kindergarten children in Sarnia, Ontario, that was linked to the consumption of unpasteurized milk. A comprehensive serolog- ical survey was not performed, but ...
JOURNAL OF CLINICAL MICROBIOLOGY, June 1994, p. 1457-1463 0095-1 137/94/$04.00+0 Copyright © 1994, American Society for Microbiology
Vol. 32, No. 6
Enzyme-Linked Immunosorbent Assay for Detection of Immunoglobulin G Antibodies to Escherichia coli Vero Cytotoxin 1 MOHAMED A. KARMALI,l.2* MARTIN PETRIC,"2 MONICA WINKLER,'2 MARTINA BIELASZEWSKA,"2t JAMES BRUNTON,2,4 NICOLE VAN DE KAR,s TATSUYA MOROOKA,6 G. BALAKRISHNA NAIR,7 SUSAN E. RICHARDSON," 2 AND GERALD S. ARBUS3 Research Institute and the Department of Microbiology, The Hospital For Sick Children,1 Departments of Microbiology,2 Pediatrics,3 and Medicine,4 University of Toronto, Toronto, Ontanio, Canada; Pediatric Department, Sint Radboud Hospital, Nijmegen, The Netherlands5; Department of Pediatrics, Chikushi Hospital, Fukuoka University, Fukuoka, Japan6; and National Institute of Cholera and Enteric Diseases, Calcutta, India7 Received 10 November 1993/Returned for modification 25 January 1994/Accepted 21 March 1994
The frequency of Vero cytotoxin 1 (VT1)-neutralizing antibody (NAb) in serum specimens from 790 age-stratified (0 to 70 years) control individuals from Toronto was 61 of 790 (7.7%), with a peak of 19%o in the 20- to 30-year-old age group and a second peak of 16.7% in the 60- to 70-year-old age group. A total of 568 serum specimens, including 538 from the 790 Toronto control subjects, 21 from patients from three outbreaks of VT-producing Escherichia coli (VTEC) infection, and 9 known VT1-NAb-positive serum specimens from patients with hemolytic-uremic syndrome (HUS), were then tested for the presence of anti-VT1 immunoglobulin G (IgG) by an enzyme-linked immunosorbent assay (ELISA). The mean ELISA values of 522 VT1-NAb-negative serum specimens and 46 VT1-NAb-positive serum specimens were 0.09 ± 0.06 (range, 0 to 0.56) and 0.78 ± 0.66 (range, 0.16 to 2.91), respectively (P < 0.001; Student's t test). With a breakpoint of 0.21 (mean ELISA value of the VT1-NAb-negative sera + 2 standard deviations), the sensitivity, specificity, positive predictive value, and negative predictive value of the VT1 IgG ELISA compared with those of the VT1-NAb assay were, respectively, 95.7, 98.7, 86.3, and 99.6%. There were nine discrepant serum specimens, of which seven were anti-VT1 IgG positive and VT1-NAb negative and two were anti-VT1 IgG negative and VT1-NAb positive. The ELISA was also used for testing 238 control serum specimens from The Netherlands, Japan, and India and acute- and convalescent-phase serum specimens from 42 Toronto patients with HUS. The frequencies of anti-VT1 IgG (with VT1-NAb frequencies in parentheses) in control sera from The Netherlands, Japan, and India were 6% (3%), 1.1% (0%o), and 12% (10%o), respectively, with no age clustering. The frequencies of anti-VT1 IgG seropositivity in HUS patients were 5 of 14 (35.7%) in patients with unknown toxin exposure, 2 of 22 (9.1%) in individuals with known exposure to VT1 plus VT2 or VT1 alone, and 0 of 6 (0%o) in patients exposed to only VT2. Development of a serum anti-VT1 IgG response appears to be the exception rather than the rule in sporadic HUS patients infected with VTEC expressing VTI. However, in two family outbreaks associated with VTEC strains expressing VT1 alone and VT1 plus VT2, respectively, the presence of anti-VT1 IgG in virtually all exposed individuals who remained symptom free suggests that the presence of antibody was associated with protection. Vero cytotoxin (VT)-producing Escherichia coli (VTEC) (16) organisms, also referred to as Shiga-like toxin (SLT)producing E. coli (20), are associated with a spectrum of disease that includes nonspecific diarrhea (13), hemorrhagic colitis (29), and the hemolytic-uremic syndrome (HUS) (14, 15). Although the role of VTs in the pathogenesis of the initial nonbloody diarrhea remains uncertain, there is growing evidence (13) that these toxins are of major pathogenetic significance in the systemic complications of VTEC infection, namely hemorrhagic colitis and HUS. Histopathologically, HUS is characterized by widespread thrombotic microangiopathy affecting the kidneys, gastrointestinal tract (as in hemor-
