In conclusion, our findings indicate that effective NTM recovery from HCU- and ECMO-derived water samples can be achieved via filtration and direct culture of the filters on NTM Elite agar. This simple procedure can speed up laboratory work and provide an improved method, successfully resulting in low contamination and high detection rate, in addition to being less time-consuming. Its sensitivity and lack of a decontamination step make this protocol particularly useful for monitoring the effectiveness of device disinfection in hospital settings, even in the presence of low NTM loads. Reading timeframes should probably be extended to 7 weeks , in order to isolate even the slow-growing mycobacteria. However, an extended incubation period is not necessary for exclusion of M. chimaera contamination of the devices, as M. chimaera isolation times do not generally exceed 3 weeks.The isolation of non-tuberculous mycobacteria (NTM) from cultures is particularly laborious due to the potential overgrowth of coexisting non-acid fast bacilli. To reduce the overgrowth of these non-mycobacterial organisms, a decontamination step with NaOH or cetylpyridinium chloride is highly recommended before plating the samples on the culture medium. However, due to their toxicity, decontamination solutions tend to decrease NTM recovery from clinical and environmental samples. Here, we tested an alternative method for NTM recovery based on the use of NTM Elite agar, a selective medium that does not require a decontamination step. Using NTM Elite agar, we were able to detect non-tuberculous mycobacteria in 27.7% (30/108) of water samples analyzed. The average time to NTM detection was 18 days, but some strains required longer to grow, perhaps due to the stressful environmental conditions . NTM Elite agar’s effectiveness in inhibiting background flora was proven by the isolation of NTM from samples with and without background flora, showing no statistically significant differences in detection rates for different total viable counts of background flora ( Mycobacterium chimaera [Evidence to date indicates that heater–cooler units (HCUs) and extracorporeal membrane oxygenation (ECMO) machines used during cardiac surgery can generate infectious aerosols due to the presence of water-borne pathogens such as chimaera ,2,3,4. TAs reported by several researchers, the isolation of non-tuberculous mycobacteria (NTM) from cultures is particularly laborious due to the potential overgrowth of coexisting non-acid fast bacilli (non-AFB) ,6,7. ThuFor microbiological surveillance of HCU and ECMO water systems, the technical document issued by the European Centre for Disease Prevention and Control (ECDC) recommends concentrating water samples by filtration or centrifugation, decontaminating them with NALC-NaOH or CPC and plating on selective media, such as Middlebrook 7H11 or 7H10 agar, or egg-based media, such as Löwenstein–Jensen agar. In addition, culture in liquid medium can be performed using the mycobacteria growth indicator tube (MGIT) 960 system ,10,11. Even though a number of different publications providing useful guidance do exist ,14,15,16Recent evidence has shown that protocols based on rapidly growing mycobacterium (RGM) media can be highly effective in recovering NTM from potable and non-potable water samples . More spM. chimaera and Mycobacterium chelonae to compare recovery efficiency of the membrane filter washing procedure [Our microbiological laboratory operates as a regional reference service for microbiological surveillance of all HCUs and ECMOs located in cardiac surgery facilities across the Piedmont region . Specifically, our lab plays a central role in diagnosing, detecting, identifying, and typing environmental infectious bacterial agents , monitoring the response to epidemics, providing scientific evidence for disease prevention and control, and assisting in the development of regional guidelines on control and risk containment of NTM infection ,21,22. Frocedure  against N-acetyl-L-cysteine sodium hydroxide decontamination treatments. We demonstrated that: (i) the washing procedure yielded a low release of both mycobacterium strains from the membrane filters (≈7%), while direct plating of membrane filters led to a 100% cell recovery; (ii) water sample pretreatment with N-acetyl-L-cysteine sodium hydroxide, despite achieving complete suppression of non-acid fast bacilli, caused a reduction in mycobacteria growth, while decontamination with cetylpyridinium chloride was found to be ineffective against some non-acid fast organisms. Moreover, we compared the efficacy of NTM Elite agar in inhibiting the growth of aquatic bacteria with that of cetylpyridinium chloride and Pseudomonas aeruginosa in collected samples, by evaluating the relationship between TVC concentration and probability of NTM isolation. Here, we sought to evaluate NTM Elite agar’s performance in recovering NTM from water samples collected from HCU and ECMO devices. Furthermore, we analyzed NTM Elite agar’s selectivity in consideration of interfering bacteria, i.e., total viable counts (TVCs) and In this study, we analyzed water samples collected between January 2021 and November 2021 during a microbiological surveillance carried out on 33 devices—26 HCUs manufactured by LivaNova or Maquet Getinge and 7 ECMOs by Maquet Getinge—from nine cardiac surgery facilities and from one sink used by healthcare facility staff to fill water tanks of the hospital’s devices.A total of 108 water samples were collected in sterile 1 L plastic bottles, containing 20 mg of sodium thiosulphate. Samples were immediately sent to the laboratory or, if not possible, stored at a controlled temperature (5 ± 3 °C for 12–15 h). All analyses were performed within 24 h of collection. P. aeruginosa, and total viable counts (TVCs). TVCs and P. aeruginosa are general indicators of water cleanliness and are used as surrogate indicators of decontamination effectiveness. Enumeration of background bacteria in HCU-derived water samples was performed according to the UNI EN ISO 6222 protocol [3/mL. When we could not determine the presence of NTM due to fungal growth covering the filter or presence of non-AFB at a concentration >3 × 102 CFU/mL, samples were defined as “unreadable plates”.Water samples were tested for NTM, protocol . BrieflyP. aeruginosa was detected and quantified by culture according to the ISO 16266 method [6 method , which cNTM isolation was performed using the following procedure: 100 mL of each water sample was filtered through mixed cellulose esters filter, and the filter was directly added to a Petri dish containing NTM Elite agar  . Subsequently, the NTM Elite agar plates were incubated at 30 °C in a sealed plastic bag to prevent dehydration. Starting from the seventh day, cultures were examined weekly for seven weeks. After realizing that mycobacteria from environmental samples needed a longer time to grow, we opted to extend the incubation time from 4 weeks—as advised by the manufacturer—to 8 weeks. All colonies growing on NTM Elite agar were confirmed as AFB by Kinyoun stain and then subcultured on Middlebrook 7H11 medium and identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). NTM were processed according to the MycoEx preparation protocol following the manufacturer’s instructions. Spectra were acquired by Bruker Microflex LT  MALDI-TOF MS and analyzed by MBT Mycobacteria RUO Library v.4.0 .The results from NTM Elite agar analyses were reported as CFU/100 mL.Mycobacterium spp. Water Scheme) distributed by the Food and Environmental Proficiency Testing Unit (FEPTU) of UK Health Security Agency (UK HSA). This microbiology scheme provides samples to laboratories that examine HCU waters for Mycobacterium spp. The FEPTU report provides the expected results  to participating laboratories [As satisfactory performance control for this new protocol, we analyzed 6 samples of proficiency test (PT) for NTM recovery in water samples . Hypothesis tests on categorical variables were performed using Fisher’s exact test. Correlation between bacterial contamination , and proportion of detected NTM-positive samples was inspected through LOESS regression. All analytical computation and plotting were carried out using the statistical software R version 4.0.5  .A total of 102 water samples from 26 HCUs—12 Stockert 3T , 14 HCU40 —and from 7 ECMOs , were collected and cultured on NTM Elite agar. These 33 devices are regularly disinfected according to the manufacturer’s instructions: 58 of the 102 samples (57%) were collected after disinfection, while 44 (43%) before disinfection. Moreover, 6 water samples were taken from one sink faucet equipped with a point-of-use (POU) filter (this sink was associated with tank-filling of 4 devices—2 HCUs and 2 ECMOs—that turned out to be NTM-positive), bringing the total number of samples to 108.As shown in P. aeruginosa results were available from 100 out of 108 samples. The results of this microbiological surveillance are summarized in Overall, NTM, TVCs, and P. aeruginosa, and NTM results. As shown in 3 CFU/mL). The difference between these proportions was not significant, as Fisher’s exact test analysis indicated that we would not have reached a higher NTM recovery rate in samples without non-AFB (p = 0.4989) or P. aeruginosa (p = 1), indicating that, under our experimental conditions, non-AFB species do not inhibit or mask the growth of NTM colonies on NTM Elite agar.In order to analyze NTM ELITE agar’s selectivity, we disaggregated the data by TVCs, Locally estimated scatterplot smoothing (LOESS) regression failed to show any particular correlation between the proportion of NTM-positive samples and the presence of different TVCs of contaminating bacteria . NotablyWhen considering the comparison between TVCs—1 mL of sample analyzed—and the presence of non-AFB on NTM Elite agar—100 mL of sample analyzed—we noticed that 1% of samples yielded non-AFB on NTM Elite agar (unreadable plates) in the absence of overgrowth . Among samples collected from Stockert 3T HCUs, nine samples did not provide reliable results on NTM Elite agar due to the presence of a large number of non-AFB, eight of them having been collected prior to disinfection and one after HCU disinfection. The results are listed in NTM prevalence among the devices was 69.2% (18/26) for HCUs—mycobacteria were isolated on more than one sample from some devices—and 28.6% (2/7) for ECMO machines.M. chimaera, Mycobacterium gordonae, Mycobacterium paragordonae, M. chelonae, Mycobacterium lentiflavum, Mycobacterium mucogenicum, Mycobacterium frederiksbergense). M. chimaera was isolated only from Stockert 3T HCUs. Specifically, we detected M. chimaera in 58.3% (7/12) of Stockert 3T