Prevalence and overlap of potential embolic sources in embolic stroke of undetermined source: a retrospective cohort

January 2017 and December 2020. Patients who met the embolic strokes of undetermined source diagnostic criteria according to the criteria of the Cryptogenic Stroke “embolic strokes of undetermined source” International Working Group were selected. The presence of each potential embolic source was assessed, and patients were categorised according to the identified potential embolic sources. The main outcome was ischemic stroke recurrence, and it was collected prospectively during follow-up after the index stroke. Results . Among 330 patients admitted between 2017 and 2020, 66 (20.6%) were classified as embolic strokes of undetermined source (68.2% were men, mean age 57 ±11 years). The three most prevalent potential embolic sources were atrial cardiopathy (N = 47/66; 71.2%), arterial atherosclerosis (N = 46/66; 69.7%) and left ventricular disease (N = 26/66; 39.4%). Most patients (N = 56/66; 84.8%) had ≥ 2 potential embolic sources. After 6-month of follow up, ischemic stroke recurrence occurred in 18 (27.3%) patients. In survival analysis, the type and the number of potential embolic sources were not statistically associated with stroke recurrence. Conclusion . Most patients with embolic strokes of undetermined source had multiple potential embolic sources, which overlap considerably. The type and number of potential sources were not associated with stroke recurrence. This finding may explain the negative results of large trials of secondary prevention in the Embolic strokes of undetermined source population.


Introduction
About 10-30% of ischemic strokes have no identifiable cause despite a thorough diagnostic evaluation (cryptogenic stroke) [1,2].In 2014, researchers proposed the concept of "embolic stroke of undetermined source" (ESUS) [2], which is defined as a non-lacunar brain infarct without a significant (≥50%) stenosis of extracranial or intracranial arteries, major cardio-embolic sources or any other specific cause of stroke such as arteritis, dissection, and vasospasm [3].As its diagnosis is based on an exclusive process, ESUS represents an etiologically heterogeneous group and may be caused by various potential sources of thromboembolism, which may respond better to anticoagulation or antiplatelet therapy [4,5].Despite the increased number of published studies focusing on the aetiologies of the ESUS, little is known about the prevalence and overlap of potential embolic sources (PES) in north African patients with ESUS [6].In this study, we, therefore, aimed to assess the prevalence and degree of overlap of different PES, and to evaluate the stroke recurrence rate by identifying PES among Tunisian patients with ESUS.

Methods
We assessed all consecutive ischemic stroke patients admitted to our department of neurology in Fattouma Bourguiba hospital (Monastir, Tunisia), between January 2017 and December 2020.

Abbreviations
ARCADIA: atrial cardiopathy and antithrombotic drugs in prevention after cryptogenic stroke CI: confidence interval COMPASS: cardiovascular outcomes for people using anticoagulation strategies CT scan: computed tomography scan ECG: electrocardiogram EF: ejection fraction ESUS: embolic stroke of undetermined source HR: hazard ratios LV: left ventricle MRI: magnetic resonance imaging mRS: modified Rankin scale NAVIGATE ESUS: rivaroxaban versus aspirin in secondary prevention of stroke and prevention of systemic embolism in patients with recent embolic stroke of undetermined source NIHSS: National Institute of Health Stroke score NT Pro-BNP: N-terminal pro-B-type natriuretic peptide PES: potential embolic source PFO: patent foramen ovale RE-SPECT ESUS: dabigatran etexilate for secondary stroke prevention in patients with ESUS SD: standard deviation TEE: trans-oesophageal echography TOAST: trial of ORG 10172 in acute stroke treatment TTE: transthoracic echography Prévalence et chevauchement des sources emboliques potentielles dans l'AVC embolique de source indéterminée : une cohorte rétrospective Introduction.Les AVC emboliques d'origine indéterminée peuvent être causés par diverses sources emboliques potentielles, qui peuvent être mieux gérées par un traitement anticoagulant ou antiplaquettaire.L'identification de ces sources peut avoir des implications diagnostiques et thérapeutiques.Nos objectifs étaient d'évaluer la prévalence et le chevauchement des différentes sources emboliques potentielles identifiées dans une population de patients présentant des AVC emboliques de source indéterminée, et d'évaluer le taux de récidive des AVC en fonction du type et du nombre de sources emboliques potentielles.

