1. STEC Disease Severity Scale Martin Bitzan, M.D. Department of Pediatrics, McGill University Pediatric Nephrology Montreal Children’s Hospital Montreal, Canada FDA Joint Advisory Committee Meeting April 12, 2007

2. Objectives Discuss the concept of Shiga Toxin Mediated Events (STME) Propose a scoring system for the severity of infections by Shiga toxin producing bacteria/STEC FDA Joint Advisory Committee Meeting April 12, 2007

3. Alternative Approach to the HUS-Centered Perspective Identify set of Stx-related clinical and laboratory changes (Shiga toxin mediated events, STME) Target reduction of STME including HUS as “proof of principle” Reduction of STME will reduce HUS and other Shiga toxin-related complications

4. Shiga Toxin Mediated Events (STME) Signs and symptoms that are directly or indirectly attributable to the biologic action of Shiga toxins, based on –In vitro studies –Animal models –Clinico-pathological observations

5. HC and HUS-like lesions in Shiga toxin injected animals SignsSpeciesReferences Watery diarrhearabbit, rat Richardson 1992, Krishnan 1999, Ritchie 2003 Bloody diarrhearabbit, greyhound, baboon Richardson 1992, Raife 2004, Taylor 1999 TMA, hemolysis, thrombocytopenia greyhound, baboon Fenwick/Cowan 1998, Fenwick Raife 2004 Taylor 1999 Renal tubular injurymouse Wadolkowski 1990, Tesh 1993, Wolski 2002 Renal glomerular injury greyhound, baboon, mouse Taylor 1999, Fenwick Raife 2004, Fernandez 2000

6. Bitzan M, Richardson SE, Karmali MA. 1994 Intravenously injected Stx 1 in rabbit. Toxin detection after 2 h in endothelium by IIF with anti-Stx Ab/FITC antibody Crypt Mucosal blood vessel Intravenous Shiga toxin injection targets mucosal blood vessels in colon and cecum

7. Ideal features of a scoring system Simple Quantitative Clinical symptoms easy to assess and record by non-professionals (families) Concordance between observers Combination of clinical and laboratory parameter Relates to important clinical outcomes

8. Development of STEC Disease Severity Scale Set of candidate clinical and laboratory signs (Shiga toxin mediated events) Grading adapted from Common Terminology Criteria for Adverse Events (CTCAE) Evaluated in retrospective analysis of large cohort of bona fide STEC O157 infections

9. STEC disease scale Enteropathy Vasculopathy/coagulation Hemolytic anemia and thrombocytopenia Nephropathy Extraintestinal and extrarenal complications

10. Enteropathy (hemorrhagic colitis) 01234 Diarrhea (daily frequency of soft stools) Baseline (no diarrhea) 1 - <55 - <1010 - <15≥15 or paralytic ileus Abdominal pain/cramps NoneMildModerateSevere, pain medication Unbearable Bloody diarrhea No visible blood Occasional/ small amounts of blood Blood mixed with stool, streaks of fresh blood Frank blood (hemorrhage) Hemorrhage requiring colonoscopy or surgery

11. Microangiopathic Hemolytic Anemia and Nephropathy 01234 Hemoglobin [g/l] ≥115<115 - 105<105 - 90<90 – 65<65 or PRBC Platelets [N/nl] ≥150<150 – 125<125 - 75<75 – 25<25 or transfusion, hemorrhage HematuriaNone or trace SmallModerateLargeAnuria Serum creatinine Normal (for age) >1 - 2x upper normal >2 - 4x upper normal >4x upper normal Dialysis

12. Evaluation of the Disease Scale Epidemiology Cohort Microbiology records 08/1992 – 07/2006 STEC isolates (E. coli O157) N = 186 Chart identification and review Hospital records available N = 164 Age6.1 ± 4.7 yrs (0.3 – 18) Non-HUS84 % HUS 16% Hospitalized33% Dialyzed6% (39 % of HUS)

13. Maximal ScorePatientsOdds ratiop Bloody diarrhea 164 1.5.140 Abdominal cramps 159 1.5.060 Diarrhea frequency 164 1.7.014 Hemoglobin 95 1858<.0001 Platelets 91 46.9<.0001 Hematuria 103 9.6<.0001 Creatinine 120 94.2<.0001 Retrospective data analysis Univariate logistic regression Association of STMEs with HUS

14. Maximal scorePatientsCorr coeff*p Bloody diarrhea 164 0.26.0008 Abdominal cramps 159 0.38<.0001 Diarrhea frequency 164 0.29.0002 Anemia 95 0.70<.0001 Thrombocytopenia 91 0.74<.0001 Hematuria 103 0.62<.0001 Creatinine 120 0.69<.0001 Correlation of STMEs with Duration of Hospitalization *Spearman rank correlation

