A DNA damage-induced phosphorylation circuit enhances Mec1ATR Ddc2ATRIP recruitment to Replication Protein A.

The cell cycle checkpoint kinase Mec1ATR and its integral partner Ddc2ATRIP are vital for the DNA damage and replication stress response. Mec1-Ddc2 "senses" single-stranded DNA (ssDNA) by being recruited to the ssDNA binding Replication Protein A (RPA) via Ddc2. In this study, we show that a DNA damage-induced phosphorylation circuit modulates checkpoint recruitment and function. We demonstrate that Ddc2-RPA interactions modulate the association between RPA and ssDNA and that Rfa1-phosphorylation aids in the further recruitment of Mec1-Ddc2. We also uncover an underappreciated role for Ddc2 phosphorylation that enhances its recruitment to RPA-ssDNA that is important for the DNA damage checkpoint in yeast. The crystal structure of a phosphorylated Ddc2 peptide in complex with its RPA interaction domain provides molecular details of how checkpoint recruitment is enhanced, which involves Zn2+. Using electron microscopy and structural modeling approaches, we propose that Mec1-Ddc2 complexes can form higher order assemblies with RPA when Ddc2 is phosphorylated. Together, our results provide insight into Mec1 recruitment and suggest that formation of supramolecular complexes of RPA and Mec1-Ddc2, modulated by phosphorylation, would allow for rapid clustering of damage foci to promote checkpoint signaling.

Competency level and determinants among infection prevention and control staff in the Middle East and North Africa region.

BACKGROUND: Competency of the staff working in infection prevention and control (IPC) is the cornerstone of successful IPC programs. The objective was to assess competency level and associated factors among IPC staff working in the Middle East and North Africa (MENA) region. METHODS: A cross-sectional study was carried out in 2019 among active IPC staff who were members of the Arab Countries Infection Control Network. The questionnaire covered demographic and professional characteristics of IPC staff and characteristics of work facilities and IPC programs. A competency score was created from self-perceived responses to 8 competency domains. The competency score was then transformed into 100-scale and categorized into two groups (≥ median and < median). RESULTS: A total of 176 participants completed the survey. Participants were mostly female (65.7%), and the mean age was 40.2 ± 8.3 years. The mean competency score was 61.4%. It was slightly variable between domains, being highest with preventing transmission of infection (65%) and lowest with sterilization and disinfection (59%). Higher (≥ median) competency score was associated with having CBIC certification (p < 0.001). It was also associated with facilities having IPC plan (p = 0.005), IPC committee (p = 0.049), regular meetings of IPC committee (p < 0.001), and IPC plan included communications with healthcare workers (p < 0.001). Domain-specific competency scores were significantly associated with receiving same domain-specific training (p < 0.05 for all). CONCLUSIONS: The competency levels of IPC staff in the MENA region is still suboptimal. The current finding calls for more regional and national investment in IPC staff by providing up-to-date customized educational and training opportunities.

Novel insights into the mechanism of cell cycle kinases Mec1(ATR) and Tel1(ATM).

DNA replication is a highly precise process which usually functions in a perfect rhythm with cell cycle progression. However, cells are constantly faced with various kinds of obstacles such as blocks in DNA replication, lack of availability of precursors and improper chromosome alignment. When these problems are not addressed, they may lead to chromosome instability and the accumulation of mutations, and even cell death. Therefore, the cell has developed response mechanisms to keep most of these situations under control. Of the many factors that participate in this DNA damage response, members of the family of phosphatidylinositol 3-kinase-related protein kinases (PIKKs) orchestrate the response landscape. Our understanding of two members of the PIKK family, human ATR (yeast Mec1) and ATM (yeast Tel1), and their associated partner proteins, has shown substantial progress through recent biochemical and structural studies. Emerging structural information of these unique kinases show common features that reveal the mechanism of kinase activity.

The 4-hour target in the emergency department, in-hospital mortality, and length of hospitalization: A single center-retrospective study.

