the united states national prospective hemolytic uremic syndrome study: microbiologic, serologic,...

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The United States National Prospective Hemolytic Uremic Syndrome Study:Microbiologic, Serologic, Clinical, and Epidemiologic Findings

Nicholas Banatvala,a Patricia M. Griffin,Katherine D. Greene, Timothy J. Barrett,William F. Bibb, James H. Green, Joy G. Wells,and the Hemolytic Uremic Syndrome StudyCollaboratorsb

Foodborne and Diarrheal Diseases Branch, Division of Bacterialand Mycotic Diseases, National Center for Infectious Diseases,

Centers for Disease Control and Prevention, Atlanta, Georgia

The frequency of Shiga toxin–producing Escherichia coli (STEC) serotypes associated withpostdiarrheal hemolytic uremic syndrome (HUS) cases among children and adults in theUnited States and the proportion with IgM or IgG lipopolysaccharide antibodies to E. coliO157 were determined by use of a nationwide sample from January 1987 through December1991. Among 83 patients, STEC were isolated from 30 (43%) of 70 whose stool cultures yieldedbacterial growth (25 E. coli O157 isolates and 5 non-O157 STEC isolates). Fifty-three (80%)of 66 patients with serum samples had positive O157 lipopolysaccharide antibody titers. Ofthe 83 patients, 60 (72%) had evidence of STEC infection, including 6 of 8 adults whoseillnesses also met criteria for thrombotic thrombocytopenic purpura. Data from a subset ofpatients suggest that E. coli O157 was the cause of >80% of the STEC infections. All 3 womenwho were postpartum had evidence of E. coli O157 infection. STEC infection should beconsidered the likely cause for all persons with postdiarrheal HUS.

Hemolytic uremic syndrome (HUS) is characterized by acuterenal failure, thrombocytopenia, and microangiopathichemolyticanemia. HUS typically develops after a prodromal diarrheal ill-ness but may occur without diarrhea and is the major cause ofacute renal failure in children. Increasing interest has focusedon the infectious etiology of postdiarrheal HUS. Shiga toxin–producing Escherichia coli (STEC) and Shigella dysenteriae type1 both have been clearly associated with HUS [1, 2].

Current evidence suggests that STEC cause all or almost allcases of postdiarrheal HUS in developed countries. Several stud-ies have suggested that E. coli O157:H7 is the major cause ofpostdiarrheal HUS in North America [3, 4]. Some adults diag-nosed with thrombotic thrombocytopenic purpura (TTP)—a syn-drome characterized by symptoms similar to those of HUS butwith the addition of fever and neurologic symptoms—also have

Received 17 March 2000; revised 28 November 2000; electronically pub-lished 1 March 2001.

Informed consent was obtained from patients or their parents or guardians.Guidelines issued by the authors’ institution were followed in the conduct ofthis work.

Financial support: London Hospital Medical College (Centers for DiseaseControl and Prevention Guest Researcher support to N.B.).

a Present affiliation: Suffolk Health Authority, Ipswich, United Kingdom.b The study collaborators are listed after the text.Reprints: Dr. Patricia Griffin, Foodborne and Diarrheal Diseases Branch,

Division of Bacterial and Mycotic Diseases, National Center for InfectiousDiseases, Centers for Disease Control and Prevention, Atlanta, GA 30333.Correspondence: Dr. N. Banatvala, Dept. of Public Health, Suffolk Health,PO Box 55, Foxhall Rd, Ipswich IP3 8NN, United Kingdom ([email protected]).

The Journal of Infectious Diseases 2001;183:1063–70q 2001 by the Infectious Diseases Society of America. All rights reserved.0022-1899/2001/18307-0010$02.00

STEC infection. Unlike most persons with TTP, those with TTP-associated STEC infection generally have preceding diarrhea [1].

The proportion of HUS patients infected with E. coli O157:H7 or other STEC serotypes can be difficult to determine, be-cause HUS is usually diagnosed several days after the onset ofdiarrhea, a time when the number of pathogens in the stool isdecreasing. This delay in diagnosis and the antimicrobial treat-ment before stool specimens are obtained in some patients de-crease the yield from stool cultures. Serology provides an al-ternative method of diagnosis. To date, the value of serologicdiagnosis of E. coli O157 infection in HUS has not been fullyassessed, and temporal changes in the E. coli O157 isotype–specific antibody response have not been described.

