Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection.

Two mycobacteriophages were administered intravenously to a male with treatment-refractory Mycobacterium abscessus pulmonary infection and severe cystic fibrosis lung disease. The phages were engineered to enhance their capacity to lyse M. abscessus and were selected specifically as the most effective against the subject's bacterial isolate. In the setting of compassionate use, the evidence of phage-induced lysis was observed using molecular and metabolic assays combined with clinical assessments. M. abscessus isolates pre and post-phage treatment demonstrated genetic stability, with a general decline in diversity and no increased resistance to phage or antibiotics. The anti-phage neutralizing antibody titers to one phage increased with time but did not prevent clinical improvement throughout the course of treatment. The subject received lung transplantation on day 379, and systematic culturing of the explanted lung did not detect M. abscessus. This study describes the course and associated markers of a successful phage treatment of M. abscessus in advanced lung disease.

Quality of Life in Patients With Chronic Limb-Threatening Ischemia Treated With Revascularization.

BACKGROUND: In the BEST-CLI trial (Best Endovascular Versus Best Surgical Therapy for Patients With Chronic Limb-Threatening Ischemia), a prespecified secondary objective was to assess the effects of revascularization strategy on health-related quality of life (HRQoL). METHODS: Patients with chronic limb-threatening ischemia were randomized to surgical bypass (Bypass) or endovascular intervention (Endo) in 2 parallel trials. Cohort 1 included patients with single-segment great saphenous vein; cohort 2 included those lacking suitable single-segment great saphenous vein. HRQoL was assessed over the trial duration using Vascular Quality-of-Life (VascuQoL), European Quality-of-Life-5D (EQ-5D), the Short Form-12 (SF-12) Physical Component Summary (SF-12 PCS), SF-12 Mental Component Summary (SF-12 MCS), Utility Index Score (SF-6D R2), and numeric rating scales of pain. HRQoL was summarized by cohort and compared within and between groups using mixed-model linear regression. RESULTS: A total of 1193 and 335 patients in cohorts 1 and 2 with a mean follow-up of 2.9 and 2.0 years, respectively, were analyzed. In cohort 1, HRQoL significantly improved from baseline to follow-up for both groups across all measures. For example, mean (SD) VascuQoL scores were 3.0 (1.3) and 3.0 (1.2) for Bypass and Endo at baseline and 4.7 (1.4) and 4.8 (1.5) over follow-up. There were significant group differences favoring Endo when assessed with VascuQoL (difference, -0.14 [95% CI, -0.25 to -0.02]; P=0.02), SF-12 MCS (difference, -1.03 [95% CI, -1.89 to -0.18]; P=0.02), SF-6D R2 (difference, -0.01 [95% CI, -0.02 to -0.001]; P=0.03), numeric rating scale pain at present (difference, 0.26 [95% CI, 0.03 to 0.49]; P=0.03), usual level during previous week (difference, 0.26 [95% CI, 0.04 to 0.48]; P=0.02), and worst level during previous week (difference, 0.29 [95% CI, 0.02 to 0.56]; P=0.04). There was no difference between treatment arms on the basis of EQ-5D (difference, -0.01 [95% CI, -0.03 to 0.004]; P=0.12) or SF-12 PCS (difference, -0.41 [95% CI, -1.2 to 0.37]; P=0.31). In cohort 2, HRQoL also significantly improved from baseline to the end of follow-up for both groups based on all measures, but there were no differences between Bypass and Endo on any measure. CONCLUSIONS: Among patients with chronic limb-threatening ischemia deemed eligible for either Bypass or Endo, revascularization resulted in significant and clinically meaningful improvements in HRQoL. In patients with an available single-segment great saphenous vein for bypass, but not among those without one, Endo was statistically superior on some HRQoL measures; however, these differences were below the threshold of clinically meaningful difference.

Surgery or Endovascular Therapy for Chronic Limb-Threatening Ischemia.

BACKGROUND: Patients with chronic limb-threatening ischemia (CLTI) require revascularization to improve limb perfusion and thereby limit the risk of amputation. It is uncertain whether an initial strategy of endovascular therapy or surgical revascularization for CLTI is superior for improving limb outcomes. METHODS: In this international, randomized trial, we enrolled 1830 patients with CLTI and infrainguinal peripheral artery disease in two parallel-cohort trials. Patients who had a single segment of great saphenous vein that could be used for surgery were assigned to cohort 1. Patients who needed an alternative bypass conduit were assigned to cohort 2. The primary outcome was a composite of a major adverse limb event - which was defined as amputation above the ankle or a major limb reintervention (a new bypass graft or graft revision, thrombectomy, or thrombolysis) - or death from any cause. RESULTS: In cohort 1, after a median follow-up of 2.7 years, a primary-outcome event occurred in 302 of 709 patients (42.6%) in the surgical group and in 408 of 711 patients (57.4%) in the endovascular group (hazard ratio, 0.68; 95% confidence interval [CI], 0.59 to 0.79; P<0.001). In cohort 2, a primary-outcome event occurred in 83 of 194 patients (42.8%) in the surgical group and in 95 of 199 patients (47.7%) in the endovascular group (hazard ratio, 0.79; 95% CI, 0.58 to 1.06; P = 0.12) after a median follow-up of 1.6 years. The incidence of adverse events was similar in the two groups in the two cohorts. CONCLUSIONS: Among patients with CLTI who had an adequate great saphenous vein for surgical revascularization (cohort 1), the incidence of a major adverse limb event or death was significantly lower in the surgical group than in the endovascular group. Among the patients who lacked an adequate saphenous vein conduit (cohort 2), the outcomes in the two groups were similar. (Funded by the National Heart, Lung, and Blood Institute; BEST-CLI ClinicalTrials.gov number, NCT02060630.).