BACKGROUND: The four-hour (4 h') rule in the emergency department (ED) is a performance-based measure introduced with the objective to improve the quality of care. We evaluated the association between time in the ED with in-hospital mortality and hospital length of stay (LOS). METHODS: This was a retrospective study performed in one public hospital with over 100,000 ED referrals per year. Hospitalizations from the ED during 2017 were analyzed. We defined time in the ED as either: until a decision was made (DED); or total time in the ED (TED). In-hospital mortality and LOS were evaluated for patients with DED or TED within and beyond 4 h'. RESULTS: Compared to patients with TED or DED within 4 h', in-hospital mortality did not increase in patients with TED beyond 4 h' (2.8% vs. 3.1%, non-significant), or DED beyond 4 h' (2.1% vs. 3.2%, p < 0.001). LOS did increase in patients with either DED or TED beyond 4 h' (p < 0.001). In-hospital mortality increased with increasing DED-TED intervals for patients hospitalized in the internal medicine departments: 3.7% (0-1 h'), 5.1% (1-2 h'), 5.7% (2-3 h'), and 7.1% (>3 h') (p < 0.001). CONCLUSIONS: In-hospital mortality was not associated with time in the ED beyond 4 h'. LOS, however, was increased in this group of patients. Decreased LOS observed in patients with time in the ED within 4 h', does not support patients' risk as a contributing factor leading to higher trends in mortality observed in this patient group. In-hospital mortality was associated with an increase in DED-TED intervals in patients hospitalized in the internal medicine departments.

Time above the MIC of Piperacillin-Tazobactam as a Predictor of Outcome in Pseudomonas aeruginosa Bacteremia.

Pseudomonas aeruginosa bacteremia is an infection associated with a high mortality rate. Piperacillin-tazobactam is a ß-lactam-ß-lactamase inhibitor combination that is frequently used for the management of Pseudomonas aeruginosa infections. The pharmacokinetic-pharmacodynamic index associated with in vitro maximal bacterial killing for piperacillin-tazobactam is the percentage of the time between doses at which the free fraction concentration remains above the MIC (%fT >MIC). However, the precise %fT >MIC target associated with improved clinical outcomes is unknown. The aim of this study was to investigate the correlation between the survival of patients with Pseudomonas aeruginosa bacteremia and the threshold of the piperacillin-tazobactam %fT >MIC This retrospective study included all adult patients hospitalized over an 82-month period with Pseudomonas aeruginosa bacteremia and treated with piperacillin-tazobactam. Patients with a polymicrobial infection or those who died within 72 h of the time of collection of a sample for culture were excluded. The %fT >MIC of piperacillin-tazobactam associated with in-hospital survival was derived using classification and regression tree analysis. After screening 270 patients, 78 were eligible for inclusion in the study; 18% died during hospitalization. Classification and regression tree analysis identified a %fT >MIC of >60.68% to be associated with improved survival, and this remained statistically significant after controlling for clinical covariates (odds ratio = 7.74, 95% confidence interval = 1.32 to 45.2). In conclusion, the findings recommend dosing of piperacillin-tazobactam with the aim of achieving a pharmacodynamic target %fT >MIC of at least 60% in these patients.

Probing the Mec1ATR Checkpoint Activation Mechanism with Small Peptides.

Yeast Mec1, the ortholog of human ATR, is the apical protein kinase that initiates the cell cycle checkpoint in response to DNA damage and replication stress. The basal activity of Mec1 kinase is activated by cell cycle phase-specific activators. Three distinct activators stimulate Mec1 kinase using an intrinsically disordered domain of the protein. These are the Ddc1 subunit of the 9-1-1 checkpoint clamp (ortholog of human and Schizosaccharomyces pombe Rad9), the replication initiator Dpb11 (ortholog of human TopBP1 and S. pombe Cut5), and the multifunctional nuclease/helicase Dna2. Here, we use small peptides to determine the requirements for Mec1 activation. For Ddc1, we identify two essential aromatic amino acids in a hydrophobic environment that when fused together are proficient activators. Using this increased insight, we have been able to identify homologous motifs in S. pombe Rad9 that can activate Mec1. Furthermore, we show that a 9-amino acid Dna2-based peptide is sufficient for Mec1 activation. Studies with mutant activators suggest that binding of an activator to Mec1 is a two-step process, the first step involving the obligatory binding of essential aromatic amino acids to Mec1, followed by an enhancement in binding energy through interactions with neighboring sequences.

The Dimeric Architecture of Checkpoint Kinases Mec1ATR and Tel1ATM Reveal a Common Structural Organization.

