Granzyme A in Patients With E. Coli Bacteremic Urinary Tract Infections
Conditions: Bloodstream Infection, Sepsis, Pathogenesis, Escherichia Coli Bacteremia
Sponsor: Instituto de Investigación Sanitaria Aragón

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Study Protocol.  
Title of the study: Granzyme A in patients with E. coli Bacteremic Urinary Tract 
Infections (GABEC) 
 
Updated on June, the 5th, 2019. 
 
1. 
Background ...................................................................................................................... 2 
2. 
Research hypothesis ........................................................................................................ 2 
3. 
Aims and objectives ......................................................................................................... 2 
3.1. Aims .............................................................................................................................. 2 
3.2. Objectives ..................................................................................................................... 3 
4.1. Design and project scope .............................................................................................. 4 
4.2. Study period: ................................................................................................................ 4 
4.3. Patients and sample size ............................................................................................... 4 
4.4. Definitions .................................................................................................................... 5 
4.5. Variables ....................................................................................................................... 5 
4.6. Study plan detailing the procedures. ............................................................................ 7 
5. Ethical Aspects ..................................................................................................................... 7 
6. Planned Schedule ................................................................................................................ 7 
7. 
References ....................................................................................................................... 9 
 
 
 

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1. Background 
 
Sepsis is a high incidence, high morbidity and mortality, life-threatening organ 
dysfunction caused by a dysregulated response to infection that constitutes a public 
health priority. Improving sepsis diagnosis and finding pathogenically-relevant targets 
for pathophysiological therapy are opportunities to reduce sepsis burden.  
 
Despite not being critical to contain microorganisms, granzyme A (GZM A), a serine 
protease enzyme that is present in cytotoxic T lymphocyte granulesmight be a 
pathogenically relevant sepsis mediator. We found that GzmA knocked-out mice had 
significantly prolonged survival as compared with wild-type mice in an induced 
polymicrobial peritonitis (cecal ligation and puncture) murine model (Arias et al., 2018. 
Unpublished data). To the best of our knowledge, the pathogenic role of GZM A in 
humans with sepsis has been barely explored.  
 
In order to ascertain the pathogenic role of GZM A in sepsis we designed a nested case-
control study in patients with bacteremic urinary tract infections caused by E. coli This 
human model of infection-sepsis homogenizes other factors (patient, infection and 
microorganism related factors)  that could be pathogenically associated with sepsis in 
order to minimize confounding.   
 
2. Research hypothesis 
 
The research team has explored the role of GZM A  
 
• Conceptual hypothesis: 
- 
Granzyme A is a pathogenic sepsis mediator.  
- 
Granzyme A gene polymorphisms determine the serum concentration of 
GZM A in patients with systemic infections.  
- 
Granzyme A gene polymorphisms determine the risk of sepsis among 
patients with systemic infections. 
 
• Operational hypothesis:  
- 
Among patients with bacteremic (E. coli) urinary tract infections (UTIs), GZM 
A levels are significantly higher in those patients who develop sepsis as 
compared with those who do not develop sepsis.  
- 
There are significant differences in the GZM A gene polymorphism profile of 
patients with bacteremic (E. coli) UTIs who develop sepsis as compared with 
those who do not develop sepsis.  
 
 
3. Aims and objectives 
 
3.1. Aims 
 
1. To assess the pathogenic role of GZM A in sepsis in patients with bacteremic (E. 
coli) UTIs. 
2. To explore the capability of GZM A polymorphisms to anticipate the risk of 

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developing sepsis in patients with bacteremic (E. coli) UTIs.  
3. To evaluate the potential usefulness of GZM A as a diagnostic biomarker of 
sepsis in patients with bacteremic (E. coli) UTIs. 
4. To characterize E. coli "virulome" among circulating uropathogenic strains. 
 
3.2. Objectives  
 
1. To evaluate the correlation between serum levels of GZM A and systemic 
inflammatory response in patients with bacteremic (E. coli) UTIs.  
2. To characterize GZM A gene polymorphisms among patients with bacteremic (E. 
coli) UTIs 
3. To assess GZM A serum kinetics among patients with bacteremic (E. coli) UTIs 
4. To phenotypically and molecularly characterize E. coli strains causing bacteremic 
UTIs, including their virulence factors ("virulome").  
 
Expected outcomes. Relevance and transformative approach of the proposal. 
 
1. Characterization of the pathogenic role of GZM A in sepsis in patients with 
systemic infections  
1.1. Evaluation of the potential association between GZM A serum levels and sepsis. 
• The association between GZM A serum levels and the extent of systemic 
inflammatory response is a non-sufficient, albeit necessary condition to 
establish a pathogenic (causal) relationship between GZM A and sepsis. 
1.2. Identification of gzmA gen polymorphisms associated with an increased risk of 
developing sepsis among patients with infections: 
• Significant differences in serum GZM A concentrations between patients with 
and without sepsis would suggest a potential pathogenic role of GZM A. 
• If certain gzmA gene polymorphisms are associated with lower sepsis risk 
through lower GZM A serum levels, gzmA based sepsis risk could be estimated 
so that certain decisions could be personalized.  
• GZM A could be a new potential target for pathophysiological therapy of sepsis.  
 
