Manuka honey inhibits the development of Streptococcus pyogenes biofilms and causes reduced expression of two fibronectin binding proteins

Streptococcus pyogenes (group A Streptococcus; GAS) is always of clinical significance in wounds where it can initiate infection, destroy skin grafts and persist as a biofilm. Manuka honey has broad spectrum antimicrobial activity and its use in the clinical setting is beginning to gain acceptance with the continuing emergence of antibiotic resistance and the inadequacy of established systemic therapies; novel inhibitors may affect clinical practice. In this study, the effect of manuka honey on S. pyogenes (M28) was investigated in vitro with planktonic and biofilm cultures using MIC, MBC, microscopy and aggregation efficiency. Bactericidal effects were found in both planktonic cultures and biofilms, although higher concentrations of manuka honey were needed to inhibit biofilms. Abrogation of adherence and intercellular aggregation was observed. Manuka honey permeated 24 h established biofilms of S. pyogenes, resulting in significant cell death and dissociation of cells from the biofilm. Sublethal concentrations of manuka honey effectively prevented the binding of S. pyogenes to the human tissue protein fibronectin, but did not inhibit binding to fibrinogen. The observed inhibition of fibronectin binding was confirmed by a reduction in the expression of genes encoding two major fibronectin-binding streptococcal surface proteins, Sof and SfbI. These findings indicate that manuka honey has potential in the topical treatment of wounds containing S. pyogenes. INTRODUCTION Streptococcus pyogenes (group A Streptococcus) colonizes the nasopharynx and skin of healthy individuals, forming part of the commensal microbiota. Under appropriate conditions, S. pyogenes can be transmitted to wounds and is especially problematic after surgery, following skin grafting and for military personal with traumatic or puncture wounds. Wounds provide a route of entry to the host and damaged tissues display a matrix of proteins including collagen, albumin, fibronectin and fibrinogen, which collectively provide a plethora of ligands to which opportunistic pathogens, including streptococci, adhere Biofilms have been associated with persistent or chronic wound infections and are a major obstacle to healing Honey has had a valued place in traditional medicine for many centuries and was reintroduced into modern medicine during the 21st century. Honey exhibits broad spectrum antibacterial activities and has been reported to inhibit more than 80 species of bacteria, including meticillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, Lancefield groups A, C and G streptococci, Pseudomonas aeruginosa and Actinomyces species METHODS Bacterial strains. S. pyogenes MGAS6180 (M28, invasive disease; Green et al., 2005) was grown in Todd-Hewitt (TH) broth (Oxoid) containing 0.5 % yeast extract; Bacto agar (Difco) was added to achieve a 1.4 % (w/v) final concentration in agar plates. Cultures were incubated at 37 uC in air supplemented with 5 % CO 2 . For analysis of aggregation properties, S. pyogenes was grown in C medium containing 0.2 % glucose (Lyon et al., 1998). Medical grade manuka honey. Sterile medical grade manuka honey (Medihoney) was kindly donated by Comvita. Medihoney was provided in sterile 50 g portions. Minimum inhibitory and bactericidal concentrations. The MIC for manuka honey against planktonically grown S. pyogenes MGAS6180 was determined by serial dilution (0-95 %; w/v) in a total volume of 5 ml iso-sensitest broth (Oxoid) (according to the British Society for Antimicrobial Chemotherapy methodology for determining MIC; Andrews, 2011). Cultures were incubated for 16 h at 37 uC in aerobic conditions with 5 % CO 2 . To establish the MBC, samples corresponding to the MIC and including three samples of a higher concentration were plated onto Todd-Hewitt agar and incubated for 16 h at 37 uC in aerobic conditions with 5 % CO 2 . Assays were done in triplicate on each of three separate occasions. Inhibition of bacterial growth. To determine the extent of growth inhibition by manuka honey, triplicate cultures of S. pyogenes MGAS6180 were grown for 8 h at 37 uC in 10 ml TH broth, in aerobic conditions with 5 % CO 2 , supplemented with manuka honey (0, 20 and 40 % w/v). Bacterial growth was monitored at OD 650 at hourly intervals. Growth experiments also utilized triplicate biological samples. Aggregation assays. S. pyogenes MGAS6180 was initially grown in C medium for 16 h (Lyon et al., 1998) and harvested by centrifugation at 5000 g for 10 min. Bacterial cell pellets were resuspended in either 1 ml C medium or an appropriate concentration of manuka honey (5 and 10 %; w/v) dissolved in C