A number of epidemiological studies have suggested an association between metabolic dysfunction-associated fatty liver disease (MAFLD) and the incidence of atrial fibrillation (AF). However, the pathogenesis leading to AF in the context of MAFLD remains unclear. We therefore aimed at assessing the impact of MAFLD and liver fibrosis status on left atrium (LA) structure and function.Patients with a Fatty Liver Index (FLI) >60 and the presence of metabolic comorbidities were classified as MAFLD+. In MAFLD+ patients, liver fibrosis severity was defined using the non-alcoholic fatty liver disease (NAFLD) Fibrosis Score (NFS), as follows: MAFLD w/o fibrosis (NFS ≦ −1.455), MAFLD w/indeterminate fibrosis (−1.455 < NFS < 0.675), and MAFLD w/fibrosis (NFS ≧ 0.675). In the first cohort of patients undergoing AF ablation, the structural and functional impact on LA of MAFLD was assessed by LA strain analysis and endocardial voltage mapping. Histopathological assessment of atrial fibrosis was performed in the second cohort of patients undergoing cardiac surgery. Finally, the impact of MAFLD on AF recurrence following catheter ablation was assessed.2 p = 0.0168). Accordingly, among the MAFLD patients, those with a high risk of F3–F4 liver fibrosis presented a higher rate of AF recurrence during follow-up (p = 0.0179). In the cardiac surgery cohort , an increase in histopathological atrial fibrosis was observed in MAFLD patients with a high risk of F3–F4 liver fibrosis .In the AF ablation cohort , MAFLD patients with high risk of F3–F4 liver fibrosis presented more LA low-voltage areas as compared to patients without MAFLD , impaired LA reservoir function assessed by peak left atrial longitudinal strain (19.7% ± 8% vs 8.9% ± 0.89% p = 0.0268), and increased LA volume (52.9 ± 11.7 vs 43.5 ± 18.0 ml/mIn conclusion, we found that liver fibrosis scoring in MAFLD patients is associated with adverse atrial remodeling and AF recurrences following catheter ablation. The impact of the management of MAFLD on LA remodeling and AF ablation outcomes should be assessed in dedicated studies. Non-alcoholic fatty liver disease (NAFLD) refers to a spectrum of liver diseases characterized by excessive hepatic fat accumulation, associated or not with liver inflammation and fibrosis with the exclusion of chronic alcohol consumption. Recently, a new concept of metabolic dysfunction-associated fatty liver disease (MAFLD) has been proposed to broaden the diagnostic criteria to meet the needs of a population not previously included in clinical studies . As suchAlthough the liver–heart interaction is well described in the context of atherosclerosis  and ventAF pathogenesis encompasses a wide spectrum of mechanisms involving electrophysiological and structural remodeling of the left atrium (LA) . FurtherWe hypothesized that MAFLD is associated with poor LA remodeling and might thus alter outcomes associated with AF catheter ablation. To explore this hypothesis, we first assessed the impact of MAFLD on LA structure and function  using two distinct cohorts of patients. Then, the impact of MAFLD on AF recurrence following catheter ablation was assessed.Between March 2018 and April 2021, all patients who were candidates for a first AF catheter ablation in the Lille University Hospital were retrospectively enrolled. As specified in the 2016 European Society of Cardiology (ESC) Guidelines for the management of AF, patients were eligible for catheter ablation if they had new-onset or under-treated paroxysmal or persistent AF (The cohort study population consisted of consecutive patients (aged ≥18 years) undergoing cardiac surgery referred to the Cardiovascular Surgery Department at the Lille University Hospital  for aortic valve replacement or mitral valve repair (with or without coronary artery bypass graft). Patients with another notable valvular disease, a medical history of previous cardiac surgery, or congenital heart diseases were excluded. The ethics committee of our institution approved the cohort protocol. Written informed consent was obtained from all patients before inclusion in this cohort.2 + 0.718 × ln + 0.053 × waist circumference, cm − 15.745. Patients with an FLI score of 60 or higher with metabolic syndrome  <0.4 g/L in men or <0.5 g/L in women; fasting glycemia > 1 g/L; systolic arterial blood pressure > 130 mmHg and/or diastolic blood pressure > 85 mmHg) (2) + 1.13 × diabetes mellitus  + 0.95 × aspartate transaminase (AST) (U/L) to alanine aminotransferase (ALT) (U/L) ratio − 0.013 × platelet count (10−9/L) − 0.66 × albumin (mg/L)]. According to the original validation work, patients with an NFS of 0.675 or higher were considered positive for advanced liver fibrosis. Patients presenting a NAFLD fibrosis score below −1.455 were considered free from advanced liver fibrosis. A score between 0.675 and −1.455 was considered indeterminate (As recommended by the current guidelines  and taki85 mmHg)  were conerminate , 26. For®, Irvine, CA, USA) electroanatomic mapping system. Mapping of the pulmonary veins was performed with a deflectable decapolar catheter . Ablation was performed using a 3.5-mm irrigated ablation catheter with contact force sensing . A third, standard quadripolar catheter was used for reference and placed into the coronary sinus. For all procedures, and according to current guidelines, pulmonary vein isolation was performed in both paroxysmal and persistent AF and cavotricuspid isthmus ablation in case of typical atrial flutter. Additional lines , voltage mapping, or recording of continuous fragmented atrial electrograms (CFAEs) to target ablation were deployed to consultant conviction. For patients with left atrial voltage mapping, data were collected at the beginning of the AF ablation procedure in sinus rhythm, using an electroanatomic mapping (EAM) system  and a mapping catheter with a 3.5-mm distal tip and a 2-mm inter-electrode spacing . Adequate endocardial contact was confirmed by stable electrograms and increased contact force values of ≥10 g. The left atrium was divided into nine regions, i.e., septum, superior, posterior, inferior, and lateral walls and the four pulmonary veins (PVs) at their antrum . First, AF recurrence was established as documented AF or atrial flutter on 12-lead electrocardiogram and/or episode >30 s during Holter monitoring. Within the first year after ablation, our in-house protocol unfolds electrocardiograms when symptoms are reported, during outpatient visits in our tertiary hospital, or in the treating cardiologist’s office . Holter monitoring at 24 to 48 h was also performed at 3 and 6 months. The treating cardiologists then followed up the patients, with the number of outpatient visits at clinician discretion. A 90-day “blanking period” was followed. Follow-up of “MAFLD” vs “NoMAFLD” subgroups was censored at 1,200 days, and follow-up of “MAFLD” patients according to liver fibrosis risk was censored at 400 days because the sample size dropped to only one patient.Atrial biopsies were recovered during the cardiac operation in POMI-AF patients. Biopsies were then processed for paraffin embedding. Paraffin-embedded sections were stained with Sirius Red (RAL ref.363440-0005), and images were captured using a ZEISS Axio Scan.Z1 slide scanner. Collagen surface assessment was performed using ImageJ software (version 2.1.0/1.53C for Windows) on the entire image surface, except for the epicardium. The area corresponding to collagen was divided by the total area of the quantified surface to obtain the ratio of fibrosis.2 test. When expected counts were lower than 5, Fisher’s exact test was used. Event-free survival was estimated by the Kaplan–Meier method. First, the association between baseline characteristics and outcomes was investigated in MAFLD and non-MAFLD patients separately. The association between liver fibrosis and AF recurrence was then investigated according to the three groups defined by the MAFLD fibrosis score . The baseline characteristics between the three groups were compared by one-way ANOVA for normally distributed variables or the Kruskal–Wallis test for not normally distributed variables. Second, the association between liver fibrosis and AF recurrence was investigated after adjustment confounders  in separate multivariate proportional hazards Cox models. A two-tailed p-value <0.05 was considered statistically significant. All analyses were performed using MedCalc v16.4 . Visual rendering of the graphics was performed using GraphPad Prism version 9.0.0 for Windows .Continuous variables were tested for normality with the Shapiro test and were represented as mean ± SD in case of normality, or median [interquartile range (IQR)] otherwise. Continuous variables with no Gaussian distribution are given as median (IQR). Categorical variables were given as percentages of individuals. Bivariate comparisons were performed using the t-test for normally distributed continuous variables or the Mann–Whitney U test for variables not normally distributed. Bivariate comparisons of categorical variables were performed with the χ2 vs 28  cm2, p < 0.0001).A total of 291 patients referred to our center for a first AF ablation were included from March 2017 to December 2021. After exclusion of patients with missing components of non-invasive hepatic fatty liver or fibrosis scores, and/or no available follow-up for AF recurrence, 245 were available for analysis. With the use of the FLI cutoff of 60 , 123 patIn the MAFLD group, the NFS was used to dichotomize patients at risk for severe liver fibrosis , patients with an undetermined risk for liver fibrosis , and patients not at risk for liver fibrosis . The full flowchart is provided in Structural and function atrial remodeling were explored in this cohort using 3D-voltage mapping and LA echographic parameters. A total of 183 patients were explored in sinus rhythm with 3D-voltage mapping. Of note, the median left atrial low-voltage area was 0% . Therefore, most patients presented no or a limited low-voltage area. However, the “MAFLD w/severe fibrosis” patients presented an increase in extra-venous low-voltage area sections , which m2 in “NoMAFLD” vs 52.9 ml/m2 in “MAFLD w/severe fibrosis”, p = 0.0168) and 50.4% of the “NoMAFLD” group vs 90% in the “MAFLD w/severe fibrosis group” presenting severe LA dilatation (p = 0.0196). Representative examples of atrial function and voltage according to MAFLD fibrosis status are provided in The LA reservoir function, as assessed by the mean peak left atrial longitudinal strain (PALS), was significantly reduced in the “MAFLD w/severe fibrosis” group compared to the “NoMAFLD” group . Thus, in line with the AF ablation cohort, an increased NFS among MAFLD patients is associated with more severe structural atrial remodeling.The above data suggest an increase in atrial fibrosis in patients presenting MAFLD with severe liver fibrosis. We therefore explored the presence of atrial fibrosis according to MAFLD status in atrial biopsies collected in the second cohort of patients undergoing cardiac surgery (POMI-AF NCT03376165). In this cohort, a total of 20 patients underwent right atrial appendage biopsies during cardiac surgery and were classified according to the same FLI and NFS cutoff levels. Their baseline characteristics are summarized in Since atrial remodeling might critically impact the outcomes of AF ablation, the impact of MAFLD status on AF recurrence following ablation in the AF ablation cohort was assessed. In 201 out of 245 patients, lone pulmonary vein isolation (PVI) was performed. For the remaining 46 patients, additional lesions were assessed and included roof lines for 19 patients, cavotricuspid isthmus lines for 19 patients, box isolation for