Ignite Creation Date:
2024-05-05 @ 11:42 PM
Last Modification Date:
2024-10-26 @ 10:38 AM
Study NCT ID:
NCT01397058
Status:
UNKNOWN
Last Update Posted:
2011-08-15
First Post:
2011-07-18
Brief Title:
Reactivation of CMV Infection in Immunocompetent Patients Under Severe Stress
Sponsor:
University of Athens
Organization:
University of Athens
Study Overview
Official Title:
Observational Study of CMV Reactivation in Immunocompetent Children and Adult ICU Patients
Status:
UNKNOWN
Status Verified Date:
2011-07
Last Known Status:
RECRUITING
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
RECYSTRESS
Brief Summary:
Background Human herpes viruses establish lifelong latency after primary infection and may reactivate in immunosuppressed patients causing significant morbidity and mortality In immunocompetent patients although reactivation may occur disease development is deterred by the competent host immune response Recent studies indicate that approximately one third of CMV seropositive immunocompetent ICU patients present with CMV reactivation associated with poor outcome potentially secondary to the stress incurred CMV reactivation among immunocompetent critically ill children has not been assessed
Study Hypothesis Identifiable risk factors associated with CMV reactivation exist and may be used for future assessment of antiviral prophylaxis administration
Aim Primary aim is to identify risk factors associated with CMV reactivation and poor outcome in immunocompetent children and adults under severe stress Whether CMV reactivation occurs in critically ill children and its clinical implications remains to be determined Secondary aim is to study the role of cellular signaling pathways of inflammation and specific adaptive immunity during this process
Work packages A multicenter observational prospective study will be conducted among CMV seropositive pediatric and adult ICU patients Patient clinical progress laboratory findings management and complications will be recorded during the 28 days following ICU admission Salivary free cortisol levels plasma catecholamines and serum cytokines levels will be measured to assess stress CMV reactivation will be evaluated weekly by detecting CMV-DNA in peripheral blood and bronchial wash samples with real-time PCR In a patient subsample the nuclear factor κB and intracellular GC receptor will be measured in peripheral blood monocytes to study cellular signaling pathways of inflammation The adaptive immune response to CMV infection following in vitro viral polypeptide stimulation will be prospectively examined in a subset of patients
Expected Results The study will provide original data on critically ill children Further knowledge regarding risk factors associated with CMV reactivation and poor outcome will be accumulated Novel information regarding the role of cellular inflammation and specific adaptive immune responses during CMV reactivation will be gathered
Study Hypothesis Identifiable risk factors associated with CMV reactivation exist and may be used for future assessment of antiviral prophylaxis administration
Aim Primary aim is to identify risk factors associated with CMV reactivation and poor outcome in immunocompetent children and adults under severe stress Whether CMV reactivation occurs in critically ill children and its clinical implications remains to be determined Secondary aim is to study the role of cellular signaling pathways of inflammation and specific adaptive immunity during this process
Work packages A multicenter observational prospective study will be conducted among CMV seropositive pediatric and adult ICU patients Patient clinical progress laboratory findings management and complications will be recorded during the 28 days following ICU admission Salivary free cortisol levels plasma catecholamines and serum cytokines levels will be measured to assess stress CMV reactivation will be evaluated weekly by detecting CMV-DNA in peripheral blood and bronchial wash samples with real-time PCR In a patient subsample the nuclear factor κB and intracellular GC receptor will be measured in peripheral blood monocytes to study cellular signaling pathways of inflammation The adaptive immune response to CMV infection following in vitro viral polypeptide stimulation will be prospectively examined in a subset of patients
Expected Results The study will provide original data on critically ill children Further knowledge regarding risk factors associated with CMV reactivation and poor outcome will be accumulated Novel information regarding the role of cellular inflammation and specific adaptive immune responses during CMV reactivation will be gathered
Detailed Description:
This is a multicenter prospective observational study of CMV seropositive patients children and adults admitted in ICU Two pediatric and four adult ICU units in tertiary teaching hospitals will be recruiting for 48 and 36 months respectively
Research plan Upon ICU admission patients group A and B will be screened for inclusionexclusion criteria and after written informed consent is obtained the patient will be enrolled Clinical severity will be estimated using standardized score systems PRISM III and APACHI II respectively All pertinent demographic medical history clinical presentation and medical interventions will be entered in a database Prior to initiating any supportive treatment inotropes or corticosteroids blood and saliva will be obtained for the determination of biological markers of stress Serum and whole blood will be collected for the determination of CMV-IgG antibodies and CMV-DNA respectively CMV seronegative patients will be excluded from further evaluation Seropositive patients will be followed prospectively for 28 days Weekly follow up 7th 14th 21st and 28th day of hospitalization will include clinical and laboratory evaluation documentation of therapeutic intervention and CMV reactivation assessment CMV-DNA detection Stress markers will be re-examined only on day 7 On day 28 end of follow up the clinical outcome will be recorded
In a random subset of patients 20 children and 20 adults the intracellular signaling pathway of inflammation and specific immune responses will be studied
