Patients were enrolled from January 2001 through December 2002 from the public tertiary-care pediatric hospitals, Hospital “Dr. Humberto Notti” in Mendoza, which serves an urban and semirural area, and Hospital “Dr. Juan P. Garrahan” in Buenos Aires, which serves an urban area. Study personnel detected STEC cases through daily review of the hospitals’ laboratory records and detected diarrhea-associated HUS cases through biweekly discussions with nephrologists.

A case of STEC infection was defined as illness in a previously healthy child <16 years old who was evaluated at a participating institution and who had either culture-confirmed O157 STEC diarrhea, culture-confirmed non-O157 STEC diarrhea, or definite diarrhea-associated HUS. For convenience, we included only children permanently residing within 15 km of each institution. Local investigators stated that the characteristics of the population within each of these areas were similar to that within 50 or 100 km of the respective hospitals. We chose these 2 areas so that the population serviced would be similar to the Argentine population living in areas with a high incidence of HUS.

Definite HUS was defined as acute microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment as determined by 1) hematocrit <30% with microangiopathic changes on peripheral blood smear (e.g., schistocytes, burr cells, helmet cells, or red cell fragments); 2) platelet count <150,000/mm³; and 3) serum creatinine concentration >2 standard deviations above the upper limit of normal for age and sex (21); or abnormal urinary sediment by dipstick, i.e., hematuria (>2+) or proteinuria (>2+). HUS was considered diarrhea-associated when a diarrheal illness preceded HUS by <3 weeks. We excluded children with a family history of HUS, secondary HUS (e.g., drug-associated), or HUS associated with pneumococcal infection. Probable HUS was defined as an illness that met only 2 of the laboratory criteria in a patient with culture-confirmed STEC infection.

For each patient enrolled, 2 age- and neighborhood-matched control children without gastrointestinal illness in the 2 weeks before the matched case’s illness onset were identified. We neighborhood-matched control children to control for socioeconomic status, which was not a factor of interest, was a possible confounder, and was quite variable in the study areas. In urban areas, a trained interviewer sought controls by walking, starting at the case-patient’s home, going to the third house from the nearest corner, and continuing to every house on the block, then to the block facing the case-patient’s residence, then to other blocks in a clockwise fashion, until 2 eligible and consenting controls were interviewed. In nonurban areas, the interviewer randomly chose a cardinal direction and then sought controls beginning from the third residence from the case patient’s house in that and the opposite direction until controls were found. Informed consent was obtained from the adult primary caregiver, who was interviewed with a standardized questionnaire, administered in person. Most eligible controls were enrolled; information on potential controls who were excluded or chose not to participate was not kept.

The questionnaire (available upon request from the corresponding author) had 89 major questions and was divided into 3 major sections: characteristics of and treatment for the illness (19 questions), exposures (55 questions), and demographics (15 questions). Exposure questions were divided into sections that dealt with human contacts , care and feeding of young children, water sources and treatment, beef, other meats, fruits and vegetables, meat handling at home, animal contact, swimming, and travel. Many exposure questions had several parts (e.g., if beef consumed, was it pink; if chicken consumed, indicate if at home, restaurant, or other location). Almost all responses were measured discretely (i.e., categorically). Responses to each question were treated independently, except for those about fruits and vegetables, which were combined and dichotomized at the median; this analysis method was checked against other choices and found robust. All exposure questions for case-patients and controls were about the 7 days before the onset of illness. Interviews of case-patients were conducted a median of 12 days (range 3–41) after diarrhea onset; control interviews were conducted a median of 15 days (range 1–41) after diarrhea onset in the matched case. Controls were age-matched to case-patients by using the following groups: <12 months; 1–5 years ± 1 year; 6–9 years ± 2 years; 10–15 years ± 3 years.

Fecal samples were plated onto sorbitol-MacConkey agar directly, and after enrichment at 37°C for 4 h in trypticase soy broth were supplemented with cefixime (50 ng/mL) and potassium tellurite (25 mg/mL). The confluent growth zone and colonies were screened for stx1, stx2, and rfbO157 genes by a multiplex PCR (22,23). Isolates with stx1 or stx2 genes were identified by standard biochemical methods. Stx-positive colonies were serotyped (24) and characterized at the Argentina National Reference Laboratory (11,25).

