Prediction Model of New Onset Atrial Fibrillation in Patients with Acute Coronary Syndrome

Objective Atrial fibrillation (AF) is one of the most common complications of acute coronary syndrome (ACS) patients. Possible risk factors related to new-onset AF (NOAF) in ACS patients have been reported in some studies, and several prediction models have been established. However, the predictive power of these models was modest and lacked independent validation. The aim of this study is to define risk factors of NOAF in patients with ACS during hospitalization and to develop a prediction model and nomogram for individual risk prediction. Methods Retrospective cohort studies were conducted. A total of 1535 eligible ACS patients from one hospital were recruited for model development. External validation was performed using an external cohort of 1635 ACS patients from another hospital. The prediction model was created using multivariable logistic regression and validated in an external cohort. The discrimination, calibration, and clinical utility of the model were evaluated, and a nomogram was constructed. A subgroup analysis was performed for unstable angina (UA) patients. Results During hospitalization, the incidence of NOAF was 8.21% and 6.12% in the training and validation cohorts, respectively. Age, admission heart rate, left atrial diameter, right atrial diameter, heart failure, brain natriuretic peptide (BNP) level, less statin use, and no percutaneous coronary intervention (PCI) were independent predictors of NOAF. The AUC was 0.891 (95% CI: 0.863–0.920) and 0.839 (95% CI: 0.796–0.883) for the training and validation cohort, respectively, and the model passed the calibration test (P > 0.05). The clinical utility evaluation shows that the model has a clinical net benefit within a certain range of the threshold probability. Conclusion A model with strong predictive power was constructed for predicting the risk of NOAF in patients with ACS during hospitalization. It might help with the identification of ACS patients at risk and early intervention of NOAF during hospitalization.

Early detection and intervention in NOAF among ACS patients during hospitalization are particularly important. As a result, many eforts have been made previously. Possible risk factors related to NOAF in ACS patients have been reported in some studies, and several prediction models have been established to identify high-risk individuals [13,[19][20][21]. However, these models had only modest predictive value with the c-statistics ranging from 0.62 to 0.79, and were not comprehensively evaluated or external validated. Owing to the lack of a specifc and practical predictive method, the development of a predictive model that has satisfactory predictive value based on clinical characteristics at admission becomes desirable. In addition, ACS includes three subtypes, of which UA is the most common one [2]. Te risk factors for NOAF in diferent subtypes of ACS may vary, and the prediction model for NOAF in UA patients is rare.
We therefore conducted this study to (1) defne risk factors for NOAF in patients with ACS during hospitalization, (2) develop a prediction model and nomogram for NOAF risk in ACS patients and comprehensively evaluate and externally validate the model, and (3) perform subgroup analysis to develop a prediction model for NOAF risk in UA patients specifcally.

Study Participants.
Retrospective cohort studies were conducted. We retrospectively enrolled 5403 consecutive ACS patients admitted to the Department of Cardiology, Te First Afliated Hospital of the Army Medical University (Tird Military Medical University) in Chongqing, China, from January 2010 to December 2019 for the training cohort. In addition, we retrospectively enrolled 2316 consecutive ACS patients admitted to the Department of Cardiology, Second Afliated Hospital of the Army Medical University, from January 2017 to December 2019 for external validation cohort. Patients who were at least 18 years old and diagnosed with ACS were eligible for this study. Patients with a history or record of AF at admission, valvular diseases, infections, malignant tumors, systemic infammatory diseases, incomplete records of echocardiography, admission heart rate, circulating brain natriuretic peptide (BNP), and other important data were excluded from the study. ACS was defned according to the guideline [22]. Continuous ECG monitoring was routinely carried out for all ACS patients throughout the period of hospitalization so that asymptomatic AF could also be detected and diagnosed. AF was defned according to the 2016 ESC Guidelines [23]. Finally, 1535 and 1635 ACS patients were eligible for the training and validation cohort, respectively. Te study was approved by the Ethics Committee of the Southwest Hospital of the Army Medical University. Te research was conducted in accordance with the Helsinki declaration guidelines and all procedures listed here were carried out in compliance with the approved guidelines. Because this is a retrospective study and all the parameters assessed were routinely obtained in the hospitals so that no additional investigations or procedures were carried out, informed consent was waived by the Ethics Committee of Southwest Hospital of the Army Medical University.

