Factors associated with father-infant bonding during the COVID-19 pandemic: an internet-based cross-sectional study in Japan.

The COVID-19 pandemic has forced lifestyles changes and affected the relationships between fathers and their infants. However, the factors associated with paternal-infant bonding have not been clarified. This study aimed to explore the factors associated with father-infant bonding during the COVID-19 pandemic in Japan. This cross-sectional study used data from a nationwide survey and the Japanese version of the Mother-to-Infant Bonding Scale (MIBS) to measure father-infant bonding. The participants were divided into two groups depending on their partners' parity. A linear regression model (Gauss-Markov-type) was used for both groups. A total of 1055 men were included in the analysis. Of these men, 521 (49.4%) had a primipara partner, and 534 (50.6%) had a multipara partner. No significant differences were found between the two groups' MIBS-J scores. Fathers' mental health, relationship with the partner and family members, abusive behavior towards children, wanted pregnancy, and the youngest child's Neonatal Intensive Care Unit admission history were associated with father-infant bonding. Regarding factors related to COVID-19, caring for the child while the partner is at home has a negative impact on bonding, while fear related to infection with COVID-19 has no negative impact on bonding.

Pulmonary Vein Stenosis After Catheter Ablation of Atrial Fibrillation Using a Cryoballoon, Hot Balloon, or Laser Balloon.

BACKGROUND: Pulmonary vein stenosis (PVS) after PV isolation (PVI) for atrial fibrillation (AF) is a severe complication that requires angioplasty. This study aimed to compare the reduction of the cross-sectional PV area (PVA) and the incidence of PVS after cryoballoon (CB)-PVI, hot balloon (HB)-PVI, or laser balloon (LB)-PVI.Methods and Results: A total of 320 patients who underwent an initial catheter ablation procedure for AF using a CB, HB, or LB in 2 hospitals were included. They underwent contrast-enhanced multidetector CT before and 3 months after the procedure. In all 4 PVs, the reduction in PVA was more significant in the LB group than in the CB or HB groups, respectively. Moderate (50-75%) and severe (>75%) PVS were observed in 5.3% and 0.5% of the PVs, respectively. Although moderate PVS was more frequently observed in the LB group than in the CB or HB groups (8.2%, 3.8%, and 5.0%; P=0.03), the incidence of severe PVS was similar in the LB, CB, and HB groups (0.3%, 0.5%, and 1.0%; P=0.46). Symptomatic PVS requiring intervention occurred in 1 (0.3%) patient. CONCLUSIONS: Although the reduction in cross-sectional PVA and the incidence of moderate PVS after LB-PVI was more significant than after CB-PVI or HB-PVI, it rarely led to severe PVS. Symptomatic PVS requiring intervention was rare after the balloon ablation of AF.

Using the pre-freezing technique results in a wider area of antral isolation during cryoballoon pulmonary vein isolation in patients with atrial fibrillation.

The "pre-freezing" technique was a method in which a fully inflated balloon after the start of freezing was pressed against the pulmonary vein (PV) during cryoballoon ablation and has been applied especially in large-size PVs. Of 556 patients who underwent cryoballoon ablation for atrial fibrillation (AF), the pre-freezing technique was applied to 48 patients. The resulting 2:1 propensity score-matched data set included 120 patients. Using the pre-freezing technique, all left-superior PVs, all left-inferior PVs, and 95% of right-superior PVs were successfully isolated. In most right-inferior PVs, complete sealing using the pre-freezing technique was challenging, and this technique was not applied. Procedure time was similar between the two groups. In the pre-freezing group, the percentage of the left atrial posterior wall isolated was larger (47.6 ± 10.3 vs. 42.8 ± 15.7%, P = 0.006), and the postoperative reduction of diaphragmatic compound motor action potentials tended to occur less frequently (2.5 vs. 12.5%, P = 0.07), and the reduction of the cross-sectional LSPV area was smaller (17.5 ± 12.2 vs. 27.2 ± 19.8%, P = 0.03) than the conventional group. The AF-free rate of the two groups was similar between the two groups (P = 0.15). The pre-freezing technique was a simple method that can isolate a wider surface area during cryoballoon PV isolation. While the postoperative AF recurrence was comparable, the postoperative reduction in the cross-sectional PV area was less than that of the conventional method, which may reduce the risk of PV stenosis.

