Calculation of ATP production rates using the Seahorse XF Analyzer.

Oxidative phosphorylation and glycolysis are the dominant ATP-generating pathways in mammalian metabolism. The balance between these two pathways is often shifted to execute cell-specific functions in response to stimuli that promote activation, proliferation, or differentiation. However, measurement of these metabolic switches has remained mostly qualitative, making it difficult to discriminate between healthy, physiological changes in energy transduction or compensatory responses due to metabolic dysfunction. We therefore present a broadly applicable method to calculate ATP production rates from oxidative phosphorylation and glycolysis using Seahorse XF Analyzer data and empirical conversion factors. We quantify the bioenergetic changes observed during macrophage polarization as well as cancer cell adaptation to in vitro culture conditions. Additionally, we detect substantive changes in ATP utilization upon neuronal depolarization and T cell receptor activation that are not evident from steady-state ATP measurements. This method generates a single readout that allows the direct comparison of ATP produced from oxidative phosphorylation and glycolysis in live cells. Additionally, the manuscript provides a framework for tailoring the calculations to specific cell systems or experimental conditions.

Fibrosis Management Algorithm in Secondary Liposculpture.

BACKGROUND: Liposuction is considered one of the most common procedures in plastic surgery. However, major postoperative complications such as visceral injury, fluid overload, and necrotizing fasciitis still occur. Likewise, minor complications such as ecchymosis, seromas, infections, and contour irregularities that do not threaten the life of the patient do generate significant dissatisfaction. Current evidence regarding the management of fibrosis after previous liposuction remains limited. OBJECTIVES: The objective of this article is to standardize a management algorithm based on the extensive experience and successful results of the primary author (G.M.). METHODS: Patients who underwent secondary liposculpture between August 2022 and May 2023 were evaluated prospectively. Inclusion criteria comprised females between 18 and 60 years old, nonsmokers, with a BMI < 35 kg/m2 and a history of previous body contouring surgeries. Identification of the patient's skin condition and subcutaneous lesions in the adipose tissue were obtained in detail. Statistical analysis of preoperative and postoperative medical photographs was also performed with the Fiji Biological image analyzer. RESULTS: Photographic analysis of preoperative and postoperative photographs showed a statistically significant difference between the areas affected by fibrosis (P < .001). The most frequent clinical findings were depressions in 99% of the females (74), followed by soft nodules in 95% (70), hard nodules in 81% (61), adhesions in 47% (35), and finally cutaneous bursas in 4%. CONCLUSIONS: Our classification system and management algorithm for fibrosis and contour irregularities is a safe and reliable tool. Results were objectively verified, yielding statistically significant outcomes.

Medical specialist distributions in Ecuador: a geographical and temporal analysis of data from 2000 to 2017.

BACKGROUND: Knowledge of medical specialists' numbers and geographical distribution are essential for planning health services and health workforce supply. However, although the distribution of physicians is a significant concern for society and policymakers in Ecuador, no studies have evaluated the distribution of specialists in the country. This study aimed to explore the geographical and temporal distribution of medical specialists in Ecuador over 18 years from 2000 to 2017 and analyse its implications for health planning and medical training. METHODS: We conducted an ecological time-series study based on the National Statistical Register of Resources and Health Activities data. This register provides administrative information for health professionals working in public and private health institutions. Rates of medical specialists by year, geographical area, and speciality were estimated. We used joint-point analyses to identify time trends for medical specialists and physicians in training. RESULTS: From 2000 to 2017, medical specialists grew from 2737 to 10,929. The rate of medical specialists per 10,000 population increased from 4 in 2000 to 10.3 in 2017. Based on Joint point analysis, two temporal trends were identified. Between 2000 to 2015, specialists increased by 4.1% per year, and between 2015 and 2017, they increased by 20% per year. For the entire study period, three cities (Quito, Guayaquil, and Cuenca) accounted for more than 50% of the specialists in the country. However, medical specialists in other cities and rural areas increased from 37% in 2000 to 46% in 2017. The provinces of Esmeraldas, Carchi, Bolívar and Los Ríos presented rates of less than 6 specialists per 10,000 population by 2017. Of the 46 medical specialities identified by 2017, three represented more than 30% of the professionals (gynaecology 12%, paediatrics 11% and family and community health 8.4%). CONCLUSIONS: This study shows that the number of medical specialists in Ecuador has increased significantly over the last two decades, although with inequalities in the distribution of specialists between provinces and regions. The results of this study provide background for the Ecuadorian health system when introducing Human Resources of Health (HRH) policies.

