Supplementary Appendix

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1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Lapeyraque A-L, Malina M, Fremeaux-Bacchi V, et al. Eculizumab in severe Shiga-toxin associated HUS. N Engl J Med 2011;364: DOI: /NEJMc

2 Case Summaries of the Three 3-year-old Patients Treated with Eculizumab Patient 1 presented with enteritis and bloody diarrhea and subsequently developed symptoms of typical HUS with thrombocytopenia (20 x 10 3 /μl), hemolytic anemia (hemoglobin level [Hgb] 4.1 g/dl), undetectable serum haptoglobin level, red blood cell fragmentation and increased LDH level (3463 IU/L), and acute renal failure (plasma creatinine level [PCr] 4.17 mg/dl [368 μmol/l]). Leukocyte count was markedly increased (32.3 x 10 3 /μl). Stx-2 producing E.coli (O157 serotype) was isolated in the stool. The child became rapidly anuric, and daily hemodialysis (HD) was initiated 7 days after the onset of the bloody diarrhea. Decreased C3 and elevated C3d serum concentrations suggested activation of the alternative complement pathway, and plasma exchanges (PE) were performed with each HD session. After 3 days of persistent hemolysis, anuria and daily HD/PE sessions, the child had a transient episode of impaired motor coordination and altered consciousness. Brain MRI showed no abnormalities. Two days later, the child developed impaired eye movements, blurred speech and soon thereafter, left sided hemiplegia and somnolence that progressed to coma. At that time, MRI showed extensive diffusion restriction in the basal ganglia and the right hemisphere (Figure). In view of the complement activation and the progressive central nervous system involvement despite PE therapy, the patient was treated with eculizumab, 600 mg, administered twice at a 7-day interval (Alexion, Cheshire, CT). Within a few hours after the first infusion, the child s neurological condition started to improve dramatically. Within 48 hours, consciousness, motor and speech control were largely regained. Platelet count normalized within 3 days, and LDH level dropped 4 days after the first eculizumab infusion (Figure). Urine production started within 24 hours of the first dose of eculizumab, and dialysis was discontinued 3 days later. Serum C3c and C3d levels returned to normal range. Further neurologic recovery ensued, the child had a few intercurrent self-limited episodes of focal seizures. MRI performed 8 days after the first eculizumab infusion showed marked regression of lesions (Figure). Nine days after the first eculizumab administration the patient was discharged with a completely normal neurologic examination. Renal function fully recovered within 2 weeks (Figure). At most recent follow-up (6 months after the acute illness), the child has normal plasma creatinine level (0.52 mg/dl [46 μmol/l]) with slight residual proteinuria (0.37 g/l) and borderline arterial hypertension. Patient 2 was admitted with bloody diarrhea. Abdominal ultrasound showed jejunal intussusception that spontaneously resolved. Stx-2 gene and E.coli O157H7 adhesion factor were detected in the stool. Four days after admission the child developed deep lethargy with visual hallucinations and agitation. Laboratory tests revealed thrombocytopenia (45 x 10 3 /μl), hemolytic anemia (Hg 10.4 g/dl, undetectable serum haptoglobin level, red blood cell fragmentation and increased LDH level [7639 IU/L]) and acute renal

3 failure (PCr 2.74 mg/dl [242 μmol/l]). Leukocyte count, C3 and C4 levels were in the normal range, but liver enzymes were elevated (AST: 511 IU/L, ALT: 291 IU/L). Prothrombin time and serum troponin levels were normal as were ECG and echocardiography. Brain CT scan was normal. Brain MRI showed lowered diffusion index in the globus pallidus and caudate nuclei and a hypersignal in the flair sequence in the globus pallidus nuclei. The child was anuric. HD was initiated 5 days after the beginning of bloody diarrhea, and changed to hemofiltration 5 days later. Six daily plasma exchanges were performed and led to discrete improvement of consciousness but no improvement in brain MRI. Five days after PE cessation, laboratory tests showed persistent hemolytic anemia (Hg 8.1 g/dl, undetectable haptoglobin level and increased LDH level [2209 IU/L), thrombocytopenia (43 x 10 3 /μl) associated with high neutrophil (30 x 10 3 /μl) and leukocyte counts (44.4 x 10 3 /μl), hyperglycemia (23 mmol/l) suggesting a pancreatic involvement and increased troponin-t (10.1 ng/ml). Echocardiography demonstrated left ventricular dysfunction, confirming myocardial involvement. Patient 2

