Preoperative Nonselective Chest Computed Tomography Prior to Primary Cardiac Surgery Results in Meaningful Change to Surgical Management: Systematic Review and Pooled Prevalence Meta-Analysis.

BACKGROUND: Routine screening chest computed tomography (CT) prior to primary cardiac surgery is advocated by some surgeons due to the purported benefits of identifying significant aortic calcification that impacts ongoing management, such as performing anaortic off-pump surgery or adjusting cannulation strategy. Additionally, axial imaging can identify incidental findings that may require concomitant or staged procedures such as ascending aortic dilatation or pulmonary lesions. The objective of this study was to quantify the impact that nonselective chest CT prior to primary cardiac surgery had on subsequent management. METHOD: A systematic review and pooled prevalence meta-analyses were performed in accordance with Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. Included studies performed non-selective chest CT prior to primary cardiac surgery. RESULTS: A total of eight studies, including 2,250 patients were included. The rate of mortality and stroke was low (1% and 2%, respectively). Calcification of the ascending aorta was identified in 15% of patients (95% confidence interval [CI] 5.0-26.0). A significant change to the surgical plan such as cannulation strategy, off-pump surgery, cancellation, or an additional procedure was required in 7% (95% CI 2.0-12.0). Clinically relevant incidental findings requiring in-patient management or follow-up were identified in 10% (95% CI 6.0-14.0). CONCLUSIONS: Nonselective CT chest prior to primary cardiac surgery identifies clinically relevant findings that result in a modification of the surgical plan in a significant population of patients to address the risk of stroke associated with aortic calcification as well as the identification of important incidental findings such as pulmonary lesions.

Survival Benefit of Multiple Arterial Revascularization With and Without Supplementary Saphenous Vein Graft.

Background It is unknown if the presence of saphenous vein grafting (SVG) adversely affects late survival following coronary surgery with multiple arterial grafting (MAG) versus single arterial grafting. Methods and Results A retrospective, observational, multicenter cohort study from 2001 to 2020 was conducted using the Australian and New Zealand Society of Cardiac and Thoracic Surgeons Database linked to the National Death Index. Patients undergoing primary isolated coronary artery bypass grafting with ≥2 grafts were included, and exclusions were patients aged <18 years, reoperations, concomitant or previous cardiac surgery, and the absence of arterial grafting. Demographics, comorbidities, medication, and operative configurations were propensity score matched between cohorts. The primary outcome was all-cause late death. Of 59 689 eligible patients, 35 113 were MAG (58.8%), and 24 576 were single arterial grafting (41.2%). Of the MAG cohort, 17 055 (48.6%) patients did not receive supplementary SVG (total arterial revascularization). Matching separately generated 22 764 patient pairs for MAG versus single arterial grafting, and 11 137 patient pairs for MAG with total arterial revascularization versus MAG with ≥1 supplementary vein grafts. At a median follow-up duration of 5.0 years postoperatively, the mortality rate was significantly lower for MAG than single arterial grafting (hazard ratio [HR], 0.79 [95% CI, 0.76-0.83]; P<0.001). The stratified MAG analysis found that MAG with total arterial revascularization had a lower risk of late death (HR, 0.85 [95% CI, 0.80-0.91]; P<0.001) compared with MAG with ≥1 supplementary vein grafts. Sensitivity analyses produced consistent outcomes as the primary analysis. Following adjustment for the presence of SVG in the Cox model, the survival advantage of incremental number of arteries was lost. Conclusions Multiple arterial grafting has significantly improved long-term survival compared with single arterial grafting. A further incremental survival benefit exists when no SVG is used.

Defining the Role of Anaortic Coronary Artery Bypass Grafting.

As the population ages and co-morbidities become more prevalent, the complexity of patients presenting for coronary artery bypass surgery is increasing. Cardiopulmonary bypass and aortic cross-clamping in these patients carry increased risk and, indeed, in some patients, with ascending aortic disease, the risks are prohibitive. Total-arterial anaortic coronary artery surgery is a technique that provides complete surgical coronary artery revascularization without cardiopulmonary bypass and without manipulating the ascending aorta. The technique essentially eliminates the risk of cerebral embolization of aortic atheroma and aortic injury. Anaortic techniques are an essential skillset for coronary artery surgery centers treating higher-risk patients.