Axon guidance genes are regulated by TDP-43 and RGNEF through long-intron removal.

Rho guanine nucleotide exchange factor (RGNEF) is a guanine nucleotide exchange factor (GEF) mainly involved in regulating the activity of Rho-family GTPases. It is a bi-functional protein, acting both as a guanine exchange factor and as an RNA-binding protein. RGNEF is known to act as a destabilizing factor of neurofilament light chain RNA (NEFL) and it could potentially contribute to their sequestration in nuclear cytoplasmic inclusions. Most importantly, RGNEF inclusions in the spinal motor neurons of ALS patients have been shown to co-localize with inclusions of TDP-43, the major well-known RNA-binding protein aggregating in the brain and spinal cord of human patients. Therefore, it can be hypothesized that loss-of-function of both proteins following aggregation may contribute to motor neuron death/survival in ALS patients. To further characterize their relationship, we have compared the transcriptomic profiles of neuronal cells depleted of TDP-43 and RGNEF and show that these two factors predominantly act in an antagonistic manner when regulating the expression of axon guidance genes. From a mechanistic point of view, our experiments show that the effect of these genes on the processivity of long introns can explain their mode of action. Taken together, our results show that loss-of-function of factors co-aggregating with TDP-43 can potentially affect the expression of commonly regulated neuronal genes in a very significant manner, potentially acting as disease modifiers. This finding further highlights that neurodegenerative processes at the RNA level are the result of combinatorial interactions between different RNA-binding factors that can be co-aggregated in neuronal cells. A deeper understanding of these complex scenarios may lead to a better understanding of pathogenic mechanisms occurring in patients, where more than one specific protein may be aggregating in their neurons.

Mitigation of TDP-43 toxic phenotype by an RGNEF fragment in amyotrophic lateral sclerosis models.

Aggregation of the RNA-binding protein TAR DNA binding protein (TDP-43) is a hallmark of TDP-proteinopathies including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). As TDP-43 aggregation and dysregulation are causative of neuronal death, there is a special interest in targeting this protein as a therapeutic approach. Previously, we found that TDP-43 extensively co-aggregated with the dual function protein GEF (guanine exchange factor) and RNA-binding protein rho guanine nucleotide exchange factor (RGNEF) in ALS patients. Here, we show that an N-terminal fragment of RGNEF (NF242) interacts directly with the RNA recognition motifs of TDP-43 competing with RNA and that the IPT/TIG domain of NF242 is essential for this interaction. Genetic expression of NF242 in a fruit fly ALS model overexpressing TDP-43 suppressed the neuropathological phenotype increasing lifespan, abolishing motor defects and preventing neurodegeneration. Intracerebroventricular injections of AAV9/NF242 in a severe TDP-43 murine model (rNLS8) improved lifespan and motor phenotype, and decreased neuroinflammation markers. Our results demonstrate an innovative way to target TDP-43 proteinopathies using a protein fragment with a strong affinity for TDP-43 aggregates and a mechanism that includes competition with RNA sequestration, suggesting a promising therapeutic strategy for TDP-43 proteinopathies such as ALS and FTD.

Characterization of cardiovascular serious adverse events after bypass or endovascular revascularization for limb-threatening ischemia in the BEST-CLI trial.

OBJECTIVE: Cardiovascular complications after revascularization to treat chronic limb-threatening ischemia (CLTI) are a major concern that guides treatment. Our goal was to assess periprocedural cardiac and vascular serious adverse events (SAEs) in the Best Endovascular vs Best Surgical Therapy in Patients with CLTI (BEST-CLI) trial. METHODS: BEST-CLI was a prospective randomized trial comparing surgical (OPEN) and endovascular (ENDO) revascularization for patients with CLTI. Thirty-day SAEs, classified as cardiac or vascular, were analyzed. Adverse events are defined as serious when they affect safety in the trial, require prolonged hospitalization, result in significant disability or incapacitation, are life-threatening, or result in death. Interventions were analyzed in a per protocol fashion. RESULTS: In the BEST-CLI trial, 850 OPEN and 896 ENDO interventions were evaluated. Forty (4.7%) and 34 (3.8%) patients experienced at least one cardiac SAE after OPEN and ENDO intervention, respectively (P = .35). Overall, there were 53 cardiac SAEs (0.06 per patient) after OPEN and 40 (0.045 per patient) after ENDO interventions. Cardiac SAEs in the OPEN arm were classified as related to ischemia (50.9%), arrhythmias (17%), heart failure (15.1%), arrest (13.2%), and heart block (3.8%); in the ENDO arm, they were classified as ischemia (47.5%), heart failure (17.5%), arrhythmias (15%), arrest (15%), and heart block (5%). Approximately half of SAEs were classified as severe for both OPEN and ENDO. SAEs were definitely or probably related to the procedure in 30.2% and 25% in the OPEN and ENDO arms, respectively (P = .2). Vascular SAEs occurred in 58 (6.8%) and 86 (9.6%) of patients after OPEN and ENDO revascularization, respectively (P = .19). In total, there were 59 (0.07 per patient) and 87 (0.097 per patient) vascular SAEs after OPEN and ENDO procedures. Vascular SAEs in the OPEN arm were classified as distal ischemia/infection (44.1%), bleeding (16.9%), occlusive (15.3%), thromboembolic (15.3%), cerebrovascular (5.1%), and other (3.4%); in the ENDO arm, they were distal ischemia/infection (40.2%), occlusive (31%), bleeding (12.6%), thromboembolic (8%), cerebrovascular (1.1%), and other (4.6%). SAEs were classified as severe for OPEN in 45.8% and ENDO in 46%. SAEs were definitely or probably related to the procedure in 23.7% and 35.6% in the OPEN and ENDO arms (P = .35), respectively. CONCLUSIONS: Patients undergoing OPEN and ENDO revascularization experienced similar degrees of cardiac and vascular SAEs. The majority were not related to the index intervention, but approximately half were severe.