medium and the OD 600 of the culture was adjusted to 1.0 (±0.05) if necessary. In both cases, manuka honey at twice the desired concentration was dissolved in double strength TH and diluted to the required concentration using TH; cell pellets were resuspended in either 5 or 10 % (w/v) honey solutions. In an untreated control, manuka honey was replaced with PBS added to maintain the appropriate volume and concentration of TH media. Aggregation assays were carried out in triplicate as described previously Static biofilm model. S. pyogenes MGAS6180 was initially grown in C medium for 16 h and these stationary phase cultures were harvested by centrifugation and adjusted to OD 650 0.1. To determine whether manuka honey prevented biofilm formation, biofilms were established in 96-well microtitre plates (Greiner) in 50 ml TH, supplemented with manuka honey (0, 10, 20 and 40 % w/v), by inoculating each well with 5 ml harvested cells. Plates were incubated at 37 uC for 24 h, aerobically with 5 % CO 2 . To estimate biomass, unattached cells were gently aspirated and discarded, and adherent cells were washed twice with PBS and stained with crystal violet (0.25 %; w/v) for 10 min; following a further two washes with PBS, cell-bound crystal violet was resolubilized with 7 % acetic acid, and absorbance was measured at 595 nm (A 595 ) Live-Dead staining. Images of bacterial cells were collected for control cells (untreated) and for cells treated with 40 % (w/v) manuka honey for 2 h, to determine the effect of manuka honey on viability. Biofilms were grown in Petri dishes in 5 ml TH media, or 5 ml 40 % (w/v) honey dissolved in TH media, as previously described; liquid was aspirated from the plates and biofilms were washed with 1 ml PBS. Biofilms were scraped from the coverslips using a cell scraper, resuspended in 1 ml PBS and stained with Live-Dead BacLight bacterial viability kit (Invitrogen), following the manufacturer's instructions. Fluorescence microscopy images were obtained using a Nikon Eclipse 80i fluorescent microscope with oil immersion and 6100 lens. For detection of SYTO 9 (green channel) a 488 nm excitation and 520 nm emission filter was used. For propidium iodide detection (red channel) a 543 nm excitation and 572 nm emission filter was used. Image analysis used Volocity Software (PerkinElmer). Biofilm disruption. To determine whether manuka honey affected biofilm biomass by facilitating the dissociation of adherent cells from S. E. Maddocks and others 782 Microbiology 158 the biofilm, assays were conducted to monitor the numbers of viable planktonic cells that were released into the liquid phase, from established biofilms following treatment with manuka honey. Biofilms were grown for 24 h as described above. The liquid was aspirated from each well, biofilms were washed twice with PBS to remove any planktonic or loosely adherent cells, and manuka honey over a range of concentrations [0, 10, 20 and 40 % (w/v), respectively] was added to the 24 h established biofilms. Following the application of honey, biofilms were incubated for a further 2 h at 37 uC as above and samples of the liquid above the biofilm were collected at 30 min intervals. Bacterial cells were enumerated using the total viable cell (TVC) counting method described by 21 . Fibronectin-and fibrinogen-binding assays. To determine the effect of manuka honey on adherence of S. pyogenes cells to immobilized fibronectin and fibrinogen, a crystal violet assay was conducted as described previously, using 1 % BSA to block wells prior to assaying cell binding RNA extraction from S. pyogenes biofilms. Large scale, static biofilms of S. pyogenes were grown in duplicate in 5 ml C medium (with 20 % honey for the 'treated' biofilms) in sterile Petri dishes for 24 h at 37 uC, as for the small scale biofilms. The liquid was aspirated and the biofilm was scraped from the surface of the Petri dish using a sterile cell scraper. Biofilms were resuspended in 500 ml PBS and vortexed for 1 min to break up cell aggregates. Honey-treated and untreated cell suspensions were equilibrated (to approximately 2.5610 9 c.f.u.) prior to treatment with mutanolysin (100 mg) and lysozyme (100 mg) for 20 min at 37 uC. RNA extraction was carried out using the SV Wizard total RNA extraction kit (Promega) according the manufacturer's instructions. RNA quantification was performed by spectrophotometric measurement using a NanoDrop ND-1000 (NanoDrop Technologies) and each RNA sample was adjusted to give a final concentration of 10 ng ml 21 . End point RT-PCR to determine the relative expression of sof and sfbI. PCR primers were designed to amplify a 1100 bp fragment of the sof gene (sof-fwd: 59-ACTTAGAAAGTTATCTGTAGGG; sofrev: 59-TCTCTCGAGCTTTATGGATAG) and 1200 bp fragment of the