritidis titers in all three control sera fell from 1:200 to 1:10 after absorption. Epidemiological aspects of E. coli 0157 LPS antibody screening. Among six family members of HUS patients investigated, the healthy sibling of patient E who excreted E. coli 0157 in stools did not mount a significant antibody response (IHA titer, 1:16). On the other hand, the asymptomatic father of patient B who also had a positive stool culture showed a good antibody response and had a high 0157 LPS antibody titer (IHA titer, 1:16,384). We examined serum samples from three of six patients involved in a recent outbreak of HUS (9). Of the six children, only two were stool culture positive for E. coli 0157:H-, and both children had antibody titers of 1:16,384 in the IHA. Of the four children with negative stool cultures, serum samples were available from one, and this child had an E. coli 0157 LPS antibody titer of 1:65,536, thus supporting the diagnosis of E. coli 0157 infection.
1176
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TABLE 1. IHA LPS antibody titers of representative serum samples from children with HUS during the acute and postacutea phases of the disease Patient (age, in yr)
A (5)
B (2)
C (5)
D (5)
E (8)
F (6)
a
IHA antibody titer (1:) for: SRBC coated with LPS from:
Time (wk after onset of diarrhea)
E. coli 091 0111
02
022
1 2 4 22
256 64 16 16
64 64 16 4
16 16 16 16
16 16 16 16
4,096 1,024 256 64
64 64 16 16
1
16
16
16
64
4,096
16
2 5 32
64 64 16
64
64 64 16
1,024 256
4
256 64 16
16 16 4
1 3 5 24
64 64 64 64
16 64 64 64
64 64 16 64
256 256 256 64
>65,536
1 2 4 32
16 16 16 64
64 64 64 256
16 64 64
64 256 16 16
>65,536 4,096
1 2 4 24
16 64 64 16
16 64 256 16
64 64 64
64 64 256 64
65,536 4,096 1,096 256
64 64 256 16
1 2 6 34
256 64 64 64
16 16 16 16
16 16 16 64
64 64 64 64
64 256 256 256
16 16 16 64
16
256 16
0157
09
64
JOURNAL OF CLINICAL MICROBIOLOGY, May 1992, p. 1174-1178 0095-1137/92/051174-05$02.00/0 Copyright C) 1992, American Society for Microbiology
Indirect Hemagglutination Assay for Diagnosis of Escherichia coli 0157 Infection in Patients with Hemolytic-Uremic Syndrome MARTIN BITZAN't AND HELGE KARCH2* Department of Pediatrics, University Hospital of Hamburg-Eppendorf, Hamburg,1 Institut for Hygiene and Microbiology, University of Wurzburg, Wurzburg, Germany Received 12 November 1991/Accepted 29 January 1992
An indirect hemagglutination assay consisting of sheep erythrocytes coated with lipopolysaccharide (LPS) from Shiga-like toxin-producing Escherichia coli 0157 was used for the serological diagnosis of E. coli 0157 infections in children with classical (enteropathic) hemolytic-uremic syndrome (HUS). One week after the onset of diarrhea (acute phase of the disease), the E. coli 0157 antibody titer was > 1:4,096 in 22 of 27 patients with HUS, compared with 4 of 249 controls, the majority of whom had 0157 antibody titers of between 1:4 and 1:256. This antibody response was observed in HUS patients with stool cultures positive and negative for E. coli 0157. Selective absorption with homologous LPS and heterologous LPS showed that the antibody response was specific for E. coli 0157. Because of its simplicity and ease of interpretation, the indirect hemagglutination assay described in this paper is recommended for the serological diagnosis of E. coli 0157 infections in patients with HUS.