Investigator attitudes on equipoise and practice patterns in the BEST-CLI trial.

OBJECTIVES: There has been significant variability in practice patterns and equipoise regarding treatment approach for chronic limb-threatening ischemia (CLTI). We aimed to assess treatment preferences of Best Endovascular vs Best Surgical Therapy in Patients with CLTI (BEST-CLI) investigators prior to and following the trial. METHODS: An electronic 60-question survey was sent to 1180 BEST-CLI investigators in 2022, after trial conclusion and before announcement of results. Investigators' preferences were assessed across clinical scenarios for both open (OPEN) and endovascular (ENDO) revascularization strategies. Vascular surgeon (VS) surgical and ENDO preferences were compared with a 2010 survey administered to prospective investigators before trial funding. RESULTS: For the 2022 survey, the response rate was 20.2% and was comprised of VSs (76.3%), interventional cardiologists (11.4%) and interventional radiologists (11.6%). The majority (72.6%) were in academic practice and 39.1% were in practice for >20 years. During initial CLTI work-up, 65.8%, 42.6%, and 55.9% of respondents always or usually ordered an arterial duplex, computed tomography angiography, and vein mapping, respectively. The most common practice distribution between ENDO and OPEN procedures was 70/30. Postoperatively, a majority reported performing routine duplex surveillance of vein bypass (99%), prosthetic bypass (81.9%), and ENDO interventions (86%). A minority reported always or usually using the wound, ischemia, and foot infection (WIfI) criteria (25.8%), GLASS (8.3%), and a risk calculator (14.8%). More than one-half (52.9%) agreed that the statement "no bridges are burned with an ENDO-first approach" was false. Intervention choice was influenced by availability of the operating room or ENDO suite, personal schedule, and personal skill set in 30.1%, 18.0%, and 45.9% of respondents, respectively. Most respondents reported routinely using paclitaxel-coated balloons (88.1%) and stents (67.5%); however, 73.3% altered practice when safety concerns were raised. Among surgeons, 17.8%, 2.9%, and 10.3% reported performing >10 annual alternative autogenous vein bypasses, composite vein composite vein bypasses, and bypasses to pedal targets, respectively. Among all interventionalists, 8%, 24%, and 8% reported performing >10 annual radial access procedures, pedal or tibial access procedures, and pedal loop revascularizations. The majority (89.1%) of respondents felt that CLTI teams improved care; however, only 23.2% had a defined team. The effectiveness of the teamwork at institutions was characterized as highly effective in 42.5%. When comparing responses by VSs to the 2010 survey, there were no changes in preferred treatment based on Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC) II classification or conduit preference. In 2022, OPEN surgery was preferred more for a popliteal occlusion. For clinical scenarios, there were no differences except a decreased proportion of respondents who felt there was equipoise for major tissue loss for major tissue loss (43.8% vs 31.2%) and increased ENDO choice for minor tissue loss (17.6% vs 30.8%) (P < .05). CONCLUSIONS: There is a wide range of practice patterns among vascular specialists treating CLTI. The majority of investigators in BEST-CLI had experience in both advanced OPEN and ENDO techniques and represent a real-world sample of technical expertise. Over the course of the decade of the BEST-CLI trial, there was overall similar equipoise among VSs.

Secondary interventions following open vs endovascular revascularization for chronic limb threatening ischemia in the BEST-CLI trial.

OBJECTIVES: Patients undergoing revascularization for chronic limb-threatening ischemia experience a high burden of target limb reinterventions. We analyzed data from the Best Endovascular versus Best Surgical Therapy in Patients with Critical Limb Ischemia (BEST-CLI) randomized trial comparing initial open bypass (OPEN) and endovascular (ENDO) treatment strategies, with a focus on reintervention-related study endpoints. METHODS: In a planned secondary analysis, we examined the rates of major reintervention, any reintervention, and the composite of any reintervention, amputation, or death by intention-to-treat assignment in both trial cohorts (cohort 1 with suitable single-segment great saphenous vein [SSGSV], n = 1434; cohort 2 lacking suitable SSGSV, n = 396). We also compared the cumulative number of major and all index limb reinterventions over time. Comparisons between treatment arms within each cohort were made using univariable and multivariable Cox regression models. RESULTS: In cohort 1, assignment to OPEN was associated with a significantly reduced hazard of a major limb reintervention (hazard ratio [HR], 0.37; 95% confidence interval [CI], 0.28-0.49; P < .001), any reintervention (HR, 0.63; 95% CI, 0.53-0.75; P < .001), or any reintervention, amputation, or death (HR, 0.68; 95% CI, 0.60-0.78; P < .001). Findings were similar in cohort 2 for major reintervention (HR, 0.53; 95% CI, 0.33-0.84; P = .007) or any reintervention (HR, 0.71; 95% CI, 0.52-0.98; P = .04). In both cohorts, early (30-day) limb reinterventions were notably higher for patients assigned to ENDO as compared with OPEN (14.7% vs 4.5% of cohort 1 subjects; 16.6% vs 5.6% of cohort 2 subjects). The mean number of major (mean events per subject ratio [MR], 0.45; 95% CI, 0.34-0.58; P < .001) or any target limb reinterventions (MR, 0.67; 95% CI, 0.57-0.80; P < .001) per year was significantly less in the OPEN arm of cohort 1. The mean number of reinterventions per limb salvaged per year was lower in the OPEN arm of cohort 1 (MR, 0.45; 95% CI, 0.35-0.57; P < .001 and MR, 0.66; 95% CI, 0.55-0.79; P < .001 for major and all, respectively). The majority of index limb reinterventions occurred during the first year following randomization, but events continued to accumulate over the duration of follow-up in the trial. CONCLUSIONS: Reintervention is common following revascularization for chronic limb-threatening ischemia. Among patients deemed suitable for either approach, initial treatment with open bypass, particularly in patients with available SSGSV conduit, is associated with a significantly lower number of major and minor target limb reinterventions.