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The scientific use of the data collected was approved by the local Ethics Committee.Data were prospectively obtained from digital hospital records including demographics, medical history and associated vascular risk factors, prior medications, time of stroke onset and hospital admission, duration of hospitalisation, stroke severity assessed by the National Institute of Health Stroke scale score (NIHSS), results of routine laboratory and imaging investigations, and initial and discharge treatments.Stroke was defined according to the World Health Organisation criteria as an episode of acute neurological dysfunction based on a new infarct visualised in brain imaging or symptoms persisting over 24 hours [7].For ischemic stroke diagnosis, work up included cerebral CT and/or MRI if available.In search of potential cardio-embolic aetiologies, in addition to the electrocardiogram, we performed a first 24-hour rhythmic holter, in the acute phase of the ischemic stroke, then we performed a second holter, during follow-up, to identify heart rhythm disorders such as atrial fibrillation, atrial flutter, atrial hyperexcitability.We also performed a transthoracic echocardiography supplemented by a trans-oesophageal echocardiography in search of major or minor cardio-embolic causes (valvular heart disease, atrial cardiopathy, etc.).The assay of the NT-pro-BNP was carried out.In search of stenotic or non-stenotic (<50%) carotid, vertebrobasilar and aortic arch atheromatous plaques, all patients initially had an ultrasound of the supra-aortic trunks, supplemented by angio-CT scan and/or angio-MRI if available.The aetiologies of ischemic strokes were classified using the modified trial of ORG 10172 in acute stroke treatment (TOAST) and ESUS criteria [3,8].

Definition of ESUS and PES
ESUS was defined according to the criteria proposed by the Cryptogenic Stroke/ESUS International Working Group [3] as a visualised non-lacunar brain infarct with an embolic infarct pattern on a brain computed tomography or MRI, in the absence of: (1) extracranial or intracranial ipsilateral atherosclerosis causing ≥50% luminal stenosis in arteries supplying the area of ischemia; (2) major-risk cardio-embolic source, and (3) any other specific cause of stroke (e.g.arteritis, dissection, migraine/vasospasm, drug misuse).Major risk sources of cardiac embolism included permanent or paroxysmal AF, sustained atrial flutter, left ventricular ejection fraction <30%, intracardiac thrombus, prosthetic cardiac valve, mitral stenosis, atrial myxoma or other cardiac tumours.Patients known to have AF, mechanical valves, and intracardiac thrombus were excluded from the ESUS group.For patients included in the ESUS group, we defined the potential embolic sources (PES) as previously reported in the literature (Table 1): atrial cardiopathy, atrial fibrillation diagnosed during follow-up, left ventricular (LV) disease, cardiac valvular disease, patent foramen ovale (PFO), arterial atherosclerotic disease and cancer [4,5].We did not include contralateral carotid atherosclerosis in these PES.For each patient, the presence/absence of each PES was determined, and patients were categorised in ≥1 groups according to the identified PES.The prevalence and degree of overlap of PES were determined.
Table 1.Definition of PES: definition is based on a secondary analysis of NAVIGATE-ESUS [9] and on the ARCADIA [10] trial inclusion criteria.

Statistical analysis
Continuous data were expressed as means ± standard deviation, median and interquartile range (IQR), and nominal variables as counts and percentages.
The association between time to stroke recurrence and PES, was assessed with a Cox proportional-hazards model.Adjustments were realized on demographics (age and sex), medical history (hypertension, dyslipidaemia, diabetes mellitus, smoking status, and coronary artery disease) and NIHSS score at admission.For patients lost during follow-up, survival data were censored at the last time known to be alive.For patients who experienced >1 recurrence during the follow-up period, the time of the first event was used in the analysis.Associations are presented as hazard ratios (HRs) with their corresponding 95% CIs, and the level of significance was set at 5% (p < 0.05).Statistical analyses were performed by a certified statistician with the Statistical Package for Social Science (SPSS Inc., version 25.0.0 for Windows, Armonk, NY).