six patients, and other lines for 15 patients. Of note, at the end of the procedure, PVI was achieved in 100% of the patients.After a median follow-up of 418 days , AF recurrence occurred in 42.8% of the patients. No significant difference in AF recurrence was observed according to MAFLD status alone  after recurrence, in comparison to MAFLD patients without severe liver fibrosis  the presence of MAFLD is associated with adverse atrial remodeling as assessed by echocardiographic, electrophysiological, and histopathological analysis. More precisely, we showed a structural remodeling as indicated by the increase in LA volume, impaired LA reservoir function, and increased low-voltage areas in MAFLD patients at risk of liver fibrosis. Accordingly, atrial fibrosis was increased in MAFLD patients at risk of liver fibrosis. ii) The liver fibrosis scoring in MAFLD patients was predictive of AF recurrence after ablation. iii) In the case of AF recurrence, MAFLD patients with high liver fibrosis scores presented a higher AF burden.Atrial remodeling can be characterized by any complex structural, architectural, contractile, or electrophysiological changes affecting the atria with the potential to produce clinically relevant manifestations . Such haIt is now well recognized that metabolic disorders critically affect the course of patients presenting AF in terms of AF incidence , stroke Although metabolic disorders were first believed to directly induce LA remodeling and AF , recent et al. using a high-fat-diet mouse model showed a vulnerability to AF linked to a shorter action potential duration caused by enhanced K-ATP current in the context of obesity , in the liver. These cells, as activated myofibroblast, contribute to the majority of collagen formation , 50. In The visceral adipose tissue was also suggested as a major actor in atrial remodeling. More particularly, epicardial adipose tissue volume has been associated with AF incidence and ablation outcome –56. The Patients presenting MAFLD with liver fibrosis display major alterations in adipokine profile such as lower adiponectin  levels oIn our study, the impact of MAFLD on AF recurrence was assessed. We first report an incidence of AF recurrence of 42.8% at 418 days of median follow-up. Such AF recurrence incidence is consistent with previous AF ablation studies including similar proportions of persistent and paroxysmal AF: in the open-label multicenter clinical trial CABANA   as compared to patients whose risk of fibrosis is excluded (31%)  Our data are therefore in accordance with those from Donnellan et al. in a different population of MAFLD patients.The impact of metabolic disorders on AF ablation outcomes has been previously demonstrated. In the LEGACY trial , 355 oveFor the first time, we provide insights regarding the characterization of AF recurrence after AF ablation in MAFLD patients. Interestingly, we observed that MAFLD patients presented a higher AF burden when recurrence occurred as shown by the increased proportion of permanent AF following AF recurrence. This clinical observation may be secondary to the atrial remodeling observed in these patients leading to long-lasting AF episodes. Therefore, a poor metabolic profile associated with a high risk of liver fibrosis could be considered a risk marker of atrial remodeling and poor related clinical outcomes.The association between MAFLD and atrial remodeling raises several questions regarding the management of patients scheduled for AF ablation. On the one hand, the impact of intensive management of the metabolic syndrome prior to AF ablation is likely beneficial to reducing recurrence risk and may even justify postponing the procedure. On the other hand, a personalized AF ablation strategy in MAFLD patients with severe liver fibrosis could be considered, with a more extensive approach , especially in the setting of persistent AF. Therefore, further clinical studies assessing the impact of AF ablation strategies after MAFLD scoring are needed to address these questions.In both cohorts, patients were classified under the MAFLD spectrum, thus including mild-to-moderate alcohol consumption and other cardio-metabolic pathologies. With the use of this classification, it is more difficult to attribute the observation specifically to the liver, but the data are certainly more relevant to clinical practice with patients suffering from metabolic syndrome . Liver sPatients presenting MAFLD at risk for liver fibrosis exhibit an increased LA remodeling with impaired hemodynamic, electrophysiological, and histopathological properties. These patients also exhibit a higher risk for AF recurrence following catheter ablation. The impact of the management of MAFLD on LA remodeling and AF ablation outcomes should be assessed in dedicated studies.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Comité de Protection des Personnes/DRN. The patients/participants provided their written informed consent to participate in this study.Patient inclusion and follow-up: TD, JR, CK, FB, AV, and DK. Echography: AC, SA, and SS. Biochemical assay: PM. Study design: RD, BS, DM, SN, LB, EW, ZG, and DD. Wrote and/or revised manuscript: RD, BS, DM, and SN. All authors contributed to the article and approved the submitted version.This work was supported by the French National Agency programs PreciDIAB grant (ANR 18-IBHU-0001), the European Genomic Institute for Diabetes , and PreciNASH (ANR-16-RHUS-0006). BS was a recipient of an ERC Advanced Grant (no. 694717).We thank the BICeL-(UMS2014-US41) cell imaging platform for the access to the equipment and the technical advice provided.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Cutaneous soft-tissue sarcoma (CSTS) of the head and neck are rare and are known to have aggressive clinical course. The current study utilizes a population-based registry in the U.S. to characterize these malignancies and explore disparities. Methods: National Cancer Institute’s (NCI) Surveillance, Epidemiology and End Result (SEER) database from 2000 to 2018 was queried to report incidence and survival data in 4253 cases in the U.S. Results: Males were 5.37 times more likely and Non-Hispanic-White people (NHW) were 4.62 times more likely than females and Non-Hispanic-Black people (NHB) to develop CSTS of the head and neck. The overall incidence was 0.27 per 100,000 persons in 2018, with a significant increase since 2000. Advanced age and stage, histologic group other than ‘fibromatous sarcoma’ and lower SES groups were independent factors for worse overall survival. Conclusions: CSTS of the head and neck demonstrate sex and racial/ethnic disparities in incidence and socioeconomic disparities in overall survival. Level of evidence: II. Cutaneous soft tissue sarcomas (CSTS) of the head and neck are rare  and reprThe National Institute of Minority Health and Health Disparities defines health disparities research as that which addresses health differences in socially disadvantaged populations related to specific outcomes . In the National Comprehensive Care Network (NCCN) guidelines recommend proper biopsy technique, followed by surgical resection with adequate margins and complete histological analysis for the treatment of CSTS of the head and neck . DisparaThe cohort of cases for the current study was isolated using the NCI’s SEER program . PresentPatient age was converted to a categorical variable . We chose this stratification to align with adolescent and young adult population demographics being defined at 15–39 ,20. StagSEER* Stat software  was used to analyze incidence rates which were age adjusted and normalized using the 2000 US Standard population using the dataset ‘18 registries 2000–2018’. Statistical analysis was performed using SPSS Statistical package version 27.0 . Log-rank test was utilized for categorical values to evaluate the effects of demographic, clinical, pathological, treatment and socioeconomic variables. A multivariable analysis was performed to determine independent predictors of outcome using the Cox proportional hazards model.A total of 4253 patients were extracted from the SEER database from 2000–2018. The demographics for the cohort are shown in p < 0.05 , ‘female’ sex (p < 0.001), ‘NHB’ racial/ethnic group (p < 0.001), ‘well differentiated’ grade (p < 0.001), ‘localized’ stage (p < 0.001), size of primary tumor less than 5 cm (p = 0.04), histologic broad group of myxosarcoma, liposarcoma and complex mixed and stromal sarcoma (p < 0.001), surgical excision (p < 0.001), surgical procedure ‘wide excision including amputation surgery’ (p = 0.025), lack of radiotherapy and chemotherapy (p < 0.001) and urban origin of patients (p = 0.038) were significantly associated with improved survival. The five- and ten-year overall survival rate for the entire cohort were 0.44 and 0.27, respectively . UnivariChi-square test of independence was performed to examine the relation between ‘SES’ and ‘surgery’/‘surgical procedure’. In the current analysis, the relation was not statistically significant (data not shown).Due to the high degree of collinearity between ‘surgical excision’ and ‘surgical procedure’, we ran multivariable analysis with each of the variables separately. The determination of independent prognostic factors using Cox P-H model yielded similar results with either of the variables. We have presented below the multivariable analysis using ‘surgical procedure’ variable . On multivariable analysis  age grouTo the best of our knowledge, this is the first population-based study for patients with CSTS of the head and neck, delineating the survival, prognostic factors and disparities in incidence and overall survival. Previous reports consist of single-center-based data focusing on soft tissue sarcoma of the head and neck ,4,5. PatThis is the first report of incidence of CSTS of the head and neck and highlights the sex and racial/ethnic disparities in incidence. Males and NHW are ~five times more likely to present with CSTS of the head and neck . RouhaniOther than ‘year of diagnosis’ and ‘insurance status’, all other factors achieved statistical significance on univariable analysis . HoweverThe broad histologic group ‘fibromatous sarcoma’ included: 8810.3 fibrosarcoma NOS (0.3%), 8811.3 fibromyxosarcoma (0.2%), 8815.3 solitary fibrous tumor, malignant (0.01%), 8825.3 myofibroblastoma, malignant (<0.1%), 8830.3 malignant fibrous histiocytoma 47.6%), 8832.3 dermatofibrosarcoma (15.8%), 8833.3 pigmented dermatofibrosarcoma (0.3%), and 8836.3 malignant angiomatoid fibrous sarcoma (<0.1%) . Others %, 8832.3Previous reports have highlighted the importance of surgical resection and local disease control in the treatment of sarcoma ,4,5,7,8.Limitations of the current study include lack of any information on specific chemotherapy or any other medical therapy in the SEER database. Similarly, no information regarding any medical history, radiological studies or serological work up is provided in the database limiting our analysis. Epidemiological studies comparing SEER areas to non-SEER areas in the U.S. conclude that their age and sex distributions are comparable except that SEER areas tended to be more affluent and more urban than non-SEER areas. Staging can be a potential pitfall in all studies based on the database as lack of any radiological record makes it impossible to verify the stage at diagnosis.Despite these limitations, the current study constitutes a significant step towards identification of independent factors associated with improved survival and highlights sex and racial/ethnic disparities in incidence and SES disparities in overall survival. The latter finding further highlights a potential issue with access to health care.