Data Collection.
We collected information on demographics (age and gender) and comorbidities (hypertension, diabetes, cardiomyopathy, cerebral infarction, chronic obstructive pulmonary disease, and chronic renal insufciency). In addition, data from physical examination (height, weight, heart rate, and blood pressure) and Killip classifcation at admission and the frst biochemical/echocardiography examination (blood routine, liver and kidney function, cardiac troponin, circulating high-sensitivity Creactive protein, circulating BNP, etc.) were collected. Te information about medication and percutaneous coronary intervention (PCI) received during hospitalization was also collected from the electronic medical records. NOAF is defned as the onset of AF during hospitalization, and there is no history or record of AF before.

Statistical Analysis.
Te descriptive statistics are presented as frequency counts and proportions for categorical data, means and standard deviation (SD) for continuous variables that were normally distributed, and medians and interquartile ranges (25th-75th percentile) for continuous variables that were not normally distributed. To test the diferences in means and proportions between two groups, we used a t-test and a chi-square test, respectively. Multiple imputation was used for the missing BMI, neutrophil count, and leukocyte count (which accounted for <10% of observations). Previous studies found that ACS patients with the admission heart rate above 80 bpm are at the highest risk of in-hospital mortality [24], suggesting that an admission heart rate above 80 bpm is of great signifcance for ACS patients. In the training cohort, the median admission heart rate of NOAF patients was over 85 bpm, and the median admission heart rate of patients without NOAF was less than 80 bpm. Terefore, heart rate at admission was transformed into dichotomous variable at 85 bpm. Killip classifcation at admission was transformed into a dichotomous variable as "heart failure" (Killip classifcation was II, III, or IV) and "no heart failure" (Killip classifcation was I). Te level of circulating BNP was log-transformed due to a heavy skew in the distribution.
To investigate the risk factors of NOAF, the signifcance of each variable in the training cohort was assessed by univariate logistic regression analysis. All variables associated with NOAF at a signifcant level were candidates for multivariable analysis. For the selection of prediction model, according to the TRIPOD statement, logistic regression is used for short-term (for example, 30-day mortality) prognostic outcomes, so multivariable logistic regression is used. Backward elimination was adopted for selection of variables entering the fnal multivariable logistic regression model. Te variance infation factor (VIF) was used to identify collinearity among the covariates. Te collinearity was negligible because the VIFs of the variables were less than 5. Te model was validated in the external cohort. Te discrimination of the models was assessed by the ROC curve and area under the curve (AUC). Te calibration curve was drawn to evaluate the model's calibration degree. Te clinical utility of the model was evaluated by decision curve analysis. Te nomogram was depicted based on the prediction model.
A two-sided P value <0.05 was considered to be statistically signifcant. All these statistical analyses were conducted using R 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria).

Results
During the study period, 1535 and 1635 ACS patients were recruited in the training and validation cohorts, respectively. Te incidence of NOAF during hospitalization was 8.21% in the training cohort and 6.12% in the validation cohort. Te UA, STEMI, and NSTEMI patients were 820 (53.42%), 248 (16.16%), and 467 (30.42%) in the training cohort, and 1288 (78.78%), 128 (7.83%), and 219 (13.39%) in the validation cohort. Te median length of hospital stay was 6 days and 7 days in the training and validation cohorts, respectively.
Te clinical and demographic characteristics of the patients are presented in Table 1. Compared with patients who did not develop AF, NOAF patients were signifcantly older, more likely to combine with heart failure, have a higher heart rate at admission, and have higher levels of circulating BNP, D-dimer, fbrinogen, serum creatinine, left atrial diameter, and right atrial diameter. NOAF patients had signifcantly lower levels of admission systolic blood pressure, lymphocytes, platelets, hemoglobin, triglycerides, albumin, and glomerular fltration rate and were less likely to receive aspirin and PCI during hospitalization in both training and validation cohorts. In addition, there was no signifcant diference in the ACS subtype between non-NOAF and NOAF patients in the training and validation cohort. Te incidence of NOAF for UA, NSTEMI, and STEMI patients were 7.44%, 8.87%, and 9.21%, respectively, in the training cohort and were 5.98%, 6.25%, and 6.85%, respectively, in the validation cohort. It indicated that NOAF was more frequent in those with STEMI than in those with NSTEMI or UA, although the diferences were not signifcant. Te variables with signifcant associations assessed by the univariate logistic regression are shown in Supplementary  (Table 2).
Te ROC curve analysis was used to investigate the discrimination of the prediction model. Te AUC was 0.891 (95% CI: 0.863-0.920) and 0.839 (95% CI: 0.796-0.883) in the training and validation cohorts, respectively ( Figure 1). Te Hosmer and Lemeshow test of the fnal model showed an efective goodness-of-ft (P > 0.05 in both cohorts). Te calibration plots presented a good agreement between the predicted probability and actual probability of NOAF ( Figure 2). Te fnal decision curve showed that for a threshold probability between 5% and 50%, the model had a positive net beneft ( Figure 3). A nomogram was constructed based on the model ( Figure 4).
We then performed a subgroup analysis to develop a prediction model for NOAF risk in UA patients specifcally. Te fnal multivariable logistic regression model included six predictors that were also predictors of ACS, namely age, heart failure, left atrial diameter, right atrial diameter, BNP level, and no PCI (Table 3). Te AUC was 0.894 (95% CI: 0.854-0.934) and 0.844 (95% CI: 0.796-0.891) in training and validation cohorts, respectively (Supplementary Figure S1), and the model passed the calibration test (P > 0.05) (Supplementary Figure S2). Te clinical utility evaluation shows that the model has a clinical net beneft within a certain range of the threshold probability (5%-50%) (Supplementary Figure S3). A nomogram was also constructed (Supplementary Figure S4).

