# Presentation: 108576

## 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

## 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

**Notes:**
For the past 20 years we, as a community, have been focusing on HUS and its treatment. 
The low incidence of HUS severe hampers the feasibility of randomized controlled treatment trials.

## 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

## 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

**Notes:**
The prototypic phenotype of STEC infection is the hemorrhagic colitis. In its complete form it presents with frequent bowel movements/diarrhea, crampy abdominal pain and intestinal hemorrhage. Extraintestinal manifestations include hemolytic anemia with schistocytes in the blood, thrombocytopenia, 

Signs and symptoms that can be directly or indirectly attributed to the biologic action of Shiga toxins, based on in vitro and animal experimental evidence and clinical observation
STMEs will be prevented or modified by the timely administration of Shiga toxin neutralizing agents

## HC and HUS-like lesions in Shiga toxin injected animals

| Signs | Species | References |
| --- | --- | --- |
| Watery diarrhea | rabbit, rat | Richardson 1992, Krishnan 1999, Ritchie 2003 |
| Bloody diarrhea | rabbit, greyhound, baboon | Richardson 1992, Raife 2004, Taylor 1999 |
| TMA, hemolysis, thrombocytopenia | greyhound, baboon | Fenwick/Cowan 1998, Fenwick Raife 2004
Taylor 1999 |
| Renal tubular injury | mouse | Wadolkowski 1990, Tesh 1993, Wolski 2002 |
| Renal glomerular injury | greyhound, baboon, mouse | Taylor 1999, Fenwick Raife 2004, Fernandez 2000 |

**Notes:**
In an attempt to understand what to include in the list of STMEs, I have summarized key lesions oberserved in animal models of Shiga toxin injection. It is well known that none of the models perfectly reproduces the full complement of human HUS. 
Interestingly, watery diarrhea is one of the features, that can be produced by parenteral injection of purified Shiga toxin alone, as is bloody diarrhea, thrombotic microangiopathy, and various renal injuries. They should therefore be considered for the score. 

## Intravenous Shiga toxin injection targets mucosal blood vessels in colon and cecum 

*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

**Notes:**
Intravenously injected Shiga toxin 1 targets small mucosal blood vessel endothelium in cecum and colon of rabbits in vivo and causes mucosal and submucosal edema and hemorrhage
Immunofluorescence detection of VT1. Two male New Zealand White rabbits weighing 2 kg were challenged intra-venously, one with a 3-mg bolus of purified VT1 (test) and one with 2.5 ml of PBS (control). Two hours later, under anesthesia and after heparinization, the blood was purged from the body by concomitant infusion of intravenous saline and bleeding from the femoral artery, until the withdrawal was clear and about 700 ml of saline had been infused. Cecal and spinal cord sections were taken and immediately frozen in optimal cutting temperature compound (Tissue-Tek; Miles) in plastic cryomolds in liquid nitrogen and stored at -70°C until sectioning. Sections were cut at 6 p,m, and adjacent sections were stained with hematoxylin and eosin and an immunofluorescent stain. For the immunofluores-cence studies, the slides were brought to room temperature, fixed in paraformaldehyde, dehydrated in ethanol gradients, and incubated with a 1:200 (cecal) or 1:100 (spinal cord) dilution of mouse anti-VT1 monoclonal antibody PH1 (1) (C. Lingwood, Toronto, Ontario, Canada) for 20 min at room temperature. After three washes in PBS, the slides were incubated with a 1:10 dilution of affinity-purified goat anti-mouse immunoglobulin G-F(ab')2-fluorescein isothiocyanate antibody (Cappel-Organon Teknika Inc.) preadsorbed with normal rabbit serum overnight at 5°C. The slides were rinsed three times in PBS, stained with 0.01% Evans blue solution, and rinsed once more with PBS; coverslips with glycerol and
p-phenylenediamine were added. The slides were examined and photographed under a Reichert-Jung fluorescence pho-tomicroscope.

Renal Injury Is a Consistent Finding in Dutch Belted Rabbits Experimentally Infected with Enterohemorrhagic Escherichia coli 
Alexis García,1 Carlos J. Bosques,2 John S. Wishnok,2 Yan Feng,1 Brad J. Karalius,3 Joan R. Butterton,3 David B. Schauer,1,2 Arlin B. Rogers,1 and James G. Fox1,2 
The Journal of Infectious Diseases    2006;193:1125-1134

1Division of Comparative Medicine and 2Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, and 3Division of Infectious Diseases, Massachusetts General Hospital, Boston 

