A Validated Composite Score Demonstrates Potential Superiority to MELD-Based Systems in Predicting Short-Term Survival in Patients with Liver Cirrhosis and Spontaneous Bacterial Peritonitis—A Preliminary Study

Background: Spontaneous bacterial peritonitis (SBP) is a severe complication in cirrhosis patients with ascites, leading to high mortality rates if not promptly treated. However, specific prediction models for SBP are lacking. Aims: This study aimed to compare commonly used cirrhotic prediction models (CTP score, MELD, MELD-Na, iMELD, and MELD 3.0) for short-term mortality prediction and develop a novel model to improve mortality prediction. Methods: Patients with the first episode of SBP were included. Prognostic values for mortality were assessed using AUROC analysis. A novel prediction model was developed and validated. Results: In total, 327 SBP patients were analyzed, with HBV infection as the main etiologies. MELD 3.0 demonstrated the highest AUROC among the traditional models. The novel model, incorporating HRS, exhibited superior predictive accuracy for in-hospital in all patients and 3-month mortality in HBV-cirrhosis, with AUROC values of 0.827 and 0.813 respectively, surpassing 0.8. Conclusions: MELD 3.0 score outperformed the CTP score and showed a non-significant improvement compared to other MELD-based scores, while the novel SBP model demonstrated impressive accuracy. Internal validation and an HBV-related cirrhosis subgroup sensitivity analysis supported these findings, highlighting the need for a specific prognostic model for SBP and the importance of preventing HRS development to improve SBP prognosis.


Introduction
Patients with liver cirrhosis often exhibit an impaired defense against bacteria, leading to reduced bacterial clearance [1]. This immune defect, coupled with increased intestinal permeability and gut bacterial overgrowth, facilitates bacterial translocation. As a result of the pathological bacterial translocation in liver cirrhosis, the bacterial infection is either present on admission or develops during hospitalization in approximately 30% of patients with cirrhosis [2]. The most prevalent form of these infections is spontaneous bacterial peritonitis (SBP) [2]. SBP is characterized by the spontaneous infection of ascitic fluid, occurring in cirrhotic patients in the absence of an intra-abdominal source of infection [3]. The presence of Figure 1. Flowchart of the study. Following the inclusion and exclusion criteria, a total of 327 patients diagnosed with the first episode of SBP were included in the study analysis.

Diagnosis, Definition, and Management of Liver Cirrhosis with Ascites and Spontaneous Bacterial Peritonitis
The diagnosis of liver cirrhosis (LC) with ascites was based mainly on the following criteria: (1) LC diagnosed based on histopathology (liver biopsy) or compatible clinical features, laboratory data, sonographic/computed tomography/magnetic resonance imaging findings typical for liver cirrhosis [24], or non-invasive testing such as FibroScan [25]; (2) ascites caused by portal hypertension (serum-ascites albumin gradient (SAAG) ≥ 1.1 g/dL) [26]; (3) exclusion of other underlying disease that can cause ascites such as malignancy (HCC or metastasis), right-sided congestive heart failure/constrictive pericarditis, Budd-Chiari syndrome, post-sinusoidal obstruction syndrome, portal or splenic vein thrombosis, and the possibility of schistosomiasis. SBP is suspected in cirrhotic patients presenting with suggestive symptoms and signs, such as fever, abdominal pain, altered mental status, abdominal tenderness, or hypotension [27]. SBP is diagnosed upon an absolute neutrophil count in ascites fluid of ≥ 250 cells/mm 3 and/or positive ascites culture, in the absence of a surgically treatable intraabdominal source of infection and other causes of elevated ascites neutrophil count, such as hemorrhage, pancreatitis, peritoneal tuberculosis, or carcinomatosis [28]. Since infections are common in cirrhotic patients with variceal bleeding [29], paracentesis should be performed upon hospital admission in all cirrhotic patients with ascites, even if admitted for reasons other than ascites [30]. Baseline laboratory data were collected when SBP was suspected, and prompt diagnosis was facilitated by efficient laboratory services. In our facility, the results of ascites fluid routine can be obtained within 2 h, enabling a timely diagnosis of SBP within 1 day.
The management of SBP follows the recommendations of the International Ascites Club [31] and the AASLD/EASL/JAMA guidelines [5,25]. In the case of HBV-related  SBP is suspected in cirrhotic patients presenting with suggestive symptoms and signs, such as fever, abdominal pain, altered mental status, abdominal tenderness, or hypotension [27]. SBP is diagnosed upon an absolute neutrophil count in ascites fluid of ≥250 cells/mm 3 and/or positive ascites culture, in the absence of a surgically treatable intra-abdominal source of infection and other causes of elevated ascites neutrophil count, such as hemorrhage, pancreatitis, peritoneal tuberculosis, or carcinomatosis [28]. Since infections are common in cirrhotic patients with variceal bleeding [29], paracentesis should be performed upon hospital admission in all cirrhotic patients with ascites, even if admitted for reasons other than ascites [30]. Baseline laboratory data were collected when SBP was suspected, and prompt diagnosis was facilitated by efficient laboratory services. In our facility, the results of ascites fluid routine can be obtained within 2 h, enabling a timely diagnosis of SBP within 1 day.
The management of SBP follows the recommendations of the International Ascites Club [31] and the AASLD/EASL/JAMA guidelines [5,25]. In the case of HBV-related cirrhotic patients, antiviral agents are administered if they meet the latest APASL guideline [32][33][34].