sfbI gene (sbfI-fwd: 59-AACTGCTTTAGGAACAGCTTC; sbfI-rev: 59-CCACCATAGCCACAATGCT). The complete genome sequence for S. pyogenes MGAS6180 was obtained from the NCBI database (www.ncbi.nlm.nih.gov) and used as a basis to design the primers used in this study. Internal control primers were designed to amplify a 900 bp internal fragment of the glr (murI; glutamate racemase) gene (glr-fwd: 59-ATGGATACAAGACCAATTGGG; glr-rev: 59-TCATAA-GGTGACATGCTCCAC), a known housekeeping gene in S. pyogenes that is commonly used for MLST (http://spyogenes.mlst.net/misc/ info.asp) RESULTS Inhibition of planktonic S. pyogenes by manuka honey The MIC of manuka honey against S. pyogenes MGAS6180 was found to be 20 % (w/v) and the MBC was found to be 45 % (w/v). Growth curves with 20 % (w/v) manuka honey resulted in a reduced growth rate and reduction in overall cell number ( S. pyogenes aggregation and biofilm development are inhibited by sublethal concentrations of manuka honey To test the capacity for manuka honey to inhibit intercellular aggregation, suspensions of S. pyogenes MGAS6180 were mixed with 10 % (w/v) manuka honey. In the absence of manuka honey, cells strongly aggregated but in the presence of 10 % (w/v) honey, aggregation was completely inhibited (P,0.05) When biofilms of S. pyogenes were initiated in the presence of 10, 20 and 40 % (w/v) manuka honey, a statistically significant reduction in biomass was observed in each case. A reduction of 75 % (P50.03) was observed with 10 % (w/ v) manuka honey; 20 and 40 % (w/v) manuka honey resulted in between 90 % (P50.02) and 96 % (P50.01) reduction in biomass, respectively Manuka honey facilitates cell death and dissociation of bacterial cells from established S. pyogenes biofilms To determine the effect of manuka honey against established biofilms of S. pyogenes MGAS6180, biofilms were grown for 24 h prior to honey treatment. Following 2 h treatment with 10 and 20 % manuka honey (w/v), a 72 % (P50.32) and 77 % (P50.08) reduction in biomass was observed, respectively. Following treatment for 2 h with 40 % (w/v) manuka honey, the observed reduction in biofilm biomass was even greater, and statistically significant at 85 % (P50.01) Live-Dead viability staining (Invitrogen) of untreated biofilms grown for 24 h at 37 uC, honey-treated (40 % w/ v) biofilms grown for 24 h at 37 uC, and biofilms that were grown for 24 h at 37 uC, then treated for 2 h with 40 % (w/ v) manuka honey, showed that in both cases, the honey treatment resulted in cell death With time, bacterial cells may dissociate from a biofilm. To determine whether manuka honey facilitated the dissociation of S. pyogenes from biofilms, the levels of planktonic cells in the liquid phase were monitored for 2 h following the application of manuka honey (the biofilm data described above showed that the biomass was reduced following 2 h treatment, so this time period was deemed appropriate for these experiments). Immediately after application of the manuka honey (0 min) the c.f.u. was approximately the same for each condition. After 30 min and throughout the duration of the experiment (120 min), higher c.f.u. values were recovered from biofilms treated with 10 and 20 % (w/v) manuka honey (subinhibitory concentrations) compared with untreated biofilms, with a maximum recovery of 1.2610 6 c.f.u. S. E. Maddocks and others 784 Microbiology To establish whether manuka honey affected binding of S. pyogenes MGAS6180 to the wound-associated proteins fibronectin and fibrinogen, 1 mg of each of these two protein ligands was immobilized to the surface of a microtitre plate. The extent of binding inhibition using a sublethal dose (20 % w/v) of manuka honey was compared with adherence observed in the absence of manuka honey. Binding of S. pyogenes to fibronectin in the presence of 20 % (w/v) manuka honey was reduced by 74 %, which was found to be statistically significant (P50.01); conversely no such reduction in binding was observed for fibrinogen (P50.38) Genes encoding the surface adhesins Sof and SbfI are differentially expressed in response to exposure to a sublethal concentration of manuka honey To determine whether the decreased binding to fibronectin was the result of differential expression of two of the major fibronectin-binding proteins (Sof and SfbI) of S. pyogenes MGAS6180 in response to sublethal concentrations of honey, end point RT-PCR was employed. The PCR products were analysed by densitometry and quantified relative to the New England Biolabs 1 kb quantitative molecular mass Streptococcus pyogenes biofilms and honey http://mic.sgmjournals.org 785 marker (thresholds for detection ¢0.15 nmol). The results confirmed that both sfbI and sof were expressed to a lesser extent in the presence of 20 % (w/v) manuka hone