Discussion
In this study, we found age, admission heart rate, left atrial diameter, right atrial diameter, heart failure, BNP level, less statin use, and no PCI were independent predictors of NOAF during hospitalization in ACS patients. Te prediction model based on these eight variables had good discrimination, calibration, and clinical utility.
Te incidence of NOAF during hospitalization in Chinese ACS patients ranged from 6.7% to 13.4%. [13,16,25,26] Te incidence variation may be due to diferent study design, participants, and treatments. In this study, the incidence was 8.21% in the training cohort and 6.12% in the validation cohort, which were similar to those in the previous studies. Early detection of high-risk patients is crucial for the intervention of NOAF.
Several studies had tried to establish prediction models for NOAF in ACS patients. Mazzone and colleagues had established a model to predict NOAF during hospitalization in STEMI patients who underwent PCI, and the model included age, leukocyte count, BNP, and obesity. Te Cstatistics were 0.734 and 0.76 in the training and validation cohorts, respectively [20]. Two studies reported the plasma BNP level in patients with STEMI is a predictor of NOAF, and the AUC was 0.623 and 0.647, respectively [13,21]. Yildirim et al. developed a Value of Syntax Score II to predict NOAF in NSTEMI patients who underwent PCI, and the AUC of the model was 0.799 [19]. However, these models had only modest discriminatory power and were not validated or evaluated in terms of their calibration or clinical utility.
In this study, our multivariable analysis revealed several predictors of NOAF. By combining these predictors, we constructed a prediction model. Interestingly, the newly constructed model demonstrated a strong discriminatory performance (with AUC over 0.8) to identify patients with an increased risk of NOAF. Tis model was externally validated and comprehensively evaluated. In addition to International Journal of Clinical Practice