Large-Scale Production of LGR5-Positive Bipotential Human Liver Stem Cells.

BACKGROUND AND AIMS: The gap between patients on transplant waiting lists and available donor organs is steadily increasing. Human organoids derived from leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-positive adult stem cells represent an exciting new cell source for liver regeneration; however, culturing large numbers of organoids with current protocols is tedious and the level of hepatic differentiation is limited. APPROACH AND RESULTS: Here, we established a method for the expansion of large quantities of human liver organoids in spinner flasks. Due to improved oxygenation in the spinner flasks, organoids rapidly proliferated and reached an average 40-fold cell expansion after 2 weeks, compared with 6-fold expansion in static cultures. The organoids repopulated decellularized liver discs and formed liver-like tissue. After differentiation in spinner flasks, mature hepatocyte markers were highly up-regulated compared with static organoid cultures, and cytochrome p450 activity reached levels equivalent to hepatocytes. CONCLUSIONS: We established a highly efficient method for culturing large numbers of LGR5-positive stem cells in the form of organoids, which paves the way for the application of organoids for tissue engineering and liver transplantation.

Collision Avoidance Resource Allocation for LoRaWAN.

The number of connected IoT devices is significantly increasing and it is expected to reach more than two dozens of billions of IoT connections in the coming years. Low Power Wide Area Networks (LPWAN) have become very relevant for this new paradigm due to features such as large coverage and low power consumption. One of the most appealing technologies among these networks is LoRaWAN. Although it may be considered as one of the most mature LPWAN platforms, there are still open gaps such as its capacity limitations. For this reason, this work proposes a collision avoidance resource allocation algorithm named the Collision Avoidance Resource Allocation (CARA) algorithm with the objective of significantly increase system capacity. CARA leverages the multichannel structure and the orthogonality of spreading factors in LoRaWAN networks to avoid collisions among devices. Simulation results show that, assuming ideal radio link conditions, our proposal outperforms in 95.2% the capacity of a standard LoRaWAN network and increases the capacity by almost 40% assuming a realistic propagation model. In addition, it has been verified that CARA devices can coexist with LoRaWAN traditional devices, thus allowing the simultaneous transmissions of both types of devices. Moreover, a proof-of-concept has been implemented using commercial equipment in order to check the feasibility and the correct operation of our solution.

Uremic toxins promote accumulation of oxidized protein and increased sensitivity to hydrogen peroxide in endothelial cells by impairing the autophagic flux.

Chronic kidney disease (CKD) is associated with high mortality rates, mainly due to cardiovascular diseases (CVD). Uremia has been considered a relevant risk factor for CVD in CKD patients, since uremic toxins (UTs) promote systemic and vascular inflammation, oxidative stress and senescence. Here, we demonstrate that uremic toxins indoxyl sulfate (IxS), p-cresyl sulfate (pCS) and indole acetic acid (IAA) are incorporated by human endothelial cells and inhibit the autophagic flux, demonstrated by cellular p62 accumulation. Moreover, isolated and mixed UTs impair the lysosomal stage of autophagy, as determined by cell imaging of the mRFP-GFP-LC3 protein. Endothelial cells exposed to UTs display accumulation of carbonylated proteins and increased sensitivity to hydrogen peroxide. Rapamycin, an autophagy activator which induces both autophagosome formation and clearance, prevented these effects. Collectively, our findings demonstrate that accumulation of oxidized proteins and enhanced cell sensitivity to hydrogen peroxide are consequences of impaired autophagic flux. These data provide evidence that UTs-induced impaired autophagy may be a novel contributor to endothelial dysfunction.

Biochemistry of Peroxynitrite and Protein Tyrosine Nitration.