A 15-year follow-up study of radiofrequency catheter ablation for atrial fibrillation in patients with tachycardia-bradycardia syndrome.

INTRODUCTION: Catheter ablation for atrial fibrillation (AF) in patients with tachycardia-bradycardia syndrome (TBS) can be a major therapeutic option to replace permanent pacemaker implantation (PMI). However, the very long-term outcome of more than 15 years in these patients has not been elucidated. METHODS: From 2002 to 2008, 25 consecutive TBS patients (62 ± 7.9 years old, 68% male) with both AF and symptomatic sinus pauses (>3.0 s) were performed radiofrequency AF ablation. These patients were followed for 15 ± 2.7 years. RESULTS: The median longest sinus pause before the ablation procedure was 6.0 s (4.4-8.0). Following 1.6± 0.8 ablation procedures, 18 (72%) patients remained free from AF. Three (12%) patients died due to noncardiovascular causes, and seven (28%) patients underwent PMI due to symptomatic sinus pause after recurrent AF in five patients and progression of sinus node dysfunction in two patients. The median duration from the first AF ablation to PMI was 6.3 years (range: 9 days to 11.0 years). Five and two patients required PMI more than 5 and 10 years after the first ablation procedure, respectively. CONCLUSION: AF ablation prevented PMI in 72% of TBS patients for a 15-year follow-up. However, in consideration of the long duration of PMI, a continuous careful long-term follow-up was warranted.

Effect of air removal with extracorporeal balloon inflation on incidence of asymptomatic cerebral embolism during cryoballoon ablation of atrial fibrillation: A prospective randomized study.

Background: It was previously reported, based on a retrospective study, that preliminary removal of air bubbles in heparinized saline water with extracorporeal balloon inflation reduced the incidence of asymptomatic cerebral embolism (ACE). The present study aims to compare the incidence of ACE between a conventional and pre-inflation method during cryoballoon ablation in a prospective randomized controlled study. Methods: A total of 98 atrial fibrillation patients were enrolled and randomized into conventional and pre-inflation groups. Patients in the pre-inflation group received balloon massaging with preliminary extracorporeal balloon inflation in saline water before the cryoballoon was inserted into the body. Results: The baseline characteristics were similar between the two groups. Post-procedural 3-Tesla MRI revealed CE in 27.6% of patients. Symptomatic CE only occurred in two patients in the pre-inflation group. One patient had transient dysarthria and mild muscle weakness in one hand; the other patient complained of transient left upper limb weakness, left lower limb paresthesia and dysarthria. The incidence of ACE detected by cerebral MRI did not differ between the two groups to a statistically significant extent (conventional vs. pre-inflation; 22.9% vs. 29.2%; P = 0.49). In the multivariable analysis, eGFR was independently associated with the presence of ACE (odds ratio 0.95; 95% confidence interval 0.907-0.995; P = 0.03). Conclusion: In this prospective randomized study, the preliminary removal of air bubbles in heparinized saline water with extracorporeal balloon inflation had no impact on the incidence of ACE.

Transition of the heart rate and atrial premature complex after cryoballoon vs. radiofrequency ablation for paroxysmal atrial fibrillation.

The temporal changes in ambulatory monitoring findings after cryoballoon (CB) ablation of atrial fibrillation (AF) have not been well elucidated. This study aims to compare the details of ambulatory monitoring after CB and radiofrequency catheter (RFC) ablation for AF. Of 724 consecutive AF patients who underwent initial ablation using a CB or RFC, 508 (254 pairs) were selected using propensity score matching. Ambulatory monitoring was performed at 1, 3, 6, 12, 24 and 36 months after the procedure. After 1, 3 and 6 months, the number of total heart beats (THBs) was larger in the CB group than in the RFC group. It gradually decreased and became significantly similar by 12 months after ablation. THBs significantly increased 1, 3, 6 and 12 months after ablation in both the RFC and CB groups and became statistically similar by 24 months after ablation. The atrial premature contraction burden was higher in the RFC group than in the CB group at 3 months after ablation. THB and APC burden after AF ablation were significantly different between the RF and CB groups. THBs returned to statistically similarity by 2 years after ablation in both groups.

Very long term outcome after linear versus electrogram guided ablation for persistent atrial fibrillation.