Results
Between January 2017 and December 2020, 330 patients with acute first-ever ischemic stroke were included in our study.Nine patients were excluded from this analysis because of missing data (cardiac explorations (n = 5) and supra-aortic vascular imaging (n = 4).Of the remaining 321 patients, 66 (20.6%) were classified as ESUS (Figure 1).

Baseline characteristics, prevalence, and overlap of PES in ESUS patients
The major baseline characteristics of patients with ESUS according to PES are summarised in  4).The prevalence of stroke recurrence was high for patients with cancer (N = 1/5; 20%), and AF (N = 2/16; 16.7%).Whereas the lowest prevalence was reported in patients with heart valvular disease (N = 1/12; 4.5%) and PFO (N = 1/16; 6.2%).In the survival analysis, the different types of PES and the absence of PES were not significantly associated with recurrence of stroke (Table 5).The rates of ischemic stroke recurrence were similar between patients with ≤1, 2 and ≥3 PES (Table 6).In the survival analysis, the number of PES was not significantly associated with recurrence of stroke (Table 6).

Discussion
This is the first description of a Tunisian ESUS cohort using the criteria proposed by the Cryptogenic Stroke/ESUS International Working Group in 2014.In our study, we found that approximately 20% of all included ischemic strokes (n = 330) were classified as ESUS type and they were more prevalent among young adults.Our data are consistent with a systematic review of published studies and trials evaluating ESUS patients, where frequency of ESUS ranged from 7% to 42% of all ischemic strokes with an average of 17% and the mean age ranged from 53 to 69 years [9,11,12].Some discrepancies between studies may result from differences in methods and frequency in providing diagnostic workup in various countries, as well as the heterogeneity of the characteristics of the patients included in these studies [5,13].therapeutic trials: The NAVIGATE ESUS and the RE-SPECT ESUS trials [9,21,22] showed that anticoagulation is not superior to aspirin to prevent stroke recurrence in patients with ESUS, showing that the lumping therapeutic approach of oral anticoagulation for the unselected population with ESUS was not the optimal strategy and indirectly validating the ESUS concept as an etiologically heterogeneous entity [5,12].
The heterogeneity of embolic sources and their remarkable degree of overlap, as reported in the present study, could explain these negative results.Over 40% of patients with ESUS have multiple PES, some of which may be associated with low-blood flow which predisposes to formation of red thrombi that may respond better to anticoagulation, whereas other embolic sources may be associated with atherosclerotic plaque ulceration which triggers the formation of white thrombi that may have responded better to antiplatelet therapy [20].
In this context, treating patients with ESUS with anticoagulants rather than antiplatelets may cause simply exchanging the type of thrombus, with the overall burden of thrombi remaining unchanged and hence, no change in the rate of stroke recurrence.
If this hypothesis is correct, it would be rational to expect that a combination of anticoagulant and antiplatelet in patients with ESUS would be associated with a substantial reduction of stroke recurrences in patients with ESUS.These thoughts are supported by the results of the COMPASS (cardiovascular outcomes for people using anticoagulation strategies) trial which showed that a combination of low-dose rivaroxaban and aspirin was associated with a large reduction of stroke risk compared with aspirin as monotherapy [23,24].Furthermore, the strategy of combining anticoagulant and antiplatelet therapy may not apply to patients with atrial cardiopathy.Meanwhile, the ARCADIA (atrial cardiopathy and antithrombotic drugs in prevention after cryptogenic stroke) trial [10], which is currently investigating whether oral anticoagulation with apixaban is a better strategy compared with aspirin in patients with atrial cardiopathy, reports positive results.We found that the risk of stroke recurrence was similar across different PES, which suggest that the thromboembolic risk across these PES is similar.This is in line with the concept of ESUS, which considers all these PES as minor-risk embolic sources [5,9].versus aspirin in secondary prevention of stroke and prevention of systemic embolism in patients with recent embolic stroke of undetermined source) [9,21] and the RE-SPECT ESUS (dabigatran etexilate for secondary stroke prevention in patients with ESUS) trials [22] as well as several observational studies [4,11], reported that two-thirds of all patients with ESUS had at least 2 potential embolic sources, and the most common ones were non-stenotic large artery disease, left ventricular disease, and atrial cardiopathy.Moreover, one-third of the patients had at least 3 such sources (each was present in nearly half of the study population) [5,9].It was also reported that most patients with ESUS have multiple PES, which could explain the neutral results of * Atrial fibrillation not initially present and identified during follow-up is considered the cause of ESUS.
We also found that the risk of stroke recurrence in patients with multiple PES was not further increased.Similar studies with longer follow-up and adequately powered cohort studies are needed to show whether these risk differences would be larger in the long-term.This is the first description of a Tunisian ESUS population providing detailed data on potential etiologic causes of ESUS and the degree of PES overlap.Another strength of this study is that the definition of ESUS was based on the criteria proposed by the Cryptogenic Stroke/ESUS International Working Group.This may allow future studies to compare other ESUS populations to the current one using standardised criteria.
Another strength of the present study is that the estimated degree of PES overlap might be the actual overlap because of the extensive panel of diagnostic work-up investigations that had been performed for most patients, regardless of their ages.Moreover, we used several measurements to define each PES, especially the atrial cardiopathy [9,10].In this context, our estimates of the prevalence of each PES and of prevalence of multiple PES are likely reasonable.Nevertheless, our study has certain limitations.It is a single-center character rather than a population-based setting, which may have introduced selection bias.In addition, a type II error due to moderate sample size and limited number of events cannot be excluded.Additionally, it is a retrospective analysis of prospectively collected data, which may have introduced collection and registration bias.
In the present study, we conclude that most ESUS patients had multiple PES with a major degree of overlap.Rather, prospective data with advanced diagnostic methods are warranted to identify the true distribution of the underlying stroke aetiology in patients with ESUS.Currently, ESUS may be considered as a developing diagnostic concept that triggers increased diagnostic studies presumably leads to increased detection rates of the underlying stroke cause and subsequently reduces the overall rate of undefined or cryptogenic strokes.