Data analysis used 3 steps: an initial univariate analysis, a second univariate analysis adjusted for highly significant factors of prior or secondary interest, and a final multivariable model-building analysis. Four factors were ultimately chosen in the second univariate analysis as a fixed set from which to explore further model-building, based on epidemiologic sensibility, strong association, and stability of subsequent adjusted associations. Single- and multiple-variable conditional logistic regression models were used to evaluate associations between the outcome and exposure variables. At each step, risk factors that were statistically significant (p<0.05) and had biologic plausibility were selected for further modeling. In multivariable model-building, we pursued forward, backward, and the best subset selection strategies, as well as manual strategies. Standard methods were used to assess model fit, including residual analyses. Maximum likelihood parameter estimates from these models were used to calculate point estimates and confidence intervals for odds ratios, referred to henceforth as matched odds ratios. Exact analysis was used where small sample size would make asymptotic analysis suspect, and Mantel-Haenszel odds ratios were computed when maximum likelihood estimates did not exist.

Exploratory and sensitivity analyses were performed on subsets of the data defined by location and subcategory of disease status. Because the subsets by site (Buenos Aires and Mendoza) and by serogroup (O157, non-O157) were small and most factors examined had already been demonstrated as risky or protective in the larger dataset, we used a p value of 0.10 to assess significance by site and serogroup. For similar reasons, we did not perform multivariable analyses on these subsets. Three patients with mixed STEC infection and 2 stx-positive HUS patients without STEC isolated were excluded from the analysis by serogroup. Data were analyzed with Epi Info 2000 (Centers for Disease Control and Prevention, Atlanta, GA, USA) and SAS 9.0 (SAS Institute, Inc, Cary, NC, USA) software. The study was approved by the hospitals’ ethics committees as well as the institutional review boards of the Ministry of Health of Argentina and CDC.

Among 157 eligible case-patients, 150 (96%) were enrolled; the parents of 1 child refused, and interviewers could not contact the parents of 6. The hospital “Dr. Juan P. Garrahan” in Buenos Aires enrolled 54% of the cases, and the hospital “Dr. Humberto Notti” in Mendoza enrolled 46%. Among the 150 enrolled cases, 17 met both entry criteria of culture-confirmed STEC infection and definite HUS, 82 met only the criterion of culture-confirmed STEC infection, and 51 met only the criterion of definite HUS. In addition to the patients with definite HUS, 10 patients with culture-confirmed STEC infection had probable HUS.

The median age of case-patients was 1.8 years (range 4 months–14 years). The median age of the 299 controls was similar (2.0 years; range 1 month–17 years); 58% of case-patients versus 43% of controls were female (p = 0.01). One hundred and thirty-four (89%) case-patients were urban residents.

Among the 150 case-patients, clinical findings included bloody diarrhea (84%) and vomiting (71%). Ninety-four (63%) were hospitalized for a median of 4.5 days (range 1–14 days). Among the 78 case-patients with definite HUS (dHUS n = 68) or probable HUS (pHUS n = 10), 37 (47%) had peritoneal dialysis (34 dHUS 50%, 3 pHUS 30%), 2 (3%) had hemodialysis (all dHUS), and 69 (88%) received erythrocyte transfusions (62 dHUS, 7 pHUS).

Among the 99 case-patients with culture-confirmed STEC infection, 96 had a single STEC isolated: 58 (60%) were O157:H7, and 38 (40%) non-O157. Among the 99 with STEC isolated, the proportion who had O157 was 82% (14 of 17) among children with definite HUS, 90% (9 of 10) among children with probable HUS, and 53% (38 of 72) among children without HUS. Among the 38 non-O157 isolates, the serotype frequency was 29% O145:NM, 11% O26:H11, 11% O113:H21, 8% O174:H21, 5% O8:H19, 5% O145:H25, 5% ONT:NM, and 3% (1 each) O2:H11, O15:H27, O25:NM, O58:H40, O91:H7, O103:H2, O103:H25, O111:NM, O121:H19, and O171:H2.