Figure 3CITED IN TEXT  |  HIGH-RESOLUTION IMAGE (462 kB)        A, Sham-inoculated normal rabbit cecum. B, O153-infected rabbit cecum with marked mucosal and submucosal edema and focal submucosal hemorrhage. C, O153-infected rabbit, with surface erosion with adherent bacteria and many infiltrating heterophils (counterparts of neutrophils). D, Detection of adherent Escherichia coli in the cecum of rabbit 5 (experiment 1) infected with O153, by fluorescent in situ hybridization using a Flu PNA probe. E, Pseudomembranous typhlitis in rabbit 5 (experiment 1) infected with O153; note the mucosal ulceration with reflected fibrinonecrotic cap (arrow). F, Thrombohemorrhagic vasculitis with serpiginous intravascular fibrin thrombi (small arrows) and obliterative leukocytoclastic vasculitis (large arrow). All slides were stained with hematoxylin-eosin except panel D, which is peptic nucleic acid–fluorescent in situ hybridization. Scale bar, 1 mm (A, B, and E), 20  m (C and D), and 200  m (F).

## 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

## 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

**Notes:**
The scale is modeled after the CTCAE, which has been originally introduced 25 years ago and which is familiar to many clinical investigators 

1. A set of clinical signs and symptoms, including laboratory signs was compiled, based on a literature review and clinical experience
2. The grading of the signs and symptoms was taken from the Common Terminology Criteria for Adverse Events and adapted, if necessary, to the clinical tableau of hemorrhagic colitis and HUS. Originally compiled in , the CTCAE scale is widely used to grade emergent adverse events in clinical research trials. Events are uniformly recorded and graded on a 1-5 scale, where 1-3 indicate mild, moderate and severe deviations from normal, 4 posibly llife threateining signs, and 5 death.
The proposed Scale was then evaluated in a retrospective analysis of a large cohort of pediatric patients with culture-proven, mostly E. coli O157:H7 infection encompassing about 150 patients wtih diarhea or colitis and 24 patients with laboratory evidence of thrombotic microangiopathy, i.e. so-called incomplete or complete HUS.

Compilation of candidate STMEs
Symptoms and laboratory signs adapted from Common Terminology Criteria for Adverse Events (CTCAE)
Evaluation of the scale
Retrospective analysis of large cohort of bona fide STEC infections

## STEC disease scale

- Enteropathy
- Vasculopathy/coagulation
- Hemolytic anemia and thrombocytopenia
- Nephropathy
- Extraintestinal and extrarenal complications

## Slide 10

| Enteropathy (hemorrhagic colitis) |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
|  | 0 | 1 | 2 | 3 | 4 |
| Diarrhea
(daily frequency of soft stools) | Baseline
(no diarrhea) | 1 - <5 | 5 - <10 | 10 - <15 | ≥15
or paralytic ileus |
| Abdominal pain/cramps | None | Mild | Moderate | Severe, 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 |

**Notes:**
Example of the scale shown 
On the Y axis are listed the various prosed items 
The x axis elaborates the severeity 
It is linear grading system rangin form 0 -4 or 5. 
In principle

## Slide 11

| Microangiopathic Hemolytic Anemia and Nephropathy |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
|  | 0 | 1 | 2 | 3 | 4 |
| 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 |
| Hematuria | None or trace | Small | Moderate | Large | Anuria |
| Serum creatinine | Normal
(for age) | >1 - 2x upper normal | >2 - 4x
upper normal | >4x
upper normal | Dialysis |

## 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**

- Age		6.1 ± 4.7 yrs (0.3 – 18)
- Non-HUS	84 %
- HUS 		16%
- Hospitalized	33%
- Dialyzed		6% (39 % of HUS)

## Retrospective data analysis Univariate logistic regressionAssociation of STMEs with HUS

| Maximal Score | Patients | Odds ratio | p |
| --- | --- | --- | --- |
| 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 regressionAssociation of STMEs with HUS

**Notes:**
Note: 1) No other variable was independently related to hospitalization when max hemoglobin score was in the model
2) No ENT symptom was independently associated with HUS when any TMaN symptom was in the model.
3) When partial HUS was not included in the definition of HUS, max diarrhea score had odds ratio 1.5, p = 0.073.
Overall presence of bloody
Disease 
Disease Scores (max)

Data AnalysisLogistic regression binary values (dependent variables are categorical: Hospitalization, HUS, Dialysis « yes or no ») versus scores (discrete variables)

## Correlation of STMEs with Duration of Hospitalization

| Maximal score | Patients | Corr 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

**Notes:**
Data Analysis: Correlations – Spearman’s correlations (Hospital days non-normally distributed)
Note: also correlated Creatinine concentrations instead of score – correlation coefficients similar to sores
Number of packed red blood cell transfusion were closely related to diagnosis of HUS.