cardioembolic sources (1) Arterial atherosclerosis disease, any of:
Patients with ESUS underwent a follow-up of at least 6 months after index stroke.Outcome of interest was ischemic stroke recurrence during follow-up.The assessment of stroke recurrence was performed by onsite patient visits or by contact with the patient and/or his primary physician.
-Mitral valve abnormalities: moderate-severe mitral annular calcification, valve prolapsus -Aortic valve abnormalities: moderate-severe aortic stenosis, valve thickening, bileaflet valve -Patent foramen ovale detected on TTE or TEE with a high risk of embolism (large shunt, atrial septal aneurysm) Non-* Atrial fibrillation not initially present and identified during follow-up is considered the cause of ESUS.** Patients with left ventricular ejection fraction <30% were excluded.PTFV1= P-wave terminal force in lead V1; NT pro-BNP = N-terminal pro-B-type natriuretic peptide; TTE = trans-thoracic echocardiography; TEE = trans-esophageal echocardiography; CT= computed tomography; mR= Magnetic resonance; Cancer: active or in remission 2.2.Assessment of outcome

Table 2 .
Repartition of each PES in the ESUS population.
* Atrial fibrillation not initially present and identified during follow-up is considered the cause of ESUS.** Patients with left ventricular ejection fraction <30% were excluded.N = number of patients; Med = median; NIHSS = National Institute of Health Stroke Scale; mRs= modified Rankin scale; PES= potential embolic source; SD= standard deviation * Atrial fibrillation not initially present and identified during follow-up is considered the cause of ESUS.** Patients with left ventricular ejection fraction <30% were excluded.N = number of patients; SD= standard deviation

Table 3 .
Baseline characteristics and outcomes of patients per PES.

Table 4 .
Index stroke characteristic and stroke recurrence during follow-up per PES.

Table 5 .
Comparison of stroke recurrence according to type of PES.Hazard ratios (HR) and 95% confidence intervals (CI) computed using a multivariable Cox proportional hazards model.Models were adjusted for age, sex, hypertension, dyslipidaemia, diabetes mellitus, smoking status, coronary artery disease and NIHSS score at admission.

Table 6 .
Comparison of stroke recurrence according to number of PES.Hazard ratios (HR) and 95% confidence intervals (CI) computed using a multivariable Cox proportional hazards model.Models were adjusted for age, sex, hypertension, dyslipidaemia, diabetes mellitus, smoking status, coronary artery disease and NIHSS score at admission.