A prospective multicenter surveillance study of pediatric patients with acute BD (first cohort) was conducted between October 10, 2018 and June 30, 2019 in seven tertiary-care hospitals (Reference Sites) and their 18 hospitals that treat patients with less complex illnesses (referral units) in Buenos Aires, La Pampa, Mendoza, Neuquén, Río Negro and Santa Fe provinces, and Buenos Aires City, Argentina (Fig. 1). Screening criteria included children of either gender between 1 and 9 years of age who presented acute BD. Acute BD was defined as loose stools by parental/caregiver report, with the presence of visible blood or blood observable only under a microscope, with less than 2 week-duration.

Figure 1.

Geographic distribution of bloody diarrhea and STEC-positive bloody diarrhea cases. Map data obtained from © OpenStreetMap contributors. Data is available under the Open Database License https://www.openstreetmap.org/copyright.

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We also studied a second cohort of HUS cases aged 1–9 years, known to be infected with STEC in the same hospitals during the study interval. HUS was defined as the triad: microangiopathic hemolytic anemia (fragmented red blood cells in peripheral blood smear – schistocytes, of non-immune origin [negative direct Coombs test]), thrombocytopenia (platelet count<150000cells/mm3), and renal dysfunction (serum creatinine concentration>2 standard deviations above the upper limit of normal for age).

The demographic, clinical and microbiological data of all cases incorporated were obtained from the medical history and related medical records, according to the standards of the Reference Site. A study-specific electronic Case Report Form (eCRF) was used as the only tool for data collection, storage and reporting. Data entered included age, gender, origin (province/locality), date of onset of bloody diarrhea, date of collection of the stool sample (identified by a code number for microbiological analysis), and history of antibiotic exposure. Progression to HUS within 28 days from the STEC diagnostic confirmation was identified and the date of the diagnosis (i.e., the date that the HUS case definition was fulfilled) was recorded.

The study was performed in compliance with the legal and regulatory requirements, as well as scientific purpose, value and rigor, and the principles established in the Declaration of Helsinki42 and in the Guide for Good Clinical Practices (ICH Harmonised Tripartite Guideline: Guideline for Good Clinical Practice E6(R2). November 9th, 2016. URL: https://database.ich.org/sites/default/files/E6_R2_Addendum.pdf).

The study protocol was approved by the Institutional Review Boards or the local Ethics Committees as required by the local regulation [Disposición ANMAT No. 6677/10. November 2010. URL: https://www.argentina.gob.ar/normativa/nacional/disposici%C3%B3n-6677-2010-174557/texto] and institutional Standard Operating Procedures. Thus, each Reference Site joined the study as soon as approval was obtained, between October 2018 (Hospital Penna of Bahia Blanca, Buenos Aires Province) and February 2019 (Hospital Molas of Santa Rosa, La Pampa).

Dates of reception and processing of the stool sample in the local laboratory, type of sample (fresh stool or rectal swab) and result of the microbiological tests were recorded.

All fecal samples were screened using the Shiga Toxin Quik Chek (STQC) test (Abbott™, TechLab®, Blacksburg, VA) according to the manufacturer's instructions and the guidelines established by the National Reference Laboratory (NRL) of Argentina directly on feces (direct method), and after overnight (16–20h) stationary incubation at 37°C in 8ml of Gram Negative (GN) Broth-Hajna (Becton Dickinson, Sparks, MD) (enrichment method). A total of 413/714 fecal samples were processed by an in-house validated multiplex PCR, according to laboratory capacity17. These samples were plated onto sorbitol-MacConkey agar (SMAC, Becton Dickinson) directly, and after enrichment at 37°C for 4h in trypticase soy broth (TSB) (Becton Dickinson), and TSB supplemented with cefixime (50ng/ml) and potassium tellurite (25mg/ml) (CT-TSB). The confluent growth zone and colonies were screened for stx1, stx2, and rfbO157 genes. Isolates positive for stx1 and/or stx2 were confirmed as E. coli by standard biochemical tests.

STEC infection in HUS patients was established by at least detecting (i) Stx1 and/or Stx2 by STQC; (ii) stx genes by mPCR and/or STEC isolation; (iii) detection of free fecal Shiga toxin (FFStx) by neutralization of the cytotoxic effect on Vero cells, using Stx1- and Stx2-specific monoclonal antibodies (MAb 13C4 and BC5BB12, respectively)16; (iv) detection of specific IgM and IgG antibodies against the O-polysaccharide of E. coli O157, O145, O121, and O103 using an indirect enzyme linked immunosorbent assay (CHEMLIS® E. coli Combi Glyco-iELISA kit, Chemtest S.A., Argentina).

Fecal samples, and stx-positive isolates and sera were sent to the NRL for confirmation and further characterization23,31.

A descriptive and exploratory analysis was performed using R software version 3.5.0 (R Foundation for Statistical Computing, Vienna, Austria). Continuous variables were summarized by mean/standard deviation and median/interquartile range (IQR), whereas frequency and proportions were applied to summarize qualitative variables. Categorical variables were compared using the Pearson chi-square test. All tests were two-sided and p-values less than 0.05 were considered statistically significant.

The following proportions and intervals were estimated: (1) proportion of STEC-positive BD by any method (STQC, direct and/or enriched culture, and mPCR) globally and by Reference Site; (2) cumulative incidence of STEC-positive BD evolving to HUS; (3) interval between the onset of BD and seeking medical attention; (4) interval between the onset of BD symptoms and sample collection; (5) interval between sample collection and processing; and (6) frequency of confirmed HUS cases. The performance of the STQC in comparison to the mPCR was also evaluated.