Laboratory Methods
A Serology for CMV Serum CMV-IgG antibodies will be measured by Enzyme Immunoassay Method Elisa Abbott Laboratories in hospital serology lab
B Estimation of biological stress indicators
B1 Measurement of free cortisol Saliva samples will be collected three times a day 800 am 200pm and 800 pm to determine the circadian pattern cortisol secretion using special synthetic swabs Salivetta-Salivette Sarsted and stored at -70oC Cortisol levels will be determined with electrochemiluminescence immunoassay Elecsys Cortisol reagent kit ROCHE
B2 Plasma catecholamines Catecholamine levels noradrenaline adrenaline and dopamine will be measured before the initiation therapy with any inotropic agent Samples will be taken from a venous catheter with the patient supine only in patients not receiving inotropes Plasma will be stored at -85oC The Liquid HPLC reverse phase High Performance Liquid Chromatography - reverse phase will be used
B3 Serum TNF-a will be measured by quantitative-sandwich-Enzyme Immunoassay method Elecsys ROCHE
C Detection and quantification of CMV-DNA from whole blood and bronchoalveolar lavage BAL in ventilated patients If a patient is discharged before day 28 salivettes will be given at home to collect saliva for CMV-DNA determination stored in home fridge and returned on day 28 Samples will be stored at -70oC and all samples from each patient will be examined at the same time DNA extraction and real-time PCR will be performed by commercial kits Nanogen Advanced Diagnostics A gene region encoding the Major Immediate Early Antigen MIEA of CMV will be detected All PCR testing will be performed at the Cytology Laboratory Attikon University Hospital Athens Greece Associate Prof P Karakitsos
D Glucocorticoid receptor GR and nuclear factor κB NFκB measurements in whole cell and nuclear extracts from peripheral blood lymphocytes will be performed by immunoblotting at the Department of Biological Chemistry Athens University Ass Prof P Moutsatsou8
E Specific immune response against CMV Specific immune responses of CD4 and CD8 T lymphocytes from peripheral blood after in-vitro stimulation with CMV polypeptides including pp65 antigen will be studied with intracellular staining Intracellular staining ICS The proliferation of CD4 CFSE and the cytotoxicity of CD8 perforin and granzyme B will be assessed
Statistical analysis For the data analysis adults and children will be examined separately Seropositive patients without CMV reactivation will be used as the control group A p-value p of 005 is the criterion for statistical significance T-test and chi-square test will be used for quantitative and qualitative analysis while the Mantel-Haenzel method will be used to calculate relative risk of different outcomes mortality intubation etc between the two groups To identify potential risk factors associated with CMV reactivation and clinical outcome logistic regression analysis using the statistical program SAS v9 will be performed
Sample calculation To calculate study sample size the statistical program EpiInfo version 6 was used Based on recent data from adults 30 may demonstrate CMV reactivation while in the ICU The mortality rate in the pediatric and adult ICU has been estimated to reach approximately 8 and 63 respectively Recent data suggest that CMV reactivation is associated with increased mortality RR193 and morbidity RR570 The sample size was calculated to meet the criteria of 99 confidence interval and 90 statistical power We estimated that our CMV population should include 165 children and 109 adults
Work packages WP1 and WP2 involve clinical research enrollment and clinical follow up of children and adults respectively All data collection will be entered in an anonymous database which will include demographic data past history clinical and laboratory evaluation clinical management and interventions upon admission and during follow up period Adults and children will be evaluated separately Once CMV results are provided WP4 risk factors associated with CMV reactivation as well as adverse clinical outcomes including both those directly ie pneumonitis febrile episodes and hepatitis and indirectly associated ie extended ICU stay or mechanical ventilation and increased rate of bacterial infections will be examined
WP3 involves the evaluation of stress from a clinical WP31 and WP 32 and basic research WP33 approach Biochemical stress markers will be evaluated upon admission and on day 7 to examine duration of stress Concomitant drug administration will be taken into account Stress markers will be correlated with CMV reactivation and clinical outcome to examine whether they can be used as identifiable risk factors for future research In WP33 cellular signaling pathways of inflammation in a subset of our cohort will be studied Specifically the interaction of NFκΒ activation and glucocorticoid receptor sensitivity in patients mononuclear cells will be evaluated and correlated with CMV reactivation and outcome
WP4 involves the prospective detection and quantification of CMV-DNA in different biological samples All data will be included in the main database and more importantly a DNA bank will be formed for possible detection of other herpesviruses in the future
WP5 will evaluate specific immune responses to CMV in a subset of patients These will be correlated with the underlying disease reactivation and stress Moreover a bank of supernatants from those experiments will be formed to measure cytokines Luminex in the future
Research plan Upon ICU admission patients group A and B will be screened for inclusionexclusion criteria and after written informed consent is obtained the patient will be enrolled Clinical severity will be estimated using standardized score systems PRISM III and APACHI II respectively All pertinent demographic medical history clinical presentation and medical interventions will be entered in a database Prior to initiating any supportive treatment inotropes or corticosteroids blood and saliva will be obtained for the determination of biological markers of stress Serum and whole blood will be collected for the determination of CMV-IgG antibodies and CMV-DNA respectively CMV seronegative patients will be excluded from further evaluation Seropositive patients will be followed prospectively for 28 days Weekly follow up 7th 14th 21st and 28th day of hospitalization will include clinical and laboratory evaluation documentation of therapeutic intervention and CMV reactivation assessment CMV-DNA detection Stress markers will be re-examined only on day 7 On day 28 end of follow up the clinical outcome will be recorded