HCUs, including two devices labeled with an M. chimaera-free certificate (claimed by the manufacturer), while all Maquet devices—both HCU 40 and ECMO—were negative. Two LivaNova devices were colonized by two different mycobacterium species in the same samples ; one Maquet device was colonized by three different mycobacterium species .A total of 43 isolates belonged to 7 mycobacterial species : for instance, three out of eight Pseudomonas species [Overall, NTM were isolated from 27.6% of non-AFB-free samples vs. 28.6% of non-AFB-containing samples . This is species , therebyBurkholderia multivorans. Therefore, the discrepancy with the results of Alexander et al. may be ascribable to the different amounts of sample analyzed in the two studies. While we cultured 100 mL of filtered samples by direct plating of membrane filter, in the study by Alexander et al., only one hundredth of the sample was plated .NTM Elite agar’s effectiveness in inhibiting non-AFB contamination is in keeping with a report by Alexander et al. , showingA significant shortcoming of our study might be the lack of a direct comparison of the described culture method for NTM to a standard culture method such as concentration by centrifugation  or filtrMycobacterium spp. Scheme” confirmed the validity of our protocol: we isolated M. chelonae from three PT distributions (M. chelonae at levels of approximately 2.2 × 102 and 5.0 × 102 CFUs (distribution number MY006B and MY008A), and all laboratories failed to detect M. chelonae at levels 5.8 × 101 CFUs (distribution number MY009A). The root cause for this may lie in the incubation temperature of MGIT: M. chelonae grows better at lower temperatures (~30 °C) compared to other mycobacterial species [M. chelonae loads  can yield a false negative if incubated at temperatures above 30° C. Moreover, according to Kim et al. [M. gordonae and M. paragordonae strains can be extremely challenging when medium seeding is performed at a temperature above 30 °C. Indeed, M. gordonae and M. paragordonae, two phylogenetically close species, grow optimally at a temperature of 30 °C—M. paragordonae can grow in macrophages at 30 °C, but not at 37 °C [The results obtained from participation in the PT “ibutions , while 7 species ,34. Therm et al. , isolatiat 37 °C . Thus, aM. chimaera contamination of the devices, as M. chimaera isolation times do not generally exceed 3 weeks.In conclusion, our findings indicate that effective NTM recovery from HCU- and ECMO-derived water samples can be achieved via filtration and direct culture of the filters on NTM Elite agar at 30° C. This simple procedure can speed up laboratory work and provide an improved method, successfully resulting in low contamination and high detection rate, in addition to being less time-consuming. Its sensitivity  and lack of a decontamination step make this protocol particularly useful for monitoring the effectiveness of device disinfection in hospital settings, even in the presence of low NTM loads. As some samples collected from devices prior to disinfection showed overgrowth, we advise that a lower amount of sample should be filtered and plated, in case higher levels of contamination from other water-borne microorganisms are expected. Moreover, according to our results, we recommend that the reading timeframes should be extended to 7 weeks , in order to isolate even slow-growing mycobacteria. However, an extended incubation period is not necessary for exclusion of


The aim of this study was to investigate a real-world heart failure (HF) cohort regarding (1) prevalence of known diabetes mellitus (DM), undiagnosed DM and pre-diabetes, (2) if hf treatment differs depending on glycaemic status and (3) if treatment of DM differs depending on HF phenotype.All patients who had received a diagnosis of HF at Umeå University Hospital between 2010 and 2019 were identified and data were extracted from patient files according to a prespecified protocol containing parameters for clinical characteristics, including echocardiogram results, comorbidities, fasting plasma glucose (FPG) and hemoglobin A1c (HbA1c) values. Patients’ HF phenotype was determined using the latest available echocardiogram. The number of patients with previous DM diagnosis was assessed. Patients without a previous diagnosis of DM were classified as non-DM, pre-diabetes or probable DM according to FPG and HbA1c levels using WHO criteria.In total, 2326 patients  with HF and at least one echocardiogram were assessed. Of these, 617 (27%) patients had a previous diagnosis of DM. Of the 1709 patients without a previous diagnosis of DM, 1092 (67%) patients had either an FPG or HbA1c recorded, of which 441 (41%) met criteria for pre-diabetes and 97 (9%) met criteria for probable diabetes, corresponding to 19% and 4% of the entire cohort, respectively. Patients with HF and diabetes were more often treated with diuretics and beta blockers compared with non-DM patients . There was no difference in DM treatment between HF phenotypes.DM and pre-diabetes are common in this HF population with 50% of patients having either known DM, probable DM or pre-diabetes. Patients with HF and DM are more often treated with common HF medications. HF phenotype did not affect choice of DM therapy. Heart failure (HF) and diabetes mellitus or pre-diabetes are commonly coexisting disorders which negatively impact patient outcome.Prevalence estimates for glycaemic disorders in HF patients differ between settings, and often come from selected populations, for example, trial participants.This single-centre study encompassing an unselected HF population finds that 27% of patients had known diabetes mellitus, 4% of patients had probable undiagnosed diabetes and 19% of patients had pre-diabetes.The high prevalence of glycaemic disorders warrants a generous approach to glycaemic testing in HF patients, and further studies on how to best manage pre-diabetes in HF.Heart failure (HF) and diabetes mellitus (DM) are two common disorders that individually increase mortality, morbidity and healthcare costs.6Pre-diabetes, defined as above normal blood glucose and below the diagnostic cut-off for DM,The aim of this study was to investigate a Northern European real-world HF population regarding:Prevalence of pre-diabetes, undiagnosed DM and known DM.If HF treatment differs depending on DM status.If DM treatment differs depending on HF status.We identified all patients who had received a diagnosis of HF  at the Heart Centre or the Department of Internal medicine at Umeå University Hospital, Sweden, between January 2010 and December 2019 and who lived within the catchment area of the hospital. The diagnosis could be from either a HF hospitalisation event or outpatient visit and includes both prevalent cases and new onset HF during the inclusion period. The hospital is the only one within its geographic catchment area and serves a mixed urban and rural population of about 150 000 people and has the only specialist cardiology clinic in the area.The patients were identified in the hospitals electronic health records system (NCS Cross). Clinical parameters were extracted from the patients’ electronic health records using a prespecified protocol encompassing lab results, echocardiogram data, ECG results, comorbidities, medication and known diabetes status. DM was categorised as either Type 1 DM (T1DM) or T2DM, according to ICD 10 codes. Coronary artery disease was defined as either a previous myocardial infarction or the presence of at least one stenosis of 50% or more in a major coronary vessel. The latest available value for each parameter was used regardless of if it was from an inpatient or outpatient setting, including echocardiogram results. HF phenotype was categorised according to left ventricle EF (LVEF) as HF with reduced EF , HF with mildly reduced ejection  or HFpEF (EF ≥50%).Data on HbA1c and fasting plasma-glucose (FPG) were recorded if there was a value from within 1 year from the otherwise latest available lab result. It was not possible to ascertain in every case if a plasma glucose measurement was fasting or not. Due to this uncertainty, we put more emphasis on HbA1c levels when classifying patients’ DM status. With that in mind, we categorised patients’ DM status using the following criteria, based on WHO recommendationsKnown DM: Presence of a clinical diagnosis of DM.Probable DM: HbA1c≥48 mmol/mol, or FPG≥7.0 mmol/L in the absence of a HbA1c-value.Pre-diabetes:Impaired fasting glucose (IFG) defined as FPG≥6.1 mmol/L if HbA1c <48 mmol/mol, or FPG 6.1–6.9 mmol/L in the absence of a HbA1c value.Intermediate hyperglycaemia defined as HbA1c 42–47 mmol/mol.Non-DM: No previous diagnosis of DM and not meeting criteria for probable DM or pre-diabetes.Unknown: No previous diagnosis of DM and no glycaemic tests available.2 test. When analysing differences between HF phenotypes, a test of homogeneity was performed between all groups. If that was significant, pairwise comparisons between groups were made. The null hypothesis used was that there are no significant differences between groups. If a group had less than five patients, Fisher’s exact test was used instead. A p<0.05 was considered significant.Extracted data on patients who were alive 1 January 2020 were analysed using SPSS V.27. Continuous parameters are presented as means with SDs. Continuous variables without normal distribution are presented as medians with IQR. Categorical variables are presented with frequencies and percentage. When analysing differences between glycaemic profiles, each group was compared with the non-DM group using Pearson’s χA total of 2433 patients with a diagnosis of HF were alive 1 January 2020. Of these, 107 did not have an echocardiogram recorded and no EF available and were excluded. Of the 2326 patients with known EF, a majority (59%) were men, 617 (27%) had known DM, 26 with T1DM and 591 with T2DM. A total of 1084 patients (47%) had HFpEF, 561 patients (24%) had HFmrEF and 681 patients (29%) HFrEF. There were differences in many clinical characteristics between men and women. See Of the 1709 patients without DM, 577 (34%) had both a HbA1c and an FPG recorded within the inclusion period, while 274 (16%) patients had an FPG but no HbA1c, and 247 (14%) patients had a HbA1c but no FPG recorded. 