Primary Outcomes and Scheduled Follow-Up Periods
Due to the high short-term mortality associated with SBP, the primary outcome of this study was defined as in-hospital mortality, as well as mortality at 3 months (3 M) Diagnostics 2023, 13, 2578 4 of 16 and 6 months (6 M) following diagnosis. Each patient was followed up at least every 3 to 6 months until the date of death or March 2018, whichever occurred first.

Statistical Analysis
The data were properly tabulated for statistical analysis. Normally distributed continuous data were presented as mean ± standard deviation (SD) and compared using an independent Student's t-test. Non-normally distributed continuous data were expressed as median and interquartile range (IQR) and compared using the Mann-Whitney U test. Categorical variables were presented as frequencies and percentages and compared using the χ 2 test or Fisher's exact test. The predictive ability of the novel model was evaluated by calculating the area under the receiver operating characteristic curve (AUROC) and compared to existing scoring systems such as CTP, MELD, MELD-Na, iMELD, and MELD 3.0. The predictive performance of each scoring system in predicting mortality was compared using the DeLong test. The best cut-off values for the new SBP scoring system were determined using the Youden Index. All statistical analyses were performed using IBM SPSS Statistics version 25 (SPSS Inc., Chicago, IL, USA) and R 4.2.2 (R Core Team, 2021; Vienna, Austria). A p-value less than 0.05 was considered statistically significant.

Construction of a Novel Prognostic Model
Using the current dataset, the primary outcomes were the dependent variable in the construction of a novel prognostic model. Initially, several independent variables were included, such as patients' demographics, etiology of cirrhosis, ascites PMN, the presence or absence of sepsis, ascites, hepatic encephalopathy (HE), HRS, serum Cr, Na, K, total bilirubin, albumin, INR, hemoglobin, WBC, and PLT. Variables with a p < 0.20 in the univariate analysis were included in a multivariate logistic regression analysis [36] to identify independent predictors that could predict primary outcomes with an AUROC > 0.8, indicating a good discriminatory ability [37].

Model Validation and Sensitivity Analysis
For model validation, internal validation was performed using 5-fold cross-validation, which involved dividing a group of 327 patients into 5 equal subsets. The model was trained 5 times, with each iteration using 4 out of the 5 subsets as the training data (261 patients), while the remaining subset (66 patients) served as the test (validation) set. The performance of the model was assessed using various metrics, including AUROC, Brier scores, and accuracy at intervals. The results of the internal validation, including AUROC, Brier scores, and accuracy, can be found in Supplementary Table S4 and Supplementary Figure S1. A subgroup sensitivity analysis was performed specifically on the HBV subgroup to further examine the performance of the predictive models.