The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1·Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signaling, and repair. Here, we present the first structural insight for dimers of Mec1·Ddc2 and Tel1 using single-particle electron microscopy. Both kinases reveal a head to head dimer with one major dimeric interface through the N-terminal HEAT (named after Huntingtin, elongation factor 3, protein phosphatase 2A, and yeast kinase TOR1) repeat. Their dimeric interface is significantly distinct from the interface of mTOR complex 1 dimer, which oligomerizes through two spatially separate interfaces. We also observe different structural organizations of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1·Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated, providing potential access of substrates to this kinase, even in its dimeric form.

Structural basis for salt-dependent folding of ribonuclease H1 from halophilic archaeon Halobacterium sp. NRC-1.

RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1 (Halo-RNase H1) requires ⩾2M NaCl, ⩾10mM MnCl2, or ⩾300mM MgCl2 for folding. To understand the structural basis for this salt-dependent folding of Halo-RNase H1, the crystal structure of Halo-RNase H1 was determined in the presence of 10mM MnCl2. The structure of Halo-RNase H1 highly resembles those of metagenome-derived LC11-RNase H1 and Sulfolobus tokodaii RNase H1 (Sto-RNase H1), except that it contains two Mn(2+) ions at the active site and has three bi-aspartate sites on its surface. To examine whether negative charge repulsion at these sites are responsible for low-salt denaturation of Halo-RNase H1, a series of the mutant proteins of Halo-RNase H1 at these sites were constructed. The far-UV CD spectra of these mutant proteins measured in the presence of various concentrations of NaCl suggest that these mutant proteins exist in an equilibrium between a partially folded state and a folded state. However, the fraction of the protein in a folded state is nearly 0% for the active site mutant, 40% for the bi-aspartate site mutant, and 70% for the mutant at both sites in the absence of salt. The active site mutant requires relatively low concentration (∼0.5M) of salt for folding. These results suggest that suppression of negative charge repulsion at both active and bi-aspartate sites by salt is necessary to yield a folded protein.

Perception of pain and distress in intubated and mechanically ventilated newborn infants by parents and health professionals.

BACKGROUND: An understanding of perceptions of parents and health caregivers who assist critically ill neonates is necessary to comprehend their actions and demands. Therefore this study aim to analyze the agreement among parents, nurse technicians and pediatricians regarding the presence and intensity of pain and distress in mechanically ventilated and intubated newborn infants. METHODS: Cross-sectional study comprising 52 infants and 52 trios of adults composed of one parent, one nurse technician, and one pediatrician who all observed the same infant. All infants were intubated and under mechanical ventilation and were not handled during the observations. Each newborn was simultaneously observed by the trio of adults for 1 minute to evaluate the presence of pain and distress. The intensity of pain and distress that the adults believed was felt by the infants was marked in a visual analogical scale. Adults' agreement about the simultaneous presence of pain and distress in each infant was analyzed by marginal homogeneity and Cochran tests. The agreement about the intensity of pain and distress in each infant was studied by Bland-Altman plot and intraclass correlation coefficient (ICC). RESULTS: The assessments of pain and distress were heterogeneous in all three investigated groups of adults as determined by the results of a Bland-Altman plot. The presence of distress was more frequently reported compared with pain (marginal heterogeneity, p < 0.01). The pain and distress scores in each adult group were not correlated as shown by ICC [parents, 0.36 (95% CI: 0.01-0.63); nurses 0.47 (0.23-0.66); pediatricians, 0.46 (0.22-0.65)]. CONCLUSIONS: Adults systematically underscore pain in comparison to distress in mechanically ventilated newborns, without recognizing the association between them.

Influence of cytochrome P450 2D6*10/*10 genotype on the risk for tramadol associated adverse effects: a retrospective cohort study.