In a random subset of patients 20 children and 20 adults the intracellular signaling pathway of inflammation and specific immune responses will be studied
Laboratory Methods
A Serology for CMV Serum CMV-IgG antibodies will be measured by Enzyme Immunoassay Method Elisa Abbott Laboratories in hospital serology lab
B Estimation of biological stress indicators
B1 Measurement of free cortisol Saliva samples will be collected three times a day 800 am 200pm and 800 pm to determine the circadian pattern cortisol secretion using special synthetic swabs Salivetta-Salivette Sarsted and stored at -70oC Cortisol levels will be determined with electrochemiluminescence immunoassay Elecsys Cortisol reagent kit ROCHE
B2 Plasma catecholamines Catecholamine levels noradrenaline adrenaline and dopamine will be measured before the initiation therapy with any inotropic agent Samples will be taken from a venous catheter with the patient supine only in patients not receiving inotropes Plasma will be stored at -85oC The Liquid HPLC reverse phase High Performance Liquid Chromatography - reverse phase will be used
B3 Serum TNF-a will be measured by quantitative-sandwich-Enzyme Immunoassay method Elecsys ROCHE
C Detection and quantification of CMV-DNA from whole blood and bronchoalveolar lavage BAL in ventilated patients If a patient is discharged before day 28 salivettes will be given at home to collect saliva for CMV-DNA determination stored in home fridge and returned on day 28 Samples will be stored at -70oC and all samples from each patient will be examined at the same time DNA extraction and real-time PCR will be performed by commercial kits Nanogen Advanced Diagnostics A gene region encoding the Major Immediate Early Antigen MIEA of CMV will be detected All PCR testing will be performed at the Cytology Laboratory Attikon University Hospital Athens Greece Associate Prof P Karakitsos
D Glucocorticoid receptor GR and nuclear factor κB NFκB measurements in whole cell and nuclear extracts from peripheral blood lymphocytes will be performed by immunoblotting at the Department of Biological Chemistry Athens University Ass Prof P Moutsatsou8
E Specific immune response against CMV Specific immune responses of CD4 and CD8 T lymphocytes from peripheral blood after in-vitro stimulation with CMV polypeptides including pp65 antigen will be studied with intracellular staining Intracellular staining ICS The proliferation of CD4 CFSE and the cytotoxicity of CD8 perforin and granzyme B will be assessed
Statistical analysis For the data analysis adults and children will be examined separately Seropositive patients without CMV reactivation will be used as the control group A p-value p of 005 is the criterion for statistical significance T-test and chi-square test will be used for quantitative and qualitative analysis while the Mantel-Haenzel method will be used to calculate relative risk of different outcomes mortality intubation etc between the two groups To identify potential risk factors associated with CMV reactivation and clinical outcome logistic regression analysis using the statistical program SAS v9 will be performed
Sample calculation To calculate study sample size the statistical program EpiInfo version 6 was used Based on recent data from adults 30 may demonstrate CMV reactivation while in the ICU The mortality rate in the pediatric and adult ICU has been estimated to reach approximately 8 and 63 respectively Recent data suggest that CMV reactivation is associated with increased mortality RR193 and morbidity RR570 The sample size was calculated to meet the criteria of 99 confidence interval and 90 statistical power We estimated that our CMV population should include 165 children and 109 adults
Work packages WP1 and WP2 involve clinical research enrollment and clinical follow up of children and adults respectively All data collection will be entered in an anonymous database which will include demographic data past history clinical and laboratory evaluation clinical management and interventions upon admission and during follow up period Adults and children will be evaluated separately Once CMV results are provided WP4 risk factors associated with CMV reactivation as well as adverse clinical outcomes including both those directly ie pneumonitis febrile episodes and hepatitis and indirectly associated ie extended ICU stay or mechanical ventilation and increased rate of bacterial infections will be examined
WP3 involves the evaluation of stress from a clinical WP31 and WP 32 and basic research WP33 approach Biochemical stress markers will be evaluated upon admission and on day 7 to examine duration of stress Concomitant drug administration will be taken into account Stress markers will be correlated with CMV reactivation and clinical outcome to examine whether they can be used as identifiable risk factors for future research In WP33 cellular signaling pathways of inflammation in a subset of our cohort will be studied Specifically the interaction of NFκΒ activation and glucocorticoid receptor sensitivity in patients mononuclear cells will be evaluated and correlated with CMV reactivation and outcome
WP4 involves the prospective detection and quantification of CMV-DNA in different biological samples All data will be included in the main database and more importantly a DNA bank will be formed for possible detection of other herpesviruses in the future
WP5 will evaluate specific immune responses to CMV in a subset of patients These will be correlated with the underlying disease reactivation and stress Moreover a bank of supernatants from those experiments will be formed to measure cytokines Luminex in the future
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None