Peroxynitrite is a short-lived and reactive biological oxidant formed from the diffusion-controlled reaction of the free radicals superoxide (O2•-) and nitric oxide (•NO). In this review, we first analyze the biochemical evidence for the formation of peroxynitrite in vivo and the reactions that lead to it. Then, we describe the principal reactions that peroxynitrite undergoes with biological targets and provide kinetic and mechanistic details. In these reactions, peroxynitrite has roles as (1) peroxide, (2) Lewis base, and (3) free radical generator. Physiological levels of CO2 can change the outcome of peroxynitrite reactions. The second part of the review assesses the formation of protein 3-nitrotyrosine (NO2Tyr) by peroxynitrite-dependent and -independent mechanisms, as one of the hallmarks of the actions of •NO-derived oxidants in biological systems. Moreover, tyrosine nitration impacts protein structure and function, tyrosine kinase signal transduction cascades and protein turnover. Overall, the review is aimed to provide an integrated biochemical view on the formation and reactions of peroxynitrite under biologically relevant conditions and the impact of this stealthy oxidant and one of its major footprints, protein NO2Tyr, in the disruption of cellular homeostasis.

The role of extracellular matrix on liver stem cell fate: A dynamic relationship in health and disease.

The liver stem cell niche is a specialized and dynamic microenvironment with biomechanical and biochemical characteristics that regulate stem cell behavior. This is feasible due to the coordination of a complex network of secreted factors, small molecules, neural, blood inputs and extracellular matrix (ECM) components involved in the regulation of stem cell fate (self-renewal, survival, and differentiation into more mature phenotypes like hepatocytes and cholangiocytes). In this review, we describe and summarize all the major components that play essential roles in the liver stem cell niche, in particular, growth factor signaling and the biomechanical properties of the ECM.

Recellularized Native Kidney Scaffolds as a Novel Tool in Nephrotoxicity Screening.

Drug-induced kidney injury in medicinal compound development accounts for over 20% of clinical trial failures and involves damage to different nephron segments, mostly the proximal tubule. Yet, currently applied cell models fail to reliably predict nephrotoxicity; neither are such models easy to establish. Here, we developed a novel three-dimensional (3D) nephrotoxicity platform on the basis of decellularized rat kidney scaffolds (DS) recellularized with conditionally immortalized human renal proximal tubule epithelial cells overexpressing the organic anion transporter 1 (ciPTEC-OAT1). A 5-day SDS-based decellularization protocol was used to generate DS, of which 100-µm slices were cut and used for cell seeding. After 8 days of culturing, recellularized scaffolds (RS) demonstrated 3D-tubule formation along with tubular epithelial characteristics, including drug transporter function. Exposure of RS to cisplatin (CDDP), tenofovir (TFV), or cyclosporin A (CsA) as prototypical nephrotoxic drugs revealed concentration-dependent reduction in cell viability, as assessed by PrestoBlue and Live/Dead staining assays. This was most probably attributable to specific uptake of CDDP by the organic cation transporter 2 (OCT2), TFV through organic anion transporter 1 (OAT1), and CsA competing for P-glycoprotein-mediated efflux. Compared with 2D cultures, RS showed an increased sensitivity to cisplatin and tenofovir toxicity after 24-hour exposure (9 and 2.2 fold, respectively). In conclusion, we developed a physiologically relevant 3D nephrotoxicity screening platform that could be a novel tool in drug development.

Naturally-Derived Biomaterials for Tissue Engineering Applications.

Naturally-derived biomaterials have been used for decades in multiple regenerative medicine applications. From the simplest cell microcarriers made of collagen or alginate, to highly complex decellularized whole-organ scaffolds, these biomaterials represent a class of substances that is usually first in choice at the time of electing a functional and useful biomaterial. Hence, in this chapter we describe the several naturally-derived biomaterials used in tissue engineering applications and their classification, based on composition. We will also describe some of the present uses of the generated tissues like drug discovery, developmental biology, bioprinting and transplantation.

Cetuximab Prevents Methotrexate-Induced Cytotoxicity in Vitro through Epidermal Growth Factor Dependent Regulation of Renal Drug Transporters.