617 (36%) of the patients without previously known DM had neither an FPG nor HbA1c recorded. When applying the stated criteria for DM status on the 1092 patients without previous DM diagnosis with available FPG or HbA1c tests, 97 (9%) patients were deemed to have probable DM and 447 (41%) patients pre-diabetes . DM status did not differ significantly between HF phenotype p=0.32, . Clinica10.1136/openhrt-2022-002133.supp1Supplementary dataNon-DM patients were less often treated with beta blockers and loop diuretics compared with patients with pre-diabetes or known DM and tended to be less often treated with ACE inhibitors or angiotensin receptor blocker. Patients with probable DM had the highest rate of loop diuretic and MRA treatment. There were no differences between groups regarding treatment with angiotensin receptor blocker-neprilysin inhibitor (ARNI) or digitalis. Sodium-glucose cotransporter 2 (SGLT2)-inhibitor treatment only occurred in probable DM (1%) and known DM (12%) .Of the 617 patients with known diabetes, 301 were treated with metformin, 245 with insulin, 106 with dipeptidyl peptidase four inhibitors, 75 with SGLT2 inhibitors, 47 with glucagon-like peptide one receptor analogues and 38 with sulphonylureas (including repaglinide). There were no differences in choice of DM treatment across HF phenotypes .In this real-world HF population, one-fourth of patients had previously known DM. Of the patients without previous DM with available glycaemic tests, 1/10 had probable DM and 4/10 pre-diabetes. Diabetes and pre-diabetes prevalence and treatment did not differ between HF phenotypes. DM and pre-diabetes patients tended to have higher rates of diuretics and beta blockers prescribed.et alThere are three ways to diagnose pre-diabetes and diabetes—FPG, HbA1c and through an oral glucose tolerance test (OGTT).Patients with known DM and pre-diabetes were more often treated with beta blockers and diuretics compared with non-DM patients. This might indicate that these patients have more symptomatic HF compared with non-DM patients, which would be in line with the worse prognosis seen in HF patients with DM.17 24The usage of SGLT2-inhibitors was generally low, at only 3%. This reflects that the data used for this study covers the time up to December 2019, which was before SGLT2-inhibitors were approved for HF treatment without DM. If this study was repeated today, SGLT2 inhibitor treatment rates would likely be markedly higher.Use of ARNI was low at only 6% of all patients, corresponding to 22% of the number of HFrEF patients, but did not differ between glycaemic groups. The prescription rate in our healthcare region was still above average compared with national rates,et alOur cohort was reasonably balanced in regard to sex, with 59% men and 41% women. However, women were significantly older with more HFpEF, less HFrEF and worse renal function, and less use of MRA, ARNI and SGLT2-inhibitors, which is commonly seen in HF cohorts.While pre-diabetes is a risk factor for adverse outcomes in HF, the benefit of identifying and treating pre-diabetes in HF is unclear. Treatment of other comorbidities in HF patients, including DM, is considered important, and choice of DM therapy should be influenced by the presence of HF.There is evidence for lifestyle and drug interventions in pre-diabetes to prevent progression to T2DM and to lower the risk of cardiovascular disease,This study is limited by being a single-centre study based on data from patient records, so the results might not be generalisable to other populations or settings. The wide patient selection criteria used has both advantages and drawbacks. The intent and the advantage of this method is that as many HF patients as possible could be included so that results are applicable to the full span of HF patients encountered in the clinic. The drawbacks include that the patient cohort is heterogeneous with regard to HF type, aetiology, duration and clinical setting, which makes the results less applicable to specific HF subpopulations. Using retrospective data from patient files meant that we did not have complete or current data on all patients. Specifically, FPG and HbA1c were missing on many patients. Additionally, some recorded FPG values could be from non-fasting patients due to missing or erroneous labelling in patient files, which is why we relied more on HbA1c levels than FPG to classify a patient as having probable DM. Of patients without previous DM with available tests, half had some level of dysglycaemia. The rate in the whole population might be lower due to bias in which patients were tested, but our results indicate that blood glucose abnormalities are very common, despite limitations. Prevalence numbers are also affected by which criteria are used. The American Diabetes Association (ADA) recommend lower limits than the WHO,NYHA class was another parameter that was rarely stated in patient files. This limits us from analysing our material regarding symptomatic severity and how it correlated to glycaemic class and choice of treatment.Another limitation is that we used the latest available echocardiogram to classify patients’ HF phenotype. Patients with HF with recovered EF would therefore be classified as HFmrEF or HFpEF, instead of HFrEF as the European Society of Cardiology guidelines recommend.17In this Northern European real-world HF population, the prevalence of known DM was 27%. Of patients without known DM with available FPG or HbA1c-measurements, 9% had probable DM and 41% pre-diabetes, corresponding to 4% and 19% of the entire cohort, respectively. The prevalence of DM, pre-diabetes or normoglycaemia or choice of DM treatment did not differ between HF phenotypes. Patients with pre-diabetes and DM were more often treated with diuretics and beta blockers than normoglycaemic patients.


Investigation of the causality between 25(OH)D deficiency and OSA would help facilitate disease prevention.We conducted a two-sample bi-directional Mendelian randomization (MR) study. For forward analysis, 237 newly identified genetic variants are used as proxies for 25(OH)D to estimate the unconfounded effect on OSA among 16,761 OSA cases and 201,194 controls of European ancestry. Reverse analysis was performed to detect the causal impact of OSA on 25(OH)D levels. The inverse variance weighted (IVW) method was used as the primary analysis. Sensitivity analysis was performed to evaluate the robustness of our results. Multivariate MR analysis was conducted to evaluate the direct link between 25(OH)D and OSA after accounting for body mass index (BMI).P = 0.01). No evidence of the causal link from 25(OH)D to OSA was detected . Sensitivity analysis suggested the MR estimates were not biased. Multivariate MR analysis indicated the effect of OSA on 25(OH)D vanished upon accounting for BMI .IVW indicated that OSA causally associated with a lower level of 25(OH)D D, which might be driven by BMI. Obesity management should be enhanced in patients with OSA to prevent 25(OH)D deficiency.The online version contains supplementary material available at 10.1186/s12890-022-02172-y. Obstructive sleep apnea (OSA) refers to a kind of sleep-related disorder characterized by a repetitive, partial, or complete obstruction of the upper respiratory tract during sleep, resulting in an intermittent drop in blood oxygen partial pressure, blood oxygen saturation, and hypercapnia. EpidemiVitamin D, the liposoluble vitamin, has two forms: D2  and D3 D. VitaminMendelian randomization (MR) is a valuable method for causality inference via human genetics because of its initial application in evaluating if low cholesterol results in cancer–16. GiveP < 5 × 10− 8); (b) genetic instruments are not associated with potential confounders; and (c) genetic instruments only affect the outcome through the exposure of interest. Forward [from 25(OH)D to OSA] and reverse [from OSA to 25(OH)D] MR analyses were performed. Rigorous sensitivity analysis was applied in this study to evaluate any violation of the MR assumptions.We conducted a bidirectional two-sample Mendelian randomization study to appraise the causal association between circulating 25(OH)D levels and OSA. MR design is based on three critical fundamental assumptions: (a) genetic instruments are robustly associated with exposure of interest , which comprised 441,291 individuals of European ancestry. 16,668,957 SNPs were tested.We obtained the GWAS data for circulating 25(OH)D from the IEU consortium (https://www.finngen.fi/en), which aims to evaluate genotype-phenotype correlations to ultimately identify novel therapeutic targets. Genotype imputation, quality control and principal component analysis were undertaken in the FinnGen GWAS. The diagnosis of OSA was based on the International Statistical Classification of Diseases (ICD) codes, including ICD-10: G47.3 and ICD‐9: 3472 A. Specifically, cases with OSA were determined through subjective symptoms, clinical examination and sleep registration applying apnea‐hypopnea index (AHI) ≥ 5 per hour or respiratory event index (REI) ≥ 5 per hour. A total of 16,380,465 SNPs were tested in the OSA GWAS.Summary-level statistic for OSA was obtained from the FinnGen consortium (Round 5), comprising 16,761 OSA cases and 201,194 controls . Full OSA GWAS was publicly available from the FinnGen research project  r 2 < 0.01 within a 5,000 kb distance] from the full GWAS data to serve as the instruments proxying circulating 25(OH)D levels. Secondly, given that body mass index (BMI) is the established risk factor for OSA  (Table P = 0.45) or the MR-Egger method . Using Cochran Q test, we did not identify any heterogeneity . Besides, test of intercept of the MR-Egger regression showed no significant horizontal pleiotropic effects for the included variants . In addition, leave one out analysis showed that the null IVW estimate was not biased by any single variant. When assessing the causal effect of 25(OH)D on BMI, no evidence of the association between them was observed , implying that BMI did not work when considering the causal role of 25(OH)D on OSA.Using IVW, we identified a null causal effect of 25(OH)D on OSA [odds ratio (OR) = 0.99, 95% confidence interval (CI) = 0.88 ~ 1.12, P = 0.11), and no horizontal pleiotropy was detected . However, leave one out analysis indicated that the null estimate was driven by rs10928560 . After discarding rs10928560, IVW (five SNPs) detected a causal association between OSA and a decreasing concentration of 25(OH)D . Similar trends were also shown in other MR methods D concentration, we conducted a reverse analysis. IVW (six SNPs) suggested a null causal effect of OSA on 25(OH)D Table /Fig. 1. P = 2.63 × 10− 23). This result indicated that genetic predicted obesity was associated with lower 25(OH)D concentration. IVW also detected that genetic predicted higher BMI was causally associated with OSA . Further MVMR analysis indicated that the observed causal effect of OSA on 25(OH)D was attenuated after accounting for BMI .To further evaluate whether this observed causality was driven by BMI, we conducted MVMR analysis to detect the direct effect of OSA on 25(OH)D. Before MVMR analysis, we evaluated the causal role