Metabolic Reprogramming Helps to Define Different Metastatic Tropisms in Colorectal Cancer

Approximately 25% of colorectal cancer (CRC) patients experience systemic metastases, with the most frequent target organs being the liver and lung. Metabolic reprogramming has been recognized as one of the hallmarks of cancer. Here, metabolic and functional differences between two CRC cells with different metastatic organotropisms (metastatic KM12SM CRC cells to the liver and KM12L4a to the lung when injected in the spleen and in the tail vein of mice) were analysed in comparison to their parental non-metastatic isogenic KM12C cells, for a subsequent investigation of identified metabolic targets in CRC patients. Meta-analysis from proteomic and transcriptomic data deposited in databases, qPCR, WB, in vitro cell-based assays, and in vivo experiments were used to survey for metabolic alterations contributing to their different organotropism and for the subsequent analysis of identified metabolic markers in CRC patients. Although no changes in cell proliferation were observed between metastatic cells, KM12SM cells were highly dependent on oxidative phosphorylation at mitochondria, whereas KM12L4a cells were characterized by being more energetically efficient with lower basal respiration levels and a better redox management. Lipid metabolism-related targets were found altered in both cell lines, including LDLR, CD36, FABP4, SCD, AGPAT1, and FASN, which were also associated with the prognosis of CRC patients. Moreover, CD36 association with lung metastatic tropism of CRC cells was validated in vivo. Altogether, our results suggest that LDLR, CD36, FABP4, SCD, FASN, LPL, and APOA1 metabolic targets are associated with CRC metastatic tropism to the liver or lung. These features exemplify specific metabolic adaptations for invasive cancer cells which stem at the primary tumour.This work was supported by grants cofounded by Fondo Europeo de Desarrollo Regional -FEDER- PI17CIII/00045 and PI20CIII/00019 from the AES-ISCIII program to RB from the Instituto de Salud Carlos III (ISCIII) and grants from Spanish Ministry of Science (Plan Nacional I+D+i PID2019-110183RBC21), Regional Government of Community of Madrid (P2018/BAA-4343-ALIBIRD2020-CM, and Y2020/BIO-6350), and Ramón Areces Foundation (CIVP19A5937) to AR. AM-C FPU predoctoral contract is supported by the Spanish Ministerio de Educació n, Cultura y Deporte. AQ-F acknowledges Comunidad de Madrid for the Garantıa Juvenil PEJD-2017-RE/BMD-3394 contract. GS-F is a recipient of a predoctoral contract (grant number 1193818N) supported by the Flanders Research Foundation (FWO).S