From January 1987 through December 1991, patients wereenrolled in a nationwide prospective study of postdiarrhealHUS. The major objective was to characterize the frequencyof different STEC serotypes in this cohort. The second objectivewas to identify the proportion of patients with IgM and IgGlipopolysaccharide (LPS) antibodies to E. coli O157 and todocument the variation in antibody response over time. Thethird objective was to describe the clinical and epidemiologicfeatures of a population of children and adults throughout theUnited States with postdiarrheal HUS.

Patients and Methods

Case ascertainment. A convenience sample of pediatric nephrolo-gists and adult hematologists were invited to participate in a prospectiveUS study of HUS. Physicians were asked to obtain stool and serumspecimens and to complete a questionnaire for patients with HUS,documenting epidemiologic, clinical, and laboratory findings. Most

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1064 Banatvala et al. JID 2001;183 (1 April)

physicians who chose to participate were pediatric nephrologists. Chil-dren were defined as persons !18 years old.

Selection criteria. Persons with physician-diagnosed post-diarrheal HUS or TTP (supported by a review of the history andlaboratory findings provided in the questionnaire) were consideredfor inclusion in the study. Requirements for inclusion were as fol-lows: (1) diarrhea with onset in the 21 days before the diagnosisof HUS or TTP; (2) anemia (hemoglobin level 1100 g/L and hema-tocrit !0.3); (3) microangiopathic changes consistent with hemo-lysis on peripheral blood smear, such as schistocytes, burr, or helmetcells; (4) platelet count !150,000/mm3; (5) and acute renal impair-ment (serum creatinine level >1.0 mg/dL if patients !13 years oldor creatinine level >1.5 mg/dL if patients >13 years old). All pa-tients accepted for the study were considered to have HUS. TTPwas defined as illness that not only met the criteria for HUS butalso included fever (temperature 1387C) and newly diagnosed sei-zure, paresis, or encephalopathy. To avoid selection bias, we didnot accept patients whose stool samples had earlier been tested forE. coli O157:H7, regardless of the result. However, at the time ofthe study, stool specimens from HUS patients in these hospitalswere not typically tested for E. coli O157:H7.

Collection and storage of specimens. Fecal samples were ob-tained from patients as soon as possible after presentation and weresent to the Foodborne and Diarrheal Disease Branch, Centers forDisease Control and Prevention (CDC) in Atlanta. By protocol,all fecal samples were to be sent to CDC on dry ice by overnightmail. However, some specimens were thawed on arrival. Manyspecimens were kept at 2707C for several weeks at CDC beforeassay. Acute- and convalescent-phase serum samples were re-quested to be obtained at the diagnosis of HUS and again 3–4weeks after onset of symptoms. Serum samples were sent to CDCand were frozen at 2707C until assayed.

Identification of STEC. Of the 79 stool specimens received, 65were plated to both MacConkey agar (MAC) and sorbitol-MAC(SMAC). Fourteen specimens were plated only on SMAC, gen-erally because only a swab was received. Sorbitol-negative colonieswere selected from SMAC and were screened with O157 antiserum.Up to 20 colonies selected from MAC were tested for gene se-quences encoding for Shiga toxin 1 and 2 by oligonucleotide DNAprobes (nonselective method); all E. coli O157 isolates also weretested using these probes [5]. Isolates with Shiga toxin 1 or 2 geneswere biochemically identified and were serotyped by use of E. coliO and H antisera. One isolate identified as E. coli ORough:NM(“Rough” indicates that the isolate agglutinates nonspecifically inO typing antisera; NM indicates nonmotile) was further charac-terized by polymerase chain reaction (PCR) assay for the uid Aand H7 alleles [6, 7]. The 5 non-O157 STEC isolates were char-acterized further by PCR primers that amplified sequences encod-ing intimin (which has a role in mediating attaching-effacing le-sions) and enterohemorrhagic E. coli hemolysin (enterohemolysin)[8–10].

Detection of E. coli O157 LPS antibody. Serum specimens weretested by ELISA for antibodies to E. coli O157 LPS, as describedelsewhere [11]; however, alkaline phosphatase–labeled monoclonalantibodies against human IgM and IgG (Zymed Laboratories) wereused as the detecting reagents. Seropositivity was defined as eitheran IgM titer >1:320 or an IgG titer >1:80. The cutoffs for bothtests were determined by methods reported elsewhere [11], using se-

rum samples from culture-positive patients and healthy control sub-jects. The IgM ELISA is 80% sensitive and 95% specific (CDC,unpublished data), and the IgG ELISA is 92% sensitive and 91%specific [11] for persons with culture-confirmed STEC O157 infection.