of BMI on 25(OH)D and found that genetic predicted BMI was associated with 25(OH)D D and OSA suggested that genetically predicted adult OSA might be causally associated with a lower 25(OH)D concentration. However, no evidence about the causal role of 25(OH)D in OSA was observed. Further MVMR analysis indicated that the causal effect of OSA on 25(OH)D was driven by BMI. To the best of our knowledge, this is the first MR study to explore the causality between 25(OH)D and OSA.P = 0.008)[Previous observational studies evaluating the association between 25(OH)D and OSA yielded inconsistent results. Some studies reported a significant relationship between a lower level of serum 25(OH)D and OSA. Goswami and colleagues found the relationship between OSA and 25(OH)D insufficiency may be mostly attributed to higher BMI and bigger neck circumference . Accordi = 0.008). Some st = 0.008), 11, 22. = 0.008). ConsideUsing MR design, this study provided evidence about the causal impact of OSA on a decreasing 25(OH)D level. The potential mechanisms underlying this causality are worth discussing. Specifically in the present study, MVMR indicated that the causal effect of OSA on 25(OH)D might be driven by BMI. This might have an implication in clinical practice that obesity management should be reinforced in patients with OSA to prevent 25(OH)D deficiency. Based on the existing literature, obesity has been identified as a major risk factor for OSA. Studies have confirmed that the excess of fat deposition surrounding the airway contributes to the narrowing of upper air way, 25. BesOur research made several signs of progress. First, to the best of our knowledge, no MR study on the relationship between 25(OH)D and OSA has yet been explored. To enhance the statistical power to detect causal relationships, we used numerous variants summarized from large-scale GWAS research on 25(OH)D and OSA to date. Second, using the MR design, our study simulated RCTs in observational settings. RCTs are regarded as the golden standard for causal inference, but the high cost makes it impractical for many scholars. MR studies can effectively avoid confounding bias because SNPs were randomly assigned at conception. Compared to other observational studies, MR can also avoid the reverse causal effect. Third, a crucial strength of our research, compared to the analysis of individual-level data from a small study, is that effect-size data were gathered from large-scale GWASs for exposure and outcome traits, allowing us to quantify effect sizes more precisely.There are several limitations to our research. First, although limiting the sample to people of European ancestry reduced population structure bias, this may restrict the generalizability of our findings to other populations. Second, population stratification could not be fully ruled out in our study. One point that should be noted is that our OSA GWAS data was derived from participants from Finland, a northern European country with low sun exposure. Therefore, national program specific to this county, like Vitamin D fortification in milk products, could potentially influence the standard 25(OH)D levels in Finland population, which might induce bias to our MR results. Third, our findings only reported the total effect of OSA on 25(OH)D concentration, but the underlying mechanisms warrant further investigation. Firth, only summary-level statistics were used in this study, making stratification analysis like severity, BMI, gender or age stratification unavailable. However, the original GWAS analysis from the FinnGen research project has adjusted for sex and age. More detailed MR analyses are warranted when GWAS with severity distribution is publicly available in the future. In addition, considering the critical role of BMI in OSA development, the present study used MVMR to adjust with BMI to detect the direct effect of OSA on 25(OH)D.In conclusion, this MR study showed that genetically predicted OSA decreased circulating 25(OH)D levels, which might be driven be BMI. These results provided a novel insight into the role of OSA in 25(OH)D metabolism and might have an implication for clinicians to enhance obesity management among the patients with OSA in clinical practice to prevent 25(OH)D deficiency, as that 25(OH)D is widely involved in multiple health conditions.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3


Ascocotyle (Phagicola) trentinii n. sp. is described based on adults from experimentally infected ducklings (Anas platyrhynchos domesticus) fed with metacercariae from the visceral serosa of the Mediterranean banded killifish, Aphanius fasciatus (Cyprinodontiformes: Aphaniidae), from coastal lagoons in northeastern Italy (Emilia-Romagna Region). The new species is placed into the subgenus Phagicola because of the presence of a single row of circumoral spines, vitelline follicles being confined between the ventral sucker and testes, and uterine loops not reaching anterior to the ventral sucker. Ascocotyle (P.) trentinii n. sp. differs from other members of the subgenus Phagicola, as well as other species of Ascocotyle, by the number (27–33) of circumoral spines which are 13.5–17 μm long and 3.5–5 μm wide, and by the morphology of a gonotyl which is composed of about 8 large refractile pockets. The occurrence of metacercariae in A. fasciatus indicates that the life cycle of the new species is completed in brackish water lagoons. It is the fourth species of Ascocotyle described in Europe and may be endemic to the Mediterranean region because its second (fish) intermediate host is endemic to this region.  •Ascocotyle (Digenea: Heterophyidae) is described from Europe after more than half a century.A new species of •The new species is characterised by a unique number (27–33) of circumoral spines.•Aphanius fasciatus(Cyprinodontiformes), from coastal lagoons is a second intermediate host.Mediterranean banded killifish,•Ascocotyle pindoramensisfrom South America, which has no circumoral spines, is most closely related to the new species. Some ofAscocotyle includes species characterized by an oral sucker armed with circumoral spines arranged usually in one or two rows (spines are absent in a very few taxa), and bearing a conical muscular prolongation (posterior appendage) of the oral sucker  (Cyprinodontiformes: Aphaniidae), the fish were found heavily infected with metacercariae of heterophyid trematodes. The metacercariae found in A. fasciatus were fed to ducklings (Anas platyrhynchos domesticus) to obtain sexually mature adults. Morphological observation revealed their affiliation to Ascocotyle because they possess a single row of circumoral spines, a conical muscular prolongation (posterior appendage) of the oral sucker, a gonotyl with refractile bodies and other characteristics typical of this genus  on the Adriatic coast of Emilia-Romagna Region, northern Italy. Metacercariae were embedded within the serosa of visceral organs (peritoneum). Previous analyses on different batches of A. fasciatus sampled from the same area showed high prevalence and high intensity  belonging to the same species on the basis of the number of circumoral spines (see Thirty-five ines see . MetacerAnas platyrhynchos domesticus var. American pekin) were fed with A. fasciatus infected with metacercariae of the new species. Fish infection was verified at dissection microscope before feeding the ducklings. Ten fish were chopped together and fed to four ducklings. The fish were previously checked under dissection microscope to verify the presence of metacercariae, always over 100 in number. Ducklings were kept in a ground stand in a facility of the State Veterinary Institute of Lombardia and Emilia-Romagna in Forlì. They were subjected daily to coprological examination until the finding of digenean eggs, i.e., 5 days p.i. Ducklings were then euthanised by cervical dislocation and their intestine was examined to collect the parasites. The adults  were then isolated from the gut of experimentally infected ducklings 5 days post-infection, washed in sterile saline and then fixed in 70% ethanol.To obtain adults, four 3 days old ducklings  and QuickPhoto . Measurements are expressed in micrometres (μm) and are presented as the range, with the mean followed by standard deviation (SD); n = number of measurements, usually also corresponding to the number of specimens measured, with few exceptions, such as size of circumoral spines. For scanning electron microscopy (SEM), adults were dehydrated through a graded alcohol series, dried by hexamethyldisilazane, coated with gold and then examined using a Phenom XL G2 Desktop SEM (Thermo Fisher Scientific) operating at 5 kV.2.2.1Total DNA, from both a metacercaria and an adult, were extracted using PureLink Genomic DNA Kit  following the manufacturer's protocol. The 28S rRNA gene was amplified with the primers U178 (forward 5′-GCACCCGCTGAAYTTAAG-3′) and L1642 (reverse 5′-CCAGCGCCATCCATTTTCA-3′) of https://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignments were constructed by ClustalW in BioEdit 7.2.5 . The sequences were compared with previously published data by BLAST tools ; natural host of metacercariae: Aphanius fasciatus.Site of infection: Small intestine.Type-locality: Cervia salt marshes, Emilia-Romagna Region, Italy , the site where metacercariae, used to experimentally infect the type host, were collected from Aphanius fasciatus.Other localities: Santa Gilla lagoon, Sardinia, Italy (Ascocotyle sp. 3).Type-material: Holotype and 13 paratypes are deposited at the Helminthological Collection of the Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, České Budějovice, Czech Republic (IPCAS D-846) and the Natural History Museum, Geneva, Switzerland .ZooBank number: urn:lsid:zoobank.org:act:A0A8E5E9-86FC-45A1-9DFA-065AB568E533.Second intermediate host: Mediterranean banded killifish, Aphanius fasciatus (Cyprinodontiformes: Aphaniidae); infected 29 of 35 killifish, i.e., prevalence of 83%, and intensity of infection reaching up to 100 metacercariae/fish; even most heavily infected fish host were sexually mature.Etymology: the species is named after Professor Massimo Trentini, our dear colleague and friend who participated in the initial study of this parasite and prematurely passed away.Representative DNA sequences: newly generated sequences of 28S rDNA  of an adult and a metacercaria were identical, which confirms they represent different ontogenetic stages of the same species.3.2Description (based on 29 mounted specimens and 2 specimens studied using SEM): Body pyriform, 440–635  long and 290–470  wide, with maximum width at mid-hindbody or slightly more anterior D, E. Bodvs VS) 1: 0.94–1.33 (1.11 ± 0.12).Oral sucker subterminal, 58–85  long and 64–100  