Synchronous Carotid Endarterectomy and Anaortic Off-Pump Coronary Artery Bypass Surgery.

BACKGROUND: There is ongoing debate regarding the optimal strategy and timing for the surgical management of patients with severe concomitant carotid and coronary artery disease. Anaortic off-pump coronary artery bypass (anOPCAB), which avoids aortic manipulation and cardiopulmonary bypass, has been shown to reduce the risk of perioperative stroke. We present the outcomes of a series of synchronous carotid endarterectomy (CEA) and anOPCAB. METHODS: A retrospective review was performed. The primary endpoint was stroke at 30 days post-operation. Secondary endpoints included transient ischaemic attack, myocardial infarction and mortality 30 days post-operation. RESULTS: From 2009 to 2016, 1,041 patients underwent anOPCAB with a 30-day stroke rate of 0.4%. The majority of patients had preoperative carotid-subclavian duplex ultrasound screening and 39 were identified with significant concomitant carotid disease who underwent synchronous CEA-anOPCAB. The mean age was 71±7.5 years. Nine patients (23.1%) had previous neurological events. Thirty (30) patients (76.9%) underwent an urgent operation. For CEA, a conventional longitudinal carotid endarterectomy with patch angioplasty was performed in all patients. For anOPCAB, total arterial revascularisation rate was performed in 84.6% and the mean number of distal anastomoses was 2.9±0.7. In the 30-day postoperative period, there was one stroke (2.63%), two deaths (5.26%), two transient ischaemic attacks (TIAs) (5.26%) and no myocardial infarction. Two patients experienced acute kidney injury (5.26%), one of which required haemodialysis (2.63%). Mean length of stay was 11.37±7.9 days. CONCLUSION: Synchronous CEA and anOPCAB is a safe and effective option for patients' severe concomitant disease. Preoperative carotid-subclavian ultrasound screening allows identification of these patients.

Lost in Translation: The Impact of Language Barriers on the Outcomes of Patients Receiving Coronary Artery Revascularization.

INTRODUCTION: Clear and effective communication is vital in discussions regarding coronary revascularization. Language barriers may limit communication in healthcare settings. Previous studies on the influence of language barriers on the outcomes of patients receiving coronary revascularization have produced conflicting results. The aim of this systematic review was to evaluate and synthesise the existing evidence regarding the effects of language barrier on the outcomes of patients receiving coronary revascularization. METHODS: A systematic review was conducted, including a search of the PubMed, EMBASE, Cochrane, and Google Scholar databases on 01/10/2022. The review was conducted in accordance with PRISMA guidelines. This review was also prospectively registered on PROSPERO. RESULTS: Searches identified 3983 articles of which a total 12 studies were included in the review. Most studies describe that language barriers result in delayed presentation, but not delays in treatment following hospital arrival with respect to coronary revascularization. The findings with respect to the likelihood of receiving revascularization have varied significantly; however, some studies have indicated that those with language barriers may be less likely to receive revascularization. There have been some conflicting results with respect to the association between language barrier and mortality. However, most studies suggest that there is no association with increased mortality. In studies that evaluated length of stay variable results have been reported based on geographical location. Namely Australian studies have suggested no association between language barrier and length of stay, but Canadian studies support an association. Language barriers may also be associated with readmissions following discharge, and major adverse cardiovascular and cerebrovascular events (MACCE). CONCLUSION: This study demonstrates that patients with language barriers may have poorer outcomes from coronary revascularization. Future interventional studies will be required to consider the sociocultural context of patients with language barriers, and may be targeted at timepoints including prior to, during, or after hospitalisation for coronary revascularization. Further examination of the adverse health outcomes of those with language barriers in fields outside of coronary revascularization are required in view of the stark inequities identified in this field.

Minimally invasive off-pump anaortic coronary artery bypass (MACAB).