Statistical methods. Clinical, epidemiologic, and laboratorydata were entered into a database. The x2 test, Fisher’s exact testof statistical significance, and Kruskal-Wallis 1-way analysis-of-variance by ranks test (H test) for comparing means were computedby using Epi Info version 6 (CDC).

Results

Epidemiology. From 1 January 1987 through 31 December1991, specimens from 83 persons with clinical and laboratorydiagnoses of HUS were included in the study. Cases were reportedmost frequently in the summer (figure 1). Patients were from 16states representing all 4 US census regions: Northeast (4 patients),South (16), Midwest (30), and West (33). The median age of thepatients was 4 years (range, 3 months to 64 years). Forty-six(55%) patients were !5 years old, 27 (33%) were 5–17 years old,5 (6%) were 18–44 years old, and 5 (6%) were >45 years old.Fifty-one patients (49%) were boys. Among children, the medianage of boys was similar to that of girls (3.5 vs. 3.0 years, re-spectively). Among adults, the median age was 45 years for menand 29 years for women. Seventy-one (86%) patients were white,5 (6%) were black, 6 (7%) were Hispanic, and 1 (1%) was Asian/Pacific Islander. One patient’s second episode of HUS occurredduring the study period. Clinical and microbiologic findings as-sociated with both this patient and another child have been re-ported in detail elsewhere [12, 13].

Clinical findings and therapy. The median interval betweenthe onset of diarrhea and the diagnosis of HUS was 5 days (range,1–21 days). Visible blood or blood streaks were present in thestool in the 21 days before diagnosis of HUS in 60 (73%) of the82 patients for whom information was recorded; 54 (78%) of 69patients had abdominal pain, and 17 (22%) of 77 had upperrespiratory tract infection symptoms. Among children, the me-dian duration of diarrhea before the diagnosis of HUS was 6.5days for patients with bloody stools and 5.5 days for those withnonbloody stools. Among adults, the median duration of diar-rhea before diagnosis of HUS was 7 days for patients with bloodystools and 3 days for those with nonbloody stools. Median lengthof hospitalization of children was 11 days (range, 1–388 days)and of adults was 20 days (range, 1–384 days).

For study subjects, the median levels of hemoglobin andserum creatinine were 65 g/L (range, 41–95 g/L) and 5.1 mg/dL (range, 1.0–15.5 mg/dL), respectively. The median plateletcount was 30,000/dL (range, 3000–95,000/dL), and the medianlevel of blood urea nitrogen was 100 mg/dL (range, 22–270 mg/dL). Twenty-one (25%) patients had major neurologic compli-cations: 18 (22%) had seizures (1 of whom also had quadri-paresis), 4 (5%) had encephalopathy (2 of whom also hadseizures), and 1 (1%) had aphasia.

Red cell transfusions were done in 31 (37%) of 83 patients.

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JID 2001;183 (1 April) US National HUS Study 1065

Figure 1. Month of onset of hemolytic uremic syndrome in 83 US patients, 1987–1991. Differential shading indicates patients who were positiveor negative for Shiga toxin–producing Escherichia coli (STEC) by all tests.

Forty-six patients (55%) required dialysis (peritoneal dialysisin 31; hemodialysis in 13; and peritoneal dialysis and hemo-dialysis in 2).

Twenty-four (31%) of 78 persons with information availablewere administered an antimicrobial agent in the 3 weeks beforediagnosis of HUS. The proportions were similar among children(21 [31%] of 68) and adults (3 [30%] of 10). Twenty-five regimenswere reported for 24 patients (b-lactams were used for 14 patients;trimethoprim-sulfamethoxazole for 6; macrolides for 2; andnitroimidazole, aminoglycoside, and quinolone for 1 each).

Four of the 5 females of childbearing age (14–45 years) werepregnant in the 2 months before the onset of diarrhea (3 werepostpartum at the onset of HUS). Illnesses in 4 of these patientsmet the criteria for TTP.

Outcomes. Of the 73 children, 8 (11%) had illnesses thatmet the criteria for TTP, and 4 (6%) died. The children whodied were 7 ( ), 9 ( ), and 14 ( ) years old (3 boysn p 1 n p 2 n p 1and 1 girl). None of the children who died had illness that metthe criteria for TTP.

Of the 10 adults, 8 (80%) had illnesses that met the criteriafor TTP. A 48-year-old woman and a 45-year-old man died;illnesses in both met the criteria for TTP.