The optimal ablation strategy for persistent atrial fibrillation (PsAF) remains to be defined. We sought to compare very long-term outcomes between linear ablation and electrogram (EGM)-guided ablation for PsAF. In a retrospective analysis, long-term arrhythmia-free survival compared between two propensity-score matched cohorts, one with pulmonary vein isolation (PVI) and linear ablation including roof/mitral isthmus line (LINE-group, n = 52) and one with PVI and EGM-guided ablation (EGM-group; n = 52). Overall, 99% of patients underwent successful PVI. Complete block following linear ablation was achieved for 94% of roof lines and 81% of mitral lines (both lines blocked in 75%). AF termination by EGM-guided ablation was accomplished in 40% of patients. Non-PV foci were targeted in 7 (13%) in the LINE-group and 5 (10%) patients in the EGM-group (p = 0.76). During 100 ± 28 months of follow-up, linear ablation was associated with superior arrhythmia-free survival after the initial and last procedure (1.8 ± 0.9 procedures) compared with EGM-group (Logrank test: p = 0.0001 and p = 0.045, respectively). In multivariable analysis, longer AF duration and EGM-guided ablation remained as independent predictors of atrial arrhythmia recurrence. Linear ablation might be a more effective complementary technique to PVI than EGM-guided ablation for PsAF ablation.

Identification and characterization of a novel Enterococcus bacteriophage with potential to ameliorate murine colitis.

Increase of the enteric bacteriophages (phage), components of the enteric virome, has been associated with the development of inflammatory bowel diseases. However, little is known about how a given phage contributes to the regulation of intestinal inflammation. In this study, we isolated a new phage associated with Enterococcus gallinarum, named phiEG37k, the level of which was increased in C57BL/6 mice with colitis development. We found that, irrespective of the state of inflammation, over 95% of the E. gallinarum population in the mice contained phiEG37k prophage within their genome and the phiEG37k titers were proportional to that of E. gallinarum in the gut. To explore whether phiEG37k impacts intestinal homeostasis and/or inflammation, we generated mice colonized either with E. gallinarum with or without the prophage phiEG37k. We found that the mice colonized with the bacteria with phiEG37k produced more Mucin 2 (MUC2) that serves to protect the intestinal epithelium, as compared to those colonized with the phage-free bacteria. Consistently, the former mice were less sensitive to experimental colitis than the latter mice. These results suggest that the newly isolated phage has the potential to protect the host by strengthening mucosal integrity. Our study may have clinical implication in further understanding of how bacteriophages contribute to the gut homeostasis and pathogenesis.

Long-term course of phrenic nerve injury after cryoballoon ablation of atrial fibrillation.

While phrenic nerve palsy (PNP) due to cryoballoon pulmonary vein isolation (PVI) of atrial fibrillation (AF) was transient in most cases, no studies have reported the results of the long-term follow-up of PNP. This study aimed to summarize details and the results of long-term follow-up of PNP after cryoballoon ablation. A total of 511 consecutive AF patients who underwent cryoballoon ablation was included. During right-side PVI, the diaphragmatic compound motor action potential (CMAP) was reduced in 46 (9.0%) patients and PNP occurred in 29 (5.7%) patients (during right-superior PVI in 20 patients and right-inferior PVI in 9 patients). PNP occurred despite the absence of CMAP reduction in 0.6%. The PV anatomy, freezing parameters and the operator's proficiency were not predictors of PNP. While PNP during RSPVI persisted more than 4 years in 3 (0.6%) patients, all PNP occurred during RIPVI recovered until one year after the ablation. However, there was no significant difference in the recovery duration from PNP between PNP during RSPVI and RIPVI. PNP occurred during cryoballoon ablation in 5.7%. While most patients recovered from PNP within one year after the ablation, PNP during RSPVI persisted more than 4 years in 0.6% of patients.

EP4 signalling is essential for controlling islet inflammation by causing a shift in macrophage polarization in obesity/type 2 diabetes.