Taking care of kidney transplant recipients during the COVID-19 pandemic: experience from a medicalized hotel.

The global overload that health systems are undergoing since the start of the COVID-19 pandemic has forced hospitals to explore sustainable alternatives to treat vulnerable patients that require closer monitoring and higher use of resources, such as Kidney Transplant Recipients (KTRs)1,2 .The use of telemedicine and hospital-like infrastructures represent a valid option for most patients with mild-moderate COVID-19, as well as for patients in the recovery phase who cannot be discharged from hospital

Durvalumab Plus Carboplatin/Paclitaxel Followed by Maintenance Durvalumab With or Without Olaparib as First-Line Treatment for Advanced Endometrial Cancer: The Phase III DUO-E Trial

PURPOSE Immunotherapy and chemotherapy combinations have shown activity in endometrial cancer, with greater benefit in mismatch repair (MMR)-deficient (dMMR) than MMR-proficient (pMMR) disease. Adding a poly(ADP-ribose) polymerase inhibitor may improve outcomes, especially in pMMR disease. METHODS This phase III, global, double-blind, placebo-controlled trial randomly assigned eligible patients with newly diagnosed advanced or recurrent endometrial cancer 1:1:1 to: carboplatin/paclitaxel plus durvalumab placebo followed by placebo maintenance (control arm); carboplatin/paclitaxel plus durvalumab followed by maintenance durvalumab plus olaparib placebo (durvalumab arm); or carboplatin/paclitaxel plus durvalumab followed by maintenance durvalumab plus olaparib (durvalumab + olaparib arm). The primary end points were progression-free survival (PFS) in the durvalumab arm versus control and the durvalumab + olaparib arm versus control. RESULTS Seven hundred eighteen patients were randomly assigned. In the intention-to-treat population, statistically significant PFS benefit was observed in the durvalumab (hazard ratio [HR], 0.71 [95% CI, 0.57 to 0.89]; P = .003) and durvalumab + olaparib arms (HR, 0.55 [95% CI, 0.43 to 0.69]; P < .0001) versus control. Prespecified, exploratory subgroup analyses showed PFS benefit in dMMR (HR [durvalumab v control], 0.42 [95% CI, 0.22 to 0.80]; HR [durvalumab + olaparib v control], 0.41 [95% CI, 0.21 to 0.75]) and pMMR subgroups (HR [durvalumab v control], 0.77 [95% CI, 0.60 to 0.97]; HR [durvalumab + olaparib v control] 0.57; [95% CI, 0.44 to 0.73]); and in PD-L1-positive subgroups (HR [durvalumab v control], 0.63 [95% CI, 0.48 to 0.83]; HR [durvalumab + olaparib v control], 0.42 [95% CI, 0.31 to 0.57]). Interim overall survival results (maturity approximately 28%) were supportive of the primary outcomes (durvalumab v control: HR, 0.77 [95% CI, 0.56 to 1.07]; P = .120; durvalumab + olaparib v control: HR, 0.59 [95% CI, 0.42 to 0.83]; P = .003). The safety profiles of the experimental arms were generally consistent with individual agents. CONCLUSION Carboplatin/paclitaxel plus durvalumab followed by maintenance durvalumab with or without olaparib demonstrated a statistically significant and clinically meaningful PFS benefit in patients with advanced or recurrent endometrial cancer

