Anti-streptokinase antibodies and outcome of fibrinolytic therapy with streptokinase for left-sided prosthetic valve thrombosis Sandeep Singh MD, DM, Shrenik Doshi MD, DM, Salman Salahuddin MD, DM, Mohamad Tarik MSc, Parag Barwad MD, DM, Lakshmy Ramakrishnan MD, Sivasubramanian Ramakrishnan MD, DM, Ganesan Karthikeyan MD, DM, MSc, Balram Bhargava MD, DM, Vinay K. Bahl MD, DM PII: DOI: Reference:
S0002-8703(14)00619-X doi: 10.1016/j.ahj.2014.10.012 YMHJ 4750
To appear in:
American Heart Journal
Received date: Accepted date:
27 February 2014 9 October 2014
Please cite this article as: Singh Sandeep, Doshi Shrenik, Salahuddin Salman, Tarik Mohamad, Barwad Parag, Ramakrishnan Lakshmy, Ramakrishnan Sivasubramanian, Karthikeyan Ganesan, Bhargava Balram, Bahl Vinay K., Anti-streptokinase antibodies and outcome of fibrinolytic therapy with streptokinase for left-sided prosthetic valve thrombosis, American Heart Journal (2014), doi: 10.1016/j.ahj.2014.10.012
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT TITLE PAGE TITLE: Anti-streptokinase antibodies and outcome of fibrinolytic therapy with
RI P
T
streptokinase for left-sided prosthetic valve thrombosis
SC
SHORT TITLE: Anti-streptokinase antibodies and fibrinolysis
MA NU
AUTHORS:
Sandeep Singh*MD, DM; Shrenik Doshi*MD, DM; Salman Salahuddin*MD, DM; Mohamad Tarik≠MSc; Parag Barwad*MD, DM; Lakshmy Ramakrishnan≠MD; Sivasubramanian Ramakrishnan*MD, DM; Ganesan Karthikeyan*MD, DM, MSc;
ED
Balram Bhargava*MD, DM; Vinay K Bahl*MD, DM
PT
From the Departments of *Cardiology and ≠Cardiac Biochemistry
CE
All India Institute of Medical Sciences, New Delhi-110029, India
AC
ADDRESS FOR CORRESPONDENCE: Sivasubramanian RamakrishnanMD, DM Additional Professor, Department of Cardiology Room No. 32, 7th Floor All India Institute of Medical Sciences, New Delhi-110029, India Email- [email protected] Ph:+ 91 1126594420, Fax: +91 11 26588663
1
ACCEPTED MANUSCRIPT
2
ABSTRACT Background: Left-sided prosthetic valve thrombosis (PVT) is a serious complication of
T
valve replacement. In developing countries, fibrinolysis with streptokinase (SK) is often used
RI P
as the first line of treatment. Anti-streptokinase (anti-SK) antibodies are widely prevalent in the general population but their effect on the efficacy and outcome of fibrinolysis with SK in
SC
patients with PVT is not known.
Methods: Patients with rheumatic heart disease and prosthetic valve replacement presenting
MA NU
with a first episode of PVT were enrolled. All patients underwent fibrinolysis with SK. An indirect ELISA assay was used to detect anti-SK antibodies prior to fibrinolysis. Relationship of these antibodies to the outcome of fibrinolysis was evaluated.
ED
Results: Forty-four patients treated for valve thrombosis were included. Thrombosis affected 33 mitral and 11 aortic prosthetic valves. On fibrinolysis with SK, 32 (73%) patients achieved
PT
complete success, while it was unsuccessful in the remaining 12 patients. There were 3 bleeding events, 1 stroke and 3 deaths. Mean anti-SK antibody levels were not significantly
CE
different between patients who had complete success and those who did not (8.81±2.43
AC
versus 7.67±1.26 Au/mL; P=0.13), and did not correlate with the outcome after adjustment with other variables. Patients in NYHA class III or IV had a greater chance of failed fibrinolytic therapy, even after adjustment for other prognostic variables (OR 9.0, 95% CI – 1.29 – 63.02; P=0.027). Conclusion: Anti-SK antibody titres are not associated with success of fibrinolytic therapy using SK in patients with left-sided PVT.