Activation of the prostaglandin E2 receptor EP4 alters polarization of adipose tissue macrophages towards the anti-inflammatory M2 phenotype to suppress chronic inflammation. However, the role of EP4 signalling in pancreatic macrophages that affect insulin secretion is unclear. We examined the role of EP4 signalling in islet inflammation in vitro and in vivo. Obese diabetic db/db mice were treated with an EP4-selective agonist or vehicle for 4 weeks. Islet morphology did not significantly differ and glucose-stimulated insulin secretion was increased, whereas the pancreatic M1/M2 ratio was decreased in the EP4 agonist-treated group compared to the vehicle group. Because EP4 activation in MIN6 cells did not affect insulin secretion, we used a MIN6/macrophage co-culture system to evaluate the role of EP4 signalling in islet inflammation and subsequent inhibition of insulin release. Co-culture with M1-polarized macrophages markedly suppressed insulin expression in MIN6 cells; however, modulation of M1 polarization by the EP4 agonist significantly reversed the negative impact of co-cultivation on insulin production. The enhanced expression levels of pro-inflammatory cytokines in co-cultured MIN6 cells were markedly inhibited by EP4 agonist treatment of M1 macrophages. Thus, EP4 activation may suppress islet inflammation and protect ß-cell function by altering inflammatory macrophages in the diabetic pancreas.

Anatomical and Procedural Factors of Severe Pulmonary Vein Stenosis After Cryoballoon Pulmonary Vein Ablation.

OBJECTIVES: This study examined the anatomical or procedural factors associated with severe pulmonary vein (PV) stenosis after cryoballoon PV isolation. BACKGROUND: PV stenosis is a complication associated with cryoballoon ablation. METHODS: The study included 170 consecutive patients with paroxysmal atrial fibrillation who underwent cryoballoon ablation. In addition to factors generally considered to be related to the occurrence of PV stenosis (PV size, cryoballoon application number and time, and minimum freezing temperature), we evaluated the following 4 factors: 1) depth of balloon position; 2) the PV angle (internal angle between each PV and horizontal line); 3) noncoaxial balloon placement (hemispherical occlusion); and 4) contact surface area between the cryoballoon and the PV wall (defined as the balloon contact ratio). RESULTS: Severe PV stenosis (≥75% area reduction) was observed in 9 (1.3%) PVs (6 left superior and 3 right superior PVs) in 9 patients. The PV size, cryoballoon application number and time, minimum freezing temperature, and the depth of cryoballoon position were not significantly associated with occurrence of severe PV stenosis, but the PV angle was significantly smaller in PVs with severe stenosis than it was in those without stenosis (25.6 ± 9.7° vs. 34.2 ± 6.4°; p < 0.001). Hemispherical occlusion was more frequently observed and balloon contact ratio was larger in PVs with severe stenosis (55.6% vs. 14.8%; p = 0.049) than in those without stenosis (0.70 ± 0.06 vs. 0.54 ± 0.08; p < 0.001). CONCLUSIONS: A horizontally connecting PV, noncoaxial placement of cryoballoon, and a larger contact surface area of the cryoballoon were predictors of the occurrence of severe PV stenosis after cryoballoon ablation.

Comparison of atrial arrhythmia recurrence after persistent atrial fibrillation ablation between patients with or without tachycardia-induced cardiomyopathy.

BACKGROUND: The presence of heart failure (HF) has been associated with poorer outcomes in patients undergoing catheter ablation (CA) for atrial fibrillation (AF). However, the effectiveness of CA amongst the subset of patients with tachycardia-induced cardiomyopathy (TIC) remains poorly defined. METHODS AND RESULTS: In a retrospective analysis we compared outcomes of first-time CA for persistent AF in a cohort of patients with previously diagnosed TIC (n = 45; age 58 ± 8 years; 91% male) to those with structurally normal hearts (non-TIC; n = 440; age 55 ± 9 years; 95% male). TIC was defined as an impaired ventricular function (left ventricular ejection function [LVEF] <50%), which was reversed after the treatment of HF. We compared atrial arrhythmias (AAs) recurrence after the CA in the TIC and non-TIC cohorts. In the TIC group, LVEF improved from 35.8% ± 8.1% to 57.5% ± 8.3% after treatment of HF. During 3.3 ± 1.5 years follow-up, AAs-free survival after CA was significantly higher in the TIC group as compared with the non-TIC group (69% vs 42%; P = .001), despite a comparable CA strategy between the two groups. In multivariable analysis, absence of HF with TIC, longer AF duration, and complex fractionated atrial electrogram ablation were independent predictors of arrhythmia recurrence (OR, 1.02; 95% CI, 1.01-1.03; P < .01; OR, 0.40; 95% CI, 0.20-0.79; P < .01 and OR, 2.29; 95%CI; 1.27-4.11; P < .01, respectively). In addition, the outcome after the last procedure was superior in the TIC cohort (89% vs 72%; P = .03) with fewer CA procedures as compared with the non-TIC cohort (1.3 ± 0.5 vs 1.5 ± 0.7; P = .01). CONCLUSIONS: Persistent patients with AF with TIC have a more favorable outcome after the CA as compared with those without.

