Diagnostic Utility of Combined 2D Ultrasonography and Contrast-Enhanced Ultrasonography in Evaluation of Carotid Plaque Vulnerability for Predicting Recurrent Ischemic Strokes


 Objective: To evaluate the sensitivity and specificity of combined 2D ultrasonography (USG) and contrast-enhanced ultrasonography (CEUS) in analyzing the carotid plaque vulnerability for predicting the recurrent ischemic strokes (IS). Methods: One hundred and fifteen patients with first IS were studied by 2D USG and CEUS. The carotid plaques were then classified on the basis of echogenicity (2D USG) and neovascularization (CEUS). The presence or absence of recurrent IS was considered as the dependent variable. Age, gender, body mass index (BMI), hypertension, hyperglycemia, hyperlipidemia, history of smoking and drinking, type of plaque echogenicity, and grade of plaque neovascularization were considered as independent variables. The risk factors of recurrent IS were analyzed by both univariate and multivariate logistic regression analysis. Finally, the sensitivity and specificity of combined 2D USG and CEUS in the diagnosis of recurrent IS was evaluated by receiver operating characteristic curve. Results: Univariate logistic regression analysis revealed that hypertension, echogenicity type, and grade of plaque neovascularization were predictors of recurrent IS. Further, multivariate logistic regression analysis revealed that the echogenicity type (OR=0.282, P=0.012) and grade of plaque neovascularization (OR=7.408, P<0.0001) were independent risk factors for recurrent IS. The sensitivity, specificity, and area under the curve of combined method were 0.865, 0.769, and 0.817, respectively (95%CI: 0.733-0.902, P<0.0001), which were higher than both 2D USG and CEUS.Conclusions: The echogenicity type and grade of plaque neovascularization are independent risk factors for recurrent IS. The combination of two methods has high sensitivity and specificity in predicting the recurrent IS.


| Introduction
Amongst strokes, ischemic type is most frequently observed and accounts for 70-80% of the total cases [1]. Ischemic stroke (IS) is characterized by high rates of incidence, disability, and recurrence. Previous studies evaluating cerebral infarcts have demonstrated a one-year recurrence rate of 32% [2]. Thus, the prognosis of patients with IS can be signi cantly improved by effective prevention of recurrent cerebral infarcts.
Carotid atherosclerotic plaque has been identi ed as an independent risk factor for IS [3].
Histopathological studies have demonstrated that plaque neovascularization is a marker of plaque vulnerability [4]. Moreover, several researchers have reported that plaque neovascularization is linked to high risk characteristics of plaque [5,6]. Therefore, early identi cation of plaque neovascularization and evaluation of its vulnerability are of great value in clinical prevention of recurrent strokes.
Conventional ultrasonography (CUS) is the most commonly employed imaging method for examination of carotid plaques and can evaluate plaque stability on the basis of echogenicity, shape, size, and integrity of brous cap. However, it cannot detect neovascularization in plaques. Contrast enhanced ultrasonography (CEUS) is a novel method to evaluate the stability of carotid plaques. It not only identi es the plaque neovascularization in real time, but can also quantitatively evaluate its density [7].
In the past, CUS or CEUS was used to evaluate the vulnerability of plaque. However, their combined ability to improve the predictive value of recurrent cerebral infarcts has not been clari ed. Thus, the aim of the present study was to assess the combined sensitivity and speci city of two dimensional (2D) ultrasonography (USG) and CEUS for evaluation of carotid plaques in predicting the recurrence of IS.