2. Characterization of GZM A as a sepsis biomarker in a human model of infection-
sepsis.  
 
2.1. Evaluation of GZM A kinetics in the aforementioned human model of infection-
sepsis:  
• If the GZM A serum level is found to be sensitive and specific enough, 
determination of GZM A could assist diagnostic and therapeutic decisions in 
patients with suspected sepsis, which indeed is a difficult-to-diagnose entity 
due to its common non-specific presentation.  
• The correlation between serum concentration of GZM A and the clinical course 

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of the infection could assist therapeutic decisions, such as antibiotic 
streamlining, the switch from sequential intravenous to oral antibiotics and 
establishing the optimal duration of antibiotic therapy.  
 
3. Phenotypical and molecular characterization of uropathogenic E. coli causing 
bloodstream infections: 
• Phenotypic and molecular characterization of E. coli isolates involved in 
bacteremic UTIs has a great epidemiological interest 
• Identification of patients infected with hypervirulent strains could potentially 
influence the monitoring and therapeutic approach. 
 
 
- 
Discrimination of E. coli isolates according to their virulence (virulome)/potential 
pathogenicity is important to exclude strain-specific factors, when trying to 
establish the causal/pathogenic relationship between GZM A and sepsis.  
 
4. Methods 
 
4.1. Design and project scope 
 
• Prospective, exploratory nested case-control study to be conducted at one 
academic hospital (Hospital Clínico Universitario Lozano Blesa) affiliated with the 
Instituto de Investigación Sanitaria Aragón (IIS Aragón). 
 
 
4.2. Study period: June 2019 – December 2020.  
  
4.3. Patients and sample size 
 
Inclusion criteria (To meet all): 
 
• Age³ 18 years old.  
• E. coli bloodstream infection 
• Urinary source. Urinary source should be considered if (any) 
- 
Urinary source is clinically suspected and both E. coli isolates (in blood and in 
urine culture) share the same phenotype (antibiogram). 
- 
Urinary source/origin is clinically suspected, and the urine culture is negative, 
but the patient had received at least one dose of any systemic antibiotic with 
antimicrobial activity against the E. coli strain causing the BSI before blood 
cultures were obtained. 
- 
Both isolates (in blood and in urine culture) share the same phenotype 
(antibiogram) and there is no alternative source.  
 
Exclusion criteria: 
 
• Use of systemic antibiotics for >48h in the two months preeeding the episode. 

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• Immunocompromised hosts 
- 
Patients receiving systemic steroid use (>10 mg prednisone/day during 10 or 
more days in the previous 2 months). 
- 
Patients receiving biological therapy (previous 2 months).  
- 
Active solid or hematological cancer.  
- 
HIV +. 
- 
Neutropenia < 500 PMN/microl.  
 
• Basal urinary tract abnormalities or locally modified vesical microbiome (any): 
- 
Ureteroileostomy, ureterosigmoidostomy, ureterostomy (Bricker) or 
nephrostomy.  
- 
Indwelling urinary catheter (in the last two months) 
- 
Urological surgery (in the last two months). 
- 
Intravesical chemotherapy (in the last two months). 
- 
Intravesical BCG instillation (in the last two months).  
 
Potential candidates will be detected daily by the microbiologists on the research team. 
Inclusion criteria will be verified in the multidisciplinary meeting that antimicrobial 
stewardship teams (AST) conduct on a daily basis at the participating hospital.  
 
Estimated size of the study population. Matching:  
• 50 patients with a sepsis/ non sepsis 1:1 ratio will be included.  
• Septic and non-septic patients will be matched on gender, age (+/- 10 years), 
comorbidity (Charlson score +/-1), time symptom onset to blood  culture (+/- 
24h) 
 
4.4. Definitions 
 
Cases (sepsis / septic shock):  
• Sepsis or shock septic are defined as life-threatening organ dysfunction caused 
by a dysregulated host response to infection according to the 2001 
SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definition Conference. 
 
Controls:  
• Absence of sepsis or septic shock according to the 2001 
SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definition Conference.  
 
4.5. Variables 
 
• Patient-related variables:  
- 
Demographical: gender, age. 
- 
Comorbidity: Modified Charlson Index 
- 
Baseline serum creatinine. 
 
• Infection-related variables.  
- 
Time from the onset of symptoms to the start of antimicrobial treatment.  
- 
Time from the onset of symptoms to the start of appropriate antimicrobial 

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treatment. 
- 
Time form the onset of symptoms to the surgical therapy (if needed).  
- 
Clinical severity at time 0 (blood culture), and day 2- 3 and day 30: sepsis score. 
 