Serology. A total of 94 serum samples were obtained from66 of the 83 patients (samples from only 65 of the 66 patientswere available for IgM assay). The age range of these patientswas 3 months to 64 years (median, 3 years), and samples werecollected between 0 and 85 days after the onset of diarrhea.Fourteen (21%) of the patients had illnesses that met the criteria

for TTP. Fifty-three (80%) of the 66 patients had IgM-positive(46 [71%] of 65) or IgG-positive (48 [73%] of 66) O157 LPSantibodies. The proportion of children either IgM or IgG sero-positive (47 [81%] of 58) was similar to that of adults (6 [83%]of 8). Both IgM and IgG were positive in 41 (63%) of the samplesfrom the 65 patients with samples available for IgM assay, andboth were negative in 13 (20%) of the samples. Testing serumsamples for IgM, in addition to IgG, increased the proportionof seropositive patients from 47 (72%) to 53 (82%) of 65. Serumsamples from 2 of the 5 patients with IgM but no IgG antibodieswere obtained within 4 days after the onset of diarrhea.

Seropositive patients were more likely to have blood in theirstool (38 [72%] of 53) than were seronegative patients (7 [54%]of 13), although this finding was not statistically significant.The proportion of seropositive patients with upper respiratorytract infections or abdominal pain was similar to that of sero-negative patients. The duration of diarrhea before HUS wasdiagnosed also was similar for both seropositive and seronega-tive patients. There was no statistical association between sero-positivity and sex, age, region of residence, or antibiotic ex-posure in the 3 weeks before the onset of diarrhea.

The proportion of serum specimens positive for IgG (78%)and IgM (81%) was highest 7–13 days after the onset of diarrhea(figure 2), although the overall proportion was not significantlydifferent at 0–6 days or 14–20 days. Convalescent-phase sero-conversion occurred in 1 (25%) of 4 persons whose initial serumspecimens were obtained in the first 6 days of illness. In samplesobtained >56 days after the onset of diarrhea, 2 patients (25%)

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Figure 2. Proportion (median and 95% confidence intervals) of hemo-lytic uremic syndrome patients with antibodies to Escherichia coli O157lipopolysaccharide at different intervals after the onset of diarrhea.

Table 1. Virulence-associated gene sequences and sorbitolfermentation of non-O157 Shiga toxin–producing Escheri-chia coli isolated from patients with hemolytic uremicsyndrome.

Serotype stx1a stx2b eaec E-hlyd

Sorbitolfermentation

at 24 h

O111:NM 1 1 1 1 1O126:H27 1 2 2 2 1O172:NM 2 1 1 1 2OX3:H21 2 1 2 1 1ORough:NM 2 1 1 1 2

NOTE. “Rough” indicates that the isolate agglutinates non-specifically in O typing antisera; NM, nonmotile; 1, positive; 2,negative.

a Gene encoding Shiga toxin 1 production [10].b Gene encoding Shiga toxin 2 production [10].c Gene encoding intimin, which plays an essential role in medi-

ating attaching-effacing lesions [8].d Gene encoding enterohemorrhagic E. coli hemolysin (entero-

hemolysin) [9].

had IgM antibodies, and 7 (70%) had IgG antibodies. Of the13 persons whose serum specimens were both IgM and IgGnegative, 7 had specimens collected between days 7 and 20, and3 only had samples taken 120 days after the onset of diarrhea.

Stool culture and characterization of isolates. Fecal speci-mens from 79 (95%) of the 83 patients were cultured for amedian of 8 days (range, 2–30 days) after the onset of symp-toms. STEC were isolated from 30 (43%) of the 70 specimenswith bacterial growth. Twenty-five (83%) of these isolates wereE. coli O157; of these, 22 had the H7 antigen, and 3 were NM.The other 5 isolates were E. coli serotypes O111:NM, O126:H27, O172:NM, OX3:H21, and OROU:NM. The OROU:NMisolate was negative for the uid A and H7 alleles characteristicof E. coli O157:H7.