ACCEPTED MANUSCRIPT
3
Left-sided prosthetic valve thrombosis (PVT) is a potentially life threatening complication that is commonly seen in developing countries. Surgery has been accepted as the
T
management of choice for obstructive PVT, however, because of its limited availability and
RI P
high cost, fibrinolytic therapy has become the first-line treatment. Streptokinase (SK), a firstgeneration fibrinolytic agent is preferred in developing countries in view of its low cost and
irrespective of the dosing regimen.[1-3]
SC
easy availability. Efficacy of SK in PVT remains suboptimal, ranging from 60-80%,
MA NU
Anti-SK antibodies have widespread presence in the general population as a consequence of previous streptococcal exposure or infection, though the levels may vary in different populations.[4,5] High levels have been reported in developing countries and in geographical
ED
areas having high endemic streptococcal infection.[6-8] The association of these antibodies to an immune reaction following treatment with SK is well known.[9-11] Further, their effect on
PT
the efficacy of SK in ST-segment elevation myocardial infarction (STEMI) has been previously studied.[5,8,12,13] However, to the best of our knowledge, impact of anti-SK
CE
antibodies on the treatment of PVT with SK has not been addressed. We hypothesized that
AC
assessing anti-SK antibody titers may be useful in predicting the efficacy of SK for the treatment of PVT.
METHODS Patients: This is a single-centre, prospective, observational study conducted from May 2010 to December 2012 at a teaching hospital in India. Patients with prosthetic mechanical heart valves implanted for the treatment of rheumatic heart disease (RHD) and presenting with objectively confirmed, first episode of left-sided PVT were included. Patients were enrolled from the out-patient clinic or emergency department of the institute. Patients with PVT with
ACCEPTED MANUSCRIPT
4
valve implanted for diseases other than RHD or patients with recurrent PVT were excluded. The diagnosis of PVT was suspected in patients presenting with new onset symptoms
T
attributable to valve thrombosis such as dyspnea, angina and congestive heart failure of less
RI P
than two weeks duration. A confirmatory diagnosis required the documentation of new onset of hypomobile or immobile valve leaflets on cine-fluoroscopy with or without evidence of
SC
increased transvalvular gradients on Doppler echocardiography. As per institutional protocol, all patients with mechanical heart valves undergo periodic cine-fluoroscopic examinations
MA NU
during their routine follow-up visits. Previous records were checked to confirm the new onset of prosthetic valve dysfunction. Patients with PVT who were less than 12 years of age, patients with a history of fibrinolysis with SK and those with contraindications to fibrinolytic
ED
therapy (previous intracranial hemorrhage, major bleeding within past 2 weeks, ischemic stroke within last 3 months or presence of a left atrial thrombus on transthoracic
PT
echocardiography) were excluded. All patients gave written informed consent and the study protocol was approved by the Institutional Ethics Committee.
CE
Demographic and clinical parameters obtained at baseline included age, sex, NYHA class,
AC
presence of atrial fibrillation (AF) and left ventricular ejection fraction (LVEF). Compliance to oral anticoagulants was assessed by self-reporting by the patients. Their INR values at presentation were recorded. Fibrinolytic Therapy: All
patients
underwent
fibrinolysis
with
SK with
serial
echocardiographic and cine-fluoroscopic monitoring. The following protocol was used for fibrinolysis with SK- 250,000 units intravenous bolus over 30 min followed by a continuous infusion of 100,000 units/hour. Cine-fluoroscopy and Doppler echocardiography were performed 8-12 hourly or after any significant change in clinical status. Criteria for stopping SK infusion were either successful fibrinolysis or any complication mandating its
ACCEPTED MANUSCRIPT
5
discontinuation. It was continued for up to a maximum of 96 hours in patients with partial or no response, at the discretion of the treating physician.