• Inflammation, sepsis mediators and biomarkers.  
The following biomarkers will be determined during patient enrollment:  
- 
White blood cell count and differential. 
- 
Platelet count. 
- 
Fibrinogen.  
- 
Prothrombin activity.  
- 
C reactive protein. 
- 
Procalcitonin. 
- 
GZM A. 
 
GZM A serum levels will be obtained in all patients during the enrollment visit, the 2-3 
day and the 30 day visits (GZM A kinetics). GZM A levels will be determined by an ELISA 
commercial assay (Human Granzyme A ELISA development kit{HRP]; Mabtech).  
 
GzmA gene polymorphisms, as well as other potentially associated mutations, will be 
screened by Whole Exome Sequencing (WES). To this aim, we will use DNA isolated from 
peripheral blood cells and the AmpliSeq technology kit on the Ion Torrent platform 
following the manufacturer’s instructions. This platform is available at the Genomics 
Central Research Unit (CRU) at CIBA from University of Zaragoza/IIS Aragon. All kits for 
isolating and analyzing DNA samples are commercially available and optimized by 
Thermo Scientific. Bioninformatic analysis will be performed by an agreement 
established between Genomics CRU and Micromics SL led by Pedro Gonzalez at CRG in 
Barcelona.  
 
• Microbiological variables  
- Minimum inhibitory concentrations (MIC) of the E. coli isolates retrieved from 
urine and blood cultures will be determined through automated microdilution 
panels as routinely performed by the Microbiology Laboratory of participating 
hospitals.  
- Whole genome sequencing (WGS) of the E. coli strains isolated in blood and 
urine cultures. The presence of E. coli known virulence factors will be analyzed 
and a virulence score for each strain will be calculated (Mora-Rillo et al., 2015).  
 
 
 
 

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4.6. Study plan detailing the procedures.  
 
 
Day 0 
Day 2 - 3 
Day 30 
Screening 
(inclusion 
and 
exclusion criteria) 
X 
 
 
Informed Consent 
X 
 
 
Anamnesis  
X 
 
 
Charlson Modified 
X 
 
 
Sepsis score 
X 
X 
 
Serum 
X 
X 
X 
Plasma 
X 
X 
X 
Microbiological 
samples 
X 
 
 
Mortality 
 
 
X 
New hospitalization 
 
 
X 
 
5. Ethical Aspects 
 
Surveillance of patients with bacteremia is a routine activity of the Antimicrobial 
Stewardship Team in our hospital, that depends on the Sistema Aragonés de Salud. 
Demographical, clinical and microbiological data registry is part of this activity.  
 
The study protocol has been reviewed and approved by the Comité Ético de 
Investigación Clínica de Aragón (CEICA): approval Number: PI19/070  
  
The enrollment of patients to determinate GZM A, gzmA gen polymorphisms will 
require signed informed consent.  
 
 
6. Planned Schedule  
 
January to Jun 2019: 
 
- 
Local Ethics Committee (CEICA) approval request: Compilation of 
administrative documentation and forms, including information sheet and 
informed consent 
- 
Statistical consultation. 
- 
Design of the case report form (CRF) and database 
- 
Biobank arrangements. 
- 
Recruitment of the study coordinator. 
- 
Training of research staff.  
- 
Clinical Trials Register.  
 
July 2019 to August 2020. 
 
- 
Screening and inclusion of patients with bacteremic (E. coli) UTIs. 
- 
Data capture (demographical, clinical and basic laboratory parameters). 

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- 
Storage of patient samples and E. coli strains (blood and urine).  
- 
GZM A determination. 
- 
Whole exome sequencing (gzmA polymorphisms). 
- 
Whole genome sequencing (E. coli strains).  
 
September 2020 to December 2020. 
 
- 
Data analysis  
- 
Manuscripts preparation. 
Manuscript submission.  
 
 
 

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7. References 
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• Arias, M., Jimenez de Bagues M.P., et al. (2014). "Elucidating sources and roles of 
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• Arias, M., Martinez-Lostao L., et al. (2017). "The Untold Story of Granzymes in 
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• Dellinger, R.P., Levy, M.M., Rhodes, A., Annane, D., Gerlach, H., Opal, S.M., et al. 
(2013). Surviving Sepsis Campaign: international guidelines for management of 
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• Iwashyna, T.J., Cooke, C.R., Wunsch, H., et al. (2012). "Population burden of long-
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• Metkar, S. S., C. Menaa, et al. (2008). "Human and mouse granzyme A induce a 
proinflammatory 
cytokine 
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presentation, diagnosis, and prognosis. Parsons PE, ed. UpToDate. Waltham, MA: 
UpToDate Inc. http://www.uptodate.com (Accessed on July 22, 2018).  
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