STEC were isolated from 28 (47%) of 60 children and 2 (20%)of 10 adults ( ). One adult’s stool sample contained non-P p .17O157 STEC, serotype OX3:H21; the other non-O157 STECisolates were from samples from children. Of the 28 children’sstool samples that yielded STEC, 24 (86%) contained E. coliO157. Among adults, 1 of 2 STEC isolates were E. coli O157.Of the 28 children with STEC, none of the 16 children >3 yearsold but 4 of the 12 children !3 years old had non-O157 STEC( ). Of the 17 persons whose stool specimens were col-P p .02lected >10 days after the onset of diarrhea, 5 (29%) had speci-mens with STEC isolates, and 3 of these were E. coli O157isolates. The interval between the onset of diarrhea and thedate when stool was obtained from patients whose specimensyielded STEC isolates (median, 7 days; range, 4–29 days) wassimilar to that of patients whose specimens yielded no STECisolates (median, 9 days; range, 2–30 days).

Of the 30 E. coli isolates, genes for both Shiga toxins 1 and2 were identified in 22 (1 of which was O111:NM), Shiga toxin2 alone was identified in 7 (3 of which were non-O157 isolatesOROU:NM, OX3:H21, and O172:NM), and Shiga toxin 1alone was identified in 1 (which was E. coli O126:H27). Of thenon-O157 STEC, 3 had genes encoding intimin and entero-

hemolysin, 1 had the genes for only enterohemolysin, and 1had genes for neither virulence factor (table 1). Two non-O157STEC (O172:NM and OROU:NM) shared the typical O157characteristic of not fermenting sorbitol within 24 h.

The proportion of persons with bloody diarrhea was higheramong those with E. coli O157 (20 [80%] of 25) than amongthose with non-O157 STEC (3 [60%] of 5) or those with no STECisolated (26 [65%] of 40), but these differences were not statis-tically significant ( ). Among those with informationP p .27available, the isolation rate of STEC was similar among personswho had (8 [42%] of 19) and had not (21 [45%] of 47) receivedantimicrobial treatment in the 3 weeks before HUS began.

To assess the relative frequency of E. coli O157, comparedwith that of the non-O157 serotypes, we examined results fromthe 65 stool specimens that were assayed by the nonselectivemethod (by which E. coli O157 and other serogroups had anequal likelihood of isolation). Eleven STEC isolates were identi-fied by this method: 8 (73%) were E. coli O157, and 3 (O111:NM, O126:H27, and OX3:H21) were non-O157 STEC isolates.All the E. coli O157 strains identified by this method also wereidentified from the SMAC plate, and additional E. coli O157strains were identified from the SMAC plate.

Combined serology and stool culture. Of the 55 persons withsamples tested both by serology and by stool culture, 45 (82%)had evidence of STEC infection (table 2). Of these 55 persons,the samples were positive for O157 LPS antibodies in all 20whose stool culture yielded E. coli O157, in 3 of the 4 whosestool culture yielded non-O157 STEC, and in 21 (68%) of the31 whose stool cultures were negative.

Ten (18%) of these 55 patients had no evidence of STECinfection; among them, the proportion with bloody diarrhea (3[30%] of 10) was lower than in those with STEC infection (30[68%] of 44; ). Eleven (48%) of the 23 persons who wereP p .03not confirmed as having STEC infection had onset of diarrheain May through September, compared with 45 (75%) of the 60

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Table 2. Relationship between Escherichia coli O157 lipopolysac-charide (LPS) serology results and Shiga toxin–producing E. coli(STEC) in 55 US patients with hemolytic uremic syndrome from whomboth serum and stool samples were obtained.

Serologic resultfor O157 LPS

E. coli O157isolated(n p 20)

Non-O157STEC isolated

(n p 4)

No STECisolated

(n p 31)

Seropositive (n p 44) 20 (100) 3 (75)a 21 (68)Seronegative (n p 11) 0 1 (25)b 10 (32)

NOTE. Data are no. (%) of patients. Seropositive is defined as either IgMor IgG seropositive; seronegative is defined as both IgM and IgG seronegative.An additional 11 patients were tested only by serology, and 10 (91%) of thesewere O157 LPS seropositive. An additional 17 patients were tested only by stoolculture: E. coli O157 was isolated from 5, and OX3:H21 was isolated from 1.

a O126:H27, O172:NM, and OROU:NM.b O111:NM.

with STEC infection ( ; figure 1). STEC-negative pa-P p .04tients were no more likely than patients with STEC infectionto have used antibiotics in the 3 weeks before the onset ofdiarrhea. Of the 2 adults in this group with no evidence ofSTEC infection, 1 was 18 weeks pregnant, and the other diedof presumptive pancreatic carcinoma.