| Patients
One hundred and fteen cases, including 76 males and 39 females, with acute stroke, hospitalized in the Department of Neurology, Gaozhou People's Hospital between January 2020 and March 2021 were included in the study. The age of the study population ranged from 47 to 91 years, with a mean age of 70.0±10.1 years. In all the patients, computed tomography (CT) or magnetic resonance imaging (MRI) was used to arrive at the diagnosis of atherosclerotic cerebral infarcts. In a calm state, the systolic (SBP) and diastolic (DBP) blood pressure of brachial artery were assessed thrice and the mean values were calculated. In the morning, fasting state venous blood sample (3ml) was collected to evaluate the fasting blood glucose, and lipid pro le.
The cases belonging to an age group of 18 years or more, with an initial stroke involving carotid artery territory con rmed by MRI or CT, an ultrasonography demonstrating plaques in carotid artery with a size of 2 mm or more, and those willing to follow-up for 15 months were included in the study. While, cases allergic to sulfur hexa uoride, and with contraindication to CEUS were excluded [8,9]. Moreover, to exclude the in uence of stenosis, patients with severe stenosis (70% or more, according to NASCET) of carotid artery were not included. The present study focused on the vulnerability of carotid plaques and the recurrence of cerebral infarct was the end point of the study.
The study protocol was reviewed and approved by the Ethics Review Committee, Gaozhou People's Hospital. As per the requirements of the National Legislation and Institutions, the written informed consent from the patients was not required.

Instruments and Methods
2.2.1 Instruments CEUS of the carotid plaque was performed with Esaote MyLab Class C scanner (MyLabClassC Advanced, Esaote, Genova, Italy), equipped with a 12-18 MHz linear transducer.

Ultrasonographic examination
The common carotid, internal carotid, external carotid, and vertebral arteries were examined by 2D USG. The stenosis of carotid artery, and the number and distribution of carotid plaques were noted. The thickest carotid plaque was identi ed and evaluated. The same plaque was studied by both 2D USG and CEUS. The CEUS examination was performed following a bolus injection of SonoVue (Bracco, Milan, Italy). Dynamic images were continuously collected for further o ine analysis.
CEUS was used to grade the neovascularization of carotid artery plaques and included four grades as follows: Grade 0 (no enhancement of plaques, denoting absence of plaque neovascularization), Grade 1 (Several punctate enhancements within adventitia or tissue surrounding the plaque suggesting limited presence of plaque neovascularization), Grade 2 (Adventitia or shoulder of the plaque was enhanced, suggesting moderate neovascularization, less neovascularization than Grade 3 but more than Grade 1), and Grade 3 (Diffuse enhancement within the plaque suggesting the presence of a pulsatile artery within the plaque) [11].

Statistical analysis
The continuous data with normal distribution was represented in terms of as mean ± standard deviation (SD), while categorical data was represented in terms of frequency (percentage). The continuous and categorical data were compared with Student's t-test and Chi-Square or Fisher's exact test, respectively. Univariate and multivariate logistic regression analysis was used to determine the independent risk factors of recurrent cerebral infarcts. Presence or absence of recurrent cerebral infarct was used as a binary dependent variable. The receiver operating characteristic (ROC) curve was used to evaluate the sensitivity and speci city of independent risk factors in predicting the recurrent cerebral infarcts. The data was analysed with SPSS (IBM, Armonk, NY, USA) version 23.0 for windows. A probability (P) of less than 0.05 was considered as statistically signi cant.

| Results
The present study involved 115 cases with acute IS, of which 37 (32.2%) had recurrent IS during the study period.
Analysis by ROC curve demonstrated that the sensitivity, speci city, and area under the curve (AUC) for predicting the recurrent cerebral infarcts with Type 1 and 2 echogenicity (maximum Youden index: 0.409)