samples were collected within 4 to 9 days (median, 7 days) after the onset of diarrhea. In addition, serum samples were collected between 1986 and 1990 from 200 hospitalized children (median age, 3.8 years) who had no diarrhea, gastrointestinal disorders, or renal diseases (negative controls). Since E. coli 0157 may be transmitted from person to person (11, 13), serum and fecal samples were collected from six family members of two HUS patients for investigation. In addition, serological testing for 0157 LPS antibodies was performed with serum samples from children involved in an outbreak of HUS in upper Bavaria (9). All fecal samples were cultured for enteropathogenic bacteria as described previously (9), and all sera were assayed for antibody titers. Preparation of LPSs from enteropathogenic bacteria. LPSs were prepared from E. coli 0157:H7 strains 933 (Centers for Disease Control, Atlanta, Ga.) and HUS-CL40 (M. A. Karmali, Toronto, Ontario, Canada); clinical isolates of E. coli of serotypes 0111:H-, 091:H-, 022:H8, and 02:H5; Yersinia enterocolitica 09 strain Ye 96; and strain 145/91 of Salmonella enteritidis 09,12:g,m: -. LPSs were extracted with hot aqueous phenol, purified by treatment with proteinase K (Sigma Chemicals, Deisenhofen, Germany), and subjected to further aqueous phenol extraction and repeated dialysis. The purity of the LPS preparations was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by immunoblotting as described previously (8). The LPS preparations were treated with sodium hydroxide (NaOH) before they were used for coating sheep erythrocytes (SRBC) and for the absorption of patient sera. The alkali treatment was done as described by Schlecht and Westphal (15), with minor modifications. In brief, 5.0 mg of each LPS preparation was suspended in 2.0 ml of distilled water, to which 0.4 ml of 1 N NaOH was added. The mixture was incubated at 56°C for 60 min, neutralized with 1 N acetic acid, and brought to 5.0 ml with distilled water. The NaOHtreated LPS preparations either were directly used to sensitize SRBC or were lyophilized to absorb patient sera. Preparation of LPS-sensitized SRBC. A 1.2-ml quantity of
In recent years, enterohemorrhagic Escherichia coli 0157:H7 and 0157:H- have emerged as pathogens of significant clinical importance to public health. They produce Shiga-like toxins (SLTs; verotoxins) and are responsible for diarrhea, hemorrhagic colitis, and occasionally classical hemolytic-uremic syndrome (HUS), which is characterized by a sudden onset of thrombocytopenia, hemolysis, and renal failure (5, 10, 12). HUS typically appears about 1 week after the onset of diarrhea, at which time E. coli 0157 is commonly absent from the stools of these patients (2, 4, 16). However, HUS patients do mount an antibody response to this organism, and two studies (2, 4) have reported elevated antibody titers to the lipopolysaccharide (LPS) of E. coli 0157 in over 70% of their cohorts. In this paper, we describe an indirect hemagglutination assay (IHA) for E. coli 0157 LPS antibody titers which can be used to improve the diagnosis of E. coli 0157 infections in patients with HUS.
MATERIALS AND METHODS Serum and fecal specimens. The clinical criteria for classical HUS have been previously described (2, 10). Serum and fecal samples were collected from 27 children with HUS (median age, 3.5 years) during the acute phase (1 week after the onset of diarrhea) and during the postacute phase (2 to 3, 4 to 6, and >6 weeks after the onset of diarrhea) of the disease. Sera from 17 of the 27 children were collected between 1986 and 1989 as part of a prospective study of SLT-producing E. coli in northern Germany. Sera from the remaining 10 were obtained during 1990 from various parts of Germany. All of the HUS children had diarrhea. In 20 of the 27 children this diarrhoea was hemorrhagic. Control serum and fecal samples were obtained from 49 children (median age, 4 years) who had diarrheal disease but who did not develop HUS (diarrheal controls). The serum *
Corresponding author.
t Present address: Department of Microbiology, The Hospital for
Sick Children, University of Toronto, Toronto, Ontario, Canada.