rhagic colitis), and, in severe cases, other organs and tissues such as the brain, the pancreas, and the lungs (9, 27). Recent evidence suggests that the thrombotic microangiopathy results from a direct toxic action of VT on vascular endothelial cells (22, 23, 28). The three VTs produced by human VTEC isolates (11, 20, 21, 30) consist of VT1 [SLT I; type strain C600(H19J)], VT2 [SLT II; type strain C600(933w)], and VT2c (SLT Ilc and SLT II vh; type strains E32511, B2F1, and 7279). All three toxins may be present alone or as a combination of two toxins (typically VT1 and VT2 or VT2c and VT2). VT1 is serologically distinct from VT2 (and VT2c), the toxins showing no cross-neutralization by homologous antisera in tissue culture assays (10, 13, 20, 21). On the other hand, VT2 is completely neutralized by antiserum to VT2c, whereas VT2c is only partially neutralized by VT2 antiserum (10). Although patients have been reported to develop rising levels of VT-neutralizing antibodies (NAbs) following VTEC infection (1, 2, 4, 14, 15, 19), little is known about the nature and duration of the serum antitoxic response following infec-
* Corresponding author. Mailing address: Department of Microbiology, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8. Phone: (416) 813-5994. Fax: (416) 8135993. t Present address: Institute of Medical Microbiology, Faculty of Pediatrics, Charles University, Vuvalu 84, 150 06, Prague 5, Motol, Czech Republic.
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MATER1ALS AND METHODS Serum specimens. (i) Control populations. A total of 790 control serum specimens from Toronto were examined for the presence of NAb against VT1. They included 323 samples from otherwise healthy children, aged 1 month to 15 years, that were submitted to our laboratory for the investigation of respiratory infections. There were 152, 80, and 91 serum specimens, respectively, from each of the three 5-year age groups (O to 5, 5 to 10, and 10 to 15 years); the remaining 467 samples were from healthy blood donors (kindly supplied by Roslyn Herst) aged 15 to 65 years (with about 40 to 50 samples in each of the 10 5-year age categories between 15 and 65 years). The presence of VT1-NAb was also investigated with 238 control serum specimens from The Netherlands, Japan, and India. The control patients in these countries consisted of individuals who had attended the hospital for reasons unrelated to diarrhea, dysentery, hemorrhagic colitis, or HUS. The Dutch control subjects consisted of 100 age-stratified persons (including 10 from each of the following age categories: 70 years) from the city of Nijmegen. The Japanese control subjects included 88 patients from Fukuoka, aged 1 month to 22 years (mean ± standard deviation, 8.3 ± 6.8 years) with the following distribution: 38 0 to 5 years, 22 6 to 10 years, 16 11 to 15 years, 2 16 to 20 years, and 10 21 to 25 years. The 50 Indian control patients from Calcutta ranged in age from 10 years to 75 years (mean ± standard deviation, 36 ± 17.5 years). (ii) Patients and outbreaks. VT1-NAb in serum specimens of 21 persons involved in three outbreaks of VTEC infection and in the acute- and convalescent-phase serum specimens of 42 patients with HUS who attended our hospital was mea-
becco A; 0.01 M, pH 7.3; UNIPATH, Inc., Basingstoke, United Kingdom]), PBS-Tween (a solution of 0.1% Tween 20 in PBS), and PBS-Tween-bovine serum albumin (BSA) (a solution of 0.05% Tween 20 and 0.5% BSA in PBS). BSA and Tween 20 were obtained from Sigma Chemical Co., St. Louis, Mo. The procedure used was a standard antibody ELISA in which the test conditions and reagent concentrations were fully optimized. Using 96-well microtiter plates (Falcon Pro-Bind assay plate 3915; Becton Dickinson, Paramus, N.J.), test wells were coated with antigen by incubation with 100 ,d of purified VT1 (3 ,ug/ml in PBS) per well overnight at room temperature, with PBS substituted for the toxin in antigen control wells. After the plates had been washed three times with PBS-Tween, 250 [lI of 2% BSA in PBS was added to each well, and the plates were incubated for 1 h at room temperature. The plates were washed three times with PBS-Tween and incubated for 1 h at room temperature with test sera and control sera (one high-positive serum specimen, two negative serum specimens, and one weak-positive serum specimen from our collection) diluted 