| Discussion
The present study suggest that the type of echogenicity and grade of carotid artery plaque neovascularization were independent risk factors for recurrent IS. The combination of 2D USG and CEUS was found to have a good sensitivity and speci city for predicting the recurrent cerebral infarcts.
At present, there are many known risk factors of recurrent cerebral infarcts. Amongst them, age, gender, hypertension, diabetes, hyperlipidemia, and history of smoking have been con rmed to be closely associated with recurrent cerebral infarcts [12,13]. Previous studies have demonstrated that this association is mainly due to the fact that these risk factors have the ability to cause the progression of atherosclerosis and further lead to recurrent cerebral infarcts [14].
Contrarily, the present study demonstrated no signi cant difference between the recurrent and nonrecurrent groups in terms of mean age, gender, BMI, diabetes, and hyperlipidemia. Thus, it may be suggested that the above risk factors have little value in predicting the recurrent cerebral infarcts.
Moreover, these risk factors may be of value in increasing the chances and triggering the events leading to cerebral infarcts. However, they may not have any role in promoting their recurrence. The only risk factor with signi cant difference between the groups was systolic hypertension. Thus, poorly controlled SBP might have resulted in altered structure of carotid artery wall and induced recurrent IS [13].
In the present study, univariate and multivariate logistic regression analysis demonstrated that grade of carotid plaque echogenicity was an independent predictor of recurrent cerebral infarcts. In ROC curve analysis, the AUC was 0.705 (95%CI: 0.602-0.807; P = 0.000) for predicting recurrent cerebral infarcts with Type 1 and 2 echogenicity. This nding is consistent with the previous study [15].
The echogenicity of carotid plaques on 2D USG is an indirect parameter re ecting their vulnerability. Previous studies have con rmed that the echogenicity of carotid plaque is closely associated with the recurrent cerebral infarcts [16]. The highly vulnerable plaques rich in lipid or those having internal bleeding are mostly hypoechoic, while the less vulnerable plaques rich in brous tissue are more hyperechoic [16]. However, the degree of plaque vulnerability cannot be accurately re ected. Intraplaque neovascularization is closely related to intraplaque hemorrhage, which is the main cause of IS [16][17][18][19].
CEUS can accurately re ect the neovascularization in carotid plaques and is an effective parameter to directly re ect their vulnerability [4,20]. CEUS can not only result in signi cantly improved imaging of blood ow and vascular wall, but also depict microvasculature. Moreover, even a single microbubble can be displayed at the capillary level [21]. For homogeneous brous tissue and mixed plaques, CEUS can also accurately evaluate their vulnerability [22]. The guidelines and recommendations of the European Federation of Societies for Ultrasound in Medicine and Biology (EFSUMB) have clearly proposed that CEUS can evaluate the stability of carotid plaques by detecting neovascularization [7].
In addition to the plaque neovascularization, CEUS can also detect ulceration on the surface of carotid plaques, and accurately display the shape, size, depth, and other characteristics of ulceration [23,24].
In the present study, CEUS had better sensitivity and speci city than 2D USG in predicting recurrent cerebral infarcts. It was found to be an independent risk predictor and closely associated with the recurrent cerebral infarcts. In multivariate logistic regression analysis, the OR of ECUS in predicting recurrent IS was much higher than that of 2D USG (7.408 vs 0.282). This could be due to the exclusion of hyperechoic and calci ed plaques.
However, in some cases, such as those with fresh thrombosis-associated carotid plaque, or vulnerable plaques without neovascularization, the reference value of 2D USG was greater than that of CEUS. The combination and cross reference of the both the methods can greatly improve the accuracy of diagnosis. The present study combined the two techniques to evaluate the carotid plaques, and provided a more reliable basis for judging their stability. The ROC curve analysis of the combined method resulted in an AUC of 0.817 (95%CI: 0.733-0.902; P < 0.0001), which was larger than that of 2D USG (AUC = 0.705; 95%CI: 0.602-0.807; P = 0.000) and CEUS (AUC = 0.737; 95%CI: 0.636-0.837; P < 0.0001).

| Limitations
In the present study, the thickest plaque rather than plaque which caused the recurrence of IS was evaluated. However, previous studies have reported that the stability of the largest plaque is signi cantly related to the occurrence of cerebral infarcts [25,26]. Moreover, the present study was retrospective in nature and whether the conclusions drawn hold the same signi cance in the prospective study needs to be evaluated further.

| Conclusion
The 2D USG-based echogenicity classi cation and CEUS-based grade of carotid plaque neovascularization were found to be the independent risk factors for recurrence of IS. The combination of the two methods had high sensitivity and speci city in predicting the recurrence of IS, which has clinical importance.

Declarations
Availability of data and materials The regarding raw data and material of this manuscript can be available through the corresponding author by drleaf@sina.com if required.