Patients' Baseline Characteristics
After applying the inclusion and exclusion criteria, a total of 327 patients diagnosed with the first episode of SBP were enrolled in the study. Table 1 presents the demographic characteristics of the patients, etiology of cirrhosis, hemogram and biochemical laboratory data measured at diagnosis, as well as the scores of the CTP and four MELD-based models. The patients were predominantly middle-aged and predominantly male. Among the patients, 69.2% had chronic hepatitis virus infections, with HBV infection being the most common etiology. Among the HBV-infected patients, 57.4% (85/148) received antiviral treatment. Sepsis was diagnosed in 12.8% of the SBP patients based on positive blood culture and evidence of systemic inflammatory response syndrome. Hepatorenal syndrome was present in 9.5% of the patients, and 25.7% had HE grade ≥ 1.  Table 1 also displays the interpretation of various abnormal laboratory data in patients with SBP. These abnormalities include renal function impairment, hyperbilirubinemia, hyponatremia, hypoalbuminemia, prolonged INR for prothrombin time, anemia, and thrombocytopenia. The mean white blood cell counts (WBCs) in blood were within the normal range, while the ascites polymorphonuclear leukocytes (PMNs) counts were >250 cells/µL. The mean CTP class was C. The scores for the four MELD-based models were displayed in Table 1.

Comparison of Score Differences among CTP and Four MELD-Based Models between
In-Hospital, 3-Month, and 6-Month Mortality and Non-Mortality Groups Table 2 presents the statistical differences between the in-hospital, 3-month, and 6-month mortality groups and the non-mortality group in terms of CTP and four MELDbased model scores. The in-hospital mortality rate was 39.4%. The primary causes of in-hospital mortality were sepsis with multiple organ failures (81 patients, 62.8%), endstage liver disease (ESLD)-related multiple organ failures (28 patients, 21.7%), massive gastrointestinal bleeding with shock (13 patients, 10.1%), and other causes (7 patients, 5.4%). Table 2. Statistical differences in scores among CTP and four MELD-based models for predicting in-hospital, 3-month, and 6-month mortalities between mortality and non-mortality groups.

Mortality
Non-Mortality p The cumulative 3-month and 6-month mortality rates were 51.4% and 60.9%, respectively. Significant differences were observed between the in-hospital, 3-month, and 6-month mortality and non-mortality groups in terms of the CTP score and the four MELD-based model scores (Table 2) To compare the predictive ability of the CTP score and the four MELD-based model scores for short-term mortality prediction (in-hospital, 3-month, and 6-month mortality), the AUROC was calculated for each score and compared.
In terms of predicting in-hospital mortality, as shown in Table 3 and Figure 2, the MELD 3.0 score exhibited the highest AUROC (0.786), followed by MELD-Na (0.783), iMELD (0.780), MELD (0.775), and CTP score (0.701). The MELD 3.0 score was significantly superior to the CTP score and non-significantly superior to MELD, MELD-Na, and iMELD scores in predicting in-hospital mortality. month mortality and non-mortality groups in terms of the CTP score and the four MELDbased model scores (Table 2).

Comparison of the Predicting Performance of the Original CTP, MELD, MELD-Na, iMELD, MELD 3.0, and the New SBP Scores for In-Hospital, 3-Month, and 6-Month Mortality Prediction Using AUROC and DeLong Test
To compare the predictive ability of the CTP score and the four MELD-based model scores for short-term mortality prediction (in-hospital, 3-month, and 6-month mortality), the AUROC was calculated for each score and compared.
In terms of predicting in-hospital mortality, as shown in Table 3 and Figure 2, the MELD 3.0 score exhibited the highest AUROC (0.786), followed by MELD-Na (0.783), iMELD (0.780), MELD (0.775), and CTP score (0.701). The MELD 3.0 score was significantly superior to the CTP score and non-significantly superior to MELD, MELD-Na, and iMELD scores in predicting in-hospital mortality. In terms of predicting 3-month mortality, Table 4 and Figure 3 demonstrate that the MELD 3.0 score exhibited the highest AUROC (0.760), followed by MELD-Na (0.755), iMELD (0.753), MELD (0.737), and CTP score (0.656). The MELD 3.0 score was significantly superior to the CTP score and MELD score, and non-significantly superior to MELD-Na and iMELD scores in predicting 3-month mortality. In terms of predicting 3-month mortality, Table 4 and Figure 3 demonstrate that the MELD 3.0 score exhibited the highest AUROC (0.760), followed by MELD-Na (0.755), iMELD (0.753), MELD (0.737), and CTP score (0.656). The MELD 3.0 score was significantly superior to the CTP score and MELD score, and non-significantly superior to MELD-Na and iMELD scores in predicting 3-month mortality.   In terms of predicting 6-month mortality, Table 5 and Figure 4 demonstrate that the iMELD score exhibited the highest AUROC (0.752), followed by MELD-Na (0.745), MELD 3.0 (0.742), MELD (0.728), and CTP score (0.640). The iMELD score was significantly superior to the CTP score and non-significantly superior to MELD-Na, MELD 3.0, and MELD scores in predicting 6-month mortality. In terms of predicting 6-month mortality, Table 5 and Figure 4 demonstrate that the iMELD score exhibited the highest AUROC (0.752), followed by MELD-Na (0.745), MELD 3.0 (0.742), MELD (0.728), and CTP score (0.640). The iMELD score was significantly superior to the CTP score and non-significantly superior to MELD-Na, MELD 3.0, and MELD scores in predicting 6-month mortality.  The iMELD score exhibited the highest AUROC, followed by MELD-Na, MELD 3.0, MELD, and CTP scores. The iMELD score was significantly superior to the CTP score and non-significantly superior to MELD-Na, MELD 3.0, and MELD scores. The newly developed SBP score displayed significant superiority over the CTP score. However, it did not show significant superiority over the MELD, MELD-Na, iMELD, and MELD 3.0 scores.

