Indirect Hemagglutination Assay for Diagnosis of Escherichia coli 0157 Infection in Patients with Hemolytic-Uremic Syndrome

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1 JOURNAL OF CLINICAL MICROBIOLOGY, May 1992, p Vol. 30, No /92/ $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 Selective absorption with homologous LPS and heterologous LPS showed that the antibody response was specific for E. coli 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. 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 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

2 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 Behring- Werke, 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.

3 1176 BITZAN AND KARCH J. CLIN. MICROBIOL. TABLE 1. IHA LPS antibody titers of representative serum samples from children with HUS during the acute and postacutea phases of the disease IHA antibody titer (1:) for: Patient Time (wk after SRBC coated with LPS from: (age, in onset of Uncoated Stool culture result yr) diarrhea) E. coli Y. enterocolitica SRBC A (5) , <4 0157:H , < < <4 B (2) , <4 Negative for enteric pathogens , < < <4 C (5) >65, Negative , , < D (5) >65, Negative , E (8) , Negative , , < <4 F (6) <4 0111:H < < <4 a Serum samples were collected at 1 week (acute phase) and 2 to 3, 4 to 6, and >6 weeks (postacute phase) after the onset of diarrhea. DISCUSSION Although E. coli 0157 has been repeatedly implicated in HUS, the organism is frequently absent from stools at the time of acute illness (2, 4, 9, 16). In the present study, E. coli 0157 was isolated from stools from 7 of 27 (25.9%) patients with HUS, a figure comparable to previously published data (2, 4, 16). Moreover, our observation of E. coli positive stool cultures from some family members of HUS patients complies with an earlier suggestion that the organism can be spread from person to person (13). In this context, we have identified other family members with E. coli 0157 infections among our cohort of HUS patients. This route of transmission could therefore pose a threat to public health, be it in households or hospitals, unless E. coli 0157 infections are diagnosed early and stringent hygiene control measures are undertaken. In contrast to the paucity of the infectious organism in stool cultures, we found high antibody titers to E. coli 0157 LPS in the serum samples of most HUS patients during the acute phase of the disease. The IHA titer was.1:4,096 in 81.5% of HUS patients, 6.1% of diarrheal controls, and 0.5% of negative controls. An antibody response of.1:4,096 is considered to be significant, particularly since most control patients had 0157 LPS antibody titers of 1:4 to 1:256. Among HUS patients, the antibody response was specific for E. coli 0157 since, upon absorption of their sera with homologous LPS, the antibody titer fell significantly from.1:4,096 to c1:4. On the other hand, absorption of the HUS patient sera with heterologous LPS did not alter the antibody titer. Jodal (7) reported a higher incidence of immune responders (>75%) to E. coli LPS among children with acute TABLE 2. IHA 0157 LPS antibody titers of serum samples from children with HUS and controls No. of the following with the indicated titer: IHA 0157 LPS antibody titersa Patients with Diarrheal Negative acute HUSb controlsc controlsd 1: : : : : : : : Total a Expressed as the highest dilution showing agglutination. Differences in titers between HUS patients and either control group were significant (P < 0.01). b Serum samples were collected from patients with HUS at 1 week after the onset of diarrhea (acute phase of the disease). c Age-matched patients with diarrhea but no HUS. d Age-matched patients with no diarrhea, gastrointestinal disorders, or renal diseases.

4 Voi- 30, 1992 IMMUNOSEROLOGY OF HUS 1177 L- 4) - Sw x week 0D week week I > 6. week FIG. 1. Mean + standard deviation IHA titers in serum samples from children with HUS, as determined by the IHA with SRBC coated with LPS from E. coli serogroups 02, 091, 022, 0111, and The serum samples were collected at 1, 2 to 3, 4 to 6, and >6 weeks after the onset of diarrhea. Twenty-seven follow-up serum samples were tested. pyelonephritis when sera were tested in the IHA with either a polyvalent antigen or the 0 antigen from the infecting strain. A high serum antibody response was also found in adults with urinary tract infections and renal involvement (3, 17). The IHA primarily reflects immunoglobulin M (IgM) antibody content, because of the greater agglutinating ability of IgM than of IgG (6). The rapid fall in the E. coli 0157 LPS antibody titer during the postacute phase of HUS may be due to the short half-life of IgM. High E. coli 0157 LPS antibody titers were observed in 3 of 49 diarrheal controls, although all three patients were infected with S. enteritidis. We were unable to demonstrate cross-reactivity between S. enteritidis and E. coli 0157 by absorption experiments. The interpretation of relationships between concentrations of antibodies to different antigens is complicated by the polyvalent nature of the antigenic presentations and the assumed polyclonal nature of the binding specificities of naturally occurring antibodies. To date we have investigated sera from 10 patients with established S. enteritidis infections, and only 3 of these children, who are reported in this study, had high E. coli 0157 LPS antibody titers. It could be that these children had E. coli 0157 infections prior to or simultaneously with the S. enteritidis infections. Children with HUS and with stool cultures positive for SLT-producing E. coli of serogroups other than 0157 did not show a good antibody response to homologous or heterologous LPS. Repeated stool cultures from the two children with E. coli 0111:H- and 055:H6 gave no evidence for a carrier state. It is therefore likely that our serological test might not be suitable for diagnosing E. coli infections due to other serogroups. With regard to serodiagnosis, Chart et al. (4) previously recommended that an enzyme-linked immunosorbent assay (ELISA) should be used in conjunction with immunoblotting for identifying E. coli 0157:H7 infections in HUS patients. These authors performed immunoblotting because 8 of 60 serum samples from HUS patients had borderline ELISA titers. However, only two of these were confirmed by immunoblotting. Moreover, they used only sera from healthy children as controls. In our opinion, sera from children with diarrhea should be used as controls, since diarrhea is a prerequisite for classical HUS. The LPS IHA described in the present study is sensitive and specific and offers advantages over immunoblotting, which is primarily a qualitative test, and criteria for the interpretation of LPS immunoblotting for routine diagnostic use have not yet been established. LPS-coated SRBC are stable for several weeks at 4 to 8 C, the IHIA is very simple to perform, and results are available within hours. ACKNOWLEDGMENTS We thank Jurgen Heesemann, Wurzburg, Germany, for help and generous support of this study. The help of colleagues and staff nurses of the pediatric units in sample collection is gratefully acknowledged. We are also indebted to Robert Lange, Berlin, Germany, for statistical advice and to Jochen Bockemuhl, Hamburg, Germany, for helpful discussions. We thank Joseph Neerunjun, Frankfurt, Germany, for critical reading and Monika Ulrich for assistance in the preparation of the manuscript. REFERENCES 1. Bitzan, M., H. J. Hack, and G. Mauff Yersinia enterocolitica serodiagnosis: a dual role of specific IgA. Evaluation of microagglutination and ELISA. Zentralbl. Bakteriol. Mikrobiol. Hyg. Ser. A 267: Bitzan, M., E. Moebius, K. Ludwig, D. E. Muller-Wiefel, J. Heesemann, and H. Karch High incidence of serum antibodies to Escherichia coli 0157 lipopolysaccharide in children with haemolytic uraemic syndrome. J. Pediatr. 119:

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