The clinical trial included 60 patients with VL randomly allocated into two groups: a test group (n = 30) treated with meglumine antimoniate plus NAC (SbV + NAC) and a control group (n = 30) treated with meglumine antimoniate only (SbV). The primary outcome was clinical cure  in 180 days. The cure rate did not differ between the groups; both groups had similar results in all readout indices. The immunological parameters of the patients treated with SbV + NAC showed higher sCD40L in sera during treatment, and the levels of sCD40L were negatively correlated with Interleukin-10 (IL-10) serum levels. In addition, data estimation showed a negative correlation between the sCD40L levels and the spleen size in patients with VL. For the in vitro experiments, peripheral blood mononuclear cells (PBMCs) or PBMC-derived macrophages from healthy donors were exposed to soluble Leishmania antigen (SLA) or infected with stationary promastigotes of Leishmania infantum in the presence or absence of NAC. Results revealed that NAC treatment of SLA-stimulated PBMCs reduces the frequency of monocytes producing IL-10 and lowers the frequency of CD4+ and CD8+ T cells expressing (pro-)inflammatory cytokines. Together, these results suggest that NAC treatment may modulate the immune response in patients with VL, thus warranting additional investigations to support its case use as an adjuvant to antimony therapy for VL.This investigation aimed to assess the effect of N-acetylcysteine (NAC) as an adjuvant treatment to alleviate visceral leishmaniasis (VL). The present work includes both blinded randomized clinical intervention and experimental  Leishmania (Leishmania) infantum or L. donovani. If left untreated, the disease can be fatal. Further compounding, the challenge is that the disease largely affects neglected populations—those without adequate healthcare and infrastructure support  is an infectious disease caused by digenetic parasites of the species  support .Leishmania parasites actively induce alterations in immune cell responses. This is most notable during parasite internalization by macrophages, which includes the inability to adequately clear the pathogen load  is a metabolite of L-cysteine amino acid widely used as antioxidant treatment . NAC admin vitro experiments involving either T cells or monocytes and alters production of immunological biomarker as adjuvant during treatment of patients with VL. These data give evidence that NAC could be further explored as adjuvant treatment to patients with VL.Therefore, knowing that VL disease is associated with a dysregulation in immunity and that NAC treatment directly regulates immune responses, these studies aimed to evaluate whether NAC treatment as an adjuvant to antimony treatment in VL could course correct VL-induced deficiencies in immunity to improve clinical outcomes. Our data demonstrate that NAC has immune modulatory effect in in vitro experimental study to evaluate the effect of antimony plus NAC in immunomodulation of monocytes, macrophages, and T-cell effector responses.This work has two interconnected parts: (1) a pilot blinded and randomized clinical intervention study, with patients with VL treated with antimony plus NAC compared with patients with VL treated with antimony; and (2) an All patients and healthy donors included in the studies were recruited and signed informed consent for participations in the studies. The Research Ethical Committee from the Federal University of Sergipe approved this study (advice number 1.353.887). The clinical trial pilot was registered in clinicaltrials.gov database (NCT01138956).All patients included in this study were invited to participate after VL diagnostics. All patients or their legal guardians signed an informed consent. Patients were consecutively recruited between 2008 and 2010 at the University Hospital of the Federal University of Sergipe in northwestern Brazil, which is an endemic area of VL, where approximately 64 new cases of VL are reported per year . On the basis of the number of new patients with VL, the participation of 60 patients was estimated for this clinical trial study, adopting a significance level of 0.05 and a power of 0.8.VL diagnosis was confirmed with positive serology to rK39 antigen and positive culture from bone marrow. The clinical criteria that are utilized included fever, liver and spleen (increased) sizes, diarrhea, epistaxis, jaundice, cough, and signs of anemia. Additional laboratory criteria included anemia, leukopenia, thrombocytopenia, hypoalbuminemia, and hyperglobulinemia. Patients with other chronic or acute diseases, who were pregnant, had severe neutropenia, or who used immunosuppressors were excluded from the study’s participation pool. All patients were HIV-negative and showed no cardiac alterations in electrocardiogram. The protocol used in the study does not present risks to the patients.® as standardized plus NAC, three doses of 600 mg/day (a dose every 8 h), oral route, for the same period (SbV + NAC); (2) control group (n = 30): received Glucantime® (SbV) as standardized by Brazilian Ministry of Health, 20 mg/kg/day for 20 days, intravenously.Two groups were defined to the study, and all patients included were randomized using a computer randomization table: (1) test group (n = 30): received meglumine antimoniate or GlucantimeThe medical team assessing outcomes was blinded during patient’s follow-up. Patients included in control group did not receive placebo treatment. A follow-up of clinical and laboratorial change over time was performed by the clinical staff for 180 days. The data included in this study were at the following time points: D0, before treatment; D10 and D20, during treatment; and D30, after treatment. Blood samples were obtained in these time points and used for the laboratorial tests: blood count, dosage of proteins, and alanine and aspartate aminotransferases; and for serum cytokines, measurement at D0, D15, and D30.The primary outcome was defined as the cure of VL 180 days after treatment and based on the following key readout indices: reduction of liver and spleen sizes, absence of fever, weight gain, and improvement of leucocytes and erythrocytes numbers.Parasite load was evaluated in blood sample of patients during treatment according to previous published works .Cytokines in sera were measured in three different time points: D0, D15, and D30. The serum levels of Tumor Necrosis Factor-α (TNF-α), IL-12p40, IL-10, and sCD40L were measured using Luminex multiplex assay, according to manufacturer’s recommendations (MILLIPLEX MAP Human Magnetic Bead Panel) , read in a Luminex 200, and analyzed by a Milliplex Analyst software.® 1077 . Cells were then washed and resuspended with supplemented RPMI 1640 media  .Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized whole blood of the healthy donors following previously published approach(es) . Brieflyin vitro experiments using human cells, the concentration of NAC was determined in 10 mM on the basis of the previously published data in literature (To development of terature .6/well. Cells were treated under different conditions: (a) unstimulated; (b) stimulated with Soluble Leishmania Antigen ; (c) stimulated with SLA (1 mg/ml) plus antimoniate meglumine (20 µg/ml); (d) stimulated with SLA (1 mg/ml) plus 10 mM of NAC  or stimulated with SLA (1 mg/ml) plus antimoniate meglumine (20 µg/ml) plus 10 mM of NAC. After 6 h of incubation, Brefeldin A  was added, and the cells were then seeded for an additional 12 h. The plates were washed with 1× PBS prior to blocking with 2% fetal bovine serum and 2% of fetal goat sera. After 30 min, plates were washed and incubated with the following fluorescent antibodies for cell surface markers: CD3 PE-Cy7 and CD4 V500, or CD14 PE-Cy5 and CD40L V500 . After 20 min, cells were washed, permeabilized, and incubated with the following fluorescent antibodies for cytokines for intracellular detection: IL-2 BV421, TNF-α Alexa Fluor 488, and IFN-γ APC or IL-10–APC and TNF-α PE . The cells were then washed and resuspended in 1× PBS. A BD FACSCanto II  flow cytometry was used to acquire a minimum of 50,000 cells. Datasets were analyzed using FlowJo software .PBMC assays were based on the previously published methods . BrieflyRNA was isolated from PBMCs using TRIzol  following the manufacturer’s recommended protocol. Total RNA was used for qPCR assays using TaqMan probes  to TBX21, GATA3, and FOXP3 genes. Amplification was performed using a 7500 Fast Real-Time PCR system .L. infantum parasite was used in the macrophage infection assays . L. infantum strain was cultivated at 24°C in B.O.D. incubator in Schneider’s Insect Medium  supplemented with 10% inactivated bovine fetal sera and 1% penicillin/streptomycin .One strain of Macrophage infection and treatment with NAC plus antimony5 cell per well. After 2 h of incubation, non-adherent cells were removed by washing. Adherent monocytes were differentiated for 6 days in an incubator (at 5% CO2 and 37°C). Next, macrophages (1 × 105 per well) were co-cultured with stationary promastigotes of L. infantum at a 10:1 ratio for 2 h. Next, plates were washed to remove the non-internalized parasites and were incubated for 2, 24, and 72 h under different conditions as afore-described. Supernatants were removed, and cells were fixed with absolute methanol prior to staining with rapid panoptic . A number of amastigotes and infected macrophages were determined at different time points by microscopy, and the parasite index was calculated . All experiments were performed in triplicate.Macrophage differentiation, infection, and treatment followed prior published works . BrieflyL. infantum parasites was tested using a dose–response curve, as previously published (L. infantum isolate used in the experiments were washed (described above) and exposed to crescent concentrations of NAC (0.1 to 80 mM) in Schneider Insect’s Medium for 48 h. Parasites’ viability was determined by counting motile promastigotes in a Neubauer chamber.The ability of NAC in killing ublished . Briefly® SPSS Statistics, we calculated one-way multivariate analysis of variance (MANOVA) to compare age and group effects in the pilot clinical trial, and effect size r of Mann–Whitney was used to compare various quantitative variables at the end of the treatment.D’Agostino-Pearson normality test was used for testing the Gaussian distribution. Differences among groups were considered statistically significant when p < 0.05. Friedman test followed by Dunn’s test was used for multiple comparisons. Wilcoxon test was used for the simple paired comparison, and Mann–Whitney test was used for simple unpaired comparison. R software was used in the construction of heatmaps (Heatmaply and ComplexHeatmap packages). Graphs were made using GraphPad Prism. Correlations were tested using Pearson’s r. Bootstrapping method was used to resample data and test correlation between cytokine levels and clinical parameters. Effect sizes of the differences between groups were calculated in the pilot clinical trial . Using ISixty-eight patients were diagnosed with VL. Sixty patients were selected to participate of the clinical trial. Thirty patients were randomly allocated in the test group that administered meglumine antimoniate plus NAC treatment (SbV + NAC). For the control group, 30 (separate) patients received standard meglumine antimoniate treatment (SbV group), without the NAC treatment , but not observed in SbV group  (data not shown in figure). Beyond that, one-way MANOVA was conducted to determine a difference between SbV + NAC groups and controls of sCD40L on days 0, 15, and 30  and by Universal, FAPITEC/SE, 2008. LM was sponsored by Postdoctoral Fellowship from INCT Imuno/CAPES. CS is sponsored by CAPES. JS, AJ, and RA are scientists supported by the Brazilian Research and Technology Council (CNPq). CS has Doctorate fellowship by CAPES.All authors are grateful to the participating patients of this study. We also recognize the contributions of the medical team in administration and clinical monitoring at HU-EBSERH/UFS and the researchers and students of LIBM from UFS for support in the experimental studies.