Chinese Guidelines for the Secondary Prevention of Ischemic Stroke and Transient Ischemic Attack 2010

Abstract

Cerebrovascular disease has become the leading cause of mortality and morbidity in China, in both urban and suburb population, with 1,500,000–2,000,000 new strokes each year. After correction for age, the prevalence of stroke is 116–219/100,000/year, and mortality is 58–142/100,000/year. The prevalence of ischemic stroke has increased as the population has aged, and the lifestyle has changed. There are over 7,000,000 stroke survivors living in China, and 70% of them have suffered ischemic stroke. Many of the survivors of ischemic stroke have multiple stroke risk factors, and are at increased risk of recurrent stroke. Many countries have stroke treatment recommendations that are based on data collected in the last decade from random controlled trials (RCTs) on stroke secondary prevention. Although the recommendations provide valuable information, there are considerable differences between Chinese and Western population in regard to genetic background of the population, health conditions, medicine tradition, cultural background, laws/regulations, and welfare systems. It is therefore necessary and urgent to establish stroke treatment guidelines that are relevant to the Chinese population. A panel of experts convened in July 2008 to develop guidelines for ischemic stroke secondary prevention that were specific for the Chinese population. It consisted of experts from neurology, cardiology, endocrinology, intensive care, respiratory, intervention, and epidemiology. The panel conducted a comprehensive review and synthesis of relevant domestic and international literature published before October 2008. When there was sufficient and reliable evidence, the evidence was used; alternatively, current best evidence and experts' opinions were applied. After wide discussion, multiple editions, and additions of the current evidence, these guidelines have been established. The crucial steps of secondary stroke prevention are to diagnose the causes and recognize the risk factors of stroke beginning with the acute phase of stroke. The risks of recurrent stroke are different, because the causes and the pathophysiological mechanisms of ischemic stroke/transient ischemic attack (TIA), the locations of vascular diseases, risk factors, and patients' compliance are so variable. It is therefore important to stratify patients into high or low-risk groups based on the causes, and the numbers and severity of risk factors by using tools such as Essen stroke risk scale or the ABCD2 score. Only after stratifying correctly, is it possible to deliver individualized effective treatment and suitable prevention. These guidelines are aimed to provide evidence-based recommendations on the secondary preventions of ischemic stroke/TIA to physicians in neurology, medicine, and other relevant departments in China. Unless specifically mentioned, the recommendations apply to both ischemic stroke and TIA. Please refer to the reference for methods used to classify the classes of evidence and the levels of certainty of the treatment effect [1]. Risk factors of stroke can be classified as modifiable and nonmodifiable ones. The goal is to control modifiable risk factors. Please refer to information on primary prevention regarding modifying lifestyle (such as smoking, alcohol excess, obesity, and limited physical activity) to reduce the risk of stroke. This guideline is to focus on the medical management of stroke secondary prevention. Hypertension is a major risk factor for stroke/TIA. The prevalence of hypertension has been rising with the rapid socioeconomic growth and lifestyle changes in China. Studies in China suggest a logistic relationship between elevated blood pressure (BP) and the risk of stroke [2]: for every 10 mmHg elevation of systolic BP from baseline, the relative risk of stroke increases 49%; for every 5 mmHg elevation of diastolic BP, the relative risk of stroke increases 46%. The impact strength of hypertension on the risk of stroke in Chinese and Japanese populations is 1.5 times that of western population. A Chinese survey in 2002 showed the diagnose rate of hypertension was 30.2%; the treatment rate was 24.7%; and control rate was only 6.1%. During the same period, the rates of diagnosis, treatment, and control of hypertension in the United States were 70%, 59%, and 34%, respectively. Lowering BP may benefit patients with stroke/TIA on secondary prevention. A systemic review of seven published RCT showed that lowering BP was associated with significant reductions of recurrent stroke, and total vascular events, without significant reductions in fatal stroke and vascular mortality [3]. However, the Prevention Regimen for Effectively Avoiding Second Stroke (ProFESS) trial found no difference in recurrent stroke in patients treated