A total of 714 children with BD were included in the study. Median age was 42 months (IQR 24–63; range, 11–119 months), 44.8% (n=320) were female and 4.2% (n=30) received antimicrobial treatment before admission to the hospital. The median interval between BD onset and seeking medical attention was 0 days (IQR 0–1 days; range, 0–17 days).

Twenty-seven STEC-HUS cases were enrolled. Median age was 37 months (IQR 25–59 months; range 16–104 months) and 51.9% (n=14) were female. For this cohort, the median interval between BD onset and STEC-HUS diagnosis was 4 days (IQR 1–6 days; range, 0–10 days).

Of the 714 patients, Hospital “Dr. José Penna” of Bahía Blanca enrolled 12.4% of the cases, Hospital “Dr. Humberto J. Notti” of Mendoza 18.8%, Hospital “Dr. Castro Rendón” of Neuquén, 16.1%, Hospital “Sor María Ludovica” of La Plata 21.9%, Hospital Italiano of Buenos Aires City 11.6%, Hospital “Dr. Orlando Alassia” of Santa Fe 11.2%, Hospital “Dr. Lucio Molas” of Santa Rosa, La Pampa 8.0%. Of the total, 61.5% patients were assisted in the Reference Sites and 38.5% in the corresponding referral units. The geographic distribution of BD cases is shown in Figure 1.

Two hundred and eighty-nine (40.5%) of stool samples were collected on the same day of BD onset, 250 (35%) one day later, while the remaining 175 (24.5%) two or more days after BD onset; 593 (83.1%) were processed by STQC within the first 24h of sampling.

A total of 29 cases were confirmed by STQC and/or mPCR, resulting in a cumulative incidence of 4.1% (95% CI: 2.7%–5.8%). Of the 29 STEC-positive BD cases, 21 (72.4%) were detected by the STQC test, 13 (44.8%) by the direct method and 8 (27.6%) by the enrichment method. By mPCR, 28 cases were stx-positive. In the remaining case that tested positive by STQC, mPCR was not performed; however FFStx was detected at the NRL as described by Karmali et al.16 Among the stool samples collected on the same day and one day later of BD onset, the rate of STEC detection was 4.3% (23/539), and 3.4% (6/175) among the samples collected 2 or more days later. The proportion of STEC-positive BD ranged from 1.3% to 8.7% (Fig. 1) and there was a summer peak (Fig. 2). Of the 29 STEC-positive BD cases detected, 17 (58.6%) were cared for in the referral units and 12 (41.4%) in the Reference Sites (Table 1).

Figure 2.

Proportion of STEC-positive bloody diarrhea cases by month.

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The greatest proportion of STEC-positive BD (8.8%, 14/160) was in children 12–23 months of age, and significant differences were observed among age groups (chi-square test for equal proportions (α=0.05), p=0.0026; pairwise comparisons: 12–23 vs. 24–59, p=0.0012; 12–23 vs. 60–120, p=0.0214; 24–59 vs. 60–120, p=0.5365) (Table 2).

Of the 29 STEC-positive BD cases, 9 (31.0%) and 6 (20.7%) were caused by O157:H7 and O145:H28, respectively. Other serotypes detected were O26:H11 (4), O121:H19 (1), ONT:H2 (1), ONT:HNT (3). In five mPCR-positive cases, STEC was not recovered. Among 24 STEC isolates, the frequency of the stx-genotypes was, stx2a-only (n=12, 50.0%), stx2a/stx2c (n=8, 33.3%), stx1a (n=2, 8.3%), stx1a/stx2a and stx1a/stx2c (n=1, 4.2%, each one). All O157:H7 strains were recovered during warm weather months.

Three (10.7%) STEC-positive BD cases, diagnosed by direct or enriched STQC and confirmed by mPCR in local laboratories from Buenos Aires, Neuquén and Santa Fe developed HUS during follow-up. Additionally, one STEC-negative BD case by direct and enriched STQC tests and mPCR at the local laboratory in Mendoza developed HUS 4 days after admission and STEC O157:H7 stx2a/stx2c, and anti-O157-specific antibodies were detected at the NRL. Thus, with the addition of this latter case, 13.8% of STEC-positive BD patients developed HUS, three of whom were ≤24 months of age. The median interval between BD onset and the progression to STEC-HUS was 4.5 days (IQR 3–5 days; range, 3–5 days) during warm weather months. STEC O157:H7 stx2a/stx2c was isolated from three patients, while a STEC ONT:H2 stx1a/stx2a was identified in the remaining one.

The 27 HUS patients of the second cohort were attended at the following hospitals: Sor María Ludovica (13), Notti (6), Penna (4), Castro Rendón (2), Italiano (1), Molas (1). The distribution by month was the following: December (2), January (8), February (9), March (5), April (1), May (1) and June (1).

The patient attended at the Hospital Italiano of Buenos Aires City was first STEC-positive in the local laboratory in Chubut Province. Of the remaining 26 cases analyzed by STQC and mPCR, 11 (44%) were Stx-positive by STQC, 5 by the direct method, and 6 after enrichment, while all 26 cases were positive by mPCR. No HUS patient received antibiotic treatment before this outcome ensued.

Among the 27 patients, STEC O157:H7 strains were recovered in 10 (37.0%), O145:H28 in 6 (22.2%). Among the O157:H7 isolates, the stx2a/stx2c genotype prevailed (n=8) and all O145:H28 strains were stx2a-only. Ten HUS cases were stx2-PCR positive without isolation and one was stx1-PCR without isolation. FFStx was detected in 9 of 22 cases analyzed; anti-O157 antibodies were identified in 8 and anti-O145 in 6 of 19 cases studied. The remaining 5 sera samples were negative for the currently available antigens (O157, O145, O121).