BACKGROUND: Minimally invasive direct coronary artery bypass has enabled coronary artery bypass graft to compete with the appeal of less invasive percutaneous coronary procedures. Favorable results of coronary artery bypass surgery performed without the use of cardiopulmonary bypass and without touching the aorta (anOPCAB) have enabled the development and use of minimally invasive methods. METHODS: Between 2016 and 2021, 112 patients underwent multivessel coronary bypass surgery performed using a minimally invasive method through a small thoracotomy in the left chest with off-pump, anaortic, and all-arterial grafts (Minimally Invasive Off-Pump Anaortic Coronary Artery Bypass [MACAB]). Patient data were collected and retrospectively analyzed. Eight series from the literature using the multivessel mini-OPCAB and MACAB technique were also evaluated. RESULTS: Collectively, from the literature, 2729 patients underwent an average of 2.4 bypasses with an early mortality rate of 0.7% and a stroke rate of 0.16%. In our MACAB case series, 112 patients underwent an average of 2.9 bypasses with a mortality rate of 1.8% and a stroke rate of 0%. CONCLUSION: MACAB can be performed safely by experienced surgeons and reduces neurological injury and surgical trauma and may be a good alternative for multivessel stenting. Simulation systems are essential for its dissemination, and teams dedicated to coronary surgery-with subspecialty expertise-are necessary to achieve good outcomes.

Coronary Artery Bypass Surgery Without Saphenous Vein Grafting: JACC Review Topic of the Week.

Approximately 95% of patients of any age undergoing contemporary, coronary bypass surgery will receive at least 1 saphenous vein graft (SVG). It is recognized that SVG will develop progressive and accelerated atherosclerosis, resulting in a stenosis, and in occlusion that occurs in 50% by 10 years postoperatively. For arterial conduits, there is little evidence of progressive failure as for SVG. Could avoidance of SVG (total arterial revascularization [TAR]) lead to a different late (>5 year) survival? A literature review of 23 studies (N = 100,314 matched patients) at a mean 8.8 years postoperative found reduced all-cause mortality for TAR (HR: 0.77; 95% CI: 0.71-0.84; P < 0.001). An expanded analysis with a new unpublished data set (N = 63,288 matched patients) was combined with the literature review (N = 127,565). It found reduced all-cause mortality for TAR (HR: 0.78; 95% CI: 0.72-0.85; P < 0.001). Additional Bayesian analysis found a very high probability of a TAR-associated reduction all-cause mortality.

Concomitant anaortic OPCAB and transfemoral TAVR for high-risk patients: A case series.

BACKGROUND: Combined on-pump coronary artery bypass (ONCAB) and surgical aortic valve replacement (SAVR) is the treatment of choice for concomitant severe aortic stenosis and coronary artery disease not amenable to percutaneous coronary intervention. Extensive aortic calcification and atheromatous disease may prohibit cardiopulmonary bypass and aortic cross-clamping. In these cases, anaortic off-pump coronary artery bypass (OPCAB) is a Class I (EACTS 2018) and Class IIA (AHA 2021) indication for surgical coronary revascularization. Transcatheter aortic valve replacement (TAVR) has similar benefits when compared with SAVR for this population (Partner 2 & 3). Herewith we describe a case series of concomitant Anaortic OPCAB and TAVR via the transfemoral approach for patients with coronary artery and valve disease considered too high risk for traditional coronary artery bypass grafting and SAVR due to severe aortic disease. METHODS/RESULTS: Eight patients underwent anaortic OPCAB and transfemoral TAVR during the same anesthetic in a hybrid operating room. Seven patients with multivessel disease had anaortic OPCAB via a sternotomy using composite grafts, one patient with LAD disease had anaortic OPCAB using a Da Vinci-assisted MIDCAB approach. All patients then had an Edwards Sapien 3 TAVR placed percutaneously via the common femoral artery. There was no 30 mortality or CVA in the series and all patients were discharged to home or a rehabilitation facility on Day 4-13. CONCLUSIONS: Combined anaortic OPCAB and transfemoral TAVR is a safe and feasible approach to treating concomitant extensive coronary artery disease and severe aortic stenosis. The aortic no-touch technique provides benefits in the elderly high-risk patients by reducing the risk of postoperative myocardial infarction and cerebrovascular stroke.

Selecting the optimal treatment for left main coronary disease: The importance of identifying subgroups of patients.

Randomized trials of stenting versus surgery for patients with unprotected left main (LM) coronary stenosis have largely shown similar survival between the two interventions. However, patients with LM stenosis represent a heterogeneous group in which subgroups likely to benefit from one therapy more than another are difficult to identify. Increasing coronary disease burden is the most accepted subgrouping for identifying optimal therapy but this can be defined in more detail allowing greater discrimination. Competitive flow reduces bypass graft patency in patients with isolated LM stenosis and complex bifurcation stenoses reduce the effectiveness of coronary stenting. The evidence for LM stenosis subgroupings is presented.