with telmisartan versus placebo [4]. Which antihypertensive medicines are more effective in the prevention of recurrent stroke? The Morbidity and Mortality After Stroke—Eprosartan versus Nitrendipine for Secondary Prevention (MOSES) trial suggested that an angiotensin II type I receptor antagonist (Eprosartan) could significantly lower the rate of recurrent cerebrovascular events when compared with a calcium antagonist (Nitrendipine) [5]. However, a review study on BP control in stroke prevention, which analyzed three head-to-head comparison trials consisted of total of 21,094 patients, suggested that calcium antagonist might be superior to angiotensin receptor blocker, diuretics, and β-blockers [6]. BP reduction is recommended for the prevention of recurrent stroke and other vascular events in patients with ischemic stroke/TIA. (Class I, Level of Evidence A). With consideration of age, baseline BP, routine medication, and tolerance, the target BP should be ≤140/90 mmHg, ideally 130/80 mmHg. (Class II, Level of Evidence B). The effects of antihypertensive treatment on the prevention of recurrent stroke/TIA were primarily achieved by lowering BP (Class I, Level of Evidence A). Either a monotherapy or a combination of medicines can be used (Class II, Level of Evidence B). The choice should be individualized. Blood glucose control is known to have protective effect on microvascular and macrovascular diseases secondary to Type II diabetes. Poor glucose control is associated with recurrence of stroke. Data on diabetic control in stroke prevention are largely based on primarily prevention. The Action Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation (ADVANCE) trial showed intensive control of glucose levels reduced glycated hemoglobin to 6.5%, resulting in a significant reduction in vascular events [7]. For patients with diabetes, intensive lowering BP may significantly improve the combine vascular outcome, including stroke. Epidemiological data have suggested when BP is further reduced to 120/80 mmHg there is a continuous reduction in cardiovascular events [8]. The Collaborative Atorvastatin Diabetes Study (CARDS) showed that statins are effective in reducing the risk of first incidence stroke in diabetic patients [9]. In addition, the Heart Protection Study (HPS) which included 5963 adults with diabetes, suggested add-on statins could reduce the rate of first major vascular events [10]. The goal of diabetic glycemic control is glycated hemoglobin of 6.5% or less. However hypoglycemia may be harmful, resulting in increased morbidity. (Class I, Level of Evidence A). For patients with diabetes and hypertension, BP should be strictly controlled below 130/80 mmHg. Specific antihypertensive agents, such as angiotensin converting enzyme inhibitors and angiotensin receptor blockers are beneficial in the reduction of vascular events (Class I, Level of Evidence A). In combination with tight glycemic and BP control, statins may decrease the risk of stroke (Class I, Level of Evidence A). Evidence showed a modest association between elevated total cholesterol levels and an increased risk of ischemic stroke. Lifestyle modifying and statins are the primary lipid-lowering methods. A meta-analysis study which included statins, clofibrate, cholestyramine resin, gemfibrozil, niacin, and diet, showed that all lipid-lowering therapies significantly reduced the incidence of stroke [11]. However, the best efficacy was with statins, especially in secondary prevention, due to their higher potency. The Stroke Prevention by Aggressive Reduction in Cholesterol Levels (SPARCL) trial found that statin treatment may significantly reduce the recurrent rate of stroke/TIA [12]. Although the incidence of hemorrhagic stroke was higher in the statin group, there was no difference in outcome. A meta-analysis involving 121,000 patients demonstrated that prolonged statin treatment provided significant primary protection in patients at high risk of vascular diseases, without significantly elevated risk of hemorrhagic stroke [13]. For elderly patients with normal liver and renal function, the doses of lipid-lowering agents do not need to be adjusted. However, the treatment should be individualized, with lower doses initially and close monitoring [14]. Patients with ischemic stroke/TIA and elevated cholesterol should be managed with a combination of treatments including lifestyle and dietary modifications, and medications. Statins are recommended lipid-lowering agents. It is reasonable to target the low-density lipoprotein Cholesterol (LDL-C) ≤ 2.59 mmol/L or a 30–40% reduction in LDL-C (Class I, Level of Evidence A). For patients with stroke/TIA and multiple risk factors (such as coronary artery disease, diabetes, current smoking, metabolic syndrome, cerebrovascular atherosclerosis without evidence of unstable plaque, arterial thrombosis or peripheral