Surgery or endovascular therapy for patients with chronic limb-threatening ischemia requiring infrapopliteal interventions.

OBJECTIVE: The recent publication of randomized trials comparing open bypass surgery to endovascular therapy in patients with chronic limb-threatening ischemia, namely, Best Endovascular vs Best Surgical Therapy in Patients with Critical Limb Ischemia (BEST-CLI) and Bypass versus Angioplasty in Severe Ischaemia of the Leg-2 (BASIL-2), has resulted in potentially contradictory findings. The trials differed significantly with respect to anatomical disease patterns and primary end points. We performed an analysis of patients in BEST-CLI with significant infrapopliteal disease undergoing open tibial bypass or endovascular tibial interventions to formulate a relevant comparator with the outcomes reported from BASIL-2. METHODS: The study population consisted of patients in BEST-CLI with adequate single segment saphenous vein conduit randomized to open bypass or endovascular intervention (cohort 1) who additionally had significant infrapopliteal disease and underwent tibial level intervention. The primary outcome was major adverse limb event (MALE) or all-cause death. MALE included any major limb amputation or major reintervention. Outcomes were evaluated using Cox proportional regression models. RESULTS: The analyzed subgroup included a total of 665 patients with 326 in the open tibial bypass group and 339 in the tibial endovascular intervention group. The primary outcome of MALE or all-cause death at 3 years was significantly lower in the surgical group at 48.5% compared with 56.7% in the endovascular group (P = .0018). Mortality was similar between groups (35.5% open vs 35.8% endovascular; P = .94), whereas MALE events were lower in the surgical group (23.3% vs 35.0%; P<.0001). This difference included a lower rate of major reinterventions in the surgical group (10.9%) compared with the endovascular group (20.2%; P = .0006). Freedom from above ankle amputation or all-cause death was similar between treatment arms at 43.6% in the surgical group compared with 45.3% the endovascular group (P = .30); however, there were fewer above ankle amputations in the surgical group (13.5%) compared with the endovascular group (19.3%; P = .0205). Perioperative (30-day) death rates were similar between treatment groups (2.5% open vs 2.4% endovascular; P = .93), as was 30-day major adverse cardiovascular events (5.3% open vs 2.7% endovascular; P = .12). CONCLUSIONS: Among patients with suitable single segment great saphenous vein who underwent infrapopliteal revascularization for chronic limb-threatening ischemia, open bypass surgery was associated with a lower incidence of MALE or death and fewer major amputation compared with endovascular intervention. Amputation-free survival was similar between the groups. Further investigations into differences in comorbidities, anatomical extent, and lesion complexity are needed to explain differences between the BEST-CLI and BASIL-2 reported outcomes.

The impact of revascularization strategy on clinical failure, hemodynamic failure, and chronic limb-threatening ischemia symptoms in the BEST-CLI Trial.

OBJECTIVE: Sustained clinical and hemodynamic benefit after revascularization for chronic limb-threatening ischemia (CLTI) is needed to resolve symptoms and prevent limb loss. We sought to compare rates of clinical and hemodynamic failure as well as resolution of initial and prevention of recurrent CLTI after endovascular (ENDO) vs bypass (OPEN) revascularization in the Best-Endovascular-versus-best-Surgical-Therapy-in-patients-with-CLTI (BEST-CLI) trial. METHODS: As planned secondary analyses of the BEST-CLI trial, we examined the rates of (1) clinical failure (a composite of all-cause death, above-ankle amputation, major reintervention, and degradation of WIfI stage); (2) hemodynamic failure (a composite of above-ankle amputation, major and minor reintervention to maintain index limb patency, failure to an initial increase or a subsequent decrease in ankle brachial index of 0.15 or toe brachial index of 0.10, and radiographic evidence of treatment stenosis or occlusion); (3) time to resolution of presenting CLTI symptoms; and (4) incidence of recurrent CLTI. Time-to-event analyses were performed by intention-to-treat assignment in both trial cohorts (cohort 1: suitable single segment great saphenous vein [SSGSV], N = 1434; cohort 2: lacking suitable SSGSV, N = 396), and multivariate stratified Cox regression models were created. RESULTS: In cohort 1, there was a significant difference in time to clinical failure (log-rank P < .001), hemodynamic failure (log-rank P < .001), and resolution of presenting symptoms (log-rank P = .009) in favor of OPEN. In cohort 2, there was a significantly lower rate of hemodynamic failure (log-rank P = .006) favoring OPEN, and no significant difference in time to clinical failure or resolution of presenting symptoms. Multivariate analysis revealed that assignment to OPEN was associated with a significantly lower risk of clinical and hemodynamic failure in both cohorts and a significantly higher likelihood of resolving initial and preventing recurrent CLTI symptoms in cohort 1, including after adjustment for key baseline patient covariates (end-stage renal disease [ESRD], prior revascularization, smoking, diabetes, age >80 years, WIfI stage, tissue loss, and infrapopliteal disease). Factors independently associated with clinical failure included age >80 years in cohort 1 and ESRD across both cohorts. ESRD was associated with hemodynamic failure in cohort 1. Factors associated with slower resolution of presenting symptoms included diabetes in cohort 1 and WIfI stage in cohort 2. CONCLUSIONS: Durable clinical and hemodynamic benefit after revascularization for CLTI is important to avoid persistent and recurrent CLTI, reinterventions, and limb loss. When compared with ENDO, initial treatment with OPEN surgical bypass, particularly with available saphenous vein, is associated with improved clinical and hemodynamic outcomes and enhanced resolution of CLTI symptoms.