Development of the New SBP Score Model
We developed a new SBP score model to enhance the accuracy of predicting in-hospital, 3-month, and 6-month mortalities. Based on the methods outlined in the Section 2, Figure 4. Comparison of the original CTP, MELD, MELD-Na, iMELD, MELD 3.0, and the new SBP scores in predicting 6-month mortality by AUROC. The iMELD score exhibited the highest AUROC, followed by MELD-Na, MELD 3.0, MELD, and CTP scores. The iMELD score was significantly superior to the CTP score and non-significantly superior to MELD-Na, MELD 3.0, and MELD scores. The newly developed SBP score displayed significant superiority over the CTP score. However, it did not show significant superiority over the MELD, MELD-Na, iMELD, and MELD 3.0 scores.

Development of the New SBP Score Model
We developed a new SBP score model to enhance the accuracy of predicting in-hospital, 3-month, and 6-month mortalities. Based on the methods outlined in the Section 2, the new SBP score model was constructed by integrating age, serum creatinine, total bilirubin, International Normalized Ratio (INR), platelet count, as well as the presence of sepsis and hepatorenal syndrome (HRS).
The formula of the new SBP score model is as follows:  According to the results presented in Table 3 and Figure 2, the newly developed SBP score model exhibited an impressive AUROC of 0.827. Notably, it demonstrated significant superiority over the CTP score, MELD, MELD-Na, and iMELD scores in predicting inhospital mortality. Furthermore, the new SBP score model displayed a marginal advantage compared to the MELD 3.0 score in its predictive capability for in-hospital mortality.
Moreover, in terms of predicting 3-month mortality, the newly developed SBP score model demonstrated significant superiority over the CTP score and MELD (Table 4 and Figure 3). However, it did not show significant superiority over the MELD-Na, iMELD, and MELD 3.0 scores.
Additionally, when considering the prediction of 6-month mortality, the newly developed SBP score model displayed significant superiority over the CTP score (Table 5 and Figure 4). However, it did not show significant superiority over the MELD, MELD-Na, iMELD, and MELD 3.0 scores and its AUROC did not surpass 0.8.

Model Validation
The model was internally validated using five-fold cross-validation. The performance of the new model was assessed using various metrics, including AUROC, Brier scores, and accuracy. The results of the model validation, specifically for in-hospital mortality, are presented in Supplementary Table S1 and Supplementary Figure S1. The model validation results showed good performance in terms of AUROC (>0.8), Brier scores, and accuracy for the primary outcome at different time points (3-and 6-month results not shown for brevity). Given the high prevalence of HBV-related cirrhosis in Taiwan, a subgroup sensitivity analysis was conducted to assess the predictive accuracy of the scores in this specific patient population with SBP. In terms of 3-month mortality prediction, the new SBP score showed significant superiority over the CTP score, MELD, and iMELD scores, as indicated in Table 6 and Figure 5. However, it did not demonstrate significant superiority over the MELD-Na and MELD 3.0 scores.