Of the 10 adults studied (including 8 tested both by serologyand by stool culture, and 2 tested only by stool culture), 7(70%) had evidence of STEC infection. Six of these 7 adultshad serology results positive for O157 LPS, and 1 of the 6 alsohad a stool culture that yielded E. coli O157. The seventh adultwith evidence of STEC infection had a stool specimen thatyielded STEC OX3:H21.

Of the 8 adults whose illnesses met the criteria for TTP (in-cluding 7 tested both by serology and by stool culture and 1tested only by stool culture), 6 (75%) had evidence of STECinfection. Five of these had positive serology for O157 LPS, and1 of these 5 also had a stool culture that yielded E. coli O157.The sixth had a stool specimen that yielded STEC OX3:H21.

Similar to findings among adults, of all 73 children studied,53 (73%) had evidence of STEC infection. Of the 8 childrenwhose illnesses met the criteria for TTP, 5 (63%) had evidenceof STEC infection.

Of all 83 patients in the study (including the 55 tested bothby serology and by stool culture, the 11 tested only by serology,and the 17 tested only by stool culture), 60 (72%) had evidenceof STEC infection. Of the 4 pregnancy-associated HUS cases,the 3 that were postpartum all had evidence of E. coli O157infection (all 3 patients had serologic evidence; of the 2 withstool cultures, 1 yielded E. coli O157, and 1 had no growth).

Discussion

To our knowledge, this study is the only reported US na-tionwide prospective study of postdiarrheal HUS. The resultsshow that, throughout the country, STEC could be implicatedin 72% of cases of HUS and that E. coli O157 was the likelycause in 180% of patients with STEC infection. Most studies

of HUS have enrolled only children [14]. This study found thatE. coli O157 caused most cases of postdiarrheal HUS amongadults and children.

Serologic testing for antibody to O157 LPS was very sensitiveand was positive for all patients whose stool cultures yieldedE. coli O157. Its usefulness derived partly from the fact that itwas much less time constrained than was stool culture—theantibody titer was often elevated at the same time as the typicalonset of HUS, and some patients still had antibody >60 daysafter the onset of illness. Other studies have measured the IgGand IgM responses to O157 LPS and have reported that an-tibody titers may remain elevated after 8 weeks [15, 16], butthe time line for the IgG antibody response for patients withHUS has not been well determined. We do not know why anIgM response was not detected in some patients. One possibilityis that the response was transient and thus was overlooked.Another possibility is that some patients had been exposed toO157 LPS in the past, so their current response was secondary.In addition, the IgM test is only 80% sensitive, so some re-sponses would not be detected. The usefulness of this assaysuggests that serologic tests for antibodies to important non-O157 STEC serogroups, such as O111 and O26, should be morewidely used. Development of commercial kits to test for theseantibodies also would be helpful. However, the identificationof the etiologic agent is more certain when an isolate is ob-tained, and this always should be attempted.

Stool cultures were less sensitive than serologic testing inidentifying patients with STEC infection. Among the 43 pa-tients with E. coli O157 infection identified by serologic testingwho also had stool culture, 47% had cultures with negativeresults. That 11% of the stool cultures yielded no growth in-dicates that some specimens were compromised either by freez-ing, shipping, or storing or by treatment of patients with anti-biotics. However, isolation rates for STEC are well known todecrease after the first week of illness, when HUS typicallybegins [17]. Despite these difficulties, the isolation of STECfrom 43% of specimens with bacterial growth is comparablewith the results of many other studies [14]. Indeed, the fact thatSTEC was isolated from almost one-third of the samples col-lected >10 days after the onset of diarrhea should encouragethe collection of stool specimens even late in the illness. Inaddition, more sensitive methods, such as ELISA kits for Shigatoxins, PCR assays to detect toxin genes, and immunomagneticseparation, are now available for detecting STEC in stools [18].However, the ELISA is not routinely used by clinical labora-tories, and the other tests are not widely used. E. coli O157 isthe only STEC serotype that can be easily and inexpensivelyscreened for by clinical laboratories using commercially avail-able media and reagents, because it does not ferment sorbitolat 24 h [18].