<0.01
Entries are n (%) for categorical variables and median (5th percentile-75th percentile) for continuous variables as appropriate. ACS, Acute coronary syndrome; NOAF, new-onset atrial fbrillation; BMI, body mass index; DBP, diastolic blood pressure; SBP, systolic blood pressure; COPD, chronic obstructive pulmonary disease; CRI, chronic renal insufciency; ACEI, angiotensin-converting enzyme inhibitors; BNP, brain natriuretic peptide; hsCRP, high-sensitivity C-reactive protein; WBC, white blood cell; TG, triglyceride; GFR, glomerular fltration rate; LAD, left atrial diameter; RAD, right atrial diameter; LVEF, left ventricular ejection fraction; PCI, percutaneous coronary intervention.      : Nomogram to predict NOAF risk during hospitalization in ACS patients. To use the nomogram, an individual patient's value was located on each variable axis, and a line was drawn upward to determine the number of points received for each variable value. Te sum of these numbers was located on the total points axis, and a line was drawn downward to the risk axis to determine the risk of NOAF presence. LAD, left atrial diameter; RAD, right atrial diameter; BNP, brain natriuretic peptide; PCI, percutaneous coronary intervention; HR, heart rate.
enhancing clinical risk prediction, simplicity, practicability, and costs of predictors should be considered. Te model included eight predictors which were more than those in previous studies, but these physiological and examination parameters were routinely obtained in the clinical setting. Te predictors of NOAF were age, admission heart rate, left atrial diameter, right atrial diameter, heart failure, BNP level, less statins use, and no PCI according to our results. It is known that age is an independent risk factor of AF. Te incidence of AF approximately doubled for every 10-year increment in age [27]. Previous studies demonstrated that age was an independent risk factor for NOAF in ACS patients [15,20,28]. According to a study based on 58 European hospitals, the heart rate at admission is a predictor of in-hospital mortality in patients with ACS [24]. Two studies showed that the NOAF patients had a higher heart rate at admission than non-NOAF [4,10]. ACS is associated with a sudden reduction in blood fow to the heart. Atrial ischemia causes strong conduction slowing in the ischemic zone, which may stabilize atrial reentry that maintains AF [29]. Heart rate is the most direct indicator of heart activity, and an increased heart rate may refect a subtle change in cardiac electrophysiology. Left/right atrial diameter is an independent predictor for NOAF occurrence according to some previous studies [9,26], and increased left/right atrial diameter is a marker of progressive dilatation and remodeling of the left atrial myocardium, which acts as a substrate for AF initiation and maintenance. In addition, heart failure is a risk factor for NOAF in our study whereas it is rarely reported in previous studies as a risk factor for NOAF during hospitalization. But it is known that heart failure and AF coincide in many patients. Heart failure and AF can cause and exacerbate each other through mechanisms such as structural cardiac remodeling, activation of neurohormonal mechanisms, and rate-related impairment of left ventricular function [30]. BNP is a neurohormone released from ventricular myocytes in response to acute volume and/or pressure overload is associated with the severity of left ventricular dysfunction, impaired hemodynamic parameters, and increased left ventricular end-diastolic pressure. Consistent with previous studies [26,31,32], we found that an increased circulating BNP level was a risk factor for NOAF in ACS patients.
Te use of statins is a protective factor for NOAF according to the results. Several meta-analyses have been published, demonstrating that the use of statins was signifcantly associated with an AF reduction around 30% in patients who experienced cardiac surgery [33][34][35] or electrical cardioversion [36], patients with coronary artery disease [37], or ACS [38]. Accumulated evidence has indicated that infammation characterized by an elevated level of CRP and IL-6 may play important role in AF occurrences [39]. Statins have an established anti-infammatory efect by inhibiting IL-6, tumor necrosis factor α (TNF-α) production, and nuclear factor kappa B (NF-κB) activation, and suppressing initiation and progression of AF [38]. PCI alters the natural history of ACS, and it is a protective factor for NOAF in this study, which is consistent with a previous study which reported PCI could reduce the risk of developing NOAF in patients with acute myocardial infarction [25].
In the training cohort, the NOAF patients were more likely to have combined with cardiomyopathy, but in the validation cohort, there were no signifcant diferences in the proportion of cardiomyopathy between non-NOAF and NOAF groups. Since the training and validation cohorts were selected from a single center, respectively, some characteristics of ACS patients might be diferent between these two hospitals, and selection bias might be introduced. Even though the proportions of patients combined with cardiomyopathy were diferent between non-NOAF and NOAF groups in training cohort, after adjusting confounding factors, cardiomyopathy was not a predictor in the fnal multivariable prediction model. Terefore, the impact of this selection bias on the study results was limited.
UA is the most common subtype of ACS, and it accounted for 46%-91.3% of the total ACS diagnosed [40,41]. In this study, we found that 66.5% of the ACS patients were UA. Te incidences of NOAF during hospitalization ranged from 6.7% to 11.4% for UA patients [13,19,42], and NOAF also increased the risk of renal failure, stroke, and mortality. UA patients had diferent risk factors compared with STEMI/NSTEMI patients [43]. According to previous studies, the incidences and prognoses of NOAF among diferent subtypes of ACS were also different. NOAF was more frequent in those with STEMI than in those with NSTEMI or UA [17,44], which was consistent with our results. Te incidence of NOAF for UA, NSTEMI and STEMI patients was 7.44%, 8.87% and 9.21%, respectively, in the training cohort, and was 5.98%, 6.25% and 6.85%, respectively, in the validation cohort, although these diferences were not signifcant. NOAF had a larger impact in NSTEMI on the risk of death, stroke, or recurrent myocardial infarction than in STEMI and UA [44,45]. So we expect the risk factors for NOAF in diferent subtypes of ACS may vary. But there were few studies aiming to build a prediction model for NOAF in UA patients specifcally. In the subgroup analysis of UA patients, we found there were only six predictors