Background: Tramadol is primarily metabolized by the highly polymorphic CYP2D6 enzyme, leading to a large spectrum of adverse events and clinical response. Ample evidence pointed a reduced CYPD26 activity score in individuals harboring the CYP2D6*10/*10 genotype, nevertheless, there is scarce studies on the impact of CYP2D6*10/*10 genetic polymorphism on long-term tramadol's adverse effects. Aim: To test the correlation between CYP2D6*10/*10 expression and the risk for tramadol-associated adverse effects. Method: Using a database of Leumit Healthcare Services in Israel, we retrospectively assessed the occurrence of adverse events in patients who were prescribed tramadol. A binary logistic regression model was applied to model the relationship between CYP2D6*10/*10 genotype and the occurrence of adverse effects. Results: Data from four hundred ninety-three patients were included in this study. Only 25 (5.1%) patients were heterozygous for the CYP2D6*10 variant, while 56 patients (11%) were tested positive to the CYP2D6*10/*10 genotype. Compared to carriers of other variants, patients with the CYP2D6*10/*10 variant exhibited a higher occurrence of adverse events (odds ratio [OR] = 6.14, 95% confidence interval 3.18-11.83); the odds ratio for central nervous system adverse events and gastrointestinal adverse events were 5.13 (95% CI 2.84-9.28), and 3.25 (95% CI 1.78-5.93), respectively. Conclusion: Among the different CYP2D6 genotypes, CYP2D6*10/*10 genotype carries the higher risk of tramadol related adverse events. Appreciating the frequency of this specific allele it seems prudent to pharmacogenetically screen patients considered for long term tramadol treatment for better tolerability and efficacy outcomes.

An emergency department intervention to improve earlier detection of community-onset bloodstream infection among hospitalized patients.

BACKGROUND: Blood cultures (BCs) are essential microbiologic tests, but blood culturing diagnostic stewardship is frequently poor. We aimed to study the process-related failures and to evaluate the effect of an emergency department (ED) intervention on BCs collection practices and yield. METHODS: We implemented an ED-quality improvement intervention including educational sessions, phlebotomists addition, promoting single-site strategy for BC-collection and preanalytical data feedback. BC-bottles collected, positive BCs, blood volumes and documentation of collection times were measured, before (December 2021-August 2022) and after (September 2022-July 2023) intervention. Results were corrected to hospitalizations admissions or days. We used interrupted-time series analyses for comparisons. RESULTS: A total of 64,295 BC bottles were evaluated, 26,261 before and 38,034 postintervention. The median ED-BCs collected per week increased from 88 to 105 BCs (P < .0001), resulting from increased early sampling (P = .0001). Solitary BCs decreased (95%-28%), documented times increased (2.8%-25%), and average blood volume increased (3 mL to 4.5 mL) postintervention. Community-onset Bloodstream infections (BSIs) increased (39.6-52 bottles/1,000 admissions, P = .0001), while Health care-associated BSIs decreased (39-27 bottles/10,000 days, P = .0042). Contamination rates did not change. CONCLUSIONS: An ED-focused intervention based on the education sessions and single-site strategy improved culturing stewardship and facilitated the early identification of BSI without an increase in contamination.

Coverage and methods of surveillance of healthcare-associated infections in Middle Eastern and North African countries.

BACKGROUND: Surveillance of healthcare-associated infections (HAIs) is a cornerstone for effective infection prevention and control (IPC) programs. The objective was to evaluate the coverage and methods of HAI surveillance in Middle Eastern and North African (MENA) countries. METHODS: A cross-sectional study targeted IPC staff working in MENA countries using the Infection Control Network electronic database of the Arab countries. The study focused on self-reported surveillance-related characteristics of IPC staff, facilities, and the IPC program. RESULTS: A total of 269 IPC staff were included. They were mainly females (68%), nurses (63%), and working in GCC countries (83%). Approximately 69% of covered facilities had surveillance activities. Hand hygiene, multidrug-resistant organisms, central line-associated bloodstream infections, and catheter-associated urinary tract infections were the most common surveillance activities (>90%). The surveillance workload consumed 27% of the average weekly working time. The scores of performing multiple surveillance, with appropriate methods and tools, were 83%, 67%, and 61% (respectively). Appropriate surveillance methods and/or tools were linked to GCC region, CBIC qualifications, surveillance training, specific setting (acute care and long term), staff-to-bed ratio, presence and active function of IPC committee, presence of IPC annual plan, communications with health care workers, and leadership support. CONCLUSIONS: While most health care facilities in the MENA region perform multiple surveillance, surveillance methods and tools are still suboptimal and their optimization should be a priority.

Effect of CYP2C19 Pharmacogenetic Testing on Predicting Citalopram and Escitalopram Tolerability and Efficacy: A Retrospective, Longitudinal Cohort Study.