Spearman's rank correlation (score vs continuous (discrete ?) variable)
Def: Spearman's rank correlation: Relationship or association between two continuous variables in a quantitative fashion (Epidm Biostat, page 201 

## Correlation of STMEs with Duration of Hospitalization (excluding HUS)

| Maximal score | Patients | Corr Coeff | p |
| --- | --- | --- | --- |
| Bloody diarrhea | 138 | 0.31 | .0002 |
| Abdominal cramps | 135 | 0.38 | <.0001 |
| Diarrhea frequency | 138 | 0.29 | .005 |
| Anemia | 70 | 0.08 | 0.502 |
| Thrombocytopenia | 66 | 0.09 | 0.483 |
| Hematuria | 82 | 0.08 | 0.497 |
| Creatinine (score) | 94 | -0.09 | 0.367 |

**Correlation of STMEs with Duration of Hospitalization (excluding HUS)**

**Notes:**
Data Analysis: Correlations – Spearman’s correlations (Hospital days non-normally distributed)
Note: also correlated Creatinine concentrations instead of score – correlation coefficients similar to sores
Number of packed red blood cell transfusion were closely related to diagnosis of HUS.

Spearman's rank correlation (score vs continuous (discrete ?) variable)
Def: Spearman's rank correlation: Relationship or association between two continuous variables in a quantitative fashion (Epidm Biostat, page 201 

## Estimated Risk of Hospitalization related to Enteropathy Scores

| Score | 0 | 1 | 2 | 3 | 4 |
| --- | --- | --- | --- | --- | --- |
| Diarrhea frequency | 3% | 7% | 14% | 27% | 46% |
| Abdominal pain | 1% | 6% | 22% | 59% | 88% |
| Bloody diarrhea | 2% | 7% | 21% | 50% | 79% |

- Excluding HUS Patients

## HUS  Case #1

| 1 | 3 | 5 | 7 | 9 | 11 | 13 | 15 | 17 | 18 | 21 | 23 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

**HUS  Case #1**

## Scoring of HUS patient

## STEC Disease Severity Scores

**HUS**

**Uncomplicated HC**

**“****Incomplete” HUS**

**Days after Onset of Diarrhea**

**Scores**

## 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

**Notes:**
collectively called diease burden
Shiga toxin mediated events (STME) are defined as measurable effects of the biologic actions of Shiga toxins
STEC Disease Severity Scale correlates with clinically relevant outcomes
Integrates disease severity and duration (disease burden)
Provides tool for standardized documentation and evaluation of STEC disease
Can be used in prospective studies and for evaluation of preventive or therapeutic intervention
“Damage underway by time of presentation, but in a potentially treatable cascade” (Tarr)

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

## 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

**Notes:**
The prototypic phenotype of STEC infection is the hemorrhagic colitis. In its complete form it presents with frequent bowel movements/diarrhea, crampy abdominal pain and intestinal hemorrhage. Extraintestinal manifestations include hemolytic anemia with schistocytes in the blood, thrombocytopenia, 

Nestoridi E, Kushak RI, Duguerre D, Grabowski EF, Ingelfinger . Up-regulation of tissue factor activity on human proximal tubular epithelial cells in response to Shiga toxin.Kidney Int. 2005 Jun;67(6):2254-66. Nestoridi E, Tsukurov O, Kushak RI, Ingelfinger JR, Grabowski EF. Shiga toxin enhances functional tissue factor on human glomerular endothelial cells: implications for the pathophysiology of hemolytic uremic syndrome.J Thromb Haemost. 2005 Apr;3(4):752-62. 
(Obrig ,Van de Kar 1994, Simon 1998, Bitzan 1998, 2004, Foster 2002, Cameron 2003, Karpman 2006, 
Harrison 2005, Nestoridi 2005, Cherla 2006, Ramos 2007

## 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

**Notes:**
The prototypic phenotype of STEC infection is the hemorrhagic colitis. In its complete form it presents with frequent bowel movements/diarrhea, crampy abdominal pain and intestinal hemorrhage. Extraintestinal manifestations include hemolytic anemia with schistocytes in the blood, thrombocytopenia, 