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer VA declared a shared affiliation with the author MR to the handling editor at the time of review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Avena sativa L.) has attracted a growing interest in the Qinghai-Tibet Plateau. The study aimed to investigate the microbial community and chemical composition of fresh and ensiling oat harvested from six different elevations of the Qinghai-Tibet Plateau.Ensiling whole-crop oat (A. sativa L. cv. Qingyin No. 1) was planted in six different sites across Qinghai-Tibet Plateau , where the elevations were in the range of 2,800–4,500 m above sea level (a. s. l.). Oat was harvested at the milk stage and ensiled for 90 days.The oat . Oat from SN exhibited the best fermentation quality although fresh oat of SN hosted the lowest LAB counts, indicating that high-efficient LAB might be present in fresh oat sampled from high altitudes.The highest crude protein (CP) and lowest water-soluble carbohydrate (WSC) were observed in fresh oat of SN and BM, respectively, however, no distinct gradient trend in WSC and CP concentrations along the elevation gradient. The lowest LAB counts in fresh oat from the highest elevational regions of SN. After 90 days of ensiling, the pH in all oat silages was lower than 4.2, and silages from SC and SN showed a lower pH and butyric acid concentration, and higher lactic acid (LA) concentration than silages of other regions. The oat silage from BR showed the lowest LA concentration and the highest pH. The bimodal distributions of fungal and bacterial richness in fresh oat along the elevation gradient were observed, while the elevation gradients did not affect the fungal Shannon index in fresh oat.  Bos grunniens) since ancient times  is a common forage crop cultivated on the Qinghai-Tibet Plateau because of its strong drought resistance, short growth cycle, high forage yield, good palatability, and high nutritional value. Ensiling whole-crop oat has attracted a growing interest in the Qinghai-Tibet Plateau, and oat silage became an important relief and emergency fodder to alleviate the forage shortage of cattle and sheep during the cold season. Ensiling fermentation is a traditional technique to preserve the original composition of nutrients based on LA fermentation under anaerobic conditions. The harsh natural environment and unique climates might contribute to the difference in the epiphytic microbial community and chemical composition of forages between the Qinghai-Tibet Plateau and other regions worldwide  were set for each site, and the oat from each plot was chopped in 1–2 cm lengths and packed into polyethylene plastic bags (dimensions 270 mm × 300 mm) followed by vacuum-sealing. The three plots served as replications for each elevation. The bags were taken to Lhasa and stored for 90 days at an ambient temperature (10–24°C). The fresh forages from each plot were sampled for chemical analyses, microbial counting, and DNA extraction.The oat was grown at six different sites across arable regions of Qinghai-Tibet Plateau , where the elevation was in the range of 2,800–4,500 m above sea level (a. s. l.). The geographic location and climate characteristics of these sites are shown in The silage bags were opened on d 90 of ensiling, and all silages of each bag were put into an ethanol-sterilized plastic container and mixed thoroughly. Then all silages were divided into 4 sub-samples. The first sub-sample was dried at 65°C in a forced-air oven for 48 h to determine DM contents, the dried samples were ground through a 1-mm sieve by a laboratory knife grinder. The ground samples were preserved for chemical analyses. Total nitrogen (TN) content was analyzed with a Kjeltec 8,400-Analyzer , and the crude protein (CP) was calculated by TN × 6.25 . The watg for 10 min at 4°C, and the supernatant was filtered through a 0.45 μm membrane for organic acid and ethanol analyses according to The second sub-sample (20 g wet basis) was extracted with 60 ml distilled water at 4°C for 24 h. The solution was filtered through four layers of medical gauze and Whatman filter paper , followed by measuring pH immediately using a pH electrode . Then silage extract (5 ml) was centrifuged at 12000× The third sub-sample (10 g) was homogenized with 90 ml of sterile sodium chloride solution (0.85%) for 1 min, followed by filtering through four layers of cheesecloth. Then the filtrate was 10-fold serially diluted for microbial counting, and another solution was used for microbial DNA extraction. The LAB was counted on deMan, Rogosa, and Sharp agar after 48 h of anaerobic incubation at 37°C. The number of yeasts and molds was determined on Potato Dextrose Agar (PDA) medium after 48–72 h of aerobic incubation at 28°C. Enterobacteriaceae were counted on purple-red bile glucose agar after 24 h of incubation at 37°C under aerobic conditions. Aerobic bacteria were counted on the nutrient agar medium  after aerobic incubation for 48 h at 37°C. All of the microbial data were transformed to log10 for presentation and statistical analysis.Microbial DNA was extracted using the Fast DNA SPIN Kit for Soil . The bacterial 16S rRNA V3–V4 and fungal ITS regions were amplified with primers 338F-806R and ITS1F-ITS2aR, respectively. After purification, the purified PCR amplicons were paired-end sequenced using the Illumina MiSeq PE300 platform . All raw reads were checked to discard low-quality sequences  using FLASH (Version 1.2.11) and QIIME (Version 1.7.0). Operation taxonomic units (OTUs) were clustered based on a 97% sequence similarity cutoff using UPARSE (Version 7.1).p < 0.05).The experiment was a completely randomized design, all data from chemical composition, microbial populations, microbial diversity, and fermentation were analyzed by one-way analysis of variance (ANOVA) using the GLM procedure of SAS . A polynomial contrast was used to test the linear or quadratic effects of elevation gradient on the parameters measured. The differences between means were assessed by Tukey’s multiple comparisons (−1 of fresh weight (FW), and the elevation gradient exhibited a linear effect on DM content (p < 0.001). Among all fresh forages, both NDF (p = 0.008) and ADF (p = 0.003) in fresh oat harvested from BR were the highest. The CP content was affected by elevation gradient with a linear and quadratic effect (p < 0.001). The elevation gradient exhibited a quadratic effect on WSC content (p < 0.001). The elevation gradient significantly affected the numbers of LAB with linear and quadratic effects (p < 0.001). The numbers of yeast and aerobic bacteria were significantly affected by the elevation gradient (p < 0.01).The chemical composition and microbial populations of fresh oat harvested from different sites of the Qinghai-Tibet Plateau are listed in p < 0.01) of elevation gradient on the DM and WSC contents. The DZ and SC silages had the lowest DM content, while the BR silage had the highest WSC content among all silages. The ammonia N concentration exhibited a quadratic (p < 0.01) response to the elevation gradient with the lowest value in BR silage. The amounts of LAB (p = 0.01) and aerobic bacteria exhibited a quadratic response to the elevation gradient (p < 0.01).The fermentation quality, microbial population, and chemical composition of oat silage are listed in p < 0.01) and quadratic (p = 0.047) manners along the elevation gradient. The LA concentration was affected by elevation gradient (p < 0.01) with the highest LA values in SC. The AA concentration exhibited a quadratic (p < 0.01) response to the elevation gradient with the highest AA concentration in DZ. The elevation gradient quadratically affected (p < 0.01) the butyric acid concentration. The ethanol concentration exhibited a quadratic (p = 0.017) response to the elevation gradient with the highest value in BY.The silage pH decreased in linear (p > 0.05) among all fresh materials while that in BM and SC silages was lower than in other silages. Fresh oat from BY and BR showed the highest and lowest Chao 1 index among all fresh materials while the elevation gradient did not affect (p > 0.05) the fungal Chao 1 index of silages.We obtained 2,662,561 and 2,876,965 reads by amplicon sequencing of bacterial 16S rRNA V3-V4 and fungal ITS regions, respectively. The greater coverage (>99%) and plateaued rarefaction curves for all samples indicated that the sequencing depth was adequate for reliable analysis of the bacterial and fungal community. The α-diversity indexes of the bacterial and fungal communities are shown in The PCoA plot on the unweighted UniFrac showed the separation of bacterial and fungal communities of fresh and ensiling oat . Two priErwinia, Paenibacillus, Pseudomonas, Leuconostoc, and Exiguobacterium dominated the bacterial community of fresh oat, while Lactobacillus and Kosakonia were the dominant bacterial genus in oat silages , and its relative abundances (RA) decreased to 4.9, 17.7, and 3.1% in BR, SC, and SN, respectively. After 90 days of ensiling, the RA of Pantoea marked declined except for the oat silage of BR. The RA of Lactobacillus in fresh oat was lower than 5%, it markedly increased and became the most dominant genus in oat silages of SC (71.2%) and SN (83.6%) after 90 days of ensiling. Serratia was the prevalent bacterial genus in fresh oat, and its RA significantly increased in oat silages of low-elevational regions  after 90 days of ensiling. Erwinia was present in all fresh oat, and its RA decreased after 90 days of ensiling. Paenibacillus, Pseudomonas, and Exiguobacterium were detectable in all fresh oat and disappeared in all silages. Leuconostoc was the most dominant genus in fresh oat from BR, while it was undetectable in fresh oat from other elevations.The bacterial community composition in fresh and ensiled oat at the genus level are shown in  silages . PantoeaDioszegia, Cladosporium, and Vishniacozyma were the prevalent fungal genus in fresh oat with different RA among elevation gradients. The RA of Dioszegia was increased from 2.6% in fresh oat of BM to 44.9% in fresh oat of SC. Cladosporium was the most dominant fungal genus in BY and decreased along the elevation gradient. Vishniacozyma was the most dominant fungal genus in BM, accounting for RA of 23.1%. After 90 days of ensiling, all 3 of Dioszegia, Cladosporium, and Vishniacozyma were still detectable in silages, however, the dominant role was replaced by other fungal genera. Wickerhamomyces, Candida, and Saccharomyces dominated the fungal communities of silages although they present in very low abundance in fresh oat  for well-fermented silages  than in other low altitudes , and they attributed the high LA concentration to the greater LA-fermentation efficiency of LAB on this region. The oat silage from BR showed the lowest LA concentration and the highest pH, which were in line with its highest residual WSC concentration after 90 days of ensiling, indicating