Female Patients Have Fewer Limb Amputations Compared to Male Patients in the BEST-CLI Trial.

OBJECTIVE: Female patients are less likely to be diagnosed with and treated for peripheral artery disease. When treated, there are also reported sex disparities in short- and long-term outcomes. We designed this study to compare outcomes after open and endovascular revascularization in the Best Endovascular versus best Surgical Therapy in patients with Critical Limb Ischemia (BEST-CLI) trial between females and males, and to examine outcomes of each revascularization type in an all-female cohort. METHODS: In a secondary analysis of cohorts 1 and 2 of the BEST-CLI Trial, patients with chronic limb threatening ischemia (CLTI) undergoing open surgical bypass (with or without adequate conduit) and endovascular therapy were stratified by sex. In addition, in a female-only cohort, we evaluated differences in outcomes between treatment arm (combined all bypasses from cohorts 1 and 2 and compared to all endovascular treatment in cohorts 1 and 2). Outcomes included major amputation, reintervention, major adverse limb event (MALE, a composite of major amputation and reintervention), all-cause death, and composite outcome of MALE or all-cause death. Univariable and adjusted Cox regressions were used to assess outcome between males and females. Similar methods were used to assess differences in outcomes between treatment arm in females. RESULTS: Among 1830 patients, females were significantly underrepresented, comprising only 28% (n=519) of the BEST-CLI cohort. Overall, the characteristics of females enrolled in the trial had some differences compared to males: females were more likely to have rest pain alone (72% vs 60%, p <.0001) and when presenting with an ischemic wound, were less likely to have a wound infection (38% vs 47%, p = .01). Females were less likely to have an adequate single segment greater saphenous vein (SSGSV) available (82% vs 89%, p = .01). Controlled for baseline clinical factors, at one year, females had significantly lower rates of major limb amputation compared to males (HR .70, p = .023), which drove better amputation- and MALE- free survival rates. All-cause death at one year was not statistically different between sexes (11.8% vs 11.2%, p=.286). In the all-female cohort, results paralleled the overall trial; open surgical bypass (with any conduit) had significantly better outcomes compared to endovascular therapy. Specifically, among females undergoing endovascular therapy, the rate of major reintervention was particularly high compared to females undergoing open surgical bypass (24.8% vs. 10.5%, p < .001). CONCLUSIONS: Despite being underrepresented in BEST-CLI, the primary results of the trial, namely, improved MALE-free survival with open surgical bypass with SSGSV, were mirrored in the all female subset. Female patients enrolled in BEST-CLI had better amputation-free survival at one year compared to male patients. These findings suggest that in treating female CLTI patients considered appropriate for both open and endovascular revascularization, surgical bypass with optimal conduit is the preferred treatment option and can potentially ameliorate poor limb preservation outcomes associated with sex.

Prosthetic conduits have worse outcomes compared with great saphenous vein conduits in femoropopliteal and infrapopliteal bypass in patients with chronic limb-threatening ischemia.

OBJECTIVE: Single segment great saphenous vein (SSGSV) traditionally has been considered the gold standard conduit for infrainguinal bypass. There are data supporting similar outcomes with prosthetic femoral-popliteal bypass. Moreover, some investigators have advocated for prosthetic conduit for femoral tibial bypass when GSV is inadequate or unavailable. We sought to evaluate long-term outcomes of infrainguinal bypass based on conduit type for treating chronic limb-threatening ischemia (CLTI). METHODS: Data from the Best Endovascular vs Best Surgical Therapy of Patients with CLTI multicenter, prospective, randomized controlled trial, comparing infrainguinal bypass with endovascular therapy in patients with CLTI, were evaluated. In this as-treated analysis, we compared outcomes of infrainguinal bypass using prosthetic, alternative autogenous vein (AAV), and cryopreserved vein (Cryo) with SSGSV bypass. Kaplan-Meier and multivariable analyses were performed to examine the associations of conduit type with major adverse limb events (MALE), reinterventions, above-ankle amputations, and all-cause death rates. RESULTS: In total, 784 bypasses were analyzed (120 prosthetic, 33 AAV, 21 Cryo, AND 610 SSGSV). For prosthetic and SSGSV, the distribution was 357 femoropopliteal (93 prosthetic and 264 GSV) and 373 infrapopliteal (27 prosthetic and 346 GSV). The mean age for the overall cohort was 67.1 years; 27.4% were female, 29.9% were non-White, and 11.5% were of Hispanic ethnicity. Patients undergoing prosthetic bypass were older (69.2 years vs 66.7 years); more likely to have chronic obstructive pulmonary disease (22.5% vs 14.0%), prior coronary artery bypass grafting (88.9% vs 66.5%), and prior stroke (23.3% vs 14%); but less often were of Hispanic ethnicity (5.8% vs 12.6%) and had diabetes (59.2% vs 71.3%) (P < .05 for all). For femoropopliteal bypass, use of prosthetic conduit was associated with increased major reinterventions at 3 years overall (19.0% vs 11.5%; P = .06) and on risk-adjusted analysis (hazard ratio [HR], 2.13; 95% confidence interval [CI], 1.09-4.2; P = .028). No significant differences in MALE or death, above-ankle amputation, or death were observed. Outcomes were similar for bypasses to above-knee popliteal targets and below-knee popliteal targets. For infrapopliteal bypass, the use of a prosthetic conduit was associated with increased major reintervention (25.3% vs 10.3%; P = .005), death (68.6% vs 34.8%; P < .001), and MALE or death (90.0% vs 48.1%; P < .001) at 3 years. After risk adjustment, infrapopliteal bypass with prosthetic conduit was associated with higher major reintervention (HR, 4.14; 95% CI, 1.36-12.6; P = .012), above-ankle amputation (HR, 4.64; 95% CI, 1.59-13.5; P = .005), death (HR, 2.96; 95% CI, 1.4-6.2; P = .004), and MALE or death (HR, 3.59; 95% CI, 1.64-7.86; P = .001) compared with bypass with SSGSV. Overall, AAV had similar outcomes at 3 years as SSGSV; however, Cryo had significantly higher above-ankle amputation (50.0% vs 12.8%) (HR, 4.2; 95% CI, 1.68-10.5; P = .002), major reintervention (41.9% vs 10.7%) (HR, 3.12; 95% CI, 1.18-8.22; P = .02), and MALE/death (88.8% vs 37.8%) (HR, 2.96; 95% CI, 1.43-6.14; P = .004). CONCLUSIONS: The use of a prosthetic conduit in infrainguinal bypass is associated with inferior outcomes compared with bypass using SSGSV, particularly for bypass to infrapopliteal targets. Cryo grafts were infrequent and also demonstrated inferior outcomes. SSGSV remains the preferred conduit of choice for infrainguinal bypass.