Background-Various antidepressant agents are metabolized by the CYP2C19 enzyme, including Citalopram and Escitalopram. Variation in CYP2C19 expression might give rise to different plasma concentrations of the active metabolites, potentially affecting both drugs' efficacy and tolerability. Aim-The aim of this study was to evaluate differences in the Escitalopram and Citalopram efficacy and tolerability between different CYP2C19 genotype-based metabolizing categories in outpatients suffering from major depressive disorder (MDD). Methods-In a retrospective, longitudinal cohort study of electronic medical-record data, 283 patients with MDD who were prescribed Escitalopram or Citalopram with the available CYP2C19-genotyping test were enrolled. The primary efficacy end point was adverse drug reactions recorded in the medical files. A proportional-odds, multilevel-regression model for longitudinal ordinal data was used to estimate the relation between the CYP2C19 genotype and adverse drug reactions, adjusting for potential confounding variables and other explanatory variables. Latent-class analysis (LCA) was utilized to detect the presence of clinically significant subgroups and their relation to an individual's metabolizing status for CYP2D6/CYP2C19. Results-With poor CYP2C19 metabolizers as a reference, for each unit difference in the activity score of the CYP2C19 phenotype, the odds ratio for drug intolerability was lowered by 0.73 (95% credible intervals: 0.56-0.89), adjusting for significant covariates. In addition, applying LCA, we identified two qualitatively different subgroups: the first group (61.85%) exhibited multiple side effects, low compliance, and frequent treatment changes, whereas the second group (38.15%) demonstrated fewer side effects, good adherence, and fewer treatment changes. The CYP2C19 phenotype was substantially associated with the group membership. Conclusions-We found a positive association between the CYP2C19 activity scores, as inferred from the genotype, and both the efficacy of and tolerability to both Es/Citalopram. LCA enabled valuable insights into the underlying structure of the population; the CYP2C19 phenotype has a predictive value that discriminates between low-adherence, low-drug-tolerance, and low-response patients and high-adherence, high-drug-tolerance, and high-response patients. Personalized medicine based on CYP2C19 genotyping could evolve as a promising new avenue towards mitigating Escitalopram and Citalopram therapy and the associated side effects and enhancing treatment success.

Barriers and facilitators of implementing interventions to improve appropriate antibiotic use in low- and middle-income countries: a systematic review based on the Consolidated Framework for Implementation Research.

BACKGROUND: Behavior change interventions that aim to improve rational antibiotic use in prescribers and users have been widely conducted in both high- and LMICs. However, currently, no review has systematically examined challenges unique to LMICs and offered insights into the underlying contextual factors that influence these interventions. We adopted an implementation research perspective to systematically synthesize the implementation barriers and facilitators in LMICs. METHODS: We conducted literature searches in five electronic databases and identified studies that involved the implementation of behavior change interventions to improve appropriate antibiotic use in prescribers and users in LMICs and reported implementation barriers and facilitators. Behavior change interventions were defined using the behavior change wheel, and the coding and synthesis of barriers and facilitators were guided by the Consolidated Framework for Implementation Research (CFIR). RESULTS: We identified 52 eligible studies, with the majority targeting prescribers practicing at tertiary facilities (N=39, 75%). The most commonly reported factors influencing implementation were found in the inner setting domain of the CFIR framework, particularly related to constraints in resources and the infrastructure of the facilities where interventions were implemented. Barriers related to the external policy environment (e.g., lack of national initiatives and policies on antibiotic use), and individual characteristics of target populations (e.g., reluctance to change prescribing behaviors) were also common, as well as facilitators related to intervention characteristics (e.g., embedding interventions in routine practice) and process (e.g., stakeholder engagement). We also provided insights into the interrelationships between these factors and the underlying causes contributing to the implementation challenges in LMICs. CONCLUSION: We presented a comprehensive overview of the barriers and facilitators of implementing behavior change interventions to promote rational antibiotic use in LMICs. Our findings suggest that facilitating the implementation of interventions to improve rational antibiotic use needs comprehensive efforts to address challenges at policy, organizational, and implementation levels. Specific strategies include (1) strengthening political commitment to prompt mobilization of domestic resources and formulation of a sustainable national strategy on AMR, (2) improving the infrastructure of health facilities that allow prescribers to make evidence-based clinical decisions, and (3) engaging local stakeholders to improve their buy-in and facilitate contextualizing interventions. TRIAL REGISTRATION: PROSPERO: CRD42021252715 .