the complex epiphytic microbial composition might retard the LA fermentation.After 90 days of ensiling, the pH in all oat silages was lower than 4.2, indicating all oat silage exhibited well fermentation quality. Of six oat silages, silages from SC and SN showed the lowest pH and butyric acid concentration, and higher LA concentration. Oat from SN exhibited the best fermentation quality although the fresh oat of SN hosted the lowest LAB counts, indicating that high-efficient LAB might be present in fresh oat sampled from high altitudes. In the present study, butyric acid was observed in all silages, indicating all oat silages had undergone a clostridial fermentation. The production of butyric acid usually results in high losses of dry matter and digestible energy . High bup > 0.05), which was consistent with the reports by The elevational patterns for plant and animal diversity generally follow a certain pattern, however, microbes do not follow similar and clear elevational diversity patterns with plants and animals . The incPantoea dominated (>41%) the fresh oat of lower elevations . This is in line with the reports of Pseudomonas and Pantoea were the dominant bacterial genera in fresh E. nutans of Tianzhu . Pantoea was the main microorganisms of fresh oat harvested from Hongyuan on the Qinghai-Tibetan Plateau, where the altitude is 3,500 m a.s.l. Pantoea (34.7%) was the dominant genera in freshly chopped wheat plants, the RA of Pantoea increased to 46.3% after 6 h of ensiling, but then decreased continuously during silage maturation and they represented only 0.02% of the overall population at the terminal stage of 90 days. After 90 days of ensiling, Pantoea in oat silages from BM, BY, and DZ decreased to the second dominant bacterial genus. Pantoea species are gram-negative bacteria from the Enterobacteriaceae family, generally associated with plants, and are recognized as undesirable bacteria of silages because they can compete for nutrients with LAB  has been isolated from soil and wheat, but there are limited reports regarding this genus in silage. It is reported that Vishniacozyma could assimilate lactic acid and D-lactose  and lowest water-soluble carbohydrate (WSC) were observed in fresh oat from SN and BM, respectively, however, no distinct gradient trend in WSC and CP concentrations along the elevation gradient. The bimodal distributions of fungal and bacterial richness in fresh oat along the elevation gradient were observed, while the elevation gradients did not affect the fungal Shannon index in fresh oat. Oat from SN exhibited the best fermentation quality although the fresh oat of SN hosted the lowest LAB counts, indicating that high-efficient LAB might be present in fresh oat sampled from high altitudes. The high-efficient LAB can be further isolated from the Qinghai-Tibet Plateau, especially the higher elevational regions where the microorganisms hold a strong tolerance to the extreme environment.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.TS: conceiving the idea, designing the experiment, and writing, reviewing, and editing. YB and ZYa: sampling and analysis, data analysis, writing, reviewing, and editing. ZYu: formal analysis, methodology, review, and editing. RS and PX: writing, reviewing, and editing. ZYu, RS, and KF: sampling, analysis, and editing. All co-authors participated in discussions and revised the manuscript, contributed to the article, and approved the submitted version.This research was supported by the National Natural Science Foundation of China (31960355 and 32160780), The Tibetan Key R&D Program (XZ202001ZY0047N), and the State Key Laboratory of Barley and Yak Germplasm Resources and Genetics Improvement  (XZNKY-2019-C-007K09).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Clinical, laboratory, and LT outcome data were collected from all the patients with CLD. 27 out 48 (56%) of the patients aged 1–16 years were sarcopenic. No differences were noted in anthropometrics, nutritional support, liver function tests, model for ESLD (MELD), or pediatric ESLD (PELD) scores between patients with and without sarcopenia. The former showed a higher prevalence of respiratory complications (66.7% vs. 42.1%) and need for inotropes (40.7% vs. 10.8%) after LT. Among patients aged 0–1 years (n: 36), those with reduced muscle mass (50%) had a longer hospitalization time (44 vs. 24 days) and higher incidences of multi-organ failure syndrome (38.9% vs. 0%) and intensive care unit-related infections (61.1% vs. 27.8%) compared to those with greater muscle mass. t-PMSA and PMI were statistically significant predictors of LT outcomes. Sarcopenia is a reliable index of frailty in children with CLD, as its presence is associated with the risk of a more challenging LT. Future studies will have to investigate the functional aspects of sarcopenia and conceive preventive measures of muscle wasting in CLD patients.Sarcopenia is a clinical condition characterized by a reduction in muscle mass, which typically affects adult patients; however, it has recently been recognized in pediatric literature. Few studies in children with chronic liver disease (CLD) undergoing liver transplantation (LT) have investigated the role of sarcopenia, with controversial results. The aim of our study was to assess the prevalence and impact of sarcopenia among children with CLD who are candidates for LT. We conducted a retrospective, single-center study at Bambino Gesù Children's Hospital  from July 2016 to July 2021, evaluating all children (0–16 years old) with CLD listed for LT with an abdomen computed tomography imaging available before LT. The total psoas muscle surface area (t-PMSA) was defined as the sum of left and right psoas muscle surface area measured at L4–L5 on axial images. The t-PMSA  Chronic liver disease (CLD) in pediatric patients is associated with progressive structural and functional deterioration of the liver due to fibrosis, cholestasis, and subsequent hepatocellular necrosis . The nutMoreover, in adults with cirrhosis, there is substantial evidence that sarcopenia is associated with increased morbidity and mortality before and after liver transplantation (LT); thus, some authors have advocated the inclusion of sarcopenia in the criteria of organ allocation for LT , 13. PooThe aim of our study was to assess the prevalence of sarcopenia among children with CLD who are candidates for LT.The secondary aim was to evaluate the impact of sarcopenia on selected transplant outcomes and identify the distinctive features of sarcopenic subjects.This was a retrospective, single-center study conducted at Bambino Gesù Children's Hospital , evaluating all children (0–16 years old) with CLD listed for LT from July 2016 to July 2021, with an abdominal CT scan performed and available up to 3 months before LT.Abdominal CT was performed in all subjects as part of the LT assessment protocol adopted at our center.Patients candidates to LT without a chronic liver disease (acute liver failure) or with conditions that may impact on sarcopenia beyond the role of liver disease  were excluded .via a nasogastric tube (NGT), parenteral nutrition (PN), or both before LT were collected. The decision to start artificial nutrition was based on the clinical assessment of nutritional status , anthropometric /model for end-stage liver disease (MELD) score), clinical , and laboratory  data were collected from patient charts. Laboratory results were registered only if they were performed within 1 month of CT imaging. Data on nutritional supplementation l status . Early aThe following outcomes were recorded during LT hospitalization: overall hospital and intensive care unit (ICU) length of stay (LOS); need for inotropes; need for re-intubation; Pediatric Index of Mortality 3 and Pediatric Organ Logistic Dysfunction 2 scores; and episodes of ICU infections and multiple organ dysfunction syndrome (MODS). MODS was defined as the presence of failure in at least two organs. ICU infections were defined as the presence of a positive bacterial culture in blood, urine, peritoneal fluid, or bronchoalveolar lavage detected during the ICU stay.The incidence of sepsis, respiratory , surgical , and graft-related  complications were recorded.Finally, mortality, number of acute rejections based on the BANFF criteria , and catWritten informed consent to participate in this study was provided by the participants' legal guardian/next of kin. The study was carried out according to the rules of Helsinki declaration.z-score was calculated according to reference data, and sarcopenia was defined as a t-PMSA z-score of ≤−2 (CT scans were recovered using the Picture Archiving and Communication System and reviewed by an experienced radiologist (LM) with experience of over 20 years in abdominal radiology. The total psoas muscle surface area (t-PMSA) was defined as the sum of the left and right psoas muscle surface areas measured at L4-L5 on axial images. The t-PMSA e of ≤−2  11)..z-score z-score available.To date, there is no standardized definition of sarcopenia for children aged <12 months as there are no reference values for t-PMSA and t-PMSA 2/(kg/m2)) and decided to stratify the population into two halves according to the median PMI. Infants with a PMI <50th percentile were conventionally considered to be sarcopenic.In this group, we calculated the psoas muscle index (PMI) as follows: PMI = (t-PMSA/(100 × BSA))  and outcomes (dependent variables).Continuous variables are expressed as median and 25th–75th centile or mean ± standard deviation, as appropriate. Categorical data are expressed as frequencies and percentages. Comparisons between continuous variables were performed by Mann–Whitney test or p < 0.05. Statistical analysis was performed using SPSS v.22.0 .Statistical significance was set at n = 60, 71%), followed by genetic disorders   who were listed for LT were evaluated. Thirty-six  were infants (<12 months old) and 48  were aged 1–16 years old . Eighty-14, 16%) . Table 1Because of the lack of a validated definition of sarcopenia for children aged <12 months, we analyzed data from the CLD populations separately according to age.z-score and mean PMI were −3.26 (±1.06) and 7.62 (6.12–8.67) compared to −0.737 (±0.989) and 10.73 (9.85–12.1), respectively (p < 0.001) . In patients with sarcopenia, the median t-PMSA < 0.001) .z-scores at LT listing. In addition, the median growth increase one year after LT was comparable between the two groups.Patients with and without sarcopenia and CLD had similar weight and height n = 18, 66.7% vs. n = 8, 38.1%) and moderate-to-severe ascites than those without sarcopenia  (p < 0.05).There were no differences in the etiology of liver diseases, liver function tests, PELD/MELD score calculated at listing or LT, and median time spent on the waiting list. Patients with sarcopenia had a higher incidence of portal hypertension (Forty-six out of 48 (96%) patients with CLD aged 1–16 years old underwent LT during the study.n = 11, 40.7% vs. n = 3, 10.8%) .During ICU stay, the proportion of patients with inotropes after surgery was higher among those with sarcopenia    . The medNo differences were observed in the incidence of MODS, ICU-related infections, or ICU mortality.n = 21, 77.8% vs. n = 8, 42.1%) (p < 0.05) , while no differences were noted in surgical, infectious, and graft-related complications.At the end of LT hospitalization, patients with sarcopenia showed an increased incidence of LT-related complications ( < 0.05) . In partPatients with and without sarcopenia showed comparable ICU and overall LOS, as well as mortality and rate of acute rejections in the 12-months following LT.z-score was a significant predictor of the need for inotropes after surgery  and the risk of developing any complications .Regression analyses revealed that the t-PMSA 2.We reviewed data from 36 infants  with CLD listed for LT with a median PMI of 9.5 (7.8–11.2) mm2) and patients without sarcopenia those and PMI of ≥50th percentile (≥9.5 mm2).To estimate the impact of sarcopenia in this population, we conventionally considered patients with sarcopenia and PMI of <50th centile , as was the duration of support.The proportion of children receiving enteral nutrition was comparable between the two groups , even if PELD scores at both listing and transplantation were similar between the two groups.Median ICU and overall hospitalization stays were longer in infants with sarcopenia  than in those without  , 4.n = 8, 44.4% vs. n = 2, 11.1%) and second postoperative days   and MODS both on the first  .No differences in the rates of mortality and acute rejection in the first year after LT were found between the two groups.