T
Serologic testing: Blood (3 ml) was collected in a plain vial for anti-SK antibody titers prior
RI P
to initiation of SK therapy, and was stored at -20°C for analysis. An indirect ELISA assay was designed for the detection of anti-SK IgG antibodies. Briefly, the microtitre plate was
SC
coated with 100µL of SK at a concentration of 500 IU/mL prepared in phosphate buffered
MA NU
saline (PBS). After an overnight incubation at 4°C and washing 3 times with wash buffer and incubated with 200µL of BSA (0.1% bovine serum albumin with PBS) for 1 hour at room temperature to block remaining binding sites. 100µL of serum samples were added in dilution of 1/800. Also serial dilutions (1:100 to 1:204800) of specific anti-SK antibody (Thermo
ED
Scientific, USA) were included in the plate. The plate was incubated for an hour at room temperature and then washed three times with the wash buffer. 100µL of HRP (horse radish
PT
peroxidase) – conjugated rabbit anti-human IgG (Jackson Immuno Research, PA, USA)
CE
diluted at 1:20000 was added to the wells. After incubation for 30 min at room temperature and washing three times with the wash buffer, 100µL of chromogen substrate solution
AC
containing TMB (Tetramethylbenzidine) reagent was added and the plate was incubated for 10 minutes at room temperature in the dark. The reaction was stopped with 100µL of 1M H2SO4 and the results were analyzed with an ELISA microplate reader at 450nm to get the optical density (OD) values of every individual serum. The titration curve of the specific antiSK antibody was used as an intra-laboratory standard. The OD value of a specified dilution of this serum 1/204800 corresponded to blank OD and therefore 1/102400 was assigned 1 arbitrary unit (Au) and subsequent dilutions as 2 Au, 3 Au and so on. The level of anti-SK antibody in all the sera was read at 1/800 dilution, and quantified using these arbitrary units.
ACCEPTED MANUSCRIPT
6
Study outcomes: Outcomes of fibrinolytic therapy were based on clinical findings, echocardiographic and cine-fluoroscopic parameters, as described previously.[1] These were
T
defined as:
RI P
Complete Success: Clinical and hemodynamic improvement with normal leaflet motion on cine-fluoroscopy and normalization of valve gradients on Doppler echocardiography (mitral
SC
valve: - mean diastolic gradient
S0002-8703(14)00619-X doi: 10.1016/j.ahj.2014.10.012 YMHJ 4750
To appear in:
American Heart Journal
Received date: Accepted date:
27 February 2014 9 October 2014
Please cite this article as: Singh Sandeep, Doshi Shrenik, Salahuddin Salman, Tarik Mohamad, Barwad Parag, Ramakrishnan Lakshmy, Ramakrishnan Sivasubramanian, Karthikeyan Ganesan, Bhargava Balram, Bahl Vinay K., Anti-streptokinase antibodies and outcome of fibrinolytic therapy with streptokinase for left-sided prosthetic valve thrombosis, American Heart Journal (2014), doi: 10.1016/j.ahj.2014.10.012
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT TITLE PAGE TITLE: Anti-streptokinase antibodies and outcome of fibrinolytic therapy with
RI P
T
streptokinase for left-sided prosthetic valve thrombosis
SC
SHORT TITLE: Anti-streptokinase antibodies and fibrinolysis
MA NU
AUTHORS:
Sandeep Singh*MD, DM; Shrenik Doshi*MD, DM; Salman Salahuddin*MD, DM; Mohamad Tarik≠MSc; Parag Barwad*MD, DM; Lakshmy Ramakrishnan≠MD; Sivasubramanian Ramakrishnan*MD, DM; Ganesan Karthikeyan*MD, DM, MSc;
ED
Balram Bhargava*MD, DM; Vinay K Bahl*MD, DM
PT
From the Departments of *Cardiology and ≠Cardiac Biochemistry
CE
All India Institute of Medical Sciences, New Delhi-110029, India
AC
ADDRESS FOR CORRESPONDENCE: Sivasubramanian RamakrishnanMD, DM Additional Professor, Department of Cardiology Room No. 32, 7th Floor All India Institute of Medical Sciences, New Delhi-110029, India Email- [email protected] Ph:+ 91 1126594420, Fax: +91 11 26588663
1
ACCEPTED MANUSCRIPT
2
ABSTRACT Background: Left-sided prosthetic valve thrombosis (PVT) is a serious complication of
T
valve replacement. In developing countries, fibrinolysis with streptokinase (SK) is often used
RI P
as the first line of treatment. Anti-streptokinase (anti-SK) antibodies are widely prevalent in the general population but their effect on the efficacy and outcome of fibrinolysis with SK in
SC
patients with PVT is not known.