arterial disease), it is reasonable to target a LDL-C level of <2.07 mmol/L or a reduction of >40% in LDL-C (Class I, Level of Evidence A). Patients with ischemic stroke/TIA and evidence of intra- and extracranial large artery atherosclerosis and unstable plaques or arterial thrombosis, are recommended early intensive statin therapy to target a LDL-C level of <2.07 mmol/L or a reduction of >40% in LDL-C (Class III, Level of Evidence C). In general, long-term use of statins is safe. Before and during statin therapy, monitoring symptoms such as muscle pain, and liver enzymes (e.g., Alanine transaminase and aspartate transaminase) and muscle enzyme (creatine kinase) is required. If those markers are persistently elevated without other identifiable causes, statins should be reduced or discontinued (Discontinuation of statins was recommended when transaminases are more than three times the normal upper limits, or creatine kinease is more than five times the normal upper limit.) (Class I, Level of Evidence A). For elderly patients with multiple organ insufficiency, or taking multiple medications, drug–drug interactions and potential side effects should be closely monitored (Class III, Level of Evidence C). If patients have a history or at high risk of intracranial hemorrhage, caution should be exercised with statin therapy (Class II, Level of Evidence B). The North American Symptomatic Carotid Endarterectomy Trial (NASCET) and the European Carotid Surgery Trial (ECST) showed carotid endarterectomy (CEA) reduced the risk of ipsilateral stroke in patients with symptomatic high-grade stenosis (70–99%) of the internal carotid artery [15, 16]. CEA is also beneficial to patients with symptomatic moderate (50–69%) ipsilateral carotid stenosis [17]. However, for patients with mild to moderate carotid stenosis (<50%), the possible benefits of CEA were outweighed by the risks of surgery. Early CEA may be associated with increased benefits, ideally within 2 weeks of their last ischemic event. Elderly patients (>75 years of age) without organ failure or severe cardiac dysfunction can also benefit from CEA. Women with severe symptomatic carotid stenosis (>70%) should consider CEA; however, women with less severe stenosis can be treated medically. It is important to prevent perioperative stroke during the procedure. The American Heart Association (AHA)/American Stroke Association Council on Stroke recommend that CEA can only be performed by a surgeon with a perioperative complication rate (combined morbidity and mortality) <6%[18]. Patients with carotid stenosis should take low-dose aspirin before and after surgery [19]. For patients with recent ischemic stroke/TIA and severe (70–99%) ipsilateral carotid artery stenosis, CEA is recommended (Class I, Level of Evidence A). For patients with recent ischemic stroke/TIA and moderate (50–69%) ipsilateral carotid stenosis, CEA is recommended depending on patient-specific factors, such as age, sex, comorbidities and the severity of stroke (Class I, Level of Evidence A). CEA may benefit the most to men ≥75 years of age with their last hemispheric ischemic events within 2 weeks (Class III, Level of Evidence C). CEA is recommended within 2 weeks of last ischemic event when indicated (Class II, Level of Evidence B). CEA is not recommended for patients with carotid stenosis <50% (Class I, Level of Evidence A). Continuation of antiplatelet therapy is recommended postoperatively (Class I, Level of Evidence A). Many reports suggested that Angioplasty and Stenting (CAS) can improve the outcome in patients with symptomatic carotid stenosis, but there is no evidence that CAS is superior to CEA. There is no clinical trial directly comparing the effectiveness of CAS with the best medical management. The Carotid and Vertebral Artery Transluminal Angioplasty Study (CAVATAS) showed CAS has comparable effectiveness as CEA on stroke secondary prevention in patients with symptomatic carotid stenosis, with less cranial neuropathy and major neck hematoma, but higher rates of restenosis [20]. A meta-analysis showed that CAS was associated with a greater 30-day risk of stroke/death when compared with CEA [21]. Intracranial angioplasty and/or stenting have been approved as safe procedures with a high degree of technical success when performed by experienced surgeons. However, the high rate of recurrent stenosis is unsolved [22]. For patients with symptomatic severe carotid stenosis (>70%), when CEA is not available, CAS may be considered, (Class IV, Level of Evidence D). For patients with symptomatic severe carotid stenosis (>70%) that are difficult to access surgically, early recurrent stenosis after CEA, and radiation-induced stenosis, CAS may be considered (Class II, Level of Evidence B). CAS may be considered in elderly patients with caution (Class II, Level of Evidence B). For patients with symptomatic