4 Because of multiorgan involvement and worsening neurologic status after PE cessation, the patient received eculizumab (600 mg) 14 days after the beginning of bloody diarrhea. At the same time, the child was vaccinated against Neisseria meningitidis and received prophylactic penicillin. The first eculizumab administration was followed by rapid improvement of neurological status within one day and resolution of myocardial dysfunction (troponin-t level dropped 2 days after infusion, and echocardiography normalized within 3 weeks). Platelet count normalized within 5 days, and LDH level dropped 5 days after infusion (Figure). Haptoglobin level normalized the day after the infusion. Urine outflow started to improve 9 days after eculizumab initiation allowing dialysis cessation 7 days later. The patient received a total of 4 weekly eculizumab injections ( mg). Thirty-five days after the first eculizumab administration the patient was discharged with completely normal neurologic status. Renal function fully recovered within one month (Figure). At last follow-up, 6 months later, the child had normal plasma creatinine level (0.58 mg/dl [52 µmol/l]) with slight residual proteinuria (0.6 g/l) without hypertension but still required insulin for persistent diabetes mellitus. Patient 3 was admitted with severe abdominal pain and bloody diarrhea. Rotavirus and E. coli O157:H7 were detected in the stool culture. Four days after onset of symptoms, the child developed deep lethargy with intermittent agitation. Laboratory tests revealed thrombocytopenia (26 x 10 3 /μl), hemolytic anemia (Hg 7.3 g/dl, undetectable serum haptoglobin level, red blood cell fragmentation and increased LDH level [2809 IU/L]), and acute renal failure (PCr 2.25 mg/dl [199 μmol/l]). Leukocyte count was increased (23.4 x 10 3 /μl) while C3 and C4 levels were normal. The child became rapidly anuric, and daily HD was initiated 7 days after the beginning of bloody diarrhea. Neurologic status progressively worsened with deepening lethargy, confusion and visual hallucinations. Meanwhile, colitis worsened towards inflammatory bowel obstruction. Laboratory investigations showed persistence of hemolytic anemia and thrombocytopenia associated with increasing neutrophil count and leukocyte count (Figure). Cardiac involvement was absent as evaluated by echocardiography and troponin level. Because of worsening neurological status and colitis, and very high leukocyte level (93.4 x 10 3 /μl) suggesting a massive inflammatory response, the child received eculizumab (600 mg) 10 days after the beginning of bloody diarrhea. At the same time, the child was vaccinated against Neisseria meningitidis and received prophylactic penicillin. The first eculizumab administration was followed by a rapid improvement in the patient s consciousness (as soon as 1 day after infusion) and symptoms of bowel obstruction gradually disappeared. Platelet count normalized within 5 days and LDH level dropped 4 days after infusion to reach normal levels 16 days later (Figure). Haptoglobin level normalized within 5 days. Leukocyte count decreased as soon as 1 day after infusion to reach normal levels within 7 days (Figure). Urine outflow started to improve 6 days after eculizumab initiation allowing dialysis cessation 7 days later. The patient received a total of 2 week-

5 ly eculizumab injections (600 and 300 mg). Twenty days after the first eculizumab administration the patient was discharged with completely normalized neurological status. Renal function recovered within one month (Figure). At last follow-up, 6 months later, the child has a plasma creatinine level in the upper limit of normal for age (0.79 mg/dl [69.8 μmol/l]) with residual proteinuria (2.35 g/l) and hypertension requiring amlodipine therapy. Mutation screening of the complement regulatory genes involved in atypical HUS (CFH, CFI, MCP, C3, CFB, and TBMD) revealed no abnormalities in any of the patients and anti-cfh antibodies were absent. Patient 3

6 DISCUSSION In each case presented here, the decision to use eculizumab as a potentially life-saving intervention was jointly made by pediatric nephrologists and intensivists after thorough consideration of the potential risks and benefits, consultation with medical experts from the manufacturer (Alexion) and intense communication with the parents, who explicitly assented to the therapy. Formal written informed consent was obtained in one case. Though eculizumab therapy induced rapid improvement in neurologic symptoms, biological thrombotic microangiopathy activity biomarkers (platelet count, LDH level, haptoglobin level) and renal function in all 3 patients, blocking the complement system, an integral component of innate immunity, presents a potential risk to host defense against pathogenic organisms. Patients with C3 deficiency are very sensitive to bacterial infections. However C3 function is not affected by eculizumab, which selectively blocks the terminal complement pathway and cell lysis via the membrane attack complex, leaving the step of pathogen opsonization by C3b fragments intact. While eculizumab creates a state of functional C5 deficiency, patients with genetic C5 deficiency rarely exhibit severe systemic infections and are selectively prone to pneumococcal and meningococcal infections. Bacteremia is rare in patients with STEC-HUS. The infection is confined to the intestinal mucosa and extraintestinal organ damage occurs mostly due to the systemic effects of the shiga toxin released by the bacteria, typically at a time when the patient is already recovering from intestinal infection. Nonetheless, great caution is indicated and the use of eculizumab in STEC-HUS here was undertaken as an extreme measure, given the clinical situation. Further systematic studies with clear inclusion and exclusion criteria and thorough clinical documentation would be required before eculizumab therapy can be recommended as a standard of care for severe HUS.