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Background Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome affects ~100 United Kingdom children each year. Around half need dialysis, a quarter develop serious complications with long-term consequences and ~3% die. No effective intervention is known; however, some studies report eculizumab, effective in atypical haemolytic uraemic syndrome, may be effective. Objectives To determine whether the severity of Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome is less in those given eculizumab. Design Randomised, double-blind, placebo-controlled, parallel-group trial with internal pilot phase and nested mechanistic laboratory studies. Setting Paediatric nephrology units in 12 United Kingdom hospitals. Participants Children aged 6 months to < 19 years weighing ≥ 5 kg, with presumed Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome, including ‘injury’ or ‘failure’ category of the acute kidney injury paediatric risk/injury/failure/loss/end criteria. Intervention Participants were randomised in a 1 : 1 ratio to receive intravenous eculizumab or placebo on day 1 and 8. All received meningococcal vaccination and prophylactic antibiotics. Main outcome measures The primary outcome measure was a multidomain clinical severity score, reflecting morbidity until day 60. Secondary outcome measures included survival, duration of renal replacement therapy, persistent neurological defect (day 60) and presence of chronic kidney disease at 1 year. Mechanistic studies assessed complement activation and vascular endothelial growth factor profiles in plasma ± urine samples. In vitro cell co-culture work assessed the effect of Shiga toxin on endothelial cells. Results Thirty-six participants from 10 sites were randomised: 17 to eculizumab and 19 to placebo. The target sample size was 134 participants – recruitment stopped early due to low recruitment (factors included reduced incidence and limited out-of-hours research infrastructure) and the COVID-19 pandemic. The mean clinical severity score for participants randomised to eculizumab was 11.5 (standard deviation 8.4) compared to 14.6 (standard deviation 7.7) for participants randomised to placebo (adjusted mean difference: −2.5, 95% confidence interval −7.8 to 2.8, p = 0.3). Five participants (three eculizumab, two placebo) experienced an adverse event; there were seven serious adverse events in six participants (five eculizumab, one placebo). Urinary complement factor H and vascular endothelial growth factor levels were high initially and subsequently normalised. Shiga toxin caused a podocyte-dependent decrease in endothelial cell factor H levels. Conclusions and limitations There was no significant difference in mean clinical severity score between eculizumab and placebo groups – since the trial was underpowered, this cannot be interpreted as evidence of no effect. No significant safety concerns were observed. With further validation, the Eculizumab in Shiga-toxin-producing Escherichia coli Haemolytic Uraemic Syndrome clinical severity score may be an outcome measure for future trials. Our results imply that Shiga toxin causes complement-dependent glomerular endothelial cell injury through its action on podocytes and subsequent cellular cross-talk. Future work We will continue to investigate cross talk between podocytes and endothelial cells after exposure to Shiga toxin and further develop plasma/urine biomarkers for diagnosis of Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome. Trial registration This trial is registered as EudraCT-2016-000997-39 and ISRCTN89553116. Funding This award was funded by the National Institute for Health and Care Research (NIHR) Efficacy and Mechanism Evaluation (EME) programme (NIHR award ref: 14/48/43) and is published in full in Efficacy and Mechanism Evaluation; Vol. 11, No. 11. See the NIHR Funding and Awards website for further award information. Plain language summary Why did we do this study? Annually, approximately 100 United Kingdom children develop Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome after infection with a diarrhoea-causing bug. The bug makes a toxin (Shiga toxin) that damages blood vessels, especially in the kidneys. Half need dialysis (artificial kidney support), about a quarter suffer fits or a stroke and about 3% die. Most children fully recover, but about a quarter suffer permanent kidney or brain damage. There are no known effective treatments. Eculizumab, a medicine which blocks part of the immune system called complement, may work. What was the question? Does eculizumab reduce the severity of Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome? What did we do? We planned to recruit 134 children, but difficulties with recruitment and the COVID-19 pandemic meant the study was stopped early after 36 children had been recruited; 17 received eculizumab, 19 received a dummy medicine (placebo). We compared children in each group using a score that measured how their kidneys and other organs were affected. We studied samples of their blood and urine, and also how Shiga toxin damages kidney cells in the laboratory. What did we find? The severity of illness was similar in both groups; however, because we only studied a small number of children, we cannot be sure this means that eculizumab does not work. Eculizumab appeared to be safe in this condition. In the blood and urine samples, we saw evidence that