Despite these obstacles, clinical laboratories must recognizethat culturing stools for STEC from patients with HUS is im-portant, because it can assist in identifying outbreaks and is

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the principal way to identify non-O157 STEC serotypes thatmay be emerging causes of diarrheal illness and HUS. Opti-mally, the initial stool specimen from all patients with HUSwould be tested for Shiga toxin. Some laboratories may alsochoose to initially screen stools using selective agar for E. coliO157, the most likely agent. However, testing initially only forE. coli O157 may decrease the likelihood of identifying a non-O157 STEC because specimens tested after being held in thelaboratory and specimens obtained from patients at a later datemay no longer contain the etiologic agent. Clinical laboratoriescan refer strains of E. coli from patients with suspected STECinfection (e.g., patients with a positive ELISA for Shiga toxin,patients with HUS, and patients with bloody diarrhea and nopathogens detected) to state health department laboratories.Referral of such strains also can help in detecting outbreaks.

Estimates of the proportion of STEC-associated HUS dueto non-O157 STEC are difficult to make. The data suggest thatnon-O157 STEC caused <20% cases, because only 20% of pa-tients tested by serology did not have antibodies to O157 LPS.This is probably a high estimate, because some patients withE. coli O157 infection do not have a serologic response [11],and some seronegative patients probably did not have STECinfection. (However, false positive serologic responses to O157LPS also occur.) Culture results provide higher estimates of theproportion of STEC-associated cases due to non-O157 STEC,but these are less reliable partly because the numbers aresmaller. Of the 30 STEC isolated, 5 (17%) were non-O157strains; however, the overall culture methodology favored de-tection of E. coli O157. Among the 65 stool specimens thatwere analyzed by the culture methodology that did not favordetection of E. coli O157, 11 STEC strains were identified, and3 (27%) were non-O157 serotypes.

Verweyen et al. [19] also reported more frequent isolation ofnon-O157 STEC from younger children than from older chil-dren, and this finding merits further study. E. coli O111:NMwas clearly responsible for 1 case of HUS, and an investigationof household members indicated that this case was part of acluster of E. coli O111 infections [20]. This serotype is a well-recognized cause of bloody diarrhea and HUS; it caused a largeoutbreak in Australia in 1995 [21] and an outbreak amongteenage campers in Texas [22]. Of 330 human non-O157 STECisolates identified by the CDC reference laboratory from 1983through 1999, 14% of strains were serogroup O111, which wassecond only to E. coli O26, which comprised 22% of isolates(N. Strockbine, CDC, personal communication). To improverecognition of the role of major non-O157 STEC serotypes, USlaboratories should consider testing E. coli isolates from pa-tients with HUS or with hemorrhagic colitis by using antiserato E. coli O111 and O26.

Our findings on the frequency of STEC and of E. coli O157in both children and adults with HUS confirm and extend pre-vious work. Studies of 150 patients with HUS that tested bothstool samples for all STEC and serum samples for antibodies

to O157 LPS also have been reported from Europe, but theyincluded only children. Similar to this study, these studies re-ported that 78% [23], 86% [16, 24], and 88% [19] of the patientshad evidence of STEC infection. A small, local US study ofpatients with postdiarrheal HUS between 1989 and 1992 alsoreported that 85% of the patients had STEC infection confirmedby either serology or by stool culture [25]. As in our findings,E. coli O157 has been identified as the predominant cause ofHUS in 2 local US studies [3, 25] and in Canada [4, 26, 27].Antibodies to O157 LPS were identified in 73% of patients withHUS tested in England [28], in 73% [16] and 66% [19] of chil-dren with HUS in Central Europe, and in 67% of children withHUS in France [24]. In Italy, a study using LPS from differentE. coli serotypes provided evidence that O157 strains were themost prevalent STEC associated with HUS in that country [29].

A relatively large proportion (18%) of patients who weretested both by serology and by stool culture had no evidenceof STEC infection. Because the assays that were used to identifyE. coli O157 infection were fairly sensitive, it is unlikely thatall of these patients had E. coli O157 infection. Some may havehad non-O157 STEC infection, and some probably did not haveSTEC infection. The diarrheal illness of these patients, likeHUS, may have been a manifestation of some other diseaseprocess. This hypothesis is supported by the fact that, comparedwith patients with evidence of STEC infection, a lower pro-portion of patients had bloody diarrhea, illness was less likelyto occur in warmer months, and 2 had conditions (pregnancyand carcinoma) that can precipitate a systemic illness with thefeatures of HUS. More studies are needed to better distinguishpatients with the clinical characteristics of diarrhea-associatedHUS who do not have STEC infection from those who haveSTEC infection and to determine the etiology of their illness.In particular, the role of pneumococcal infection should beconsidered, because it is an important cause of HUS, althoughpatients with pneumococcal-associated HUS have not been re-ported to have preceding diarrhea [30].