The Use of Intraoperative Transit Time Flow Measurement for Coronary Artery Bypass Surgery: Systematic Review of the Evidence and Expert Opinion Statements.

Transit time flow measurement (TTFM) allows quality control in coronary artery bypass grafting but remains largely underused, probably because of limited information and the lack of standardization. We performed a systematic review of the evidence on TTFM and other methods for quality control in coronary artery bypass grafting following PRISMA standards and elaborated expert recommendations by using a structured process. A panel of 19 experts took part in the consensus process using a 3-step modified Delphi method that consisted of 2 rounds of electronic voting and a final face-to-face virtual meeting. Eighty percent agreement was required for acceptance of the statements. A 2-level scale (strong, moderate) was used to grade the statements based on the perceived likelihood of a clinical benefit. The existing evidence supports an association between TTFM readings and graft patency and postoperative clinical outcomes, although there is high methodological heterogeneity among the published series. The evidence is more robust for arterial, rather than venous, grafts and for grafts to the left anterior descending artery. Although TTFM use increases the duration and the cost of surgery, there are no data to quantify this effect. Based on the systematic review, 10 expert statements for TTFM use in clinical practice were formulated. Six were approved at the first round of voting, 3 at the second round, and 1 at the virtual meeting. In conclusion, although TTFM use may increase the costs and duration of the procedure and requires a learning curve, its cost/benefit ratio seems largely favorable, in view of the potential clinical consequences of graft dysfunction. These consensus statements will help to standardize the use of TTFM in clinical practice and provide guidance in clinical decision-making.

Five-Year Survival of Transcatheter Aortic Valve Implantation in High-Risk Patients.

BACKGROUND: Although transcatheter aortic valve implantation (TAVI) has become the standard treatment for severe aortic stenosis in high-risk patients in Australia, there is still limited data on long term survival. METHODS: All patients undergoing TAVI at a single tertiary institution between September 2009 and December 2015 were included. The primary outcome was survival, by linkage of patients with the National Death Index of the Australian Institute of Health and Welfare. Post-procedure data and echocardiographic measurements were retrospectively analysed for all patients. RESULTS: A total of 186 patients were included. It was a high-risk patient population (mean EuroSCORE 31.5±20.5, mean age 83.0±8.2 years). Valve prostheses used were Edwards SAPIEN (ES) (Edwards, Irvine, CA, USA) in 16.1%, Edwards SAPIEN XT (ESXT) in 74.2%, and Medtronic CoreValve (MCV) (Medtronic, Minneapolis, MN, USA) in 9.7%. Median survival time for the entire cohort was 68.2 months (95% Confidence Interval [CI]; Lower Limit [LL] 58.0 months, Upper Limit [UL] not defined). The 2- and 5-year estimates of survival were 85% (LL 80%, UL 90%) and 56% (LL 48%, UL 66%), respectively. There was no statistically significant difference in median survival between the ES and ESXT valves, or implantation approach. Survival was greater in patients with creatinine <200 µmol/L compared to >200 µmol/L (68.8 months [LL 61.4, UL n/a] vs 48.0 months [LL 25.5, UL n/a]). Over the study period, there was a statistically significant trend in increasing mean transvalvular gradient (ES: 1.66 mmHg/yr, p=0.0058; ESXT: 2.50 mmHg/yr, p≤0.001) and maximum velocity (ESXT: 0.16 m/s/yr, p=0.004) and decreasing valve area (ESXT: -0.07 cm2/yr, p<0.001). There was substantial attrition of patient echocardiographic follow-up (number of echocardiograms followed up at 5 years=6, number at risk=41). CONCLUSIONS: This study has demonstrated acceptable survival in a high-risk cohort of patients undergoing TAVI, with comparable results to larger international experiences. There was a trend for worsening haemodynamics that needs to be monitored. Future studies need to examine patient quality of life and the performance of newer generation prostheses.

Studying the Endothelial Glycocalyx in vitro: What Is Missing?