complement is involved in the illness. We also discovered a new way that Shiga toxin damages kidney cells. What does this mean? We have been unable to show whether eculizumab is a worthwhile treatment for children with this condition. However, we have learnt lots about how the illness is caused and hope these results can be combined with other studies to give us a clearer answer. Scientific summary Background Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome affects around 100 UK children each year, following gastrointestinal infection with Shiga-toxin-producing E. coli. Around half of affected children will need dialysis, about a quarter develop serious complications with long-term consequences and about 3% die. All patients require long-term follow-up because of the risk of developing chronic kidney disease (CKD). No intervention has definitively been shown to reduce morbidity or mortality in this condition, and therefore treatment is supportive. Case reports and case series suggest that eculizumab (Soliris®, Alexion Pharmaceuticals, Boston, MA), an inhibitor of the complement system and an effective treatment for the related condition, atypical haemolytic uraemic syndrome, may be effective in this condition. Until very recently, there were no published data regarding the efficacy and safety of eculizumab in Shiga-toxin-producing E. coli haemolytic uraemic syndrome, and yet despite this its use has risen globally. A recent randomised phase 3 clinical trial reported comparison of eculizumab with placebo in 100 children with Shiga-toxin-producing E. coli haemolytic uraemic syndrome (Garnier A, Brochard K, Kwon T, Sellier-Leclerc A-L, Lahoche A, Allain Launay E, et al. Efficacy and safety of Eculizumab in pediatric patients affected by Shiga Toxin-Related Hemolytic and Uremic Syndrome: a randomized, placebo-controlled trial. J Am Soc Nephrol 2023;34:1561–73). Patients with severe multi-organ involvement were excluded. Four patients in the placebo group were withdrawn and subsequently received eculizumab. There was no difference between treatment groups in the proportion of children who required renal replacement therapy 48 hours after randomisation, In addition, no differences between groups were seen in the secondary outcome measures of extra-renal manifestations, duration of hospitalisation and mortality. During follow up, there was a slight difference in the proportion who exhibited renal sequelae at 12 months post randomisation (20 patients in the eculizumab group (43.48%) and 29 patients (64.44%) in the placebo group (P = 0.04). The authors concluded that eculizumab seemed to have no impact on the course of acute kidney injury and interpreted the 12-month follow-up data with caution. No data have been published which include the role of eculizumab in patients with severe manifestations of disease in a controlled setting. Objectives of the main trial The Eculizumab in Shiga-toxin-producing Escherichia coli Haemolytic Uraemic Syndrome (ECUSTEC) trial was designed to test the hypothesis that treatment with eculizumab reduces the severity of Shiga-toxin-producing E. coli haemolytic uraemic syndrome in children aged 6 months–< 19 years. We also wanted to assess the safety of eculizumab and test the hypothesis that treatment with eculizumab reduces the incidence of CKD following Shiga-toxin-producing E. coli haemolytic uraemic syndrome. Objectives of the mechanistic substudies The mechanistic component of the trial had the following objectives: to investigate the time course of systemic complement activation in Shiga-toxin-producing E. coli haemolytic uraemic syndrome and its relation to the severity of disease to determine whether thrombotic microangiopathy (TMA) in Shiga-toxin-producing E. coli haemolytic uraemic syndrome occurs via a Shiga-toxin-mediated reduction in podocyte vascular endothelial growth factor (VEGF) production, leading to loss of complement regulation to test whether neutrophils derived from patients with acute Shiga-toxin-producing E. coli haemolytic uraemic syndrome deliver Shiga toxin to podocytes to assess whether any genetic variations in patients with Shiga-toxin-producing E. coli haemolytic uraemic syndrome point to novel pathogenic mechanisms. Methods Design The trial was a randomised, double-blind, placebo-controlled, parallel-group trial of two doses of eculizumab in children (aged 6 months–< 19 years) with Shiga-toxin-producing E. coli Haemolytic Uraemic Syndrome. The trial had an internal pilot phase and included nested mechanistic laboratory studies and a cost-effectiveness evaluation, although the latter was not undertaken following the trial being stopped early. The mechanistic substudies were optional; all participants in the main trial were offered the opportunity to participate in the substudies, which involved providing blood and urine samples over the first 30 days of the trial. Participant blood and urine samples were used to explore the evidence for, and time course of, complement activation in this condition. Using both patient samples and an in vitro cell co-culture model, evidence was sought to support the hypothesis that Shiga toxin causes a glomerular TMA as a consequence of its effect on podocyte VEGF production. This included measurement of patient urine and plasma complement factor H (CFH) and VEGF and plasma complement activation products [by both enzyme-linked immunosorbent assay (ELISA) and a novel degradomics technique]. In