Notes from the Field: Potential Outbreak of Extrapulmonary Mycobacterium abscessus subspecies massiliense Infections from Stem Cell Treatment Clinics in Mexico - Arizona and Colorado, 2022.

Mycobacterium abscessus is an intrinsically drug-resistant, rapidly growing, nontuberculous mycobacterium; extrapulmonary infections have been reported in association with medical tourism (1). During November-December 2022, two Colorado hospitals (hospitals A and B) treated patient A, a Colorado woman aged 30-39 years, for M. abscessus meningitis. In October 2022, she had received intrathecal donor embryonic stem cell injections in Baja California, Mexico to treat multiple sclerosis and subsequently experienced headaches and fevers, consistent with meningitis. Her cerebrospinal fluid revealed neutrophilic pleocytosis and grew M. abscessus in culture at hospital A. Hospital A's physicians consulted hospital B's infectious diseases (ID) physicians to co-manage this patient (2).

Association of plasma biomarkers with cognition, cognitive decline, and daily function across and within neurodegenerative diseases: Results from the Ontario Neurodegenerative Disease Research Initiative.

INTRODUCTION: We investigated whether novel plasma biomarkers are associated with cognition, cognitive decline, and functional independence in activities of daily living across and within neurodegenerative diseases. METHODS: Glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL), phosphorylated tau (p-tau)181 and amyloid beta (Aß)42/40 were measured using ultra-sensitive Simoa immunoassays in 44 healthy controls and 480 participants diagnosed with Alzheimer's disease/mild cognitive impairment (AD/MCI), Parkinson's disease (PD), frontotemporal dementia (FTD) spectrum disorders, or cerebrovascular disease (CVD). RESULTS: GFAP, NfL, and/or p-tau181 were elevated among all diseases compared to controls, and were broadly associated with worse baseline cognitive performance, greater cognitive decline, and/or lower functional independence. While GFAP, NfL, and p-tau181 were highly predictive across diseases, p-tau181 was more specific to the AD/MCI cohort. Sparse associations were found in the FTD and CVD cohorts and for Aß42/40 . DISCUSSION: GFAP, NfL, and p-tau181 are valuable predictors of cognition and function across common neurodegenerative diseases, and may be useful in specialized clinics and clinical trials.

Phase Separation of SARS-CoV-2 Nucleocapsid Protein with TDP-43 Is Dependent on C-Terminus Domains.

The SARS-CoV-2 nucleocapsid protein (N protein) is critical in viral replication by undergoing liquid-liquid phase separation to seed the formation of a ribonucleoprotein (RNP) complex to drive viral genomic RNA (gRNA) translation and in suppressing both stress granules and processing bodies, which is postulated to increase uncoated gRNA availability. The N protein can also form biomolecular condensates with a broad range of host endogenous proteins including RNA binding proteins (RBPs). Amongst these RBPs are proteins that are associated with pathological, neuronal, and glial cytoplasmic inclusions across several adult-onset neurodegenerative disorders, including TAR DNA binding protein 43 kDa (TDP-43) which forms pathological inclusions in over 95% of amyotrophic lateral sclerosis cases. In this study, we demonstrate that the N protein can form biomolecular condensates with TDP-43 and that this is dependent on the N protein C-terminus domain (N-CTD) and the intrinsically disordered C-terminus domain of TDP-43. This process is markedly accelerated in the presence of RNA. In silico modeling suggests that the biomolecular condensate that forms in the presence of RNA is composed of an N protein quadriplex in which the intrinsically disordered TDP-43 C terminus domain is incorporated.

SARS-CoV-2, aging, and Post-COVID-19 neurodegeneration.