Analysis of single variable associations, when the fixed adjustment factors were controlled for, identified dietary habits and animal exposures linked to illness (Table 1). General dietary habits linked to STEC illness included eating at a social gathering, eating any meal prepared at home, and drinking from a baby bottle left at room temperature for >2 hours. Many beef-related exposures were significantly associated with STEC infection (Tables 1, 2). Eating beef outside the home and eating undercooked beef (described as uncooked, red and juicy, or pink) anyplace was associated with illness. Eleven percent of case-patients but only 5% of controls consumed jugo de carne (liquid squeezed from a tender, usually lightly cooked piece of beef, and spoon-fed); case-patients with this exposure ranged from 7 months to 9 years old. Living in or visiting a place with farm animals, contact with farm animals (including horses, pigs, poultry, and cattle), and contact with cattle manure were associated with illness. Risky exposures that suggest person-to-person transmission from young children included contact with a child <5 years of age, attending daycare or kindergarten, and contact with a child <5 years of age with diarrhea. Wearing diapers was also linked to illness. No significant differences between case-patients and controls were found in the distribution of most variables that relate to socioeconomic status (e.g., number of bedrooms, water supply, garbage disposal, educational level of parents). However, case households were more likely to have nonparental income.

Four protective factors were identified, all related to beef (Table 2). These were the child eating meatballs at home; the child eating empanadas (fried or baked pastries with ground beef filling) at home; the child eating meat pie at home; and the respondent always washing hands with soap and water after handling raw beef. The factors controlled for in the adjusted univariate analysis were eating more than the median number of fruits and vegetables, male sex, having a nonparent respondent, and the respondent always washing hands after handling raw beef; all were protective (Tables 1, 2).

On multivariable logistic regression analysis, significant risk factors for STEC infection that remained were eating undercooked beef outside the home (odds ratio [OR] 17.63, 95% confidence interval [CI] 1.6–197.4, p = 0.02), living in or visiting a place with farm animals (OR 6.61, 95% CI 1.5–28.8, p = 0.01), contact with a child <5 years of age with diarrhea (OR 3.29, 95% CI 1.0–10.4, p = 0.04), and having nonparental household income (OR 2.21, 95% CI 1.2–4.0, p = 0.01). Eating ground beef at home (meatballs, empanadas, or meat pie) remained protective (OR 0.36, 95% CI 0.1–0.9, p = 0.03). With this model, the fixed adjustment factors had significant estimated protective associations as follows: eating more than the median number of fruits and vegetables (OR 0.31, 95% CI 0.1–0.6, p = 0.0007), male sex (OR 0.34, 95% CI 0.2–0.7, p = 0.001), having a nonparent respondent (OR 0.34, 95% CI 0.2–0.7, p = 0.001), and the respondent always washing hands after handling raw beef (OR 0.23, 95% CI 0.1–0.6, p = 0.001).

We performed a univariate adjusted analysis for all variables by site (Tables 1, 2). For every variable that was significantly risky or protective in the combined analysis, the OR went in the same direction in the site-specific analysis (Buenos Aires 81 cases, Mendoza 69 cases), although the association was not always statistically significant. Many dietary habits, most of which were beef associated, were significantly associated with illness in Buenos Aires, whereas fewer reached statistical significance in Mendoza. Consuming jugo de carne was significantly associated with illness in both sites; however, 19.1% of case-patients from Mendoza consumed this item compared with only 4.9% from Buenos Aires.

Risk and protective factors were also analyzed separately for patients with culture-confirmed O157 or non-O157 STEC infection (Table 3). These 2 groups were similar in age, sex, and site distribution. The risk and protective factors among these 2 groups were similar to those of all study participants. Among patients with O157 STEC infection, illness was significantly associated with eating at a social gathering, with many meat-related dietary habits, exposures related to farm animals and their environment, wearing diapers, and having a nonparental household income. Protective factors included several related to eating beef at home and buying beef less than once a week. Among the smaller group of patients with non-O157 STEC infection, the only risk factors significantly linked to illness were drinking from a bottle left at room temperature, drinking formula (a factor not identified in the full group), eating a piece of beef outside the home, teething on undercooked beef at home, contact with a child <5 years of age with diarrhea, wearing diapers, and living in an overcrowded condition. Eating meat pie at home was protective for this group.