The Cut-Off Values for the New SBP Score Model
The new SBP model demonstrated a cut-off value of −0.4697 for predicting in-hospital mortality, with mortality rates of 18.7% and 73.3% below and above this threshold, respectively (Supplementary Table S2). For 3-month mortality prediction, the new model's cut-off value was 0.3712, with mortality rates of 33.6% and 84.9% below and above this value, respectively (Supplementary Table S3). Similarly, for 6-month mortality prediction, the cut-off value was 0.4690, corresponding to mortality rates of 40.8% and 83.6% below and above this threshold, respectively (Supplementary Table S4). Table 6. Comparison of the predicting ability of the original CTP, MELD, MELD-Na, iMELD, MELD 3.0, and the new SBP scores in predicting 3-month mortality in the HBV-related cirrhosis subgroup.  Table 6. Comparison of the predicting ability of the original CTP, MELD, MELD-Na, iMELD, MELD 3.0, and the new SBP scores in predicting 3-month mortality in the HBV-related cirrhosis subgroup.

The Cut-Off Values for the New SBP Score Model
The new SBP model demonstrated a cut-off value of −0.4697 for predicting in-hospital mortality, with mortality rates of 18.7% and 73.3% below and above this threshold, respectively (Supplementary Table S2). For 3-month mortality prediction, the new model's cutoff value was 0.3712, with mortality rates of 33.6% and 84.9% below and above this value, respectively (Supplementary Table S3). Similarly, for 6-month mortality prediction, the cut-off value was 0.4690, corresponding to mortality rates of 40.8% and 83.6% below and above this threshold, respectively (Supplementary Table S4).

Discussion
SBP is a common and severe complication in cirrhotic patients with ascites [38], with a prevalence ranging from 10% to 30% in hospitalized patients [4,39]. Despite improvements in early recognition and effective antibiotic treatment, in-hospital mortality rates remain high, ranging from 20% to 40% [40,41]. Early identification of high-risk patients could potentially improve survival outcomes [42]. However, specific prediction models for SBP are currently unavailable. Therefore, our study aimed to compare the predictive ability of commonly used cirrhotic prediction models, including the CTP score and four MELD-based scores (MELD, MELD-Na, iMELD, MELD 3.0), for short-term mortality prediction (in-hospital, 3 and 6 month). Additionally, we sought to develop a novel model to enhance mortality prediction compared to conventional models. In this retrospective cohort study, we analyzed data from 327 cirrhotic patients who experienced their first SBP episode between January 2006 and August 2017. Clinical data were collected at the time of diagnosis, and patients were followed until March 2018 or death. Mortality prediction was assessed using AUROC analysis. HBV infection accounted for 45.3% of cirrhosis etiologies. In-hospital mortality was 39.4%, with cumulative 3-month and 6-month mortality rates of 51.4% and 60.9%, respectively. Among the existing prediction models, MELD 3.0 showed the highest AUROC in predicting in-hospital and 3-month mortality, surpassing iMELD, MELD, CTP, and MELD-Na. For 6-month mortality, iMELD showed the best performance. Our novel SBP model, incorporating age, serum Cr, total bilirubin, INR, PLT, sepsis, and HRS, demonstrated superior predictive accuracy for in-hospital mortality in all patients (AUROC 0.827) and 3-month mortality in HBV cirrhosis (AUROC 0.813), with HRS exhibiting the highest odds ratio. Internal validation and subgroup sensitivity analysis, explicitly focusing on HBV-related cirrhosis, further corroborated these findings. Our results emphasize the importance of developing a dedicated prognostic model for SBP and highlight the significance of preventing HRS development to improve the prognosis of SBP.
The CTP score has been extensively utilized for prognostic evaluation in cirrhotic patients for over four decades [43]. However, it possesses certain limitations, including the subjectivity of certain clinical parameters and its limited discriminative ability [44][45][46]. On the other hand, MELD has demonstrated superior accuracy in predicting 3-month survival compared to the CTP scoring system for cirrhotic patients awaiting liver transplantation in the United States [9]. Since February 2002, MELD has served as the primary reference system for organ allocation in liver transplantation in the United States [47]. Furthermore, MELD has shown effectiveness in predicting mortality across various chronic liver diseases and certain complications of portal hypertension, such as hepatic encephalopathy and acute variceal bleeding [48,49].
Several MELD-based models have been developed to enhance the accuracy of predicting wait-list mortality for liver transplantation. One such model is MELD-Na, which incorporates serum sodium (Na) as an additional predictor of wait-list mortality [50,51]. MELD-Na has been shown to accurately predict 6-month mortality in cirrhotic patients awaiting liver transplantation in a multicenter study [11]. Another model, the iMELD score, was developed to improve survival prediction in cirrhotic patients [12]. These MELD-based models have been compared and exhibited strong prognostic capabilities for outcome prediction in cirrhotic patients [16,17]. Recently, MELD has been updated to version 3.0 with the aim of reducing deaths while patients are on the liver transplantation waitlist [15]. However, there is limited information on the applicability of these models in the subgroup of patients with liver cirrhosis-related complications, particularly SBP, in regions with a high prevalence of chronic hepatitis B, such as Taiwan [22,52].
In our study, the MELD 3.0 score demonstrated significant superiority over the CTP score and non-significant superiority over MELD, MELD-Na, and iMELD scores in predicting in-hospital mortality. However, its AUROC of 0.786 did not reach the desired threshold of 0.8. To overcome this limitation, we developed a new SBP score model with an impressive AUROC of 0.827, surpassing the critical threshold of 0.8. The new SBP score model significantly outperformed the CTP score, MELD, MELD-Na, and iMELD scores, and slightly outperformed the MELD 3.0 score in predicting in-hospital mortality. For the 3-month mortality prediction, the new SBP score model demonstrated significant superiority over the CTP score and MELD, with an AUROC of 0.789. Subgroup sensitivity analysis focusing on HBV-related cirrhosis and SBP further improved the AUROC to 0.813. However, the performance of the new SBP score model in predicting 6-month mortality was not satisfactory, with an AUROC of 0.762, similar to that of the iMELD score. These findings suggest that the new SBP score model is particularly effective in predicting in-hospital and 3-month mortality, aligning with the current policy of assessing the risk of death within 90 days for liver transplants [53].
It is noteworthy that our study's findings align with previous research emphasizing the detrimental impact of HRS on SBP prognosis. This is supported by the highest odds ratio in our SBP score model, highlighting the significant role of HRS in predicting SBP outcomes. These findings are consistent with a well-known study demonstrating the benefits of intravenous albumin treatment in conjunction with antibiotics, which reduces the incidence of renal impairment and mortality in cirrhotic patients with SBP, compared to antibiotic treatment alone [54]. This finding underscores the importance of preventing HRS development and highlights the potential value of interventions targeting renal function in improving SBP prognosis.
The study has several limitations. Firstly, it is a retrospective study with a relatively small number of patients. Secondly, external validation was not conducted. Thirdly, being a single-center study, a multi-center study would be beneficial in the future. Despite these limitations, the findings underscore the need for a specific prognostic model for SBP and emphasize the importance of conducting a prospective and larger case series for validation.