As the world continues to experience the effects of SARS-CoV-2, there is evidence to suggest that the sequelae of viral infection (the post-COVID-19 condition; PCC) at both an individual and population level will be significant and long-lasting. The history of pandemics or epidemics in the last 100 years caused by members of the RNA virus family, of which coronaviruses are a member, provides ample evidence of the acute neurological effects. However, except for the H1N1 influenza pandemic of 1918/1919 (the Spanish flu) with its associated encephalitis lethargica, there is little information on long-term neurological sequelae. COVID-19 is the first pandemic that has occurred in a setting of an aging population, especially in several high-income countries. Its survivors are at the greatest risk for developing neurodegenerative conditions as they age, rendering the current pandemic a unique paradigm not previously witnessed. The SARS-CoV-2 virus, among the largest of the RNA viruses, is a single-stranded RNA that encodes for 29 proteins that include the spike protein that contains the key domains required for ACE2 binding, and a complex array of nonstructural proteins (NSPs) and accessory proteins that ensure the escape of the virus from the innate immune response, allowing for its efficient replication, translation, and exocytosis as a fully functional virion. Increasingly, these proteins are also recognized as potentially contributing to biochemical and molecular processes underlying neurodegeneration. In addition to directly being taken up by brain endothelium, the virus or key protein constituents can be transported to neurons, astrocytes, and microglia by extracellular vesicles and can accelerate pathological fibril formation. The SARS-CoV-2 nucleocapsid protein is intrinsically disordered and can participate in liquid condensate formation, including as pathological heteropolymers with neurodegenerative disease-associated RNA-binding proteins such as TDP-43, FUS, and hnRNP1A. As the SARS-CoV-2 virus continues to mutate under the immune pressure exerted by highly efficacious vaccines, it is evolving into a virus with greater transmissibility but less severity compared with the original strain. The potential of its lingering impact on the nervous system thus has the potential to represent an ongoing legacy of an even greater global health challenge than acute infection.

Benchmarking the empirical accuracy of short-read sequencing across the M. tuberculosis genome.

MOTIVATION: Short-read whole-genome sequencing (WGS) is a vital tool for clinical applications and basic research. Genetic divergence from the reference genome, repetitive sequences and sequencing bias reduces the performance of variant calling using short-read alignment, but the loss in recall and specificity has not been adequately characterized. To benchmark short-read variant calling, we used 36 diverse clinical Mycobacterium tuberculosis (Mtb) isolates dually sequenced with Illumina short-reads and PacBio long-reads. We systematically studied the short-read variant calling accuracy and the influence of sequence uniqueness, reference bias and GC content. RESULTS: Reference-based Illumina variant calling demonstrated a maximum recall of 89.0% and minimum precision of 98.5% across parameters evaluated. The approach that maximized variant recall while still maintaining high precision (<99%) was tuning the mapping quality filtering threshold, i.e. confidence of the read mapping (recall = 85.8%, precision = 99.1%, MQ ≥ 40). Additional masking of repetitive sequence content is an alternative conservative approach to variant calling that increases precision at cost to recall (recall = 70.2%, precision = 99.6%, MQ ≥ 40). Of the genomic positions typically excluded for Mtb, 68% are accurately called using Illumina WGS including 52/168 PE/PPE genes (34.5%). From these results, we present a refined list of low confidence regions across the Mtb genome, which we found to frequently overlap with regions with structural variation, low sequence uniqueness and low sequencing coverage. Our benchmarking results have broad implications for the use of WGS in the study of Mtb biology, inference of transmission in public health surveillance systems and more generally for WGS applications in other organisms. AVAILABILITY AND IMPLEMENTATION: All relevant code is available at https://github.com/farhat-lab/mtb-illumina-wgs-evaluation. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Relationship between WIfI stage and quality of life at revascularization in the BEST-CLI trial.

OBJECTIVE: The WIfI (Wound, Ischemia, foot Infection) stage measures the extent of wounds, ischemia, and foot infection in patients with chronic limb threatening ischemia (CLTI) and has been associated with the risk of major amputation. Patients with CLTI have impaired health-related quality of life (HRQoL), which can be multifactorial. We hypothesized that the severity of the limb threat (WIfI stage) would be associated with poor HRQoL among patients with CLTI presenting for revascularization. METHODS: The dataset of the BEST-CLI (best endovascular versus best surgical therapy in patients with CLTI) trial, a prospective, randomized trial comparing open and endovascular revascularization strategies, was queried for HRQoL assessments at patient enrollment. The HRQoL assessments included (1) Vascular Quality of Life; (2) 12-item short form survey (SF-12), containing the utility index score (short-form six-dimension R2 utility index, incorporating physical, emotional, and mental well-being) and mental and physical components; and (3) the EQ-5D. Multivariable regression analysis was used to identify the independent associations with the baseline HRQoL assessments. RESULTS: A total of 1568 patients with complete WIfI data were analyzed, of whom 71.5% were men. The WIfI distribution was 35.5% with stage 4, 29.6% with stage 3, 28.6% with stage 2, and 6.3% with stage 1. Patients presenting with WIfI stage 4, compared with stage 1 to 3, were more often men (74.9% vs 69.6%) and current smokers (25.4% vs. 17.6%), had had end-stage renal disease (13.3% vs 8.5%) and diabetes (83.6% vs 60.2%), were not independently ambulatory (56.8% vs 38.5%), and had had higher median morbidity scores (4 vs 3; P < .05 for all). On multivariable analysis, WIfI stage 4, compared with stage 1 to 3, was associated with lower SF-12 mental component scale scores (estimate, -2.43; 95% confidence interval, -3.73 to -1.13; P < .001) and short-form six-dimension R2 utility index scores (estimate, -0.02; 95% confidence interval, -0.03 to 0.001; P = .04). The WIfI stage was not independently associated with the baseline Vascular Quality of Life, SF-12 physical component scale, or EQ-5D assessments. CONCLUSIONS: WIfI stage was independently associated with poorer quality of life because of mental, rather than physical, health for patients with CLTI. Clinicians should be aware of the burden of mental stress borne by those with the greatest limb impairment.