1174
VOL. 30, 1992
each NaOH-treated LPS preparation was added dropwise to 100 ml of 0.5% SRBC solution (vol/vol) in 0.9% saline. The mixture was incubated at 37°C for 30 min with gentle agitation, after which the SRBC were washed three times with 0.9% saline to remove unabsorbed LPS. The sensitized SRBC were suspended at a concentration of 0.6% (vol/vol) in 0.9% saline for the IHA. Control SRBC were prepared identically, except for the addition of LPS. Preparation of LPS-absorbed sera. The NaOH-treated LPS preparations were also used to absorb patient sera as follows. One milligram of each NaOH-treated LPS preparation was added to 0.5 ml of heat-inactivated (56°C for 20 min), undiluted, individual patient serum, and the mixture was incubated at 37°C for 30 min, cooled, and kept at 4°C for 1 day. The precipitate was removed by centrifugation at 1,000 x g for 20 min. The absorbed serum was kept for antibody titer measurements. LPSs prepared from E. coli of serogroups 02, 022, 091, 0111, and 0157 and from S. entenitidis were used for the absorption experiments. IHA. For the IHA, 30 RI of heat-inactivated patient serum diluted fourfold in the range of 1:2 to 1:32,768 was mixed in 96-well curved microtiter plates (Nunclon Delta, Roskilde, Denmark) with 30 ,pl of LPS-sensitized SRBC, yielding a final serum dilution of 1:4 to 1:65,536. The plates were incubated at room temperature for 3 h. The assay was carried out with absorbed and unabsorbed sera from HUS patients and controls. The highest dilution giving a clear agglutination pattern was considered the end point. Direct bacterial microagglutination assays. Y. enterocolitica and Brucella abortus have been reported to share common antigenic epitopes with 0157 LPS (14). To assess the possible relevance of cross-reacting antibodies, we performed bacterial microagglutination assays as described previously (1). Y. enterocolitica 09 strain Ye 96 and E. coli 0157:H7 strain 933 were grown in brain heart infusion broth and heated for 2 h at 100°C to prepare the respective 0 antigens. B. abortus suspensions were obtained from the Bundesgesundheitsamt, Berlin, Germany, and S. enteritidis 09,12:g,m:- suspensions were obtained from BehringWerke, Marburg, Germany. The assays were standardized with rabbit immune sera to the respective antigens. Rabbit antisera prepared against S. enteritidis 09,12: g,m: -, E. coli 0157:H7, and other 0 groups of E. coli were kindly provided by S. Aleksic (Hamburg, Germany) or were obtained from Behring-Werke. Antisera to B. abortus were purchased from the Bundesgesundheitsamt, and antisera to Y. enterocolitica 09 were purchased from the Robert Koch
Institute, Berlin, Germany. Statistical analysis. The differences in antibody titers were evaluated statistically by the Wilcoxon rank test and the Fisher exact test as appropriate. RESULTS Recovery of enteropathogenic bacteria from stool cultures of HUS patients and controls. SLT-producing E. coli was isolated from stool samples from 9 of the 27 patients with HUS (E. coli 0157:H7, 1 patient; E. coli 0157:H-, 6 patients; E. coli 055:H6, 1 patient; and E. coli 0111:H-, 1 patient). Apart from the SLT-producing E. coli, no other enteropathogens were found. Stool samples from six family members of two patients with HUS (patients B and E; Table 1) were investigated for enteropathogens. The father of patient B and one of the three siblings of patient E excreted E. coli 0157 in stools, but both were asymptomatic.