β coefficient of −4.303 [95% confidence interval (CI) −8.09 to −0.52] (p = 0.027) and MODS  (p = 0.036) in the population of children aged 0–12 months.Regression analysis revealed that PMI was a statistically significant predictor of length of hospitalization with a Intervention on modifiable prognostic factors is part of the management of patient candidates for LT to improve their overall outcome. Recent literature has highlighted that sarcopenia is a common feature of adult patients with CLD, with a significant negative impact on both LT waitlist mortality and outcomes , 23.Our study evaluated the prevalence and impact of sarcopenia in pediatric patients with CLD who were candidates for LT.Various techniques are currently available for the assessment of skeletal muscle mass. Dual-energy x-ray absorptiometry and bioelectrical impedance analyses have been widely used in the past, but they may be affected by the patient's hydration status and lack reproducibility among different vendors or accuracy compared with other methods . InsteadThe main limitation of CT is the exposure to ionizing radiation, which is a major issue, mostly in pediatric patients. In our center, CT scan is performed in all patient candidates for LT as a part of the local listing protocol; therefore, we could retrospectively review the scans in our patients in order to assess skeletal muscle mass.In our study population aged 1–16 years, we found a sarcopenia prevalence of 56%. This proportion is higher than those reported in previous studies, but comparable to that described in a recent Dutch study (55.6%) conducted on a population of 34 children with CLD –19. TablMalnutrition is a known predictor of poor outcomes in children undergoing LT . Howeverz-scores, which may explain the higher proportion of patients requiring nutritional support  than those without sarcopenia (15.6%); however, the population with sarcopenia also showed worse weight and height  support .z-scores are a more sensitive and reliable marker for muscle mass and, therefore, are a more accurate method for assessing nutritional status in children with CLD.We suggest that t-PMA The most recent guidelines for nutritional management of children with CLD are focused on advising an increased caloric intake to 130%–150% of dietary reference values but lack a clear indication on how to prevent or adjust nutritional support in case of sarcopenia . In thisOur study highlighted that both patients with and without sarcopenia had similar PELD and MELD scores. In adults, the evidence that sarcopenia is associated with the onset of unfavorable outcomes, both before and after LT, led to the development of special MELD models corrected for including sarcopenia (“Sarco-MELD”) , 28, 29.r = −0.3, p = 0.01) and ICU LOS   . Similar = 0.01) . In chil = 0.01) , 32.According to the European Working Group on Sarcopenia in Older People (EWGSOP), frailty is a multidimensional syndrome that increases vulnerability to poor health outcomes. It has been historically labeled as a “geriatric” condition as it is strictly related to aging, even if recent studies have begun to translate this concept in pediatric patients with chronic diseases , 33–35. 2 decrease in PMSA in a population of 57 children (0–18 years old) listed for LT. Moreover, in our study, indices of sarcopenia (t-PMSA and PMI) were independent predictors of some LT outcomes; therefore, we may speculate that the presence of sarcopenia negatively impacts the evolution of LT, and thus is considered as a paradigm of frailty in CLD pediatric patients.In the analysis of LT outcomes, we found that patients with sarcopenia were more prone to developing complications  than patients without, while the presence of sarcopenia did not increase the risk of death or graft failure after LT. Similarly, previous pediatric studies demonstrated worse clinical evolution in sarcopenic children with various LT outcomes . Regarding mortality, only two works identified a correlation between t-PMSA and survival after LT , 19. In This study bears several limitations. First, it has a retrospective design. The timing of CT imaging was heterogeneous (<3 months before LT), and further variations in muscle mass may have occurred before LT. The secondary limitation is the lack of reference values for t-PMSA in children aged <12 months, as the only available data refer to a population aged 1–16 years old . MoreoveIn conclusion, reduced muscle mass is now recognized as a frequent feature of children and adolescents with CLD and is related to a more complicated post-transplant course. Early identification of sarcopenia is crucial to enable targeted treatment and prevention across pediatric clinical populations, especially for those requiring LT.In the future, it will be necessary to identify new noninvasive, reliable, and reproducible techniques for diagnosing and monitoring muscle mass with widespread use in clinical practice.


Adolescence is a critical period for both the development of overweight and the transition toward a later chronotype, often accompanied by an increase in social jetlag. This study assessed whether changes in chronotype and social jetlag, are linked to changes in body composition during adolescence.N = 572). Chronotype was assessed with the Munich Chronotype Questionnaire and defined as: midpoint of sleep corrected for sleep-debt (MSFsc) accumulated over the week (later MSFsc represents later chronotype). Social jetlag (SJL) defines the difference between midpoint of sleep during week and weekend. Calculations for Fat Free Mass Index (FFMI [kg/m2)]) and Fat Mass Index (FMI) [kg/m2)]) were based on body fat percentage, weight, and height. To analyze the associations, we used linear mixed-effect regression models. Finally, the total cohort was split into three biologically relevant age groups .We used data from the DONALD open cohort study, collected between 2014 and 2019, from 213 adolescents  having at least two measures of chronotype and anthropometry (p for interaction: <0.001). No relationship was found with FFMI.Median follow-up was 2.1 years. Overall, change toward a later chronotype was significantly related with an increase in FMI . A 1 h increase in social jetlag predicted an increase in BMI-SDS of 0.08 SDS units (95% CI: 0.01–0.14) and in FMI of 0.04 kg/m2 (95% CI: 0.003–0.08). Associations were stronger for the age group ≥12 to ≤15 years (Changes in MSFsc and SJL during adolescence were associated with concurrent changes in BMI-SDS and FMI. The age ≥12 to ≤15 years appears to be a sensitive period in which chronobiological changes were clearly associated with increasing body fatness. Chronotype describes the individual preference in sleep and wake timing . MisaligHence, our hypothesis was that changes toward later chronotype or an increased social jetlag (SJL) during adolescence are associated with concurrent detrimental changes in body composition that might differ across age groups. We used repeated measurements of the DONALD Study over a median time span of 2.1 years (Q1–Q3:1.9–3.3) to define change in chronotype or SJL and body compositional changes of BMI-SDS, FMI and FFMI.The DONALD study is an ongoing, prospectively designed open cohort study conducted in Dortmund, Germany. Since 1985, data on diet, growth, developmental and metabolic factors are continuously collected from infancy (age 3 months) to adulthood. Approximately 30–35 healthy infants from Dortmund and surrounding communities, whose mothers and/or fathers have a sufficient level of the German language, are recruited every year either via personal contacts, maternity wards, or pediatric practices. The examination schedule includes quarterly examinations in infancy, half-yearly examinations in the 2nd year of life and annual examinations thereafter until young adulthood. Examinations include anthropometric measurements, lifestyle questionnaires and a 3-day food record. Chronotype and SJL assessment in the DONALD study started in 2014 for participants from 9 years onwards by use of the Munich Chronotype Questionnaire (MCTQ) . Detailen = 620 study participants completed the MCTQ several times during the study (N = 1163). Those questionnaires collected during the 2 weeks after the time change in Germany from standard to summer time or vice versa [N = 89). 378 questionnaires were completed during adulthood  leaving 696 (n = 311) completed questionnaires during adolescence. Two participants with incomplete questionnaires (N = 3) were also excluded from the dataset. Anthropometric measurements within 1 year before or after completing the MCTQ were missing for 35 questionnaires (n = 10 subjects).Among the participants of the DONALD study, those adolescents (aged 9–18 years) with at least two completed MCTQs and parallel collected anthropometric measurements were selected. From 2014 until July 2019, hence considering 5 years of follow-up, n = 95 females, 45%) out of 181 families providing 572 questionnaires  was calculated as the Midpoint of Sleep (MSF), i.e., the half-way point between sleep-onset and sleep-end on free days  correcteAdolescents were measured annually by trained nurses according to standard procedures, dressed in underwear and barefoot. Standing height is measured to the nearest 0.1 cm using a digital stadiometer. Weight was measured to the nearest 0.1 kg with an electronic scale . Skinfold thickness was measured on the right side of the body at the biceps, triceps, subscapular, and suprailiac sites to the nearest 0.1 mm with a Holtain caliper  . Sex- anParents are interviewed regarding family characteristics . Every 4 years maternal body weight and height were measured with the same equipment as for the children on the child’s admission to the study center.Adolescents’ pubertal status was assessed in different ways: Tanner stage (breast and penis development)  was visuIndividual total energy intake was calculated from weighed dietary records as individual means of three record days , collect® procedures . The significance level was set at p < 0.05. The computer codes used for the analyses of the data during the current study are available from the corresponding author on reasonable request.All statistical analyses of the present evaluation were performed using SASLinear mixed effects regression models (PROC MIXED in SAS), including both fixed and random effects, were used to analyze the association between change in MSFsc or SJL  and concurrent