wide, with conical, short posterior appendage, 37–45  long, reaching anterior margin of pharynx posteriorly. Oral sucker surrounded by single row of 29–33 (30 ± 1) massive, spearhead-like circumoral spines , Fig. 2FTestes double, unlobed, widely oval to subspherical, symmetrical, situated close to posterior margin of body, lateral to excretory bladder, 43–107  long and 37–74  wide  and 58–78  long and 48–78  wide  A. SeminaOvary spherical 34–55  long and 32–69  wide, median, at level of testes. Seminal receptacle 43–74  long and 39–104  wide, voluminous, transversely oval, median, posterior to ovary. Laurer's canal not observed. Vitellarium formed by irregularly-shaped follicles grouped in two posterolateral groups between posterior margin of ventral sucker to posterior half of testes. Common vitelline ducts slightly sinuous, ventrally passing over anterior part of seminal receptacle, medially joined and widened to form vitelline reservoir situated posterolateral to ovary.Uterus tubular, forming numerous loops between anterior margin of ventral sucker and posterior margin of testes, reaching slightly posterior to testes. Metraterm opening sinistrally into ventrogenital sinus. Eggs thick-walled, operculate, 17–21  long and 11–12  wide. Excretory vesicle V-shaped, with short and wide stem.SEM observation of adults revealed the presence of tegumental spines with different shape: bifid in almost whole-body surface H, with s3.3Aphanius fasciatus). Encysted metacercariae oval, 173–250 long (213 ± 26) and 140–160 (154 ± 8). Cyst wall double, with hyaline, elastic outer wall 3.5 μm thick and transparent membraneous inner layer. Excysted metacercariae pyriform, covered with tiny tegumental spines. Oral sucker bearing 27–33  circumoral spines and muscular posterior appendage (posterior prolongation). Intestinal caeca reaching posteriorly to level of ventral sucker; they do not contain spherical discs. Region between pharynx and intestinal bifurcation filled with large concentration of gland cells with darker content. Primordia of genital organs  well-developed; primordium of gonotyl visible in some specimens. Excretory vesicle Y-shaped, with short and wide stem, filled with dark excretory granules.Description  trentinii n. sp. differs from the other members of this subgenus as well as other species of Ascocotyle by the number (29–33) of circumoral spines 12–18 μm long and 3–6 μm wide (see Ascocotyle), and by the morphology of the gonotyl, which is composed of about 8 large refractile pockets.A. (P.) cameliae Hernández-Orts, Georgieva, Landete et Scholz, 2019 from the Magellanic penguin, Spheniscus magellanicus (Forster), in Argentina, possesses pectinate tegumental spines, but the number of their terminal points decreases continuously from three on the anterior third of the body to a single point in the posterior part of body surface (Ascocotyle (P.) pindoramensis shows digitiform tegumental spines and is devoid of circumoral spines (A. (P.) pindoramensis is the sister taxa of A. (P.) trentinii n. sp. as showed in the ML tree of the 28S rDNA. Both these species are reported in cyprinodontiform fish, Poecilia vivipara Bloch et Schneider and Aphanius fasciatus, respectively, and live in similar habitats (brackish waters), but in different continents (South America versus Europe).The tegumental spination pattern of the new species observed by SEM differs from that described in other congeneric species that were studied using SEM. The most recently described species,  surface . Ascocotl spines . Despite3.5Ascocotyle as Ascocotyle (Phagicola) sp. 3 from A. fasciatus sampled in Santa Gilla lagoon in Sardinia, Italy. Because of a similar number of circumoral spines (>30) and their arrangement in a single row, it is likely that the specimens were conspecific with A. (P.) trentinii n. sp.3.6Ascocotyle clade (including species placed in the subgenus Phagicola), with A. (P.) pindoramensis  as its sister taxon and A. (P.) longa  basal to both species (The ML tree showed the new species forming a well-supported cluster within the  species .4Ascocotyle (P.) trentinii n. sp. is the first new species of Ascocotyle described from Europe after more than a half century. This may be caused by little attention paid to search for these tiny trematodes, which mature in fish-eating birds; these host are usually protected in most European countries and thus difficult to examine for parasites. Another reason may be relatively rare occurrence of heterophyids in Europe and their low species diversity in temperate zones, especially compared with that in subtropical and tropical regions  in Brazil. In fact, both species are morphologically similar to those of Ascocotyle .Molecular phylogenetic analysis placed the new species within a well-supported clade composed from all but two species of cocotyle . The onlA. trentinii n. sp. is somewhat surprising considering extraordinarily high prevalence and intensity  of its infection in the Mediterranean banded killifish in Italy. This cyprinodontiform fish is a short-lived, non-commercial species widely distributed in the Mediterranean basin .Since A. trentinii n. sp. is still unknown. The Cervia salt marshes, where the Mediterranean banded killifish were sampled, are characterised by a very rich fauna of fish-eating birds, such as several species of the Ardeidae, Laridae and Anatidae, which may serve as possible definitive hosts of heterophyid trematodes, including the new species. Especially birds of the families Ardeidae are common definitive hosts of trematodes linked to aquatic environments that include fish.The natural definitive host of Ardea cinerea, Ardea alba, Egretta garzetta), larids  and anatids . Since several years there is also a stable population of flamingos (Phoenicopterus roseus). Considering the eating behaviour of the above mentioned species, the herons seem the most promising potential definitive hosts of A. trentinii. However, they live in a protected area, which makes their parasitological examination hardly possible.However, most lagoons are protected areas and it is difficult to examine fish-eating birds to obtain adult worms in natural definitive hosts, to better catalogue the biodiversity and to understand better ecological connections in this peculiar ecosystem. The Cervia salt pans host more than 30 sedentary species of birds , among which herons (Some non-invasive methods such as eDNA screening might help to detect foci of the parasite transmission by detecting its DNA from the eggs, miracidia or cercariae in water samples. Monitoring of the local malacofauna might also help to identify the natural first intermediate host of the new species.AscocotyleThere is no conflict of interest in the submitted manuscript entitled “A new species of


Compared to DD− group, DD+ group had significantly higher incidence of LSCEs  and shorter survival time . Kaplan–Meier analysis revealed significantly higher risks of primary endpoint (p = 0.039), and LSCEs (p = 0.040) in DD+ group. Multivariate hazard analysis showed that DD+ group had significantly higher risk of primary endpoint , and LSCEs . Ultrasound-assessed diaphragm dysfunction predicts clinical outcomes in HD patients.Skeletal muscle atrophy is prevalent and remarkably increases the risk of cardiovascular (CV) events and mortality in hemodialysis (HD) patients. However, whether diaphragm dysfunction predicts clinical outcomes in HD patients is unknown. This was a prospective cohort study of 103 HD patients. After assessment of diaphragm function by ultrasonography and collection of other baseline data, a 36-month follow-up was then initiated. Participants were divided into diaphragm dysfunction (DD+) group and normal diaphragm function (DD−) group, according to cutoff value of thickening ratio (i.e. the change ratio of diaphragm thickness) at force respiration. The primary endpoint was the first nonfatal CV event or all-cause mortality. A secondary endpoint was less serious CV events . 98 patients were eligible to analysis and 57 (58.16%) were men. 28 of 44 patients(63.64%) in DD+ group and 23 of 54 patients (42.59%) in DD− group had at least one nonfatal CV event or death (www.chictr.org.cn) as ChiCTR1800016500 on Jun 05, 2018.Trial registration: This study was registered with Chinese Clinical Trials Registry ( Low muscle mass or strength is associated with poor physical function, frailty, and higher risk of all-cause mortality in CKD patients6. Furthermore, individuals who have low muscle mass are more likely to have cardiovascular (CV) events, such as coronary artery disease, cerebrovascular disorders, heart failure, peripheral artery disease, arrhythmias10. CV diseases are the principal complication in CKD patients, and CV mortality increases as glomerular filtration rate decreases11. Therefore, skeleton muscle atrophy maybe an important target to improve the clinical outcomes in hemodialysis (HD) patients.Skeleton muscle atrophy is a common body composition abnormality in patients with chronic kidney disease (CKD), especially end stage renal disease12. Diaphragm dysfunction is associated with dyspnea, decreased exercise endurance and quality of life13. Ultrasound is considered as an optimal tool to evaluate the diaphragmatic thickness and function in multiple diseases because of its high sensitivity and specificity, combined with zero radiation and low cost17. Ultrasound-assessed diaphragm dysfunction is reliable in early identifying diaphragm atrophy and stratifying patients’ risk of worse prognosis22. Our research group has previously reported that the ultrasound-assessed diaphragm function of HD patients is worse than that of healthy individuals, and is associated with dyspnea, fatigue and hiccup in HD patients23.Diaphragm is the primary respiratory muscle of human body and accounts for 70% work of inspirationHowever, the relationship of diaphragm function and occurrence of CV events and mortality in HD patients remains unknown. The purpose of this prospective longitudinal study is to identify whether ultrasound-assessed diaphragm dysfunction predicts the clinical outcomes in HD patients.23. A total of 103 maintenance HD patients, aged 24 to 81 years old, were recruited from May 2018 to November 2018. Follow up work started from the date of enrollment for 36 months.The study was conducted at the Institute of Nephrology, Southeast University, an academic medical HD center in Nanjing, Jiangsu, China. This cohort came from a previous study conducted by our team and the detail of these patients have been published7, we calculated the minimal sample size (N = 97) with a power test  by software PASS15.According to the following assumption: 3 years follow-up, 20% probable loss during follow-up, the 3 years rate