1:100 in PBS-Tween-BSA; each serum specimen was tested in triplicate in antigen-coated wells as well as in antigen control wells. The plate was then assayed (with appropriate controls) for the presence of anti-VT1 IgG by incubation for 1 h at room temperature with 100 [lI of goat anti-human IgG conjugated with horseradish peroxidase (Bio-Rad, Richmond, Calif.) per well diluted 1:2,000 in PBS-Tween-BSA. After three washes in PBS-Tween, the substrate solution (o-phenylenediamine dihydrochloride; Sigma) was added to each well. The plates were incubated for 30 min at room temperature in darkness, and the reaction was terminated by the addition of 2 N sulfuric acid. The optical density values at 490 nm (OD490s) were measured in a microplate ELISA reader. For each serum specimen, mean OD490 readings were calculated for the test wells and for the antigen control wells, the latter being subtracted from the former to obtain the net ELISA value. Comparison of ELISA for detection of anti-VT1 IgG with the VTl-NAb assay. An initial comparative study of the ELISA and the VT1-NAb assay was conducted with 568 of the serum specimens, including 538 of the 790 control serum specimens from Toronto and the 21 serum specimens from individuals involved in three outbreaks of VTEC infection as well as 9 serum specimens from patients with HUS that were known to be positive for VT1-NAb. The anti-VT1 IgG ELISA was also used subsequently for testing acute- and convalescent-phase serum specimens from 42 patients with HUS attending our hospital and control serum specimens from The Netherlands, Japan, and India that had previously been tested by the VT1-NAb assay. Characterization of VTEC strains and toxin types. Serotypes of VTEC isolates were confirmed by A. Borczyk (Central Public Health Laboratory, Toronto, Canada) and H. Lior (Laboratory Centre for Disease Control, Ottawa, Canada). Selected VTEC isolates were tested for VT phenotype and genotype as described previously (11).
sured. VT1-NAb assay. The VT1-NAb assay was performed as described previously (14). A cut-off point of 21l >21l >21'
a VTEC 0157:H7 isolate produced VT1 and VT2. "Patients 2, 3, and 4 developed HUS. Patients 1 and 5 to 8 developed uncomplicated diarrhea. c ELISA value is the difference between the OD490 reading for the test well and the OD4,0 reading for the antigen control well. d Number of days between onset of diarrhea and collection of serum. eAlthough the exact dates of collection of these sera could not be determined, they were probably collected between 3 and 4 weeks after the onset of symptoms.
not consume any hamburger, although some family members had done so. The farm residents were investigated about 1 month after onset of symptoms in the index patient. None of the residents gave a history of diarrhea. However, one farm resident was positive for VTEC serotype O111:nonmotile (toxin phenotype and genotype VT1), similar to the isolate from the index patient. In contrast to the absence of antibody in the index patient and her sister and parents, seven of eight farm residents were positive for anti-VT1 IgG and VT1-Nab
(Table 2). (ii) Outbreak B. The results for outbreak B are presented in Table 3. In 1987, there was an outbreak of VTEC 0157:H7 (toxin types VT1 and VT2) illness (8) affecting 42 of 63 kindergarten children in Sarnia, Ontario, that was linked to the consumption of unpasteurized milk. A comprehensive serological survey was not performed, but sera (kindly provided by A. Borczyk and L. Duncan) from a subset of microbiologically confirmed symptomatic individuals were available for study. The results with these single serum specimens suggest a relationship between the anti-VT1 IgG response and the time postdiarrhea that the serum was collected. None of the sera collected within the first week of diarrhea had demonstratable antibody, whereas those collected after this period did (Table 3). (iii) Outbreak C. The results for outbreak C are presented in Table 4. Table 4 shows serological responses from a family
No. of patients
No. (%) VTI-NAb positive
Unknown VT1 and VT2 VTI VT2
14
6 (42.9)
18 4c 6
2 (11.1)
5 (35.7) 1 (5.6)
1(25.0)
1 (25.0) 0 (0)
(age) Index female (4 yr) Mother (43 yr) Brother (17 yr) Sister (15 yr) Sister (11 yr)
Symptom
HUS None None None None
VTEC/FVT
+/+
-/+ -/+ -/+ -I-
VTI-NAb titer
(days)'