Conclusions
In our study of cirrhotic patients with the first episode of SBP, the MELD 3.0 score outperformed the CTP score and showed a non-significant improvement compared to other MELD-based scores (MELD, MELD-Na, iMELD) in predicting in-hospital and 3-month mortality, although the AUROC values were below the desired threshold of 0.8. Our newly developed SBP model, incorporating age, serum Cr, total bilirubin, INR, PLT, sepsis, and HRS, achieved an impressive AUROC value exceeding 0.8 for in-hospital mortality in all patients and 3-month mortality in HBV cirrhosis. Internal validation and subgroup sensitivity analysis focusing on HBV-related cirrhosis supported these findings. These results highlight the need for a specific prognostic model for SBP and underscored the importance of preventing HRS development to improve SBP prognosis.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/diagnostics13152578/s1. Table S1: Model internal validation by 5-fold cross-validation. Table S2: The cut-off values for the new SBP score model in predicting in-hospital mortality. Table S3: The cut-off values for the new SBP score model in predicting 3-month mortality. Table S4: The cut-off values for the new SBP score model in predicting 6-month mortality. Figure S1: The AUROC curve of the training set and the test (validation) set for predicting in-hospital mortality.  Informed Consent Statement: Patient consent was waived by the IRB as this is a retrospective cohort study. The study involved only medical chart reviews without any patient intervention or the disclosure of personal information. Hence, the IRB did not require patients' signatures.
Data Availability Statement: Data and study materials will be made available to other researchers upon request via email, along with their IRB approval document.

Conflicts of Interest:
The authors declare no conflict of interest.