of primary endpoint in CKD with low sarcopenia score of 20%, and events ratio between high sarcopenia score group and low sarcopenia score group of 3 based on the study conducted by Hanatani et al.Demographic features, comorbid diseases and drugs associated with CV events were recorded through detailed interview after obtaining written informed consent. CV diseases included coronary artery disease, peripheral vascular disease, stroke and arrythmia. The serum level of fasting blood-glucose (FBG), hemoglobin, albumin, low density lipoprotein cholesterol (LDLC) and left ventricular ejection fraction  of the recruited patients were collected, and the results within 1 month closest to enrollment were used.24. During the process of ultrasound examination, participants remained supine position26 and breathed according to ultrasound technician’s instructions. A 6–13 MHz linear high-frequency ultrasound transducer with B-mode was placed perpendicular to the chest wall on the midaxillary line at diaphragmatic zone of apposition near costophrenic angle. The diaphragm thicknesses at functional residual capacity (FRC), tidal volume (VT), residual volume (RV) and total lung capacity (TLC) were recorded, respectively. Muscle thickness of diaphragm (Tdi) was a hypoechoic structure between the bilateral hyperechoic structures (pleura and peritoneum) and final value was averaged over three continuous respiratory cycles. Diaphragm excursions (DE), the distances diaphragm motioned from cephalad side to caudal side at eupnea and forced respiratory maneuver, were measured by a 1–5 MHz M-mode ultrasound transducer. Consequently, velocity of diaphragm excursion was obtained from dividing DE by motioned time, which also averaged over three continuous respiratory cycles. Tdi and mobility of diaphragm has been associated with pulmonary function27. Change of Tdi (ΔTdi) at eupnea was the absolute difference between TdiFRC and TdiVT values. ΔTdi at forced respiratory was calculated by TdiTLC minus TdiRV. Change ratio of Tdi at forced respiratory, termed as thickening ratio, was calculated by the following formula: (TdiTLC − TdiRV)/TdiRV, which reflects the maximum contractile capability of diaphragm. Thickening ratio at forced respiration has been also associated with lung function. It can help clinicians identify patients at high risk of nocturnal hypoxia, who hadn’t obvious clinical symptom related to hypoxia at early stage28. Therefore, thickening ratio at forced respiration might be an early indicator of diaphragm dysfunction.Ultrasound was used to evaluate diaphragm function of HD patients. Measurement was taken after dialysis to minimize the impact of water accumulation. Right hemidiaphragm was used, which can be more conveniently and reproducibly measured in comparison to the leftEvents collection were conducted every 2 months, either face-to-face or telephone interviews until up to 36 months. If the patients had hospitalization or received outpatient treatment in our hospital during the visit interval, we also manually checked the electronic medical records system to verify any nonfatal CV event or the causes of death. If electronic medical records were not available, we would review the discharge records or outpatient records. The primary endpoint was a composite of first nonfatal CV event and all-cause mortality. Causes of deaths were categorized as CV deaths or non-CV deaths. CV deaths included sudden death, which caused by CV events in nature unless non-CV causes were defined. Considering that some patients had more than one adverse CV event, we subsequently divided the primary endpoint into three subgroups to explore which subgroup may play a major role. These three subgroups were as follows: major adverse CV events , other major adverse CV events  and less serious CV events . The secondary endpoints were MACEs; MACEs+; and LSCEs.www.chictr.org.cn, identification number ChiCTR1800016500, 05/06/2018). The protocol of this study was performed in accordance with the Declaration of Helsinki and relevant regulations. The informed consent was obtained from all participants.This study was approval by the ethics committee of Zhong Da Hospital affiliated to Southeast University and the study was registered with Chinese Clinical Trials Registry (All participants were divided into two groups—diaphragm dysfunction (DD +) group and normal diaphragm function (DD−) group, based on the cutoff values of thickening ratio at forced respiration. The correlation between the primary endpoint and thickening ratio through ROC analysis, and the cutoff value was 0.79 . Subgroup analyses were also performed with ROC analysis. The cutoff value was also 0.79 when LSCEs as the endpoint. Lower than cutoff value indicates diaphragm dysfunction. We compared the impact of DD+ or DD− on endpoints by time-to-event analysis. In addition, we also analyzed the characteristics of participants between event group (who occurred target endpoints) and non-event group.p values are two-tailed and difference denotes statistical significance at P < 0.05. Data were analyzed by the statistical software SAS 9.4 , survival curves were drawn by R 4.0.3 , histograms were drawn by GraphPad Prism 8.0.1.Continuous variables were expressed as means ± standard deviation or medians . Categorical variables were expressed as counts(percentage). Continuous variables, according to the normality and homogeneity of variance, were compared by Wilcoxon test, Student’s t-test, or t’ test, respectively. Categorical variables were appropriately compared using chi-square test or Fisher’s exact test. The potential prognostic factors were screened one by one (α = 0.10) in Cox proportional hazard model, and then these screened out factors as covariates were taken into multivariate regression analysis by bootstrap. The results are shown as hazard ratios (HR) and 95% confident intervals (CI). All The flowchart of this study is shown in Fig. p < 0.05), while other indexes of laboratory test and demography characteristics were not significantly different between DD+ group and DD− group , TdiFRC (p = 0.040), and TdiRV (p = 0.010) (Table Tdi0) Table .vs. 26.74 ± 12.59 months, p = 0.046). 28 of the 44 patients(63.64%) in DD+ group and 23 of the 54 patients (42.59%) in DD− group had at least one nonfatal CV event or all-cause mortality  died, and 10 10.20%) died of CV events. Among the other 7 (7.14%) patients, 2 (2.04%) died of tumor, 2 (2.04%) died due to delayed dialysis during COVID-19 pandemic, 2 (2.04%) died of septic shock, 1 (1.02%) died of gastrointestinal and urinary tract bleeding. Compared to the DD− group, the DD+ group had significantly shorter survival time , and serum level of LDLC.29. In this study, diaphragm dysfunction was particularly associated with LSCEs, majority of which was heart failure readmission. Inspiratory muscle dysfunction had been found to correlate with CHF and as an independent predictor of prognosis in CHF patients30. Decreased caudal displacement of diaphragm could directly reduce lung volume and negative intrathoracic pressure, leading to imbalance of ventilation and perfusion ratio of lung, sympathetic activation, and decreased cardiac output33. During this pathophysiology, heart failure, arrythmia and myocardial ischemia are all apt to occur. Recently, dyspnea which cannot be explained by cardiopulmonary disease was named as “residual exertional dyspnea”, and one of its possible causes was diaphragm dysfunction34. In addition to nutritional intervention, simple resistance training can ameliorate diaphragm dysfunction35. However, diaphragm dysfunction has not attached enough attention of clinicians.Diaphragmatic movement not only affects respiratory, but also cardiac output. As respiratory work reduced, stroke volume and cardiac output decreased37. CKD expedites atherosclerosis by augmenting inflammation, dyslipidemia, vascular calcification, immune disorders and other mechanisms38. Skeleton muscle can communicate with heart by molecular factors39. However, the slow progression of atherosclerotic plaque to occur CV events often requires a relatively long time, and clinicians generally have attached importance to secondary prevention of atherosclerotic diseases. Prolonged follow-up time may be needed to explore the underlying relationships of diaphragm function with MACEs and MACEs+.There is no association of diaphragm dysfunction with MACEs or MACEs+ in our cohort. The major underlying pathology of cardiovascular diseases included in MACEs and MACEs+ is atherosclerosis40, and TdiTLC below 4.0 mm was defined as impaired diaphragm function in HF patients41. In this study, TdiTLC is significantly thinner in DD+ group than DD− group (Table TLC was used to differentiated DD+ group and DD− group (Data not shown). The reason of which may be that muscle mass and muscle function are incongruent in CKD patients. Impaired muscle function in uremic milieu predated muscle atrophy43. Compared Tdi of event group with non-event group, several parameters are even thicker  patients were underweight and the average serum level of albumin was > 35 g/L], however, a part of participants had appeared a relatively decline in diaphragm function evaluated by thickening ratio at forced respiration. Thus, ultrasound-assessed thickening ratio at forced respiration is a potential indicator of early identification of diaphragm dysfunction in HD patients.We should declare several limitations of our study. First, expanded sample size was needed to find the appropriate cutoff value of thickening ratio at forced respiration, having ideal sensitivity and specificity, to differentiate DD− and DD+. Second, statins and anti-platelet drugs were not used by all patients at risk of cardiovascular diseases, and the type and dosage of similar drug varied. This would bias the incidents of MACEs and MACEs+. Third, it did not perform pulmonary function tests and diaphragm ultrasound at the same time, so this study didn’t show the relationship between diaphragm function and pulmonary function in HD patients directly. Fourth, correlation analysis does not imply causation, longitudinal trial with specific intervention for improving diaphragm dysfunction to observe the effect on prognosis would be necessary.Ultrasonography is an ideal tool and easy to be operated for evaluating diaphragm function. Low thickening ratio at forced respiration is an early indicator of diaphragm dysfunction and is associated with adverse clinical outcomes in HD patients. These findings suggests that routine screening for diaphragm dysfunction is advisable in HD patients and intervention of diaphragm dysfunction is expected to be a novel strategy for improving the clinical outcomes in HD patients.Supplementary Information.