intracranial carotid stenosis, endovascular intervention may be effective (Class II, Level of Evidence B). Combination of aspirin and clopidogrel should be given before stenting, and continued for at least 1 month after surgery. Clopidogrel alone is also recommended thereafter for at least 12 months (Class IV, Level of Evidence D). A Cochrane review in 1995 showed that warfarin reduced the odds of recurrent stroke by two-thirds, with no reported intracranial hemorrhage [23]. In 2002, collaborative meta-analysis from Antithrombotic Trialists' Collaboration suggested that antiplatelet agents reduced the risk of nonfatal stroke by about a quarter [24]. In 2005, the Atrial Fibrillation Clopidogrel Trial with Irbesartan for Prevention of Vascular Events (ACTIVE W) demonstrated warfarin is superior to clopidogrel plus aspirin in prevention of vascular events in patients with atrial fibrillation, with comparable risk of major bleeding [25]. Later (2009) ACTIVE A showed that in patients with atrial fibrillation who were unsuitable to warfarin, the addition of clopidogrel to aspirin significantly reduced the risk of major vascular events compared with aspirin alone [26]. However, the risks of major hemorrhage, including intracranial bleeding were increased in the dual antiplatelet group, although not significant in fatal hemorrhage. In 2006, an RCT, comparing warfarin and aspirin in the prevention of embolic stroke in Chinese patients with atrial fibrillation without valvular diseases, showed warfarin had significantly better primary outcomes when compared with aspirin, although not significant in mortality [27]. Warfarin is effective, but it has a narrow treatment range, requiring frequent monitoring international normalized ratio (INR) and adjusting doses. Internationally, when warfarin is used for stroke prevention in patients with atrial fibrillation, the effective and safe target INR range of 2.0–3.0 is recommended. A study on the safety and the effectiveness of warfarin in Chinese patients with atrial fibrillation suggested that a target INR range of 1.5–3.0 [28]. However, this result requires further verification. For patients with ischemic stroke/TIA and atrial fibrillation (including paroxysmal atrial fibrillation), oral anticoagulation with warfarin (target INR range of 2.0–3.0) is recommended to prevent secondary stroke (Class I, Level of Evidence A). For patients unable to take oral anticoagulants, antiplatelet agents were recommended (Class I, Level of Evidence A). In addition, combination of clopidogrel and aspirin may be superior to aspirin alone (Class I, Level of Evidence A). Patients with acute MI and complicated with stroke are at very high risk of recurrent vascular events. The major risk factors for stroke in the setting of acute MI are anterior MI, hypertension, atrial fibrillation, previous stroke, and advanced age. The American College of Cardiology (ACC)/AHA guidelines for the management of patients with ST-elevation myocardial infarction (STEMI) recommended daily long-term aspirin (clopidogrel, if aspirin is contraindicated) as secondary prevention [29]. In clinically indicated patients (e.g., atrial fibrillation and LV thrombus), warfarin is recommended with a target INR range of 2.0–3.0. For patients with LV thrombus following MI and complicated with ischemic stroke/TIA, warfarin is recommended for 3 months to 1 year. Patients with ischemic stroke/TIA in the setting of acute MI, aspirin 75– 325 mg daily is recommended (Class I, Level of Evidence A). Patients with ischemic stroke/TIA in the setting of acute MI complicated with LV mural thrombus, should be treated with warfarin for at least 3 months (up to 1 year) with target INR of 2.0–3.0 (Class II, Level of Evidence B). Rheumatic Mitral Valve disease: Patients with rheumatic mitral valve disease and previous embolic event, have a high risk of recurrent embolism. Long-term anticoagulant therapy may effectively reduce embolic events in those patients. It has been demonstrated by transesophageal echocardiogram that long-term anticoagulant therapy may result in disappearance of left atrial thrombus [30]. ACC/AHA guidelines for the management of patients with valvular heart disease recommended anticoagulant therapy in patients with mitral stenosis and one of the following conditions: atrial fibrillation (paroxysmal, persistent or permanent), a history of embolic event (even in sinus rhythm), or left atrial thrombus [31]. Nonrheumatic Mitral Valve diseases: There are two nonrheumatic mitral valve diseases associated with stroke: mitral valve prolapse and mitral annular calcification. To date there have been no clinical trials addressed the efficacy of therapies for patients with mitral valve suggested antiplatelet therapy for patients with mitral valve prolapse and a thrombus and anticoagulation or antiplatelet for patients with mitral annular and