in the fnal model. Except for heart rate at admission and less statin use, the other predictors were the same as those of ACS patients. In all ACS patients, the median heart rate was 78 bpm in the non-NOAF group and 87.5 bpm in the NOAF group. And in UA patients, the median heart rate was also 78 bpm in the non-NOAF group but 82 bpm in the NOAF group. Tus, UA patients had a lower admission heart rate, and the diferences in admission heart rate between NOAF and non-NOAF groups were smaller in UA. When included in the multivariable prediction model, admission heart rate was not signifcant. In all ACS patients, the rate of statins use was 87.4% and 70.6% in the non-NOAF and NOAF groups, respectively. While in the UA patients, the rate of statin use was signifcantly higher (96.4% and 85.2% in the non-NOAF and NOAF groups, respectively). Moreover, the pathology between UA and STEMI/NSTEMI patients were diferent. Compared with STEMI/NSTEMI patients, individuals with UA do not experience acute cardiomyocyte injury/necrosis, have a substantially lower risk of death, and appear to derive less beneft from intensifed antiplatelet therapy as well as an invasive strategy within 72 h [46]. Te driving infammation process for AF between UA and STEMI/NSTEMI patients might be diferent. Several studies have investigated the efect of prior statin therapy on NOAF in ACS, but the efect in UA patients was seldom demonstrated. More evidence are needed to elucidate the efect of statin on NOAF among diferent subtypes of ACS. Some predictors of NOAF in this model are also identifed predictors of ACS such as increased age, size of the atria, and BNP [43,[47][48][49][50]. However, the risk factors that this study mainly focuses on are the characteristics of ACS patients at admission, such as Killip classifcation II-IV at admission, a heart rate over 85 bpm at admission, statins, and PCI during hospitalization. Tese are specifc factors for hospitalized ACS patients and can help clinicians classify high-risk patients accurately in the early stages of hospitalization.
In this study, the rates of ACS patients receiving PCI were 51.53% and 44.04% in the training and validation cohorts, respectively. According to the registration data of coronary intervention in China's mainland, the rate of STEMI patients received PCI increased from 30.72% in 2010 to 67.45% in 2019. Another study enrolled ACS patients from 11 tertiary hospitals in Chengdu which is a city near Chongqing from 2017 to 2019, and the rates of patients received PCI were 80.6%, 50.8%, and 26.9% for STEMI, NSTEMI, and UA patients, respectively [51]. In our study, most of the ACS patients were UA patients, accounting for 53.4% and 78.8% of the training and validation cohorts, respectively. So the rate at which ACS patients received PCI in our study was comparable to that of the whole country and neighboring city. But compared with the percentage of 70%-80% in European and American countries [52], the rate of ACS patients receiving PCI in our study was relatively low. For the medications, the rates of patients receiving statins and aspirins were relatively high in this study, while the rate of patients received ACEI was lower than those of other medications, and even lower than that of the whole country (66.4% in 2011) and neighboring city Chengdu (54.3% in 2017-2019) [51]. It is probably because clinicians were more cautious about prescribing ACEI due to their potential adverse efects on the blood pressure. Te treatments of ACS in this study indicated that there were still gaps between the treatment in clinical practice and guidelines. Te implementation of guidelines for ACS patients still needs to be further strengthened.
In this study, based on a "real world" retrospective cohort, we identifed the most signifcant predictors for NOAF among all known AF risk factors and then constructed a prediction model with a strong discriminatory performance to identify ACS patients with an increased risk of NOAF during hospitalization. Te predictors in this model were routinely examined after admission. Tey can be easily obtained at the early stage of admission. Tis model is easy to use in the daily clinical practice and might help clinicians classify high-risk patients accurately in the early stage of hospitalization and improve the intervention of NOAF.
As for the clinical utility of our prediction model, if an ACS patient was predicted to have an increased risk of NOAF during hospitalization, intensive ECG monitoring should be used to detect and diagnose early. Ten, preventive therapy might be used for the high-risk individuals. Firstly, statin and ranolazine might reduce the risk of NOAF in ACS patients, according to some studies. An analysis by Bang et al. of ACS patients showed that the absolute risk reduction of NOAF in patients with ACS receiving statin therapy was 5% (10% vs. 15%), with a calculated relative risk reduction (RRR) of 33% [53]. A meta-analysis of statin therapy for prevention of NOAF pooled data from six trials of over 160,000 patients. ACS patients who were taking a statin at baseline had a 35% reduction in NOAF (RR 0.65 (95% CI 0.55 to 0.77)) [38]. In a retrospective analysis of the MERLIN-TIMI36 trial, those ACS patients assigned to ranolazine had a lower incidence of NOAF after one year [54]. Secondly, as for the use of antiarrhythmic agents, there were limited evidences. In a Danish RCT examining those with LV dysfunction and recent MI on dofetilide or placebo, they found that treatment with dofetilide was not associated with a signifcant reduction in the risk of NOAF [55]. Tirdly, increased risk of thrombo-embolic events in ACS patients with NOAF has been described by several studies. Studies also found reduced mortality in ACS patients with new AF treated with oral anticoagulant (OAC) as compared with treatment without OAC [56,57]. Despite the clear guideline recommendations for OAC treatment for AF patients, undertreatment remains a serious issue. Some studies found less OAC prescription for new AF as compared with known AF [57,58]. Possibly, the knowledge that adding OAC to the indicated ACS antiplatelet therapy increases the risk for bleeding leads to withholding the International Journal of Clinical Practice prescription. All in all, the reduction of events with statin, antiarrhythmic agents, and OAC in these specifc scenarios has never been well studied in a dedicated randomized controlled trial, and more evidence are needed. We believe with the accumulation of evidence, and after full consideration of the risks and benefts, some preventive therapies might be used for the high-risk patients. And the prediction model provided by our study might be a practical tool in the clinical practice.