Perioperative complications following open or endovascular revascularization for chronic limb-threatening ischemia in the BEST-CLI Trial.

OBJECTIVE: Anticipated perioperative morbidity is an important factor for choosing a revascularization method for chronic limb-threatening ischemia (CLTI). Our goal was to assess systemic perioperative complications of patients treated with surgical and endovascular revascularization in the Best Endovascular vs Best Surgical Therapy in Patients with CLTI (BEST-CLI) trial. METHODS: BEST-CLI was a prospective randomized trial comparing open (OPEN) and endovascular (ENDO) revascularization strategies for patients with CLTI. Two parallel cohorts were studied: Cohort 1 included patients with adequate single-segment great saphenous vein (SSGSV), whereas Cohort 2 included those without SSGSV. Data were queried for major adverse cardiovascular events (MACE-composite myocardial infarction, stroke, death), non-serious (non-SAEs) and serious adverse events (SAEs) (criteria-death/life-threatening/requiring hospitalization or prolongation of hospitalization/significant disability/incapacitation/affecting subject safety in trial) 30 days after the procedure. Per protocol analysis was used (intervention received without crossover), and risk-adjusted analysis was performed. RESULTS: There were 1367 patients (662 OPEN, 705 ENDO) in Cohort 1 and 379 patients (188 OPEN, 191 ENDO) in Cohort 2. Thirty-day mortality in Cohort 1 was 1.5% (OPEN 1.8%; ENDO 1.3%) and in Cohort 2 was 1.3% (2.7% OPEN; 0% ENDO). MACE in Cohort 1 was 4.7% for OPEN vs 3.13% for ENDO (P = .14), and in Cohort 2, was 4.28% for OPEN and 1.05% for ENDO (P = .15). On risk-adjusted analysis, there was no difference in 30-day MACE for OPEN vs ENDO for Cohort 1 (hazard ratio [HR] 1.5; 95% confidence interval [CI], 0.85-2.64; P = .16) or Cohort 2 (HR, 2.17; 95% CI, 0.48-9.88; P = .31). The incidence of acute renal failure was similar across interventions; in Cohort 1 it was 3.6% for OPEN vs 2.1% for ENDO (HR, 1.6; 95% CI, 0.85-3.12; P = .14), and in Cohort 2, it was 4.2% OPEN vs 1.6% ENDO (HR, 2.86; 95% CI, 0.75-10.8; P = .12). The occurrence of venous thromboembolism was low overall and was similar between groups in Cohort 1 (OPEN 0.9%; ENDO 0.4%) and Cohort 2 (OPEN 0.5%; ENDO 0%). Rates of any non-SAEs in Cohort 1 were 23.4% in OPEN and 17.9% in ENDO (P = .013); in Cohort 2, they were 21.8% for OPEN and 19.9% for ENDO (P = .7). Rates for any SAEs in Cohort 1 were 35.3% for OPEN and 31.6% for ENDO (P = .15); in Cohort 2, they were 25.5% for OPEN and 23.6% for ENDO (P = .72). The most common types of non-SAEs and SAEs were infection, procedural complications, and cardiovascular events. CONCLUSIONS: In BEST-CLI, patients with CLTI who were deemed suitable candidates for open lower extremity bypass surgery had similar peri-procedural complications following either OPEN or ENDO revascularization: In such patients, concern about risk of peri-procedure complications should not be a deterrent in deciding revascularization strategy. Rather, other factors, including effectiveness in restoring perfusion and patient preference, are more relevant.

Baseline modern medical management in the BEST-CLI trial.

OBJECTIVES: The use of optimal medical therapy (OMT) in patients with chronic limb-threatening ischemia (CLTI) has not been well-studied. The Best Endovascular vs Best Surgical Therapy in Patients with CLTI study (BEST-CLI) is a multicenter, randomized, controlled trial sponsored by the National Institutes of Health comparing revascularization strategies in patients with CLTI. We evaluated the use of guideline-based OMT among patients with CLTI at the time of their enrollment into the trial. METHODS: A multidisciplinary committee defined OMT criteria related to blood pressure and diabetic management, lipid-lowering and antiplatelet medication use, and smoking status for patients enrolled in BEST-CLI. Status reports indicating adherence to OMT were provided to participating sites at regular intervals. Baseline demographic characteristics, comorbid medical conditions, and use of OMT at trial entry were evaluated for all randomized patients. A linear regression model was used to identify the relationship of predictors to the use of OMT. RESULTS: At the time of randomization (n = 1830 total enrolled), 87% of patients in BEST-CLI had hypertension, 69% had diabetes, 73% had hyperlipidemia, and 35% were currently smoking. Adherence to four OMT components (controlled blood pressure, not currently smoking, use of one lipid-lowering medication, and use of an antiplatelet agent) was modest. Only 25% of patients met all four OMT criteria; 38% met three, 24% met two, 11% met only one, and 2% met none. Age ≥80 years, coronary artery disease, diabetes, and Hispanic ethnicity were positively associated, whereas Black race was negatively associated, with the use of OMT. CONCLUSIONS: A significant proportion of patients in BEST-CLI did not meet OMT guideline-based recommendations at time of entry. These data suggest a persistent major gap in the medical management of patients with advanced peripheral atherosclerosis and CLTI. Changes in OMT adherence over the course of the trial and their impact on clinical outcomes and quality of life will be assessed in future analyses.