15. Maximal scorePatientsCorr Coeffp Bloody diarrhea 138 0.31.0002 Abdominal cramps 135 0.38<.0001 Diarrhea frequency 138 0.29.005 Anemia 70 0.080.502 Thrombocytopenia 66 0.090.483 Hematuria 82 0.080.497 Creatinine (score) 94 -0.090.367 Correlation of STMEs with Duration of Hospitalization (excluding HUS)

16. Estimated Risk of Hospitalization related to Enteropathy Scores Score01234 Diarrhea frequency3%7%14%27%46% Abdominal pain1%6%22%59%88% Bloody diarrhea2%7%21%50%79% Excluding HUS Patients

17. Platelets (N/nL) Creat (μmol/L) Hemoglobin (g/L) 1357911131517182123 HUS Case #1 Bloody diarrhea Abd cramps Diarrhea Fever Oliguria/edema/U hem Irritability PRBC transfusion Hospital ER

18. Scoring of HUS patient

19. STEC Disease Severity Scores HUSUncomplicated HC“Incomplete” HUS Days after Onset of Diarrhea Scores

20. Conclusions Shiga toxin mediated events (STME) defined as measurable, biological effects of Shiga toxins STEC Disease Severity Scale is associated with clinically relevant outcomes Allows to integrate disease severity and duration Tool for standardized documentation and evaluation of STEC disease Useful for prospective studies, preventive or therapeutic intervention

21. “Damage (is) underway by time of presentation, but in a potentially treatable cascade” (Phil Tarr)

23. Biological actions of Shiga toxin Endothelial cell injury and activation –Vasoactive mediators, adhesion molecules, prothrombotic/antifibrinolytic phenotype Platelet activation –Release of platelet factors, microparticles Monocyte activation –Cytokine/chemokine induction Renal tubular epithelial and mesangial cell stimulation and injury/apoptosis

24. Clinico-Pathological Findings in Humans with STEC Infection (1) Hemorrhagic/ischemic colitis –Frequent, painful bowel movement –Abdominal cramps –Hematoschezia Nephropathy –Hematuria, proteinuria –Renal dysfunction/oligoanuria Hematological changes –Non-immune acute hemolytic anemia –Thrombocytopenia –Neutrophilia Other organ involvement –CNS, pancreas, myocardium, others MB_FDA April 12, 2007

25. Evaluation of the Disease Scale Study Design Microbiology records 08/1992 – 07/2006 STEC isolates (E. coli O157) N = 186 Chart identification and review Hospital records available N = 164 Diagnoses Non-HUSN = 138 HUS (all forms) N = 26 Laboratory studies CBCN = 95 BiochemistryN = 120 UrinalysisN = 103 No follow-up visitsN = 103 ER follow-upN = 43 Hospitalized (initially or later) N = 54 Treated in practice N = 22 MB_FDA April 12, 2007

26. Study Population take out Age6.1 ± 4.7 yrs (range 0.3 – 18.0) Bloody diarrhea at presentation 127/16477.4% HUS all26/186 (n = 12 hospital transfer) Dialysis10/2638.5 % HUS at MCH14/1748.0 % Complete 11 6.3 % Incomplete 3 1.7 % Dialysis3/1127.3 % MB_FDA April 12, 2007

27. Worst ScorePatientsORp Bloody diarrhea 164 2.4.0001 Abdominal cramps 159 2.6<.0001 Diarrhea frequency 164 2.1<.0001 Hemoglobin 95 5.6<.0001 Platelets 96 242<.0001 Hematuria 103 3.1<.0001 Creatinine 120 7.8<.0001 Retrospective data analysis Univariate logistic regression Association of STMEs with Hospitalization

28. NUnivariateMultivariate Scores 1 Odds ratio p Odds ratio p Odds ratio p Bloody diarrhea1642.4.00013.3.005 Abdominal cramps1592.6<.00014.0 <.0001 2.7.0004 Diarrhea frequency1642.1<.00011.7.023 Hemoglobin955.6<.0001 Platelets91∞<.0001 Hematuria1033.1<.00016.2<.0001 Creatinine1207.8<.000110.2<.0001 STME are associated with Hospitalization Logistic regression

29. STEC Enteropathy Scale ER study

30. Distribution of Enteropathy Scores at Emergency Room Presentation MB_FDA April 12, 2007

31. Distribution of Extraintestinal TMA and Nephropathy scores Emergency Room Presentation MB_FDA April 12, 2007

32. Risk of Hospitalization due to Worsening of Enteropathy Scores Score01234 Diarrhea frequency7%13%24%40%58% Abdominal pain6%16%36%62%83% Bloody diarrhea9%18%35%56%75% Including HUS

33. Estimated Risk of Hospitalization related to Enteropathy Scores 1 Score01234 Diarrhea frequency3%7%22%27%46% Abdominal pain1%6%22%59%88% Bloody diarrhea2%7%21%50%79% Excluding HUS Patients 1 from univariate logistic regression models