The combination of methotrexate with epidermal growth factor receptor (EGFR) recombinant antibody, cetuximab, is currently being investigated in treatment of head and neck carcinoma. As methotrexate is cleared by renal excretion, we studied the effect of cetuximab on renal methotrexate handling. We used human conditionally immortalized proximal tubule epithelial cells overexpressing either organic anion transporter 1 or 3 (ciPTEC-OAT1/ciPTEC-OAT3) to examine OAT1 and OAT3, and the efflux pumps breast cancer resistance protein (BCRP), multidrug resistance protein 4 (MRP4), and P-glycoprotein (P-gp) in methotrexate handling upon EGF or cetuximab treatment. Protein kinase microarrays and knowledge-based pathway analysis were used to predict EGFR-mediated transporter regulation. Cytotoxic effects of methotrexate were evaluated using the dimethylthiazol bromide (MTT) viability assay. Methotrexate inhibited OAT-mediated fluorescein uptake and decreased efflux of Hoechst33342 and glutathione-methylfluorescein (GS-MF), which suggested involvement of OAT1/3, BCRP, and MRP4 in transepithelial transport, respectively. Cetuximab reversed the EGF-increased expression of OAT1 and BCRP as well as their membrane expressions and transport activities, while MRP4 and P-gp were increased. Pathway analysis predicted cetuximab-induced modulation of PKC and PI3K pathways downstream EGFR/ERBB2/PLCg. Pharmacological inhibition of ERK decreased expression of OAT1 and BCRP, while P-gp and MRP4 were increased. AKT inhibition reduced all transporters. Exposure to methotrexate for 24 h led to a decreased viability, an effect that was reversed by cetuximab. In conclusion, cetuximab downregulates OAT1 and BCRP while upregulating P-gp and MRP4 through an EGFR-mediated regulation of PI3K-AKT and MAPKK-ERK pathways. Consequently, cetuximab attenuates methotrexate-induced cytotoxicity, which opens possibilities for further research into nephroprotective comedication therapies.

Central role for hydrogen peroxide in P2Y1 ADP receptor-mediated cellular responses in vascular endothelium.

ADP activates a family of cell surface receptors that modulate signaling pathways in a broad range of cells. ADP receptor antagonists are widely used to treat cardiovascular disease states. These studies identify a critical role for the stable reactive oxygen species hydrogen peroxide (H2O2) in mediating cellular responses activated by the G protein-coupled P2Y1 receptor for ADP. We found that ADP-dependent phosphorylation of key endothelial signaling proteins--including endothelial nitric oxide synthase, AMP-activated protein kinase, and the actin-binding MARCKS protein--was blocked by preincubation with PEG-catalase, which degrades H2O2. ADP treatment promoted the H2O2-dependent phosphorylation of c-Abl, a nonreceptor tyrosine kinase that modulates the actin cytoskeleton. Cellular imaging experiments using fluorescence resonance energy transfer-based biosensors revealed that ADP-stimulated activation of the cytoskeleton-associated small GTPase Rac1 was independent of H2O2. However, Rac1-dependent activation of AMP-activated protein kinase, the signaling phospholipid phosphatidylinositol-(4, 5)-bisphosphate, and the c-Abl-interacting protein CrkII are mediated by H2O2. We transfected endothelial cells with differentially targeted HyPer2 H2O2 biosensors and found that ADP promoted a marked increase in H2O2 levels in the cytosol and caveolae, and a smaller increase in mitochondria. We performed a screen for P2Y1 receptor-mediated receptor tyrosine kinase transactivation and discovered that ADP transactivates Fms-like tyrosine kinase 3 (Flt3), a receptor tyrosine kinase expressed in these cells. Our observation that P2Y1 receptor-mediated responses involve Flt3 transactivation may identify a unique mechanism whereby cancer chemotherapy with receptor tyrosine kinase inhibitors promotes vascular dysfunction. Taken together, these findings establish a critical role for endogenous H2O2 in control of ADP-mediated signaling responses in the vascular wall.

Spontaneous intramural esophageal dissection: an unusual onset of eosinophilic esophagitis.

A 35-year-old man, with a history of rhinitis, eczema and a dubious achalasia was admitted due to chest pain and sialorrhea. Upper endoscopy showed a little hole and a narrowing of the distal esophagus. A CT-scan with oral contrast exposed a discontinuity of the lumen of the middle third of the esophagus and a dissection of submucosal space 16 cm long. The patient recovered after parenteral nutrition. After four months, an esophageal endoscopic showed transient whitish exudates, longitudinal furrows and esophageal lacerations. The biopsies illustrated significant eosinophilic inflammation, eosinophilic microabscesses and basal cell hyperplasia.

Uncoupling between core genome and virulome in extraintestinal pathogenic Escherichia coli.