Limitations
Several limitations of this study should be considered when interpreting our results. Firstly, this study was based on retrospective cohorts, and selection bias might be introduced due to collecting data retrospectively. A total of 5403 consecutive ACS patients were recruited in the training cohort, but 3868 of them were excluded, mainly due to the lack of BNP or echocardiography. Secondly, training and validation cohorts were selected from a single center, respectively. Although we validated the prediction model externally and evaluated it comprehensively, more cohorts in other medical centers are needed to further validate it. Tirdly, due to the limitation of retrospective collection of data, we did not have the data of some other previous histories such as heart failure or myocardial infarction, treatment histories before admission, the reason for PCI not performed, and the type of cardiomyopathy in patients. Bias from unmeasured confounding factors may exist. Fourthly, this is an observational study, and observational studies can be used to determine the association rather than ascertain the causal relationship. To ascertain the causal relationship, more prospective clinical studies with long follow-up are needed to support the causality. Finally, we only focus on the NOAF occurrence during hospitalization, and the results of this study need to be validated by well-designed studies with long-termfollowup.

Conclusion
In the present study, we demonstrated that age, admission heart rate, left atrial diameter, right atrial diameter, heart failure, BNP level, less statins use, and no PCI were independent predictors of NOAF during hospitalization in ACS patients. A prediction model based on these eight variables was constructed. It might help with the identifcation of ACS patients at risk and early intervention by NOAF during hospitalization.

Data Availability
Te research article data used to support the fndings of this study are available from the corresponding author upon request.

Ethical Approval
Te study was approved by the Ethics Committee of Southwest Hospital of Army Medical University. Te research was conducted in accordance with the Helsinki declaration guidelines and all procedures listed here were carried out in compliance with the approved guidelines. Because this is a retrospective study, and all the parameters assessed were routinely obtained in the hospitals so that no additional investigations or procedures were carried out, informed consent was waived by the Ethics Committee of Southwest Hospital of Army Medical University.

Disclosure
Te funders had no role in the design and conduct of the study or in collection, management, analysis and interpretation of the data, the preparation, review, and submission of the manuscript for publication.

Conflicts of Interest
Te authors declare there are no conficts of interest.