Clinical significance of early recurrence of atrial fibrillation after cryoballoon vs. radiofrequency ablation-A propensity score matched analysis.

OBJECTIVES: One of the mechanisms of early recurrence of atrial fibrillation (ERAF) after AF ablation is considered to be the inflammatory reaction of the atrial tissue. The aim of this study is to compare the clinical significance of ERAF at each stage for true AF recurrence between cryoballoon (CB) and radiofrequency (RF) ablation. METHODS: Among 798 paroxysmal AF patients who underwent an initial ablation, 460 patients (CB, n = 230; RF, n = 230) were selected by propensity score matching. Very ERAF (VERAF), ERAF-1M, ERAF-3M and true AF recurrence were defined as AF recurrence at 0-2, 3-30, 31-90 days and more than 90 days after the procedure, respectively. RESULTS: The patient characteristics of the two groups were similar. ERAF was observed 21% and 27% in the CB and RF groups, respectively. In both the CB and RF group, VERAF, ERAF-1M and ERAF-3M were more frequently observed in patients with true AF recurrence than in those without. In a multivariable analysis, ERAF-1M and ERAF-3M were found to be independent predictors of true AF recurrence in both the CB (P = 0.04 and P<0.001, respectively) and RF groups (P = 0.02 and P = 0.001, respectively). However, while VERAF was associated with true AF recurrence after RF ablation (P = 0.03), it was not associated with true AF recurrence after CB ablation (P = 0.19). CONCLUSION: The relationship between ERAF and true AF recurrence differed between the RF and CB ablation groups. While VERAF was associated with true AF recurrence after RF ablation, it was not a predictor of true AF recurrence after CB ablation.

Radiofrequency needle for transseptal puncture is associated with lower incidence of thromboembolism during catheter ablation of atrial fibrillation: propensity score-matched analysis.

Atrial fibrillation (AF) ablation requires transseptal puncture to access the left atrium. Recently, a radiofrequency (RF) needle was developed. The purpose of this study was to compare the incidence of MRI-confirmed acute cerebral embolism (ACE) during AF ablation procedures performed with RF needle versus mechanical needle transseptal puncture. This study consisted of 383 consecutive patients who underwent catheter ablation for AF that required transseptal puncture with mechanical or radiofrequency transseptal needles. Of those, 232 propensity score-matched patients (116 with each needle type) were included in the analysis. All patients had cerebral MRI performed 1 or 2 days after the procedure. Baseline characteristics were similar between the two groups. Total procedure time was significantly shorter in Group RF than Group non-RF (167 ± 50 vs. 181 ± 52 min, P = 0.01). ACE was detected by MRI in 59 (25%) patients. All patients with ACE were asymptomatic. Incidence of ACE was lower in Group RF than Group non-RF (19 vs. 32%, P = 0.02). B-type natriuretic peptide level was higher in the patients with ACE as compared to those without ACE (65.2 ± 68.7 vs. 44.7 ± 55.1 pg/ml, P = 0.02). In multivariable analysis, the use of RF needle and BNP level was related to the incidence of ACE (OR = 0.499, 95% CI 0.270-0.922, P = 0.03 and OR = 1.005, 95% CI 1.000-1.010, P = 0.03). Use of RF needle for transseptal puncture was associated with lower total procedure time and risk of ACE during catheter ablation of AF.

Atrial fibrillation diagnosed by a medical checkup is associated with a poor outcome of catheter ablation.