Familial hypercholesterolaemia in children and adolescents from 48 countries: a cross-sectional study

Background: Approximately 450 000 children are born with familial hypercholesterolaemia worldwide every year, yet only 2·1% of adults with familial hypercholesterolaemia were diagnosed before age 18 years via current diagnostic approaches, which are derived from observations in adults. We aimed to characterise children and adolescents with heterozygous familial hypercholesterolaemia (HeFH) and understand current approaches to the identification and management of familial hypercholesterolaemia to inform future public health strategies. Methods: For this cross-sectional study, we assessed children and adolescents younger than 18 years with a clinical or genetic diagnosis of HeFH at the time of entry into the Familial Hypercholesterolaemia Studies Collaboration (FHSC) registry between Oct 1, 2015, and Jan 31, 2021. Data in the registry were collected from 55 regional or national registries in 48 countries. Diagnoses relying on self-reported history of familial hypercholesterolaemia and suspected secondary hypercholesterolaemia were excluded from the registry; people with untreated LDL cholesterol (LDL-C) of at least 13·0 mmol/L were excluded from this study. Data were assessed overall and by WHO region, World Bank country income status, age, diagnostic criteria, and index-case status. The main outcome of this study was to assess current identification and management of children and adolescents with familial hypercholesterolaemia. Findings: Of 63 093 individuals in the FHSC registry, 11 848 (18·8%) were children or adolescents younger than 18 years with HeFH and were included in this study; 5756 (50·2%) of 11 476 included individuals were female and 5720 (49·8%) were male. Sex data were missing for 372 (3·1%) of 11 848 individuals. Median age at registry entry was 9·6 years (IQR 5·8-13·2). 10 099 (89·9%) of 11 235 included individuals had a final genetically confirmed diagnosis of familial hypercholesterolaemia and 1136 (10·1%) had a clinical diagnosis. Genetically confirmed diagnosis data or clinical diagnosis data were missing for 613 (5·2%) of 11 848 individuals. Genetic diagnosis was more common in children and adolescents from high-income countries (9427 [92·4%] of 10 202) than in children and adolescents from non-high-income countries (199 [48·0%] of 415). 3414 (31·6%) of 10 804 children or adolescents were index cases. Familial-hypercholesterolaemia-related physical signs, cardiovascular risk factors, and cardiovascular disease were uncommon, but were more common in non-high-income countries. 7557 (72·4%) of 10 428 included children or adolescents were not taking lipid-lowering medication (LLM) and had a median LDL-C of 5·00 mmol/L (IQR 4·05-6·08). Compared with genetic diagnosis, the use of unadapted clinical criteria intended for use in adults and reliant on more extreme phenotypes could result in 50-75% of children and adolescents with familial hypercholesterolaemia not being identified. Interpretation: Clinical characteristics observed in adults with familial hypercholesterolaemia are uncommon in children and adolescents with familial hypercholesterolaemia, hence detection in this age group relies on measurement of LDL-C and genetic confirmation. Where genetic testing is unavailable, increased availability and use of LDL-C measurements in the first few years of life could help reduce the current gap between prevalence and detection, enabling increased use of combination LLM to reach recommended LDL-C targets early in life