All human cells are coated by a surface layer of proteoglycans, glycosaminoglycans (GAGs) and plasma proteins, called the glycocalyx. The glycocalyx transmits shear stress to the cytoskeleton of endothelial cells, maintains a selective permeability barrier, and modulates adhesion of blood leukocytes and platelets. Major components of the glycocalyx, including syndecans, heparan sulfate, and hyaluronan, are shed from the endothelial surface layer during conditions including ischaemia and hypoxia, sepsis, atherosclerosis, diabetes, renal disease, and some viral infections. Studying mechanisms of glycocalyx damage in vivo can be challenging due to the complexity of immuno-inflammatory responses which are inextricably involved. Previously, both static as well as perfused in vitro models have studied the glycocalyx, and have reported either imaging data, assessment of barrier function, or interactions of blood components with the endothelial monolayer. To date, no model has simultaneously incorporated all these features at once, however such a model would arguably enhance the study of vasculopathic processes. This review compiles a series of current in vitro models described in the literature that have targeted the glycocalyx layer, their limitations, and potential opportunities for further developments in this field.

Minimally-invasive versus transcatheter aortic valve implantation: systematic review with meta-analysis of propensity-matched studies.

BACKGROUND: Minimally invasive aortic valve replacement (MiAVR) and transcatheter aortic valve implantation (TAVI) provide aortic valve replacement (AVR) by less invasive methods than conventional surgical AVR, by avoiding complete sternotomy. This study directly compares and analyses the available evidence for early outcomes between these two AVR methods. METHODS: Electronic databases were searched from inception until August 2019 for studies comparing MiAVR to TAVI, according to predefined search criteria. Propensity-matched studies with sufficient data were included in a meta-analysis. RESULTS: Eight studies with 9,744 patients were included in the quantitative analysis. Analysis of risk-matched patients showed no difference in early mortality (RR 0.76, 95% CI, 0.37-1.54, P=0.44). MiAVR had a signal towards lower rate of postoperative stroke, although this did not reach statistical significance (OR 0.42, 95% CI, 0.13-1.29, P=0.13). MiAVR had significantly lower rates of new pacemaker (PPM) requirement (OR 0.29, 95% CI, 0.16-0.52, P<0.0001) and postoperative aortic insufficiency (AI) or paravalvular leak (PVL) (OR 0.05, 95% CI, 0.01-0.20, P<0.0001) compared to TAVI, (OR 0.42, 95% CI, 0.13-1.29, P=0.13), while acute kidney injury (AKI) was higher in MiAVR compared to TAVI (11.1% vs. 5.2%, OR 2.28, 95% CI, 1.25-4.16, P=0.007). CONCLUSIONS: In patients of equivalent surgical risk scores, MiAVR may be performed with lower rates of postoperative PPM requirement and AI/PVL, higher rates of AKI and no statistical difference in postoperative stroke or short-term mortality, compared to TAVI. Further prospective trials are needed to validate these results.

Toward stroke-free coronary surgery: The role of the anaortic off-pump bypass technique.

Surgical coronary revascularization remains the preferred strategy in a significant portion of patients with coronary artery disease due to superior long-term outcomes. However, there is a significant risk of perioperative neurologic injury that has influenced guideline recommendations. These complications occur in 1%-5% of patients, ranging from overt neurologic deficits with permanent disability, to subtle cerebral defects noted on neuroimaging that may result in slow cognitive and functional decline. The primary mechanism by which these events occur is thromboembolism from manipulation of the ascending aorta. This occurs during cardiopulmonary bypass, aortic cross-clamping, and partial occlusion clamping (side clamp). Elderly patients and patients with aortic atheroma are, therefore, at significantly increased risk. Initial surgical techniques addressed this by aggressively debriding or replacing the ascending aorta during coronary artery bypass grafting (CABG). Strategies then moved toward minimizing aortic manipulation through pump-assisted beating heart surgery and off-pump surgery with partial occlusion clamping or proximal anastomosis devices. Finally, anaortic off-pump CABG aims to avoid all manipulation of the ascending aorta through advanced off-pump grafting techniques combined with in situ and composite grafts. This has been demonstrated to result in the greatest reduction in risk. Establishing successful anaortic off-pump CABG programs requires subspecialization and focused interest groups dedicated to advancing CABG outcomes.

Assessing potential for aortoiliac vascular injury from venoarterial extracorporeal membrane oxygenation cannulae: An in vitro particle image velocimetry study.