Staphylococcus aureus. Microbiologically, no coliform was detected in all the samples, confirming the honey's quality. The amount of lipid peroxidation was raised in the diabetic group with no treatment, 1% silver sulfadiazine group, and JS group, while no significant reduction was observed in other groups. Differences in wound contraction were significantly notable on various days of measurement, day 3 (p < 0.002), day 6 (p < 0.046), and day 9 (p = 0.00). The catalase level in the different treatment groups increased significantly (p < 0.05), implying an antioxidant potential of the different honey samples except for Jos honey. The study concludes that honey infused with moringa was faster and more efficient in healing diabetic wounds than other honey samples and silver sulfadiazine.There is an increase in drug-resistant strains causing infection, thus making available therapeutics less effective. As resistance increases, modern medicine focuses on the antibacterial potential of natural products, which can aid in wound healing. The present study determined Nigeria honey's antibacterial efficacy in treating diabetes-induced wound infections in Wistar albino rats. 54 Wistar rats randomly divided into 9 groups of 6 each were used for the study: group I , group II , group III (diabetes treated with 1% silver sulfadiazine), and groups IV–IX (diabetes treated with different honey samples). Physiochemical analysis and microbiological and antibacterial activity of the honey samples were determined. The treatments were carried out for 17 days, and wound contraction, malondialdehyde (MDA) levels, and catalase activity were measured. Results obtained showed that the most effective honey was DCH (21.5 ± 2.12), followed by HBP + M (15 ± 2.12) and TRB, JS, and HBP  for antibacterial activity on  Diabetes is a severe metabolic disorder that causes physiological changes in tissues and cells, leading to diabetic wounds in the lower extremities in some patients. Diabetes can cause bacterial infection and microbial resistance complications, leading to wound healing and amputation delays. The need for alternative antimicrobial techniques has increased the interest in the therapeutic use of traditional medicines such as plants and plant-based products such as honey, ginger, garlic, and moringa. Plant extract-derived products have been shown to promote wound healing by lowering inflammatory cell influx in situ, boosting angiogenesis and fibroblastic proliferation, or speeding up reepithelization. They also encourage a combination of different pathological actions . Honey iResearch has led to the development of honey-based adhesives for wound care and other biomedical applications. Honey is helpful in wound healing because it can accelerate dermal repair and epithelialization, promote angiogenesis and immune response, and reduce healing-related infections with pathogenic microorganisms . MicrobiEleven honey samples were used for this study. These honey were obtained from different beehive keepers in different geographical regions of Nigeria, namely, Nsukka south-east (NSK), Kaduna north-western (KAD), Taraba north-eastern (TRB), Jos north-central (JS), Obudu south-south (OBD), Imo south-east (IMS), Lagos south-west (DCH), US grade A branded honey (KL), honey bee pure honey (HBP), honey bee pure honey infused with moringa (HBP + M), and honey bee pure honey infused garlic (HBP + G). All samples were kept at room temperature with tight-fitting lids.Staphylococcus aureus stock isolates from the wound were acquired from the Medical Microbiology and Parasitology Laboratory at the University of Port Harcourt Teaching Hospital in Rivers State, Nigeria. Staphylococcus aureus isolates were reidentified by cultural and biochemical methods. In brief, a colony was picked from the nutrient agar slant containing Staphylococcus aureus obtained from the culture bank and streaked onto mannitol salt agar (MSA). This was incubated at 37°C for 24 hours, and characteristic Staphylococcus aureus was purified. Identification was based on colony characteristics, Gram reaction, catalase, and coagulase test.This research was conducted at the University of Port Harcourt's Medical Microbiology Laboratory. The spread plate technique was used to determine the microbial population of honey samples. Honey samples were diluted in sterile distilled water, and different concentrations of honey were generated in 70, 50, 20, and 10%, and plates were incubated for 24 hours at 37°C .8 CFU/ml) for opacity. A sterile swab was dipped into the isolate solution, pressed clear of excess fluid against the edge of the tube, and then distributed over the agar plate. The test organism was evenly distributed on the Mueller-Hinton agar (Oxoid) surface, and the plates were left on the bench to absorb the excess moisture. Wells were formed in the agar medium with a sterile cork borer (6-millimeter (mm) diameter), and 0.1 ml of honey in various concentrations was applied to the wells on the plate. The plates were incubated for 24 hours at 37°C. The inhibition's mean diameters were measured in mm.The agar well diffusion technique was used to evaluate susceptibility . The inoSelected physicochemical parameters such as pH, moisture content, ash, fibre, crude protein, fructose, glucose, sucrose, tannin, phenol, and flavonoid were determined using the methods of Agbagwa .ad libitum. Before the start of the investigation, the vital parameters of the animals, including temperature, weight, and fasting blood glucose level, were evaluated and determined to be consistent with excellent health [n = 6) and distinguished with dyes on the head, neck, midbody, tail, and right leg and no dye. The 9 experimental groups formed are presented in Adult albino rats (120.4–300.5 g) with a mean weight of 185.4 g were housed in a conventional and regulated laboratory environment with a 12 : 12 h dark and light cycle to acclimatize. The rats were fed on rodent laboratory chow and had access to water and Diabetes was developed in 48 Wistar rats after 14 days of acclimation by intraperitoneal injection (150 mg/kg body weight) of freshly produced alloxan monohydrate  in normal saline after overnight fasting . Fasting8 CFU/ml of Staphylococcus aureus prepared for the purpose.The experimental animals' dorsal skin was shaved and swabbed with ethanol under anaesthesia with an intramuscular injection of 5% ketamine and 2% xylazine. Following anaesthetic recovery, the experimental animals were placed in disinfected cages in groups for continued wound monitoring. An entire thickness punch biopsy wound was produced on the dorsum of each rat, and each wound was infected with 1 × 10The wounds were left untreated for 24 hours before infection. Additionally, swab samples of wound exudate were taken and cultured on mannitol salt agar plates and were verified using Gram reaction, catalase, and coagulase test . The effEach animal was evaluated for gross wound healing indices  . The redOn days 3, 5, and 7, swabs were taken from the wound exudates for bacterial culture and colony quantification.2 complex that can be detected spectrophotometrically at 532 nm. Tissue homogenate (10%) of granulation tissues was produced in 0.02 M phosphate-buffered saline and used to calculate catalase (CAT). The sample was combined in a test tube containing tissue homogenate (0.1 ml) and hydrogen peroxide (0.5 ml) and incubated in a water bath for 60 seconds at 37°C before being measured using a spectrophotometer at 480 nm [The glucose level was tested to confirm diabetes induction. Blood samples were taken, and the blood glucose level was determined using a glucometer (ACCU-CHEK Active®). The TBA reaction was used to assess the quantity of malondialdehyde (MDA) in wound tissue and tissue homogenates to determine the rate of lipid peroxidation. The reaction of MDA with thiobarbituric acid (TBA) in acidic circumstances and at higher temperatures results in a pink MDA-(TBA)t 480 nm , 26.SOP was followed to assure data quality at all stages of the investigation . The culture media were produced following the manufacturer's instructions.p < 0.05 was considered significant [t-test was used to compare the sample mean to the population mean.The results were statistically evaluated following double sampling, and the mean values and standard deviations were given. One-way ANOVA was employed to examine the differences across groups, and nificant . One sam3 to 7.3 × 104 CFU/ml. The least TVC was observed in honey sample KL and the highest in NSK, as shown in Total viable count (TVC) and total coliform count (TCC) were carried out on the selected honey samples to ascertain the microbiological quality of the samples. Total coliform counts were not detected in all the honey samples studied, and TVC ranged from 3.5 × 10Staphylococcus aureus was determined by the well-in-agar method at different concentrations  (p < 0.002), day 6 (p < 0.046), and day 9 (p=0.00).Similar to silver sulfadiazine treatment, honey generated a time-dependent increase in wound contraction compared to the control groups, as seen by wound photographs taken on different days . The per > 0.05) . At day p > 0.05).All groups had significant wound exudation , which dp < 0.05), it was present in both the diabetes control group (50%) and the nondiabetic control group (33.3%). Compared to the diabetes control and SS, rats in the honey therapy groups healed their wounds with noticeably greater granulation tissue (p < 0.05). The pattern from days 6 to 9 was HBP > NSK > DCH > KAD > TRB > SS > JS  in the SS group and the honey-treated groups was significantly higher than that in the diabetic untreated control group (positive control) (p < 0.05). The honey and SS treatment reduced the dense growth of Staphylococcus aureus on the culture plate on the different experimental days. Untreated diabetic wounds allowed dense growth of Staphylococcus aureus on the culture plates on days 1, 3, and 5 and then declined on days 7 and 9. On subsequent days 3, 5, 7, and 9, the honey treatment groups showed a significant reduction, allowing sparse Staphylococcus aureus on the culture plate. On day 9, the honey treatment groups and reference topical cream inhibited the growth of the Staphylococcus aureus colony, but the untreated wounds in the controls still showed dense growth . The mean baseline of the fasting blood glucose in normal control rats was between 4.7 and 6.4 mmol/L. After induction, animals that had fasting blood glucose level > 200 mg/dL (11.1 mmol/L) were grouped as diabetic for treatment.p < 0.05) was observed in the CAT levels and MDA in HBP + M, JS, NSK, TRB, DCH honey, and 1% SS. MDA level was high in people with diabetes without treatment (14.55 ± 1.2) and with SS (11.62 ± 1.29). The oxidative stress marker MDA levels in the honey group exhibited a significant change compared to the positive control (the diabetic group with no treatment). Of the honey studied, JS, TRB, and KAD had  MDA levels. MDA levels on day 17 were significantly lower in the honey groups and negative control group than in the positive control group (p = 0.037).The groups determined the antioxidants and oxidant levels by measuring MDA and CAT levels . SignifiP < 0.05) was observed in CAT levels for HBP + M, TRB, and NSK  when compared to the person with the diabetes-induced infection without treatment. No significant difference was observed in JS (20.34 ± 3.8) compared to diabetic and SS (p=0.117).The least was observed in NSK and HBP + M , while a significant difference , and other resistant organisms [p > 0.05). These findings show that this honey has high storage capacity. The ash content in pure honey reflects the overall amount of inorganic minerals. It should not be more than 0.6%. Variations were observed in the present study, which might be attributed to the various sources [A wound is said to occur when the typical structure and function of the outer tissue are altered, it can be healed by a biological process such as inflammatory response, proliferation, and remodelling to bring the injured tissue close to its original state. Diabetes can interfere with the normal healing process of the wound , 29. Prerganisms , 31. In rganisms , 33. Phyrganisms  range. Trganisms . The acirganisms . Honey crganisms . The honrganisms  specificrganisms –43. No v sources . The fib sources .Staphylococcus aureus) was susceptible to honey samples infused with plant extracts (moringa and garlic). The diverse zones of inhibition point to the distinct honey samples with the greatest DCH and lowest IMS having differing levels of effectiveness and different