RICORS2040 : The need for collaborative research in chronic kidney disease

Chronic kidney disease (CKD) is a silent and poorly known killer. The current concept of CKD is relatively young and uptake by the public, physicians and health authorities is not widespread. Physicians still confuse CKD with chronic kidney insufficiency or failure. For the wider public and health authorities, CKD evokes kidney replacement therapy (KRT). In Spain, the prevalence of KRT is 0.13%. Thus health authorities may consider CKD a non-issue: very few persons eventually need KRT and, for those in whom kidneys fail, the problem is 'solved' by dialysis or kidney transplantation. However, KRT is the tip of the iceberg in the burden of CKD. The main burden of CKD is accelerated ageing and premature death. The cut-off points for kidney function and kidney damage indexes that define CKD also mark an increased risk for all-cause premature death. CKD is the most prevalent risk factor for lethal coronavirus disease 2019 (COVID-19) and the factor that most increases the risk of death in COVID-19, after old age. Men and women undergoing KRT still have an annual mortality that is 10- to 100-fold higher than similar-age peers, and life expectancy is shortened by ~40 years for young persons on dialysis and by 15 years for young persons with a functioning kidney graft. CKD is expected to become the fifth greatest global cause of death by 2040 and the second greatest cause of death in Spain before the end of the century, a time when one in four Spaniards will have CKD. However, by 2022, CKD will become the only top-15 global predicted cause of death that is not supported by a dedicated well-funded Centres for Biomedical Research (CIBER) network structure in Spain. Realizing the underestimation of the CKD burden of disease by health authorities, the Decade of the Kidney initiative for 2020-2030 was launched by the American Association of Kidney Patients and the European Kidney Health Alliance. Leading Spanish kidney researchers grouped in the kidney collaborative research network Red de Investigación Renal have now applied for the Redes de Investigación Cooperativa Orientadas a Resultados en Salud (RICORS) call for collaborative research in Spain with the support of the Spanish Society of Nephrology, Federación Nacional de Asociaciones para la Lucha Contra las Enfermedades del Riñón and ONT: RICORS2040 aims to prevent the dire predictions for the global 2040 burden of CKD from becoming true

Evolving trends in the management of acute appendicitis during COVID-19 waves. The ACIE appy II study

Background: In 2020, ACIE Appy study showed that COVID-19 pandemic heavily affected the management of patients with acute appendicitis (AA) worldwide, with an increased rate of non-operative management (NOM) strategies and a trend toward open surgery due to concern of virus transmission by laparoscopy and controversial recommendations on this issue. The aim of this study was to survey again the same group of surgeons to assess if any difference in management attitudes of AA had occurred in the later stages of the outbreak. Methods: From August 15 to September 30, 2021, an online questionnaire was sent to all 709 participants of the ACIE Appy study. The questionnaire included questions on personal protective equipment (PPE), local policies and screening for SARS-CoV-2 infection, NOM, surgical approach and disease presentations in 2021. The results were compared with the results from the previous study. Results: A total of 476 answers were collected (response rate 67.1%). Screening policies were significatively improved with most patients screened regardless of symptoms (89.5% vs. 37.4%) with PCR and antigenic test as the preferred test (74.1% vs. 26.3%). More patients tested positive before surgery and commercial systems were the preferred ones to filter smoke plumes during laparoscopy. Laparoscopic appendicectomy was the first option in the treatment of AA, with a declined use of NOM. Conclusion: Management of AA has improved in the last waves of pandemic. Increased evidence regarding SARS-COV-2 infection along with a timely healthcare systems response has been translated into tailored attitudes and a better care for patients with AA worldwide

Height and body-mass index trajectories of school-aged children and adolescents from 1985 to 2019 in 200 countries and territories: a pooled analysis of 2181 population-based studies with 65 million participants