Extraintestinal pathogenic Escherichia coli (ExPEC) are among the most frequently isolated bacterial pathogens in hospitals. They are considered opportunistic pathogens and are found mostly in urinary and bloodstream infections. They are genetically diverse, and many studies have sought associations between genotypes or virulence genes and infection site, severity, or outcome, with varied, often contradictory, results. To understand these difficulties, we have analyzed the diversity patterns in the core genomes and virulomes of more than 500 ExPEC isolates from 5 different collections. The core genome was analyzed using a multilocus sequence type-based single-nucleotide polymorphism (SNP) pyrosequencing approach, while the virulence gene content (the virulome) was studied by polymerase chain reaction detection of 25 representative genes. SNP typing showed a similar population structure in the different collections: half of the isolates belong to a few sequence types (5 to 8), while the other half is composed of a large diversity of sequence types that are found once or twice. Sampling analysis by rarefaction plots of SNP profiles showed saturation curves indicative of a limited diversity. Contrary to this, the virulome shows an extremely high diversity, with almost as many gene profiles as isolates, and linear, nonsaturating, rarefaction plots, even within sequence types. These data show that genetic exchange rates are very heterogeneous along the chromosome, being much higher in the virulome fraction of the genome than in the core genome.

Point-of-care system for detection of Mycobacterium tuberculosis and rifampin resistance in sputum samples.

Early detection of Mycobacterium tuberculosis complex (MTBC) and markers conveying drug resistance can have a beneficial impact on preventive public health actions. We describe here a new molecular point-of-care (POC) system, the Genedrive, which is based on simple sample preparation combined with PCR to detect MTBC and simultaneously detect mutation markers in the rpoB gene directly from raw sputum sample. Hybridization probes were used to detect the presence of the key mutations in codons 516, 526, and 531 of the rpoB gene. The sensitivities for MTBC and rpoB detection from sputum samples were assessed using model samples spiked with known numbers of bacteria prepared from liquid cultures of M. tuberculosis. The overall sensitivities were 90.8% (95% confidence interval [CI], 81, 96.5) for MTBC detection and 72.3% (95% CI, 59.8, 82.7) for rpoB detection. For samples containing ≥1,000 CFU/ml, the sensitivities were 100% for MTBC and 85.7% for rpoB detection, while for samples containing ≤100 CFU/ml, the sensitivities were 86.4% and 65.9% for MTBC and rpoB detection, respectively. The specificity was shown to be 100% (95% CI, 83.2, 100) for MTBC and rpoB. The clinical sputum samples were processed using the same protocol and showed good concordance with the data generated from the model. Tuberculosis-infected subjects with smear samples assessed as scanty or negative were detectable by the Genedrive system. In these paucibacillary patients, the performance of the Genedrive system was comparable to that of the GeneXpert assay. The characteristics of the Genedrive platform make it particularly useful for detecting MTBC and rifampin resistance in low-resource settings and for reducing the burden of tuberculosis disease.

Infections caused by OXA-48-producing Klebsiella pneumoniae in a tertiary hospital in Spain in the setting of a prolonged, hospital-wide outbreak.

OBJECTIVES: We describe clinical and microbiological features of infections caused by OXA-48-producing Klebsiella pneumoniae (O48KP) in the setting of a prolonged, hospital-wide outbreak detected in January 2011. METHODS: Clinical, demographic and microbiological data of patients with growth of O48KP in clinical specimens were collected until December 2011. PCR was used to detect carbapenemase and ß-lactamase genes. The genetic relationships were determined by automated repetitive-sequence-based PCR. RESULTS: Seventy-one patients with clinically guided cultures showing growth of O48KP were identified. Nine were considered to be colonizing rather than causing infection. The most frequent source of infection was the urinary tract (22/62), followed by surgical site infections (17/62). Blood cultures were positive in 23/62 patients. Many patients had significant comorbidity and prolonged hospital stays. In-hospital mortality among patients with O48KP infections was 43.5%. The MIC(90)s of ertapenem, imipenem and meropenem were >32, 16 and 16 mg/L, respectively. No single antimicrobial was active against all the isolates. The antibiotics most active against O48KP were amikacin (97.2% susceptible), colistin (90.1%), tigecycline (73%) and fosfomycin (66.2%). Although eight clones were identified, a predominant clone caused 73.2% of the infections. Multilocus sequence typing (MLST) of the predominant clone gave sequence type (ST) 405 and bla(TEM-1), bla(SHV-76), bla(CTX-M-15) and bla(OXA-1) genes and the insertion sequence IS1999 of the Tn1999 transposon were associated with bla(OXA-48) in this clone. CONCLUSIONS: To our knowledge, this is the largest reported series of infections caused by O48KP in the setting of a single-centre outbreak and provides further input on the clinical relevance of infections caused by O48KP and the difficulties associated with its detection and control.