Hum Pathol. 1988 Sep;19(9):1102-8. The histopathology of the hemolytic uremic syndrome associated with verocytotoxin-producing Escherichia coli infections. Richardson SE, Karmali MA, Becker LE, Smith CR. Department of Pathology, Hospital for Sick Children, Toronto, Ontario, Canada. Verocytotoxin-producing Escherichia coli (VTEC) infection was present in three cases of hemolytic uremic syndrome (HUS), two fatal and one non-fatal, in which detailed histopathologic investigations were conducted. Two patients had a prodrome of bloody diarrhea, one of whom required a hemicolectomy for severe bleeding. The renal histopathology was characterized primarily by glomerular thrombotic microangiopathy (TMA) with greater than 95% of glomeruli showing changes of capillary wall thickening, endothelial cell swelling, and narrowing or thrombosis of the capillary lumen. Preglomerular arterioles were frequently thrombosed, and abnormalities of the medium-sized vessels, including endothelial cell damage and thrombosis, were also commonly observed. Gastrointestinal involvement was prominent in all three cases. The colon was most severely involved, with marked mucosal and submucosal edema and hemorrhage, in the absence of significant inflammation or widespread ulceration. Microvascular angiopathy was present in all cases, with changes ranging from endothelial cell damage to overt thrombosis. Similar pathology was seen throughout the small bowel, including the presence of TMA. In one patient, typical morphologic changes of pseudomembranous enterocolitis were found in the absence of infection with Clostridium difficile. The nature of vascular involvement in the kidneys and intestinal tract supports the hypothesis that HUS is mediated by systemic toxemia, and that endothelial cells are the primary target cells for the action of verocytotoxin. 

## 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-HUS	N = 138
- HUS (all forms) 	N = 26

***Laboratory studies***
- CBC		N = 95
- Biochemistry	N = 120
- Urinalysis	N = 103

- No follow-up visits	N = 103
- ER follow-up	N = 43
- Hospitalized (initially or later)
- 		N = 54

- Treated
- in practice
- N = 22

- MB_FDA
- April 12, 2007

## Study Population take out

- 	Age			6.1 ± 4.7 yrs (range 0.3 – 18.0)
- 	Bloody diarrhea at presentation
- 				127/164	77.4%
- 	HUS all		26/186 	(n = 12 hospital transfer)
- 		*Dialysis*		10/26		38.5 %
- 	HUS at MCH	14/174	8.0 %
- 		Complete	 11 		6.3 %
- 		Incomplete	  3  		1.7 %
- 		*Dialysis**	*	3/11		27.3 %

- MB_FDA April 12, 2007

##  Retrospective data analysis Univariate logistic regressionAssociation of STMEs with Hospitalization

| Worst Score | Patients | OR | p |
| --- | --- | --- | --- |
| 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 regressionAssociation of STMEs with Hospitalization

**Notes:**

Overall presence of bloody
Disease 
Disease Scores (max)

Data AnalysisLogistic regression binary values (dependent variables are categorical: Hospitalization, HUS, Dialysis « yes or no ») versus scores (discrete variables)

## STME are associated with Hospitalization Logistic regression

|  | N | Univariate |  | Multivariate |  | Multivariate |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Scores1 |   | Odds
ratio | p | Odds
ratio | p | Odds
ratio | p |
| Bloody diarrhea | 164 | 2.4 | .0001 | 3.3 | .005 |  |  |
| Abdominal cramps | 159 | 2.6 | <.0001 | 4.0 | <.0001
 | 2.7 | .0004 |
| Diarrhea frequency | 164 | 2.1 | <.0001 |  |  | 1.7 | .023 |
| Hemoglobin | 95 | 5.6 | <.0001 |  |  |  |  |
| Platelets | 91 | ∞ | <.0001 |  |  |  |  |
| Hematuria | 103 | 3.1 | <.0001 | 6.2 | <.0001 |  |  |
| Creatinine | 120 | 7.8 | <.0001 |  |  | 10.2 | <.0001 |

**STME are associated with Hospitalization**** **Logistic regression

**Notes:**

Overall presence of bloody
Disease 
Disease Scores (max)

Data AnalysisLogistic regression binary values (dependent variables are categorical: Hospitalization, HUS, Dialysis « yes or no ») versus scores (discrete variables)

## STEC Enteropathy Scale ER study

## Distribution of Enteropathy Scores at Emergency Room Presentation

- MB_FDA April 12, 2007

## Distribution of Extraintestinal TMA and Nephropathy scores Emergency Room Presentation

- MB_FDA April 12, 2007

## Risk of Hospitalization due to Worsening of Enteropathy Scores

| Score | 0 | 1 | 2 | 3 | 4 |
| --- | --- | --- | --- | --- | --- |
| Diarrhea frequency | 7% | 13% | 24% | 40% | 58% |
| Abdominal pain | 6% | 16% | 36% | 62% | 83% |
| Bloody diarrhea | 9% | 18% | 35% | 56% | 75% |

- Including HUS

## Estimated Risk of Hospitalization related to Enteropathy Scores1

| Score | 0 | 1 | 2 | 3 | 4 |
| --- | --- | --- | --- | --- | --- |
| Diarrhea frequency | 3% | 7% | 22% | 27% | 46% |
| Abdominal pain | 1% | 6% | 22% | 59% | 88% |
| Bloody diarrhea | 2% | 7% | 21% | 50% | 79% |

- Excluding HUS Patients

- 1 from univariate logistic regression models