Limb ischemia is a major complication associated with peripheral venoarterial extracorporeal membrane oxygenation (VA-ECMO). The high velocity jet from arterial cannulae can cause "sandblasting" injuries to the arterial endothelium, with the potential risk of distal embolization and end organ damage. The aim of this study was to identify, for a range of clinically relevant VA-ECMO cannulae and flow rates, any regions of peak flow velocity on the aortic wall which may predispose to vascular injury, and any regions of low-velocity flow which may predispose to thrombus formation. A silicone model of the aortic and iliac vessels was sourced and the right external iliac artery was cannulated. Cannulae ranged from 15 to 21 Fr in size. Simulated steady state ECMO flow rates were instituted using a magnetically levitated pump (CentriMag pump). Adaptive particle image velocimetry was performed for each cannula at 3, 3.5, 4, and 4.5 L/min. For all cannulae, in both horizontal and vertical side hole orientations, the peak velocity on the aortic wall ranged from 0.3 to 0.45 m/s, and the regions of lowest velocity flow were 0.05 m/s. The magnitude of peak velocity flow on the aortic wall was not different between a single pair versus multiple pairs of side holes. Maximum velocity flow on the aortic wall occurred earlier at a lower pump flow rate in the vertical orientation of distal side holes compared to a horizontal position. The presence of multiple paired side holes was associated with fewer low-velocity flow regions, and some retrograde flow, in the distal abdominal aorta compared to cannulae with a single pair of side holes. From this in vitro visualization study, the selection of a cannula design with multiple versus single pairs of side holes did not change the magnitude of peak velocity flow delivered to the vessel wall. Cannulae with multiple side holes were associated with fewer regions of low-velocity flow in the distal abdominal aorta. Further in vivo studies, and ideally clinical data would be required to assess any correlation of peak velocity flows with incidence of vascular injury, and any low-velocity flow regions with incidence of thrombosis.

Cryobiopsy for Identification of Usual Interstitial Pneumonia and Other Interstitial Lung Disease Features. Further Lessons from COLDICE, a Prospective Multicenter Clinical Trial.

Rationale: Transbronchial lung cryobiopsy (TBLC) is an emerging technique for interstitial lung disease diagnosis. Good histopathologic agreement between TBLC and surgical lung biopsy (SLB) was demonstrated in the COLDICE (Cryobiopsy versus Open Lung Biopsy in the Diagnosis of Interstitial Lung Disease Alliance) study; however, diagnostic confidence was frequently lower for TBLC than SLB. Objectives: To characterize specific features of TBLC predictive of usual interstitial pneumonia (UIP) in corresponding SLB and to identify clinical indices predictive of biopsy concordance. Methods: The COLDICE study was a prospective, multicenter study investigating diagnostic agreement between TBLC and SLB. The participants underwent both procedures with blinded pathologist analysis of specimens, applying international guideline criteria. The TBLC features predictive of UIP in the paired SLB and predictive features of overall concordance were analyzed. Measurements and Main Results: A total of 65 patients (66.1 ± 9.3 yr; FVC, 84.7 ± 14.2%; DlCO, 63.4 ± 13.8%) participated in the COLDICE study. UIP was identified in 33/65 (50.8%) SLB, and 81.5% were concordant with corresponding TBLC (κ, 0.61; 95% confidence interval [CI], 0.38-0.77). The UIP guideline criteria of "predominantly subpleural or paraseptal fibrosis" was infrequently reported in TBLC (8/33, 24.2%), whereas "patchy fibrosis," "fibroblast foci," and the "absence of alternative diagnostic features" were frequently observed in TBLC. The combination of these three features strongly predicted UIP in paired SLB (odds ratio [OR], 23.4; 95% CI, 6.36-86.1; P < 0.0001). Increased numbers of TBLC samples predicted histopathologic concordance with SLB (OR, 1.8; 95% CI, 1.08-3.01; P = 0.03). The predictors of discordance included older age, family history, and radiologic asymmetry. Conclusions: Subpleural and/or paraseptal fibrosis were not essential for diagnosing UIP in TBLC, provided that other guideline criteria features were present. The diagnostic accuracy of TBLC was strengthened when increased numbers of samples were taken. Clinical trial registered with www.anzctr.org.au (ACTRN12615000718549).