thrombus events. ACC/AHA guidelines for the management of patients with valvular heart disease recommended aspirin mg for patients with mitral valve prolapse and [31]. Warfarin is also recommended for stroke patients with mitral valve and complicated with mitral atrial fibrillation, or left atrial Valve Disease: Although have reported systemic in about of with there have been no clinical trials on patients with stroke/TIA and valve disease Valve and heart There have been no random clinical trials to specifically secondary stroke prevention in patients with cardiac valve or heart The European of Cardiology published guidelines on the anticoagulation management of patients with valvular heart disease which suggested INR should take into patients' risk factors and the of the For patients with ischemic stroke/TIA and rheumatic mitral valve disease, with or without atrial fibrillation, long-term anticoagulation with warfarin with a target INR range of 2.0–3.0 is recommended (Class III, Level of Evidence C). agents should not be to warfarin to bleeding risk (Class III, Level of Evidence C). For patients with ischemic stroke/TIA and rheumatic mitral valve disease, who suffered recurrent ischemic stroke/TIA on addition of antiplatelet to warfarin is reasonable (Class III, Level of Evidence C). For patients with ischemic stroke/TIA and mitral valve antiplatelet therapy may be considered (Class III, Level of Evidence C). For patients with ischemic stroke/TIA and mitral valve insufficiency, atrial fibrillation or left atrial thrombus, long-term warfarin therapy is recommended (Class III, Level of Evidence C). For patients with ischemic stroke/TIA and mitral annular antiplatelet or warfarin may be considered (Class IV, Level of Evidence D). For patients with ischemic stroke/TIA and valve disease, antiplatelet therapy is recommended (Class III, Level of Evidence C). For patients with ischemic stroke/TIA who have heart anticoagulation with warfarin is recommended with a target INR range of (Class II, Level of Evidence B). For patients with ischemic stroke/TIA who have heart or heart valve with anticoagulation with warfarin with a target INR range of 2.0–3.0 is recommended (Class II, Level of Evidence B). For patients with ischemic stroke/TIA and treated with warfarin, who have recurrent ischemic stroke/TIA with a target addition of antiplatelet may be considered (Class III, Level of Evidence C). Patients with have a high risk of mural thrombus For patients with atrial fibrillation or are at high risks of thrombus events such as thrombosis, aspirin mg daily may be used to prevent mural Patients with ischemic stroke/TIA and mural should be treated with long-term oral anticoagulation with warfarin (target INR of For patients with heart the Warfarin and in Heart trial not significant difference between warfarin and aspirin or clopidogrel at primary vascular outcome, including stroke. However, there were more bleeding events in the warfarin For patients with ischemic stroke/TIA and antiplatelet therapy or warfarin 2.0–3.0) may be considered to prevent recurrent ischemic events (Class III, Level of Evidence C). For patients with ischemic stroke/TIA and heart antiplatelet may be considered (Class III, Level of Evidence C). are ischemic events by disease, of or therapy may significantly reduce the risks of vascular nonfatal MI, and nonfatal stroke, in patients with previous stroke/TIA [24]. mg provide prevention for recurrent stroke, of the However, high doses of aspirin have a higher risk of with aspirin, clopidogrel may be more effective in reducing the risk of ischemic stroke, MI, and vascular The Clopidogrel for Risk and and trial suggested that clopidogrel may be more beneficial than aspirin in patients with symptomatic disease however, it may not be so in patients with multiple risk factors. of may reduce cerebrovascular events, but not from vascular events. There is no evidence that is more effective than and The European Stroke Prevention Study showed that combination therapy of aspirin mg a and mg a significantly reduced the risk of stroke or from vascular events when compared with aspirin alone The trial showed no difference in the recurrence rate of stroke between the clopidogrel and the and aspirin combination However, major bleeding events (including intracranial were more in the combination is also a more side effect in the combination group, which compliance in this Clopidogrel and The management of with Clopidogrel in Patients with or Stroke trial showed no significant benefit of clopidogrel mg plus aspirin mg combination therapy when compared with clopidogrel mg alone in reducing major vascular events The risk of major bleeding was significantly increased in the combination therapy However, for patients with acute coronary or coronary artery stenting, clopidogrel and aspirin combination therapy is recommended for to 