Methods: Patients with rheumatic heart disease and prosthetic valve replacement presenting
MA NU
with a first episode of PVT were enrolled. All patients underwent fibrinolysis with SK. An indirect ELISA assay was used to detect anti-SK antibodies prior to fibrinolysis. Relationship of these antibodies to the outcome of fibrinolysis was evaluated.
ED
Results: Forty-four patients treated for valve thrombosis were included. Thrombosis affected 33 mitral and 11 aortic prosthetic valves. On fibrinolysis with SK, 32 (73%) patients achieved
PT
complete success, while it was unsuccessful in the remaining 12 patients. There were 3 bleeding events, 1 stroke and 3 deaths. Mean anti-SK antibody levels were not significantly
CE
different between patients who had complete success and those who did not (8.81±2.43
AC
versus 7.67±1.26 Au/mL; P=0.13), and did not correlate with the outcome after adjustment with other variables. Patients in NYHA class III or IV had a greater chance of failed fibrinolytic therapy, even after adjustment for other prognostic variables (OR 9.0, 95% CI – 1.29 – 63.02; P=0.027). Conclusion: Anti-SK antibody titres are not associated with success of fibrinolytic therapy using SK in patients with left-sided PVT.
ACCEPTED MANUSCRIPT
3
Left-sided prosthetic valve thrombosis (PVT) is a potentially life threatening complication that is commonly seen in developing countries. Surgery has been accepted as the
T
management of choice for obstructive PVT, however, because of its limited availability and
RI P
high cost, fibrinolytic therapy has become the first-line treatment. Streptokinase (SK), a firstgeneration fibrinolytic agent is preferred in developing countries in view of its low cost and
irrespective of the dosing regimen.[1-3]
SC
easy availability. Efficacy of SK in PVT remains suboptimal, ranging from 60-80%,
MA NU
Anti-SK antibodies have widespread presence in the general population as a consequence of previous streptococcal exposure or infection, though the levels may vary in different populations.[4,5] High levels have been reported in developing countries and in geographical
ED
areas having high endemic streptococcal infection.[6-8] The association of these antibodies to an immune reaction following treatment with SK is well known.[9-11] Further, their effect on
PT
the efficacy of SK in ST-segment elevation myocardial infarction (STEMI) has been previously studied.[5,8,12,13] However, to the best of our knowledge, impact of anti-SK
CE
antibodies on the treatment of PVT with SK has not been addressed. We hypothesized that
AC
assessing anti-SK antibody titers may be useful in predicting the efficacy of SK for the treatment of PVT.
METHODS Patients: This is a single-centre, prospective, observational study conducted from May 2010 to December 2012 at a teaching hospital in India. Patients with prosthetic mechanical heart valves implanted for the treatment of rheumatic heart disease (RHD) and presenting with objectively confirmed, first episode of left-sided PVT were included. Patients were enrolled from the out-patient clinic or emergency department of the institute. Patients with PVT with
ACCEPTED MANUSCRIPT
4
valve implanted for diseases other than RHD or patients with recurrent PVT were excluded. The diagnosis of PVT was suspected in patients presenting with new onset symptoms
T
attributable to valve thrombosis such as dyspnea, angina and congestive heart failure of less
RI P
than two weeks duration. A confirmatory diagnosis required the documentation of new onset of hypomobile or immobile valve leaflets on cine-fluoroscopy with or without evidence of
SC
increased transvalvular gradients on Doppler echocardiography. As per institutional protocol, all patients with mechanical heart valves undergo periodic cine-fluoroscopic examinations
MA NU
during their routine follow-up visits. Previous records were checked to confirm the new onset of prosthetic valve dysfunction. Patients with PVT who were less than 12 years of age, patients with a history of fibrinolysis with SK and those with contraindications to fibrinolytic
ED
therapy (previous intracranial hemorrhage, major bleeding within past 2 weeks, ischemic stroke within last 3 months or presence of a left atrial thrombus on transthoracic
PT
echocardiography) were excluded. All patients gave written informed consent and the study protocol was approved by the Institutional Ethics Committee.