Atrial fibrillation (AF), especially asymptomatic cases, is often detected by medical checkups. We investigated the outcome of AF ablation in cases detected by medical checkups. We reviewed the data of 735 patients with AF (56 ± 10 years, paroxysmal: 441 patients) who underwent initial catheter ablation. All patients were divided into two groups based on their AF being diagnosed either by a medical checkup (group M) or not (group NM). AF was diagnosed by medical checkups in 263 (36%) patients. In Group M, the age was younger, time from the diagnosis to ablation shorter, left atrium dimension larger, and left ventricular ejection fraction lower than in Group NM. Male gender, persistent AF, and asymptomatic AF were more frequently seen in Group M than in Group NM. A mean of 13 ± 11 months after the initial ablation procedure, AF recurrence was more frequently observed in group M compared to group NM (P = 0.018). While the AF recurrence rate was similar in both groups in persistent AF patients (P = 0.87), it was more frequently observed in Group M than in Group NM in paroxysmal AF patients (P = 0.005). AF diagnosed by medical checkups was often associated with a worse outcome of catheter ablation, especially in paroxysmal AF patients.

Spiral activation of the superior vena cava: The utility of ultra-high-resolution mapping for caval isolation.

BACKGROUND: Little is known about the manner in which the superior vena cava (SVC) is activated during sinus rhythm. OBJECTIVE: The purpose of this study was to assess the manner of caval activation with an ultra-high-density mapping system (Rhythmia, Boston Scientific, Marlborough, MA) and its utility for SVC isolation. METHODS: Forty patients with atrial fibrillation (mean age 55 ± 12 years; paroxysmal atrial fibrillation in 23[58%]) who underwent SVC mapping with Rhythmia were studied. The location of the sinus node (SN), phrenic nerve (PN), and the manner of caval activation during sinus rhythm were analyzed. The SVC was isolated by radiofrequency applications at electrical connections, shown as breakthroughs on the Rhythmia map. RESULTS: The SN location varied widely (lateral/posterior/anterior 60%/8%/32%), while the PN was mostly located in the lateral segment (lateral/anterior 82%/18%). In 36 patients who underwent SVC isolation, the mean number of breakthroughs was 2.5 ± 0.8. The first breakthrough predominantly located in the anterior aspect (anterior/septal/posterior/lateral 78%/14%/5%/3%), and atrial activation was conducted superiorly and clockwise from the SN (referred to as spiral activation) in 32 patients (89%). The mean rotation angle from the SN to the first breakthrough was 79° ± 41°. In addition, 10 patients (25%) showed the SN within the SVC. Although radiofrequency applications were needed at the PN capture site in 11 patients (31%) the SVC was successfully isolated without any complications in all patients. CONCLUSION: The SN location showed great heterogeneity; however, atrial activation predominantly showed a clockwise spiral form. This is the first report to use ultra-high-resolution mapping to demonstrate the manner of spiral activation, which is useful for the safe and efficient isolation of the SVC.

Pulmonary vein re-mapping after cryoballoon ablation for atrial fibrillation.

Aims: Establishment of pulmonary vein isolation (PVI) during cryoballoon (CB) ablation is generally confirmed by use of an octapolar inner-lumen mapping catheter (Achieve®). The aim of this study is to evaluate the residual PV potential (PVP) using the conventional circular catheter after CB-PVI. Methods and results: A total of 105 consecutive patients (418 PVs) with paroxysmal AF who underwent the initial CB-PVI were prospectively included in this study. Of those, 305 (73%) PVs with real-time recordings of PVP elimination by Achieve® catheter during successful PVI were included. After isolation of all 4 PVs, PV antral remapping by conventional circular mapping catheter was performed. After CB-PVI, residual PVP was detected in 4.3% (13/305) of PVs (1.2% of left-superior PV, 2.5% of left-inferior PV, none of right-superior PV, and 20% of right-inferior PV). Almost 60% of residual PV potential was located around the bottom portion of the right-inferior PV. In PVs with residual potential, PV trunk was shorter (12.7 ± 5.7 mm vs. 18.7 ± 7.9, P = 0.001), minimal balloon temperature was higher (-46.6 ± 5.9 °C vs. -50.9 ± 8.2, P = 0.02), and balloon warming time was shorter (35.6 ± 17.8 s vs. 50.0 ± 22.8, P = 0.006) than those without. All residual potentials were eliminated by additional touch up ablation. After the initial ablation procedure, 1-year AF-free rate was 79.5%. Conclusion: PV remapping after CB-PVI revealed residual antral PVP in 4.3% of PVs and in 20% of RIPVs in particular. The Achieve® catheter sometimes fails to detect complete PV antral isolation.