Infection prevention and control staffing and programs in Middle Eastern Countries.

INTRODUCTION: Infection prevention and control (IPC) programs in the Middle Eastern and North African (MENA) countries are evolving. The objective was to characterize IPC personnel and programs in MENA countries, with special emphasis on the differences between Gulf Cooperation Council (GCC) and non-GCC countries. METHODOLOGY: A cross-sectional online survey was conducted in 2019 among IPC members of the Arab Countries Infection Control Network (AcicN). The survey focused on three domains; demographic and professional characteristics, organizational structure, and IPC program characteristics. RESULTS: A total of 269 participants aged 39.9 ± 8.4 years were included in the study. Majority of the participants were females (67.7%), nurses (63.7%), and of Middle-Eastern origin (57.3%). 32.2% of the participants were certified by the Certification Board of Infection Control (CBIC). Only 22.7% of participants were satisfied with their current compensation. Surveillance was the most time-consuming task (26.6%), followed by isolation (12.4%), and investigation of outbreaks (12.1%). Majority of the facilities had at least one IPC personnel per 100 beds (60.9%), supported IPC program (63.9%), a formal IPC committee (93.7%), and an IPC plan (91.4%). Compared with non-GCC countries, GCC countries had significantly more frequent CBIC certification (p = 0.003), training in cleaning/sterilization (p = 0.010), supported IPC program (p = 0.010), formal IPC committee (p = 0.001), IPC plan (p = 0.001), and higher number of IPC personnel per 100 beds (p = 0.047). CONCLUSIONS: MENA countries had generally satisfactory IPC programs and to a lesser extent staffing, with considerable variability between countries with different resources.

Mechanism of auto-inhibition and activation of Mec1ATR checkpoint kinase.

In response to DNA damage or replication fork stalling, the basal activity of Mec1ATR is stimulated in a cell-cycle-dependent manner, leading to cell-cycle arrest and the promotion of DNA repair. Mec1ATR dysfunction leads to cell death in yeast and causes chromosome instability and embryonic lethality in mammals. Thus, ATR is a major target for cancer therapies in homologous recombination-deficient cancers. Here we identify a single mutation in Mec1, conserved in ATR, that results in constitutive activity. Using cryo-electron microscopy, we determine the structures of this constitutively active form (Mec1(F2244L)-Ddc2) at 2.8 Å and the wild type at 3.8 Å, both in complex with Mg2+-AMP-PNP. These structures yield a near-complete atomic model for Mec1-Ddc2 and uncover the molecular basis for low basal activity and the conformational changes required for activation. Combined with biochemical and genetic data, we discover key regulatory regions and propose a Mec1 activation mechanism.

Quantifying the Hawthorne effect using overt and covert observation of hand hygiene at a tertiary care hospital in Saudi Arabia.

INTRODUCTION: Although direct human observation of hand hygiene (HH) is considered the gold standard for measuring HH compliance, its accuracy is challenged by the Hawthorne effect. OBJECTIVES: To compare HH compliance using both overt and covert methods of direct observation in different professional categories, hospital settings, and HH indications. METHODS: A cross-sectional study was conducted in 28 units at King Abdulaziz Medical City, Riyadh, Saudi Arabia, between October 2012 and July 2013. Compliance was defined as performing handrubbing or handwashing during 1 of the World Health Organization 5 Moments for HH indications (ie, opportunities). Overt observation was done by infection preventionists (IPs) who were doing their routine HH observation. Covert observation was done by unrecognized temporarily hired professionally trained observers. RESULTS: A total of 15,883 opportunities were observed using overt observation and 7,040 opportunities were observed using covert observation. Overall HH compliance was 87.1% versus 44.9% using overt/covert observations, respectively (risk ratio, 1.94; P < .001). The significant overestimation was seen across all professional categories, hospital settings, and HH indications. CONCLUSION: There is a considerable difference in HH compliance being observed overtly and covertly in all categories. More work is required to improve the methodology of direct observation to minimize the influence of the Hawthorne effect.