phytoconstituents. According to the findings, Moringa oleifera is responsible for the honey's bacteriostatic impact on the tested isolate. This shows a synergistic antibacterial effect evident in the increased zone of inhibition.The honey samples used in this study had degree Brix ranging from 72 to 83.4. According to reports, the Brix number is proportional to the honey's sugar amount. Brix readings will be more significant in honey with higher sugar content. Honey's acid and mineral content also contribute to the total soluble solids. This characteristic provides information about the honey's ripeness and nutritional worth , 46. Thep < 0.05). The early complete wound closure in the honey diabetic subgroup treatment showed a remarkable contraction equivalent to the SS treatments, which was related to the contraction of the wound and possibly enhanced fibroblastic response and reepithelization. These findings demonstrate that honey, when administered as a contracting agent, may reduce wound areas as effectively as SS, the topical medication employed as a standard. The study by Eyarefe et al. [Staphylococcus aureus, which could cause soft tissue reactions and an extended wound.Wound contraction increased time-dependent in the honey samples, 1% SS, and control groups. These findings demonstrate that honey is a potent wound-healing agent capable of decreasing wound diameter as effectively as 1% SS. The different honey treatment groups (Group IV–Group IX) showed remarkable statistically significant wound closure ratios on days 3, 6, and 9 of treatment , honey therapy had lower oxidative stress marker MDA levels on day 17. Additionally, the rate of bacterial clearance in the honey therapy was directly correlated with the pace of wound healing. As a component of the cellular defence system, Nuclear Related Factor 2 (Nrf2) separates from its cytoplasmic repressor Keap 1 in response to oxidative stress and binds to cytoprotective genes that encode antioxidant enzymes in the nucleus . Due to Staphylococcus aureus play in diabetic sores when left untreated, as well as how their actions may be capable of impairing the healing process and causing damage that is greater than anticipated. According to the findings of this study, honey is an effective alternative for treating wound infections since it possesses the qualities necessary to be used as a “standard to treat against diabetic wounds.” It is regarded as affordable and secure. Our study revealed that the inflammatory phase of wound healing began to decline early in the experimental groups with honey-dressed wounds. However, adding the plant extract moringa to the honey caused a miraculous recovery that included reduced wound exudation and hyperemia, fast contraction without scarring, and lack of inflammation (edge edema). The study showed that the phenolic content played a role in wound bacterial clearance, antioxidant activity, and antibacterial activity. The study concludes that honey from different zones in Nigeria possessed varying efficacy. Our findings suggest that honey infused with moringa is a suitable alternative option. It is acceptable due to its physicochemical properties, microbiological safety, antibacterial, antioxidant, and anti-inflammatory properties, and the promotion of tissue development and bacterial wound clearance found in this study. It is advantageous for managing wounds and being safe and economical. However, due to the study's findings that treatment response differs from host to host, depending on the peculiarities of the wound and the individual metabolism, care should be taken.This study investigated the effect


Even after transplantation of favourable donor lungs, some recipients require prolonged weaning from mechanical ventilation, indicating a poor prognosis. We investigated the effects of prolonged mechanical ventilation (PMV) for >14 days on the recovery and survival of patients who underwent cadaveric lung transplantation in relation to their physical traits.We retrospectively reviewed patients who underwent cadaveric lung transplantation (age ≥15 years) at a single centre between April 2015 and December 2020 and classified them into PMV and non-PMV groups . The factors predicting PMV comprised clinical factors  and physical features, namely flat chest, narrow fourth intercostal space , mediastinal shift, thoracic mediastinal-occupying ratio (TMOR) >40% and sarcopenia, according to the logistic regression analysis. The log-rank test was used to examine the association between TMOR >40% and 3-year prognosis.P = 0.023) was an independent preoperative predictive factor for PMV postoperatively. Stepwise analysis revealed intraoperative extracorporeal membrane oxygenation and reoperation as postoperative predictive factors in addition to TMOR >40%. Recipients with TMOR >40% had significantly worse 3-year survival than other recipients .The PMV group comprised 17 (33%) of 51 recipients. Multivariable logistic analysis showed that the TMOR >40% (odds ratio, 7.3; 95% confidence interval, 1.3–40.1; Recipients with a TMOR >40% may be long-term ventilator dependent and have a poor prognosis. Lung transplantation is an established procedure for end-stage lung failure [1]. Lung transplantation is an established procedure for end-stage lung failure . The 5-yhttp://cts.m.u-tokyo.ac.jp/thoracic-surgery/clinical_study), and patients who declined to participate were excluded. This study was approved by the Institutional Review Board of The University of Tokyo Hospital [approval number: # 2406-(6)].For this retrospective study, informed consent was obtained in the form of an opt-out clause on our hospital’s website  between January 2015 and December 2020. Eleven patients who could not undergo pulmonary function tests or computed tomography (CT) before lung transplantation, because they were under 15 years of age, and 8 patients who underwent living-donor lobar lung transplantation, were excluded. Preoperative chest CT results and perioperative clinical data for the remaining 51 patients were retrospectively reviewed. To analyse the cause of PMV relative to the recipients’ physical traits, as the primary outcome, the patients were categorized into 2 groups: PMV (early recovery) and non-PMV (late recovery) groups. Regarding the management of patients in the intensive care unit, we usually conduct tracheostomy in patients on mechanical ventilation over 14 days to prevent ventilator-associated pneumonia. Thus, PMV was defined as mechanical ventilation provided for >14 days after transplantation with or without tracheostomy. Finally, we investigated the relationship between important physical traits and 3-year prognosis as the secondary outcome.The recipients’ preoperative thoracic traits were evaluated using plain or contrast-enhanced CT images the day before CLT Fig. . The ima2 in men and <5.7 kg/m2 in women) and (iii) low physical performance . Sarcopenia was diagnosed when the criterion (i) and criteria (ii) and/or (iii) were met. A short physical performance battery score of ≤9 or a five-repetition sit-to-stand test result of ≥12 s for low physical performance was not routinely assessed at our centre.Preoperative sarcopenia was diagnosed according to the Asian Working Group for Sarcopenia 2019 consensus ) for CLT, most recipients  had sarcopenia and low performance scores, especially for the 6-m walk distance/second . Regarding preoperative nutritional status, the mean value of serum albumin was 4.0 ± 0.4 g/dl , which was not significantly different between patients, with or without sarcopenia .The evaluated physical traits are shown in Table ex vivo perfusion. There was no significant difference in the usage of marginal lungs  or size matching : 99% vs 101%, respectively; P = 0.704) between the PMV and non-PMV groups. In addition, the results of size matching according to the transplant procedure  did not differ significantly between the 2 groups .According to the criteria for marginal donors , 35 (69%P = 0.005), with a longer total ischaemic time , and more frequent use of intraoperative extracorporeal membrane oxygenation (ECMO)  than the non-PMV group. Regarding the continuous use of ECMO after transplantation, the PMV group had a significantly higher usage rate . Patients on ECMO were treated with continuous unfractionated heparin with activated clotting and activated partial thromboplastin time target ranges of 160–200 and 40–60 s, respectively.Regarding the transplant procedure, the PMV group underwent bilateral CLT more frequently . Grade 3 primary graft dysfunction was observed minimally in both groups . Six months after CLT, both groups (PMV vs non-PMV) showed similar elevation in forced VC and forced expiratory volume in 1 s .Within 14 days after CLT, more recipients underwent reoperation for postoperative Haemorrhage in the PMV group than in the non-PMV group  with ECMO use  and reoperation .Table P = 0.008; Fig. P = 0.020).The median follow-up period was 617 days , during which 4 patients died. The survival curves differed significantly (P = 0.149). Furthermore, there was no significant difference in the remaining primary diseases of the 2 groups.Twelve of 20 (60%) and 11 of 31 (35%) patients had idiopathic lung disease in the group with TMOR >40% and that with TMOR ≤40%, respectively. There was no significant difference between the 2 groups  among recipients who underwent CLT. TMOR >40% was a significant risk factor for PMV in the multivariable analysis. Furthermore, TMOR may be associated with a poor prognosis. These results support our hypothesis that a high relative mediastinal volume promotes dependence on mechanical ventilation after lung transplantation and results in higher mortality.In this study, we proposed TMOR for the first time as a physical trait that could be used to evaluate lung transplant candidates. In Japan, severe donor shortage results in prolonged waiting periods for lung transplantation, leading to nearly 50% mortality on the waiting list, which is much higher than the 14.6% death rate reported in the United States in 2019 , 12. PatTMOR, measured using pretransplant CT images, may reflect a relatively small chest cavity, relatively large heart, or both, although thoracic cavity volume alone or mediastinal tissue volume alone did not predict the clinical outcomes in this study. A small thoracic cavity in lung transplant recipients may be observed because of interstitial lung diseases or other underlying mechanisms that result in lung and/or thoracic shrinkage. However, no single factor, namely underlying lung disease, Haller index, flat chest or narrow intercostal space (<5 mm) was associated with poor clinical outcomes in this study. Cardiomegaly may be due to primary or secondary pulmonary hypertension (PH) or other underlying mechanisms. Although PH alone did not predict the clinical outcomes in this study, 73% of the patients had TMOR >30%, which was higher than that reported in normal individuals  [The reason lung transplant recipients with high TMOR had poor clinical outcomes is unclear. The hazard rate for TMOR on survival was 1.1, where with every 1% increase in TMOR, 10% experienced poor survival. For example, a 5% decrease in TMOR results in a 61% increased risk in the survival hazard. One possibility is a mismatch between the graft and the thoracic cavity. In the lung allocation system in Japan, size-matched recipients are prioritized; however, these matchings use predicted VC based on the age, sex and height , and recIn the stepwise regression model analysis, in addition to TMOR as a preoperative risk factor, 2 postoperative factors that were associated with PMV after CLT included ECMO use and reoperation within 14 days. Intraoperative ECMO use may reflect the poor cardiopulmonary status of recipients before transplantation. Most of the recipients waited a long time for a transplant; therefore, the overall rate of ECMO use was high . Furthermore, the need for reoperation appeared to increase with ECMO use, which might have resulted in PMV . HoweverThis study had limitations. First, the number of enrolled patients was small; thus, a part of results might have not been robust. Second, a selection bias might have been introduced in the results as this was a single-centre and retrospective study. For example, the patient population may be different from that of other centres. In particular, many marginal donors were used for CLT Table . Third, Recipients with TMOR >40% may be long-term ventilator-dependent and have a poor prognosis 3 years after CLT. TMOR may have to be considered when selecting lung transplant recipients.