Summary Background Comparable global data on health and nutrition of school-aged children and adolescents are scarce. We aimed to estimate age trajectories and time trends in mean height and mean body-mass index (BMI), which measures weight gain beyond what is expected from height gain, for school-aged children and adolescents. Methods For this pooled analysis, we used a database of cardiometabolic risk factors collated by the Non-Communicable Disease Risk Factor Collaboration. We applied a Bayesian hierarchical model to estimate trends from 1985 to 2019 in mean height and mean BMI in 1-year age groups for ages 5–19 years. The model allowed for non-linear changes over time in mean height and mean BMI and for non-linear changes with age of children and adolescents, including periods of rapid growth during adolescence. Findings We pooled data from 2181 population-based studies, with measurements of height and weight in 65 million participants in 200 countries and territories. In 2019, we estimated a difference of 20 cm or higher in mean height of 19-year-old adolescents between countries with the tallest populations (the Netherlands, Montenegro, Estonia, and Bosnia and Herzegovina for boys; and the Netherlands, Montenegro, Denmark, and Iceland for girls) and those with the shortest populations (Timor-Leste, Laos, Solomon Islands, and Papua New Guinea for boys; and Guatemala, Bangladesh, Nepal, and Timor-Leste for girls). In the same year, the difference between the highest mean BMI (in Pacific island countries, Kuwait, Bahrain, The Bahamas, Chile, the USA, and New Zealand for both boys and girls and in South Africa for girls) and lowest mean BMI (in India, Bangladesh, Timor-Leste, Ethiopia, and Chad for boys and girls; and in Japan and Romania for girls) was approximately 9–10 kg/m2. In some countries, children aged 5 years started with healthier height or BMI than the global median and, in some cases, as healthy as the best performing countries, but they became progressively less healthy compared with their comparators as they grew older by not growing as tall (eg, boys in Austria and Barbados, and girls in Belgium and Puerto Rico) or gaining too much weight for their height (eg, girls and boys in Kuwait, Bahrain, Fiji, Jamaica, and Mexico; and girls in South Africa and New Zealand). In other countries, growing children overtook the height of their comparators (eg, Latvia, Czech Republic, Morocco, and Iran) or curbed their weight gain (eg, Italy, France, and Croatia) in late childhood and adolescence. When changes in both height and BMI were considered, girls in South Korea, Vietnam, Saudi Arabia, Turkey, and some central Asian countries (eg, Armenia and Azerbaijan), and boys in central and western Europe (eg, Portugal, Denmark, Poland, and Montenegro) had the healthiest changes in anthropometric status over the past 3·5 decades because, compared with children and adolescents in other countries, they had a much larger gain in height than they did in BMI. The unhealthiest changes—gaining too little height, too much weight for their height compared with children in other countries, or both—occurred in many countries in sub-Saharan Africa, New Zealand, and the USA for boys and girls; in Malaysia and some Pacific island nations for boys; and in Mexico for girls. Interpretation The height and BMI trajectories over age and time of school-aged children and adolescents are highly variable across countries, which indicates heterogeneous nutritional quality and lifelong health advantages and risks

Rising rural body-mass index is the main driver of the global obesity epidemic in adults

Body-mass index (BMI) has increased steadily in most countries in parallel with a rise in the proportion of the population who live in cities(.)(1,2) This has led to a widely reported view that urbanization is one of the most important drivers of the global rise in obesity(3-6). Here we use 2,009 population-based studies, with measurements of height and weight in more than 112 million adults, to report national, regional and global trends in mean BMI segregated by place of residence (a rural or urban area) from 1985 to 2017. We show that, contrary to the dominant paradigm, more than 55% of the global rise in mean BMI from 1985 to 2017-and more than 80% in some low- and middle-income regions-was due to increases in BMI in rural areas. This large contribution stems from the fact that, with the exception of women in sub-Saharan Africa, BMI is increasing at the same rate or faster in rural areas than in cities in low- and middle-income regions. These trends have in turn resulted in a closing-and in some countries reversal-of the gap in BMI between urban and rural areas in low- and middle-income countries, especially for women. In high-income and industrialized countries, we noted a persistently higher rural BMI, especially for women. There is an urgent need for an integrated approach to rural nutrition that enhances financial and physical access to healthy foods, to avoid replacing the rural undernutrition disadvantage in poor countries with a more general malnutrition disadvantage that entails excessive consumption of low-quality calories.Peer reviewe