This study had several limitations. The age- and geographic-specific variation in the enrolled patients reflected the variableparticipation of physicians in different parts of the country. Thedata cannot be used to compare regional variations or the pro-portions of HUS patients who are children or adults, but theydo indicate that STEC-associated HUS occurred throughoutthe United States. Because the study was conducted from 1987through 1991, the relative frequency of STEC serotypes identi-fied may have since changed. However, documenting the majorserotypes during different time periods is important, and recentdata suggest that E. coli O157 is still the predominant cause ofHUS in the United States [31].

Several findings in this study merit further study. Four ofthe 5 women of childbearing age in the study were pregnantor postpartum; identification of STEC infection in all 3 womenwho were postpartum suggests that STEC may be an importantcause of postpartum HUS. To our knowledge, a clear link be-

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tween STEC and postpartum HUS has not been previouslymade and has not been discussed in reviews of postpartumHUS, although STEC O6:H12 was isolated from the stool of1 case who did not have gastrointestinal symptoms [32], andbloody diarrhea preceded HUS in another case report [33].Adults were more likely to meet the criteria for TTP than werechildren, and all patients who died of TTP were adults. Thisinformation suggests that the pathophysiologic characteristicsof STEC-associated HUS in adults is different from that inchildren.

Active surveillance for HUS in selected population-basedsites in the United States started in 1997 and is still beingdeveloped [31, 34]. By monitoring trends in the incidence ofHUS over time, this system will provide critical informationfor evaluating the impact of measures to decrease contami-nation of food and water by STEC. By monitoring trends overtime in the proportion of HUS cases due to different STECserotypes, this system will alert the public health communityto emerging STEC serotypes.

Hemolytic Uremic Syndrome Study Collaborators

Richard L. Siegler and Andrew Pavia (University of UtahMedical Center, Department of Pediatrics, Salt Lake City); Ka-trina Hedberg, Kristine MacDonald and Edward Belongia(Minnesota Department of Health, Minneapolis); Julie Kul-hanjian, Donald Janner and Rose-Ellen Morrell (Oakland Chil-dren’s Hospital, Oakland, CA); Kenneth Miller and BarbaraAtkin (Lutheran General Hospital, Park Ridge, IL); MartinDe Beukelaer (Medical College of Ohio, Department of Pe-diatrics, Toledo); Bryson Waldo (Children’s Hospital, Bir-mingham, AL); Mark T. Houser, Teri Wead, and Pamela Wen-dell (University of Nebraska Medical Center, Department ofPediatrics, Omaha); Charles Knupp (East Carolina UniversitySchool of Medicine, Greenville, NC); Russell A. Gerber andHarvey Frohlich (Department of Pediatrics, Egleston Hospital,Atlanta, GA); Timothy Bunchman and Ellen Wood (CardinalGlennon Children’s Hospital, St. Louis, MO); Daniel Ornt andAlice Loveys (Strong Memorial Hospital, Rochester, NY);Keith Powell (Department of Pediatrics, University of Roch-ester Medical Center, Rochester, NY); Barbara Baetz-Green-walt (Cleveland Clinic Foundation, Cleveland, OH); John Bas-tian (Children’s Hospital and Health Center, San Diego, CA);Dean Blumberg and James D. Cherry (University of Californiain Los Angeles Medical Center); John Foreman (Richmond,VA); Bernard Gauthier, Howard Trachtman and Rachel Frank(Schneider Children’s Hospital, New Hyde Park, NY); JackRogers (Harbin Clinic, Rome, GA); Ludwig Lettau (Greenville,SC); Farrin A Manian (St. John’s Mercy Medical Center, St.Louis, MO); Juan Alejos (University of California, Los Angeles);Thomas E. Calk (Humana Hospital, Snellville, GA); ColeenCunningham (State University New York Upstate Medical Uni-versity, Syracuse); Daniel Guyton (Akron General Medical Cen-

ter, Akron, OH); Stephen Ross (Kalamazoo, MI); Coral Ha-nevold (Cedars Sinai Medical Center, Los Angeles, CA).

Acknowledgments

We would like to thank the following, all of whom were at the Centersfor Disease Control and Prevention (Atlanta) during the study: Evan-geline Sowers for laboratory assistance; Cecile Ivey and Meghan Deyfor help with data handling; Paul Mead and Larry Slutsker for helpfulcomments on the manuscript; and Paul Blake and Robert Tauxe fortheir encouragement of this work when resources for such efforts wereminimal.

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