12 months to reduce the risk of recurrent vascular event. antiplatelet The trial of and aspirin following acute myocardial showed that significantly reduced the incidence of nonfatal cerebrovascular events when compared with aspirin, with significantly lower bleeding A study in Chinese patients with ischemic stroke/TIA, versus for Secondary Stroke Prevention found that might be a more effective and to aspirin in stroke secondary prevention However, these a phase trial to For patients with ischemic stroke/TIA, antiplatelet agents is recommended to reduce the risk of recurrent events (Class I, Level of Evidence mg or Clopidogrel monotherapy are as antiplatelet therapy (Class I, Level of Evidence A). antiplatelet therapy is not recommended for routine secondary stroke prevention (Class I, Level of Evidence A). However, for patients with acute coronary artery diseases or recent coronary artery stenting, clopidogrel and aspirin combination therapy is recommended (Class I, Level of Evidence The Warfarin Stroke Study showed no significant difference in the prevention of recurrent ischemic stroke or between aspirin mg and warfarin The comparison of Warfarin and for Symptomatic Intracranial study showed no significant difference in stroke or vascular in patients with stroke/TIA between aspirin versus warfarin (target INR 2.0–3.0) for patients with ischemic stroke/TIA, anticoagulation therapy not provide better protection when compared with it also increases the risk of bleeding For patients with stroke/TIA, anticoagulation is For patients with stroke/TIA, warfarin may be after ischemic event. However, for patients with large than of artery warfarin should not be a weeks (e.g., It should be individualized. anticoagulation may also benefit patients with artery or carotid For patients with ischemic stroke/TIA, oral anticoagulation is not recommended as a first therapy for the prevention of recurrent stroke/TIA (Class I, Level of Evidence A). For patients with ischemic stroke/TIA, oral anticoagulation may be considered specific conditions, artery carotid or with thrombosis or atrial (Class IV, Level of Evidence D). therapy has been used in patients with arterial the without clinical trials A meta-analysis showed no significant difference between anticoagulation and antiplatelet therapy in risk of or recurrent stroke The risks of recurrent stroke are high in the first months of arterial is anticoagulation therapy for patients without (e.g., large with severe or intracranial of or intracranial may be considered in the acute phase of with at a target range of and to oral warfarin 2.0–3.0) or 3 months after arterial If of the is anticoagulation can be anticoagulation should be continued for 3 months If is months after anticoagulation can be to long-term antiplatelet agents. therapy (e.g., or vascular surgery (e.g., may be considered for patients with stroke/TIA secondary to extracranial who have recurrent ischemic events For patients with ischemic stroke/TIA and arterial without for at range of as an treatment is recommended. can be to warfarin therapy for months is If at treatment should be to long-term antiplatelet treatment (Class III, Level of Evidence C). For patients with ischemic stroke/TIA and arterial and for anticoagulation therapy, antiplatelet therapy for months is recommended. If at long-term antiplatelet treatment is (Class III, Level of Evidence C). For patients with stroke/TIA and arterial who medical therapy, endovascular therapy or vascular surgery may be (Class III, Level of Evidence C). Although is not an risk factor of ischemic stroke/TIA, significantly associated with stroke. For patients with ischemic stroke and for thrombus is required. If thrombus, or atrial are anticoagulation therapy or is recommended. antiplatelet is For patients with or atrial therapy is not recommended. of is a safe and effective It can reduce the rate of recurrent events However, there are data to a regarding to prevent secondary stroke. For patients years of age) with stroke/TIA, for is recommended (Class III, Level of Evidence C). For patients with stroke/TIA and a antiplatelet therapy is recommended. For those patients complicated with thrombosis or atrial warfarin is recommended (Class III, Level of Evidence C). For patients with stroke/TIA and who have recurrent ischemic events medical therapy, may be considered (Class III, Level of Evidence C). suggested is associated with increased risk of stroke. of or can to The Heart Prevention Evaluation study and the Trial showed that treatment not lower the recurrent risk of vascular events, on the there was a of primary outcome, especially nonfatal myocardial The for Stroke Prevention study showed that therapy reduced levels but not reduce vascular events For patients with ischemic stroke/TIA and level daily and may reduce the levels of (Class II, Level of Evidence B). The have no of