change of Δ BMI-SDS, Δ FFMI or Δ log-FMI during adolescence (Change-on-Change-Models) , 23. Mix2) as important covariates. The joint inclusion of the msas represented the final model. To address potential biological differences by age arising from pubertal [Model building was driven by the log-likelihood criterion to define the final crude model. Covariates for model adjustment were selected according to known predictors of BMI, body composition, and chronotype or SJL , 26, 8. pubertal , chronobpubertal , 9 or bepubertal  driven cpubertal , 5 as tupubertal . Due to The Akaike Information Criterion and the Bayesian Information were used to select the correlation structure best describing the correlated nature of the data. Tests for effect modification were performed by the inclusion of interaction terms between chronotype or SJL, age (years) and sex (boys/girls).n = 58, 10%) and maternal BMI . Regression results were combined using the MIANALYZE procedure in SAS. Final models were tested regarding multicollinearity, heteroscedasticity, and normal distribution of residuals.To manage missing data we undertook multiple imputations, using the MI procedure in SAS and explored the pattern of missingness. A comparison of differences in baseline characteristics between those with complete and incomplete data has been performed . Besides stratification according to chronological age, we applied stratification by puberty stages  as suggested previously by Baird et al. [We performed the following sensitivity analyses: inclusion of total energy intake and physical activity, education of the mother, season of chronotype assessment as well as early life factors e.g., mothers’ age at birth and weight gain during pregnancy. Furthermore, we excluded participants who used an alarm clock during the weekend  and calDuring a median follow-up of 2.1 years (Q1–Q3:1.9–3.3 years) adolescents aged 9–18 years completed 3 MCTQs (Q1–Q3: 2–3). Table 2 over time , p = 0.01). Considering the median FMI at baseline as the reference, this estimate would translate into a 0.2 kg/m2 increase in FMI due to increasing lateness in chronotype during adolescence. Change in SJL was associated with BMI-SDS as well as log FMI. Associations for BMI-SDS and FMI were rather robust concerning confounder inclusion (Table p for interaction <0.001) revealed that the overall association reported for the complete sample including all adolescents aged 9–18 years is confined to the age group of 12–15-year-old adolescents . Another study analyzed the relevance of long term exposure to later bedtimes, which may reflect a later chronotype, from adolescence to adulthood in 3342 participants (age 12–32 years) for changes in BMI [2 per 6 years. Regarding SJL, Zwart et al. [z-scores), the length in follow-up, study population, sample size and differences in confounder adjustments  and the use of a single question regarding bedtime instead of a validated questionnaire assessing chronotype and SJL.To the best of our knowledge there is currently no comparable study that assessed change-on-change associations. However, among the longitudinal studies, Culnan et al. , investis in BMI . In thatt et al.  may haveBased on Roenneberg et al.  we wouldMany mechanisms are potentially involved in the associations between chronotype and body composition change  children , measuren = 63, 11%). Full case analyses would have likely biased our results differentially, meaning bias toward or away from the null. Therefore, multiple imputations were the best option to take care of missing data for the current analyses.Finally, the amount of missing covariables was low (Our results contribute to the formulation of public health strategies regarding the risk reduction of overweight development during adolescence. Medical doctors should monitor adolescents carefully, especially from approximately age 12 years onwards, regarding changes in chronotype, since this change may contribute to circadian disruptions  leading To conclude, changes in chronotype and SJL were associated with changes in body composition particularly with body fat during adolescence and between 12 and 15 years of age. Improvements of the social environment to support adolescents in line with their late internal clock, would be helpful to reduce their risk for unfavorable changes in body composition in this critical life stage.Appendix Figures and Tables


Hepatozoon spp., Babesia spp. and Leishmania infantum are common parasites of dogs in Mediterranean countries and are less frequent in cats, particularly Babesia spp. and L. infantum. Moreover, there is limited information on coinfections between these parasites and on L. infantum’s distribution in blood, skin and lymphoid tissue in cats. We used PCR and DNA sequencing to investigate the prevalence of these parasites and the aetiology of Hepatozoon spp. and Babesia spp., in blood, skin, spleen and lymph node samples from up to 212 stray cats and 82 abandoned dogs in southeast Spain. All except 2 dogs were healthy; instead, 112 cats had clinical signs. The estimated PCR prevalences  were 25% (19–31%) Hepatozoon felis in cats, 13% (6–21%) Hepatozoon canis in dogs, 1% (0–4%) Babesia vogeli in dogs, 0% Babesia spp. in cats and 21% (15–26%) and 44% (33–55%) L. infantum in cats and dogs, respectively, and infections were not associated with each other. Leishmania infantum prevalence in lymphoid tissue was significantly higher in dogs than in cats (p < 0.001), and dogs had higher parasite loads than cats (p = 0.012). Moreover, L. infantum prevalence was significantly higher in the skin and lymphoid tissue compared to blood in infected, asymptomatic animals but it was similar in cats with clinical signs, which also had higher parasite loads compared to infected, asymptomatic cats (p < 0.05). The study highlights significant differences between sympatric dogs and cats with respect to the parasite infections investigated, as well as the need to examine both lymphoid tissue and skin samples to maximise the sensitivity of L. infantum infection diagnosis. Leishmania infantum, transmitted by phlebotomine sand flies, is a relevant veterinary problem with major public health implications in Mediterranean countries. The parasite targets mononuclear phagocyte system cells. Dogs are the domestic reservoir and the most susceptible species, and the disease may be associated with a wide range of clinical signs  analysis of blood, skin and lymphoid tissue samples, (ii) assessing the aetiology and prevalence of Hepatozoon spp. and Babesia spp. in stray cats and abandoned dogs by PCR and DNA sequence analysis and (iii) investigating the statistical relationship between infections by the three parasites. We expected to provide clinical, diagnostic and epidemiological information on these infections in dogs and cats and on the extent to which Hepatozoon spp. and Babesia spp. species are shared between domestic animals and wildlife.The present study builds on the previously described work on leishmaniosis, hepatozoonosis and babesiosis in wildlife in southern Spain with the following aims: (i) estimating The study was performed on blood, skin, spleen and/or lymph node DNA from samples from 294 animals, including 212 stray cats and 82 abandoned dogs from the city of Murcia metropolitan area in southeast Spain . Abandoned dogs were animals whose owners refused to keep them and were either left in the street or taken to the municipal kennel. Those left in the streets are quickly removed by the local authorities and taken to the municipal kennel. In contrast, stray cats were animals born and raised in the street that had never been under human care.Leishmania infantum results from dogs were reported elsewhere  and 287 samples from dogs   and reverse (5′-ACA ATA AAG TAA AAA ACA YTT CAA AG-3′) primers targeting a 574-bp 18S rRNA fragment of Hepatozoon spp.  and a commercial kit , respectively. Apicomplexan infection was firstly assessed by a generic end-point PCR amplifying a 360-base pair (bp) fragment of the 18S ribosomal RNA (rRNA) with forward Piroplasmid F (5′-CCA GCA GCC GCG GTA ATT C-3′) and reverse Piroplasmid R (5′-CTT TCG CAG TAG TTY GTC TTT AAC AAA TCT-3′) primers  assay with a Taqman probe, targeting kinetoplast minicircle DNA (kDNA). The protocols used were those of Mary et al.  was used to compare with reference sequences in GenBank. They are now available in the GenBank database under the accession numbers MZ424831–MZ424837.Sarcocystis cymruensis (MG564723) and Plasmodium falciparum (accession number MF155937) were used as outgroups in Hepatozoon spp. and Babesia spp. phylograms, respectively.Phylogenetic trees were constructed using the MEGA X software  was used for all analyses.Prevalence was defined as the percentage of PCR-positives and was used to calculate 95% confidence intervals (95% CI). Fisher’s exact tests were used to compare the proportion of PCR-positives across independent demographic and clinical variables. Median CT of The cat population (percentage) included 105 males (50%) and 106 (50%) females and comprised 38 kittens (18%), 167 young adults (79%), 5 mature adults (2%) and 2 senior cats (1%). The dog population included 46 males (57%) and 35 females (43%) and was formed by 4 puppies (5%), 52 young adults (67%), 21 mature adults (27%) and 1 senior dog (1%). Concerning the size of cats and dogs, 99 (47%) and 23 (35%) were small, 95 (45%) and 21 (32%) were median and 17 (8%) and 22 (33%) were big, respectively. Finally, pure breeds and mongrels were present in 204 (96%) and 8 (4%) of cats and in 36 (44%) and 45 (56%) of dogs, respectively.The following data were missing: sex data for one cat and one dog, age data for 4 dogs, size data for one cat and 16 dogs and breed data for one dog.L. infantum diagnosis. Clinical signs in dogs were alopecia, ulcerative dermatitis, lymphadenomegaly, low body condition and muscular atrophy in one dog, and pale mucous membranes and spleen lesions in the other dog.Clinical signs were found in 112/211  cats and 2/81  dogs. The percentage (95% CI) of clinical signs in cats were low body condition in 33% (26–39%), lymphadenomegaly in 16% (11–21%), cutaneous lesions in 15% (10–20%), diarrhoea in 10% (6–13%), oral lesions in 7% (3–10%), ocular signs in 3% (1–5%), respiratory signs in 2% (0–4%), jaundice in 1% (0–1%) and pale mucous membranes in 1% (0–1%). Cutaneous clinical signs in cats included open skin lesions , scabs , exfoliative dermatitis  and alopecia . Whilst a formal post-mortem examination was not performed, splenomegaly and/or spleen lesions were detected in 5% (2–8%) of cats when samples from this organ were collected for Hepatozoon spp. PCR positives was 25% (19–31.0%) in cats and 13% (6–21%) in dogs. Babesia spp. was only detected in one dog , a 4-year-old female Chow-Chow sampled in spring (Table The percentage (95% CI) of  was 25% 9–31.0% iHepatozoon spp. infection in cats and dogs did not vary significantly according to gender, age, breed, size or the presence of clinical signs and was significantly higher in the summer in cats (p < 0.05) and in the winter in dogs (p < 0.05). No samples from dogs were collected in the summer.The prevalence of L. infantum was 21% (15–26%) in cats and 44% (33–55%) in dogs (Table p < 0.05). Prevalence overall, in the skin and in tissue (both lymphoid tissue and skin) samples, was higher in cats without clinical signs compared to those with clinical signs (p < 0.05). In blood samples, however, the prevalence was marginally higher in cats with clinical signs than in those without (p = 