the co-culture experiments, glomerular endothelial cells were exposed to Shiga toxin in the presence and absence of podocytes. Setting The trial was conducted in 12 sites in NHS hospital settings across the UK with the support of 88 Patient Identification Centres. Participants Informed consent was sought from parents/guardians of eligible children (those aged 6 months–< 19 years weighing ≥ 5 kg, with a clinical diagnosis of Shiga-toxin-producing E. coli haemolytic uraemic syndrome, including acute kidney injury (AKI) equivalent to the ‘injury’ or ‘failure’ category of the paediatric risk/injury/failure/loss/end criteria). Eligible young people aged 16–18 years provided their own consent for participation in the trial, with assent from younger children if appropriate (according to age). Screening and randomisation Screening began as soon as possible after a diagnosis of Shiga-toxin-producing E. coli haemolytic uraemic syndrome was suspected. Once eligibility was confirmed and informed consent obtained, the participants were commenced on prophylactic antibiotics, and unless contra-indicated or already administered, participants were also vaccinated against meningococcal infection. Participants were then randomised into the ECUSTEC trial via a secure online central randomisation system. Participants were randomised at the level of the individual in a 1 : 1 ratio to either eculizumab or placebo, which was commenced as soon as possible after randomisation. A minimisation algorithm was used to ensure balance in the treatment allocation over the following variables: centre, severity of AKI and hydration status. To avoid predictability in the randomisation, a random element was included in the minimisation algorithm, so that each patient had a probability (unspecified here), of being randomised to the opposite treatment that they would have otherwise received. Intervention Each participant received two intravenous infusion doses of either eculizumab (dose according to first two doses of induction regimen for atypical haemolytic uraemic syndrome) or placebo. The first dose was given as soon as possible after randomisation (designated day 1), with the second dose given 7 days later (i.e. on day 8). They also received vaccination against meningococcal disease and an 8-week course of antibiotic prophylaxis. The participants, parents/guardians, clinical staff and research teams were blind to randomised treatment allocation throughout the study. Outcome measures Primary The ECUSTEC clinical severity score (CSS), a purpose-developed, multidomain score comprising severity of AKI and extrarenal events. A single score is assigned at day 60 to reflect cumulative morbidity up until that point. The score ranges from 1 to 69 with higher scores indicating greater disease severity. Secondary Overall survival. Duration of renal replacement therapy (days). Duration of thrombocytopenia (number of consecutive days until platelet count > 150 × 109/l). Duration of haemolysis (number of days until lactate dehydrogenase within local centre reference range). Number of packed red blood cell transfusions required and volume (ml/kg). Duration markers of inflammation present (number of days until neutrophil cell count and C-reactive protein are in normal range for that centre). Persistent neurological defect at day 60 measured by structured expert assessment to include central nervous system examination, vision, hearing and neuropsychological assessment. CKD at 52 weeks (a composite end point of the presence of hypertension, albuminuria or estimated glomerular filtration rate (eGFR) < 90 ml/minute/1.73 m2 at 52 weeks). eGFR measurement using a centralised cystatin C assay at 52 weeks. Mechanistic studies Urine CFH levels. Urine VEGF levels. Presence of urine markers of podocyte damage (nephrin and Wilms tumour-1). Plasma VEGF and factor H levels. Plasma complement activation products (Bb, C3a, C4a and sC5b9 by ELISA and C3 and C4 activation markers by degradomics). Glomerular endothelial surface levels of factor H and C3d (a marker of complement activation) in a co-culture models of human conditionally immortalised podocytes and glomerular endothelial cells exposed to Shiga toxin. Whole exome sequencing and serum anti-factor H antibody levels. Results The target sample size was 134 participants, but recruitment was stopped early due to low recruitment and the impact of the COVID-19 pandemic. At the point the trial was stopped, 108 children had been screened for participation, of whom 87 were deemed eligible to participate. Thirty-six children were consented and randomised; 17 were randomised to eculizumab and 19 were randomised to placebo. One participant withdrew from the trial and one participant died. The majority of baseline data of the participants were comparable across the two groups; however, the participants in the placebo group were slightly older and consequently heavier than those in the eculizumab group. Reasons for slow recruitment included a fall in the incidence of Shiga-toxin-producing E. coli haemolytic uraemic syndrome during the trial period (up to a 37% reduction) and a lack of out-of-hours infrastructure for undertaking acute interventional clinical trials in children. The mean CSS at day 60 for participants randomised to eculizumab was 11.5 [standard deviation (SD) 8.4] compared to 14.6 (SD 7.7) for participants randomised to placebo [adjusted mean difference: −2.5, 95% confidence interval (CI) −7.8 to 2.8, p = 0.3]. Five participants (three eculizumab, two placebo) experienced an adverse event, and there were seven serious adverse events (SAEs) in six participants (five eculizumab, one placebo). None of the SAEs were considered related to the trial treatment. Mechanistic substudies Of the 36 participants recruited to the main trial, 32 consented to take part in the mechanistic studies and provided blood and/or urine samples. In anuric patients, only blood samples were collected. Urine factor H and vascular endothelial growth factor levels in serial samples The highest urine factor H levels were at day 1 (150 ng/ml), diminishing by day 4 (30 ng/ml), and completely normalising by day 30 (undetectable). The highest urine VEGF levels were at day 1 (average 1300 ng/ml) and by day 30 the levels were below 20 ng/ml. Markers of podocyte damage Western blots of urine cell pellets showed acute podocyte loss during active disease, which recovered by day 8. Plasma factor H and vascular endothelial growth factor levels No difference was seen for plasma levels of either factor H or VEGF at day 1 or day 30. Plasma degradomics analysis In a sample of five patients, N-termini consistent with complement C3 and C4 activation were much more abundant at day 1 compared with day 3. In vitro cell co-culture In response to Shiga toxin, there was a reduction in glomerular endothelial factor H levels, accompanied by evidence of complement activation (increased C3d levels) and this was critically dependent on the presence of podocytes. Shiga toxin had no effect when added to endothelial cells alone. Plasma complement activation products Mean plasma levels of Bb were elevated in both groups at baseline (4.38 mcg/ml in the eculizumab group and 10.38 mcg/ml in the placebo group, normal range 0.48–1.62 mcg/ml). They were also elevated at day 2 (5.91 mcg/ml in the eculizumab group and 4.09 mcg/ml in the placebo group) and day 4 (4.90 mcg/ml in the eculizumab group and 3.16 mcg/ml in the placebo group). At day 6 and day 8, Bb levels remained elevated in the placebo group (6.21 and 3.47 mcg/ml respectively) but were normal in the eculizumab group. In both groups, mean Bb levels were in the normal range at day 30. Mean plasma levels of C3a were elevated in both groups at baseline and at days 2 and 4. At days 6 and 8, mean levels remained elevated in the placebo group while mean levels in the eculizumab group were in the normal range. Levels were in the normal range for both groups by day 30. Mean plasma levels of C4a were elevated at all time points in both groups but fell significantly at day 30. Mean levels were 3852, 3026, 3423, 3067, 3425 and 1623 ng/ml at days 1, 2, 4, 6, 8 and 30 in the eculizumab group (normal range 110–699 ng/ml) and 3970, 3573, 2673, 4348, 2844 and 1992 ng/ml at the same respective time points in the placebo group. Mean plasma levels of sC5b9 were normal in both groups at all time points with the exception of day 4 and day 6 in the placebo group, which were elevated (487 and 514 ng/ml respectively, normal range 95–467 ng/ml). In the placebo group, a linear relationship was not established between CSS and baseline Bb (r = 0.43, p = 0.2); C3a (r = −0.16, p = 0.7); C4a (r = 0.15, p = 0.7) or sC5b9 (r = −0.17, p = 0.7). Similarly, a linear relationship was not established between CSS and the maximum value of Bb (r = 0.45, p = 0.1); C3a (r = 0.15, p = 0.6); C4a (r = 0.23, p = 0.4) or sC5b9 (r = −0.22, p = 0.5). Delivery of Shiga toxin to podocytes from patient-derived neutrophils Insufficient patient samples were obtained to complete this part of the work. Genetic variations in patients with Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome Data from whole exome sequencing have been obtained and analysis is ongoing. Conclusions In children with Shiga-toxin-producing E. coli haemolytic uraemic syndrome, the mean CSSs at day 60 were similar between those randomised to eculizumab and those randomised to placebo. However, since the trial was stopped early and did not recruit to the planned sample size, this cannot be interpreted as evidence of no effect. In order to deliver successful clinical trials of investigational medicinal products in acutely unwell children, a review of out-of-hours paediatric research infrastructure may be required. In the mechanistic substudies, we have established that urine factor H and VEGF levels are sensitive measures of early disease activity, and have demonstrated complement activation in patient serum using both ELISA and sophisticated proteomics technology. Urine factor H and VEGF levels and plasma degradomics for C3 and C4 proteins could all be further explored as new biomarkers of acute Shiga-toxin-producing E. coli haemolytic uraemic syndrome. Our co-culture cell work has demonstrated that podocyte cross-talk is responsible for factor H and complement activation levels on endothelial cells. Collectively this strongly supports the mechanistic hypothesis of a complement-mediated disease driven via the podocyte as the target cell. Trial registration This trial is registered as EudraCT2016-000997-39 and ISRCTN89553116. Funding This award was funded by the National Institute for Health and Care Research (NIHR) Efficacy and Mechanism Evaluation (EME) programme (NIHR award ref: 14/48/43) and is published in full in Efficacy and Mechanism Evaluation; Vol. 11, No. 11. See the NIHR Funding and Awards website for further award information. |