CE
Demographic and clinical parameters obtained at baseline included age, sex, NYHA class,
AC
presence of atrial fibrillation (AF) and left ventricular ejection fraction (LVEF). Compliance to oral anticoagulants was assessed by self-reporting by the patients. Their INR values at presentation were recorded. Fibrinolytic Therapy: All
patients
underwent
fibrinolysis
with
SK with
serial
echocardiographic and cine-fluoroscopic monitoring. The following protocol was used for fibrinolysis with SK- 250,000 units intravenous bolus over 30 min followed by a continuous infusion of 100,000 units/hour. Cine-fluoroscopy and Doppler echocardiography were performed 8-12 hourly or after any significant change in clinical status. Criteria for stopping SK infusion were either successful fibrinolysis or any complication mandating its
ACCEPTED MANUSCRIPT
5
discontinuation. It was continued for up to a maximum of 96 hours in patients with partial or no response, at the discretion of the treating physician.
T
Serologic testing: Blood (3 ml) was collected in a plain vial for anti-SK antibody titers prior
RI P
to initiation of SK therapy, and was stored at -20°C for analysis. An indirect ELISA assay was designed for the detection of anti-SK IgG antibodies. Briefly, the microtitre plate was
SC
coated with 100µL of SK at a concentration of 500 IU/mL prepared in phosphate buffered
MA NU
saline (PBS). After an overnight incubation at 4°C and washing 3 times with wash buffer and incubated with 200µL of BSA (0.1% bovine serum albumin with PBS) for 1 hour at room temperature to block remaining binding sites. 100µL of serum samples were added in dilution of 1/800. Also serial dilutions (1:100 to 1:204800) of specific anti-SK antibody (Thermo
ED
Scientific, USA) were included in the plate. The plate was incubated for an hour at room temperature and then washed three times with the wash buffer. 100µL of HRP (horse radish
PT
peroxidase) – conjugated rabbit anti-human IgG (Jackson Immuno Research, PA, USA)
CE
diluted at 1:20000 was added to the wells. After incubation for 30 min at room temperature and washing three times with the wash buffer, 100µL of chromogen substrate solution
AC
containing TMB (Tetramethylbenzidine) reagent was added and the plate was incubated for 10 minutes at room temperature in the dark. The reaction was stopped with 100µL of 1M H2SO4 and the results were analyzed with an ELISA microplate reader at 450nm to get the optical density (OD) values of every individual serum. The titration curve of the specific antiSK antibody was used as an intra-laboratory standard. The OD value of a specified dilution of this serum 1/204800 corresponded to blank OD and therefore 1/102400 was assigned 1 arbitrary unit (Au) and subsequent dilutions as 2 Au, 3 Au and so on. The level of anti-SK antibody in all the sera was read at 1/800 dilution, and quantified using these arbitrary units.
ACCEPTED MANUSCRIPT
6
Study outcomes: Outcomes of fibrinolytic therapy were based on clinical findings, echocardiographic and cine-fluoroscopic parameters, as described previously.[1] These were
T
defined as:
RI P
Complete Success: Clinical and hemodynamic improvement with normal leaflet motion on cine-fluoroscopy and normalization of valve gradients on Doppler echocardiography (mitral
SC
valve: - mean diastolic gradient