0.06). Moreover, overall prevalence was also marginally higher in big cats compared to other cats, and it was not associated with the animal’s gender, age or breed, and it was significantly higher in those sampled in winter compared to other times of the year (p < 0.01). Furthermore, L. infantum PCR prevalence and CTs in positive animals differed significantly depending on the sample type and between species for the same sample type.The overall PCR prevalence (95% CI) of p < 0.05) (Table p < 0.05).In cats, PCR prevalence (95% CI) was 6% 2–10%) in blood, 14% (0–28%) in lymph nodes, 16% (10–22%) in the skin and 18% (12–24%) in the spleen (Table 0% in blok = 0.21–0.30), and either fair or moderate agreement between the skin and lymphoid tissues and between spleen and lymph nodes (k = 0.31–0.46)  of the animals, specifically in 6/82 (7%) of dogs and 6/123 (5%) of cats, and no animal was infected with all three parasites.There were no significant associations between parasitic infections in either dogs or cats. Mixed Hepatozoon spp. sequences differing from one to three nucleotides were obtained from 11 PCR amplicons from dogs and were 100% identical to H. canis reference sequences  and were named HC16, HC17 and HC18. Similarly, three Hepatozoon spp. sequences differing from one to three nucleotides from 31 PCR amplicons from cats were 99.7–100% identical to H. felis  and coded HF4, HF5 and HF6 .Three different Hepatozoon canis isolate HC16 was in the same cluster as isolates HC1 and HC9 from foxes in southern Spain, including Murcia Region, and also with H. canis found in foxes from France and in dogs from Algeria and India (71% bootstrap)  and a beech marten from Andalucía (HC3), Spain (70% bootstrap)  and a wild cat from Andalucía, Spain (HF1). Isolates HF5 and HF6 grouped with H. felis found in cats from Italy and Angola and in wild cats from Bosnia and Herzegovina, and also with Hepatozoon ingwe from an African leopard from South Africa, and separate from isolate HF2 found in wild cats from Murcia  clustered with those in dogs from France, Malta, Colombia, Algeria and China, cats from Qatar, China and Brazil and foxes from France (99% bootstrap)  Fig. .Fig. 2PhHepatozoon spp. DNA sequences in abandoned dogs and stray cats were in most cases identical to those in foxes, beech martens and wild cats in southern Spain (Ortuño et al. L. infantum (Ortuño et al. Hepatozoon spp. infection was not associated with L. infantum. Potential synergism between leishmaniosis and other vector-borne infections in dogs and cats has been postulated (Baxarias et al. Leishmania coinfections have not been established.The Hepatozoon canis and B. vogeli are transmitted by Rhipicephalus sanguineus, the most common tick in dogs in Spain (Checa et al. Babesia spp. in dogs and cats suggests a low circulation of these parasites in the studied area and not because of low exposure to R. sanguineus since H. canis was found in 13% of dogs. Other articles have similarly reported low prevalences of Babesia canis, B. vogeli and Babesia gibsoni in dogs in Spain (Baxarias et al. Babesia vulpes in dogs and foxes was more prevalent in northwest Spain and Portugal (Cardoso et al. B. vogeli PCR-positive cats in Portugal, and Babesia spp.-positive cats have been sporadically reported elsewhere in Europe and Asia (Penzhorn & Oosthuizen H. canis in dogs and H. felis in cats reported here is higher than in most previous studies in the Iberian Peninsula, where it ranged from 0.7 to 3.3% for H. canis and 1.4 to 16% for H. felis (Díaz-Regañón et al. H. canis and H. felis prevalence, respectively. The prevalence of these two apicomplexan species in other European countries was similarly variable (Cimpan et al. H. canis or Hepatozoon silvestris in cats.The prevalence of Hepatozoon spp. depends on exposure to ticks and is conditioned by the animal’s lifestyle, use of ectoparasiticides and contact with wildlife (Pacifico et al. H. felis transmission routes are not known yet (Baneth et al. H. canis in dogs (Baneth et al. Hepatozoon americanum in dogs feeding on rodents and rabbits infected with a quiescent cystozoite form of the parasite (Johnson et al. Hepatozoon spp. infections might be accounted for by transmission at this time of the year since R. sanguineus in Spain may also be active in the winter (Estrada-Peña et al. Hepatozoon spp. were most prevalent in cats during the winter (Baneth et al. Hepatozoon spp. in dogs was highest in the summer (Barati & Razmi The risk of infection with Leishmania infantum was also strongly seasonal, and winter peaks in both dogs and cats would be associated with leishmaniosis’ long incubation period (Hernández et al. L. infantum in cats was slightly lower than the 26% to 30% found by Maia et al. (L. infantum prevalence in different cat samples have reported variable results. In stray and colony cats in Italy, PCR prevalence in blood was lower than in conjunctival swabs (Morganti et al. L. infantum infection prevalence in cat and dog populations.a et al. , Martín-a et al.  and Milla et al.  and higha et al. . Prevalea et al. , and accL. infantum infection in cats was not associated with clinical signs typical of FeL (Abramo et al. L. infantum transmission requires further investigation given that they are able to transmit the parasite to the vector, the prevalence may be relatively high, and cat populations are large. An objective assessment may only be possible in natural environments in the absence of dogs or other reservoirs of infection (Zanet et al. The greater prevalence of infection in lymphoid tissue in dogs compared to cats and the similar prevalence in skin samples suggest that parasite visceralitation is lower in cats than in dogs and is compatible with cutaneous leishmaniosis being the most common clinical manifestation of the disease in cats (Solano-Gallego et al. L. infantum and Hepatozoon spp. studies support a common domestic and sylvatic transmission cycle for these parasites in southeast Spain (Ortuño et al. Our Hepatozoon spp. and L. infantum but not Babesia spp. are common parasites of stray cats and abandoned dogs in south-east Spain, and Hepatozoon spp. transmission cycles are likely to be shared with wildlife. A better demographic and sanitary control of this vulnerable population is required. Compared to dogs, cats have a lower probability of L. infantum infection and the parasite becoming latent in lymphoid tissue, and when it does, parasite concentration is lower. Differences between cats and dogs in L. infantum infection prevalence and body distribution have important epidemiological, diagnostic and clinical implications.


This nonclinical study used quantitative whole-body autoradiography (QWBA) and qualitative tape-transfer microautoradiography (MARG) to investigate lefamulin distribution into urogenital tract tissues down to a cellular level in male and female rats. A single intravenous dose (30 mg/kg) of [14C]-lefamulin was administered to 3 male and 3 female Sprague-Dawley rats. At 0.5, 6, and 24 h post dose, rats were euthanized and [14C]-lefamulin distribution was investigated using QWBA and MARG of sagittal planes. [14C]-lefamulin was well distributed throughout the carcasses of male and female rats, with the highest concentrations observed in male bulbourethral gland, urethra, prostate in female clitoral gland, uterus (particularly endometrium), and ovary. In these areas, concentrations were similar to or higher than those observed in the lungs. Concentrations peaked at 0.5 h post dose, remaining detectable in the urogenital tract up to 24 h post dose. [14C]-lefamulin in rats showed rapid, homogeneous distribution into urogenital tissues down to a cellular level, with high tissue:blood ratios in tissues relevant to STI treatment. These results, and the potent in vitro activity of lefamulin against multidrug-resistant bacteria known to cause STIs, will help inform further assessment of lefamulin, including potential clinical evaluation for treatment of STIs.Lefamulin, a semisynthetic pleuromutilin antibiotic approved in the United States, Canada, and Europe for intravenous and oral treatment of community-acquired bacterial pneumonia, is highly active  Chlamydia trachomatis and Neisseria gonorrhoeae, for which incidence rates appear to be increasing  have a major impact on reproductive health . The Worcreasing . STIs cacreasing –6. In woertility , 5. Mycoin women , 7. Althnitalium  and parttibiotic . In somehromycin , and reshromycin .Lefamulin is the first pleuromutilin antibiotic approved in the United States, Canada, and Europe for intravenous (IV) and oral use in adults with community-acquired bacterial pneumonia (CABP) –12. Lefa–in vitro activity of lefamulin against a variety of pathogens, including those commonly associated with STIs such as N. gonorrhoeae, C. trachomatis, and M. genitalium   .14C]-lefamulin at 0.5 h post dose were higher in the bulbourethral gland and urethra than in other areas  and tissues involved in elimination   . Radioac14C]-lefamulin concentrations were observed in the clitoral gland, urethra, and uterus  increases in tissue:blood concentration ratios at 6 h post dose compared with the 0.5-h time point , N. gonorrhoeae , and C. trachomatis   , 16. TheI models , a transSafety and tolerability of lefamulin have been established in patients with CABP , 14. Cli14C]-lefamulin was administered to male and female rats. All procedures were conducted in accordance with the standards set forth in the Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes  of  microscale standards  against imaging plates at ambient temperature in the dark for 7 days. The distribution of radioactivity ([14C]-lefamulin) was determined using a Fuji FLA-5100 fluorescent image analyzing system  with associated Tina version 2.09  and SeeScan version 2.0  software. For each exposure, a standard curve was produced in SeeScan using data from the autoradiographic microscale standards from which tissue concentrations of radioactivity (nCi/g) were determined.QWBA analyses were conducted based on procedures previously described ; in briein situ from sections of the frozen whole-body carcasses described above versus excision of individual organs/tissues for subsequent freezing and sectioning. Specifically, 10-μm tissue sections were collected onto tape, transferred to glass microscope slides using the CryoJane tape transfer system , air dried, and coated with nuclear emulsion. Slides were exposed in light-tight cassettes at −20°C for up to 6 weeks, then subjected to development and fixation procedures followed by hematoxylin and eosin (H&E) staining. Slides were inspected using an Axio Imager.A2 microscope  under both bright and dark field. Images were captured using the Retiga 2000R  digital camera. Interpretation of MARG results was purely qualitative and supported by images captured with the digital camera.MARG analyses were performed as previously described  and modi14C]-lefamulin into relevant tissues, ratios of concentrations in the target tissue to concentrations in blood (tissue:blood) and lung (tissue:lung) were calculated at each individual time point for both male and female animals. The choice to include tissue:lung was made based the relative amount of clinical and pharmacokinetic data surrounding the lung compartment in patients and volunteers as a point of reference. In situations where concentrations in blood were below the limit of detection , the tissue:blood denominator was assumed to be a maximum of 0.062 μg equivalents/g.To evaluate the relative penetration of [