IMMUNOSEROLOGY OF HUS
1175
Enteropathogenic bacteria were cultured from the stools of 29 of the 49 diarrheal controls (Campylobacter jejuni, 5 patients; Y enterocolitica 09, 5 patients; S. enteritidis, 10 patients; Salmonella typhimurium, 9 patients). None of these 49 controls developed HUS. E. coli 0157 LPS antibody titers in HUS patients and controls. None of the serum samples from HUS patients or controls agglutinated uncoated SRBC significantly. The IHA titer was between 1:4 and 1:16. Likewise, there was no spontaneous agglutination of LPS-coated SRBC with saline. E. coli 0157 LPS antibody titers in serum samples taken during the first week of illness (acute phase of HUS) were >1:4,096 in 22 of 27 (81.5%) patients with HUS, 3 of 49 (6.1%) patients with diarrheal illness but without HUS, and 1 of 200 (0.5%) patients with no diarrhea, gastrointestinal disorders, or renal disease (Table 2). Over 97% of control patients had E. coli 0157 LPS antibody titers below 1:1,024. HUS patients had significantly higher antibody titers to E. coli 0157 LPS than to E. coli 02, 022, 091, or 0111 LPS (Fig. 1 and Table 1). Moreover, antibody titers were in the same range, i.e., >1:4,096, in patients with positive and negative stool cultures. The two HUS patients who excreted E. coli 055:H6 and 0111:H- had E. coli 0157 LPS antibody titers of 1:64, and these titers did not change when serum samples were tested against homologous 055:H6 and 0111:H- LPSs. E. coli 0157 LPS antibody titers fell rapidly during the postacute phase of HUS (Fig. 1). Antibody titers of representative serum samples are given in Table 1. Specificity of E. coli 0157 LPS antibody titers. Sera from three patients with HUS were absorbed with E. coli 0157 LPS. The IHA titer fell from .1:65,336 to 51:4. Absorption of the same sera with LPS from E. coli of serogroup 02, 022, 091, or 0111 or with LPS from S. enteritidis did not reduce the 0157 LPS antibody titer. Prior to absorption, these sera had antibody titers of 1:10 to 1:20 to B. abortus and Y enterocolitica 09 and 1:40 to 1:160 to the 0-antigen preparation of E. coli 0157 in the direct bacterial microagglutination assay. These sera did not react with S. enteritidis somatic antigens. Sera from three control patients with diarrhea associated with S. enteritidis had high antibody titers to E. coli 0157 LPS. Absorption of these control sera with S. enteritidis LPS did not alter the E. coli 0157 LPS antibody titer in the IHA or direct microagglutination assay. However, in the direct microagglutination assay, S. enteritidis titers in all three control sera fell from 1:200 to 1:10 after absorption. Epidemiological aspects of E. coli 0157 LPS antibody screening. Among six family members of HUS patients investigated, the healthy sibling of patient E who excreted E. coli 0157 in stools did not mount a significant antibody response (IHA titer, 1:16). On the other hand, the asymptomatic father of patient B who also had a positive stool culture showed a good antibody response and had a high 0157 LPS antibody titer (IHA titer, 1:16,384). We examined serum samples from three of six patients involved in a recent outbreak of HUS (9). Of the six children, only two were stool culture positive for E. coli 0157:H-, and both children had antibody titers of 1:16,384 in the IHA. Of the four children with negative stool cultures, serum samples were available from one, and this child had an E. coli 0157 LPS antibody titer of 1:65,536, thus supporting the diagnosis of E. coli 0157 infection.
1176
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TABLE 1. IHA LPS antibody titers of representative serum samples from children with HUS during the acute and postacutea phases of the disease Patient (age, in yr)
A (5)
B (2)
C (5)
D (5)
E (8)
F (6)
a
IHA antibody titer (1:) for: SRBC coated with LPS from:
Time (wk after onset of diarrhea)
E. coli 091 0111
02
022
1 2 4 22
256 64 16 16
64 64 16 4
16 16 16 16
16 16 16 16
4,096 1,024 256 64
64 64 16 16
1
16
16
16
64
4,096
16
2 5 32
64 64 16
64
64 64 16
1,024 256
4
256 64 16
16 16 4
1 3 5 24
64 64 64 64
16 64 64 64
64 64 16 64
256 256 256 64
>65,536
1 2 4 32
16 16 16 64
64 64 64 256
16 64 64
64 256 16 16
>65,536 4,096
1 2 4 24
16 64 64 16
16 64 256 16
64 64 64
64 64 256 64
65,536 4,096 1,096 256
64 64 256 16
1 2 6 34
256 64 64 64
16 16 16 16
16 16 16 64
64 64 64 64
64 256 256 256
16 16 16 64
16
256 16
0157
09
64