A systemic framework of energy efficiency in schools: experiences from six European countries.

Schools are complex physical and social institutions within national education systems. They account for significant energy consumption and like other buildings can demonstrate inefficient patterns of energy use. Poor energy performance of educational facilities is an intricate issue driven by complex causality of interconnected and dynamic factors. Addressing this issue requires a systemic approach, which is heretofore lacking. The aim of this research is to present and describe a systemic framework to facilitate energy reduction in schools across different European contexts. This transdisciplinary approach to sustainable energy use has been piloted in 13 post-primary schools located in six countries in northwest Europe. The research implements a series of planned activities and interventions, which help to unveil a systemic approach to improving energy efficiency in schools. The findings demonstrate how this approach, together with its ensuing methodologies and strategies, can contribute to reducing carbon emissions and improve knowledge and awareness around sustainable energy.

Heart rate variability in children: methodological issues and clinical applications. / Variabilidad del ritmo cardíaco en pediatría: aspectos metodológicos y aplicaciones clínicas.

Heart rate variability (HRV) corresponds to variations in heart rate or beat-beat time interval. This parameter reflects the status of the autonomic mechanisms of cardiovascular control, which may be influenced by changes characteristic of the life cycle of people, sedentary lifestyle, and various disease processes that directly or indirectly generate changes in the HRV and shift the autonomic balance, either towards greater sympathetic or parasympathetic influence on the heart. These antecedents support the use of HRV indices for the diagnosis and monitoring of various nosological entities in adults. However, there has been limited progress in the study of temporal variations in heart rhythm in the pediatric population. This article aims to describe the physiological and technical aspects of HRV in the pediatric population to provide a background that allows optimizing the application of these parameters in this age group.

La variabilidad del ritmo cardíaco (VRC) corresponde a las variaciones temporales de la frecuencia cardíaca o del intervalo latido-latido. Este parámetro refleja el estado de los mecanismos autonómicos de control cardiovascular. Los cambios propios del ciclo vital de las personas, el sedentarismo y diversos procesos de enfermedad han mostrado inducir cambios en la VRC y desplazar el balance autonómico hacia una mayor influencia simpática o parasimpática sobre el corazón. Estos antecedentes apoyan el uso de los índices de VRC para el diagnóstico y seguimiento de diversas entidades nosológicas en el adulto. Sin embargo, en la población pediátrica ha existido un limitado avance en el estudio de las variaciones temporales del ritmo cardíaco. Este artículo tiene por objetivo describir los aspectos fisiológicos y técnicos de la VRC en población pediátrica. El propósito es aportar antecedentes que permitan optimizar la aplicación de estos parámetros en este grupo etario.

Behavioural intention of hand hygiene compliance in an average Ecuadorian hospital.

OBJECTIVE: This study aimed to characterize hand hygiene behavioural intention by hospital services clusters in a medium-sized hospital in an Ecuadorian city. METHODS: This is a cross-sectional study based on the World Health Organization Hand Hygiene Knowledge Questionnaire for Health-Care Workers. The responses on hand hygiene behavioural intention for the Five Moments for hand hygiene according to the World Health Organization were recorded in three categories: before patient contact, before and after sterile technique and management of body fluids, and after contact with the environment of the patient. The variables were the knowledge regarding the source of germs causing nosocomial infections, the optimal time to achieve disinfection with alcohol, hospital services clusters (clinical medicine, surgery, and therapeutic services), and history of previous formal hand hygiene training. The variables in each moment were analysed using a saturated log-linear model. RESULTS: The average age of participants was 34 years (Q1 32.1-Q3 36.4). Of them, 62% belonged to the clinic cluster and 87.6% had previous formal hand hygiene training. The incorrect response rates for before and after sterile technique and management of body fluids, before patient contact, and after contact with the environment of the patient were 30.2, 88.4, and 99.2%, respectively. In before patient contact, the incorrect responses for optimal time depended on the department (worse surgery cluster situation), and in before and after sterile technique and management of body fluids and after contact with the environment of the patient, the incorrect responses for source of germs depended on the previous formal hand hygiene training and the department (worse surgery and clinic clusters). CONCLUSION: The incorrect answer related to hand hygiene behavioural intention was high compared to other reports, and the worse situation was found in after contact with the environment of the patient and before patient contact. These data suggest the need of strengthening permanently the hand hygiene programme.