A model using continuous predictors and a simple point-scoring system (PCR index) was developed. For the PCR index, the area under the ROC curve was 0.7686 (P < 0.0001). The sensitivity of the scoring system was 78.6% and specificity was 67.8%. The PCR index identified four severity grades , which were associated with a 3-month mortality of 7.7%, 29.4%, 54.8%, and 80%, respectively. The present PCR models using commonly measured clinical and radiologic variables predicted 3-month mortality in patients with AE-IPF.Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) is often fatal. A straightforward staging system for AE-IPF would improve prognostication, guide patient management, and facilitate research. The aim of study is to develop a multidimensional prognostic AE-IPF staging system that uses commonly measured clinical variables. This retrospective study analyzed data from 353 consecutive patients with IPF admitted to our hospital during the period from January 2008 through January 2018. Multivariate analysis of information from a database of 103 recorded AE-IPF cases was used to identify factors associated with 3-month mortality. A clinical prediction model for AE-IPF was developed by using these retrospective data. Receiver operating characteristic (ROC) analysis was used to evaluate the diagnostic performance of this model. Logistic regression analysis showed that PaO The annual incidence of AE-IPF among patients with IPF is about 5% to 15%, and AE-IPF can occur at any time during the clinical course of IPF. AE-IPF can lead to death within a few weeks to a few months4; however, clinical course and survival vary considerably. Thus, predicting outcomes in patients with AE-IPF is an important challenge for clinicians.Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) does not respond to most conventional therapies and is thus frequently fatal5, lung cancer6, and IPF8. Although some variables are known to be associated with AE-IPF mortality, no single variable accurately predicts outcomes11, and one clinical prediction model that combines variables has been proposed for AE-IPF9. A straightforward staging system for AE-IPF might improve prognostication, help guide management decisions, and allow for appropriate supportive care. Such a system could also improve future research on AE-IPF by identifying patients at high risk for clinically important outcomes.Staging systems have proven useful for determining prognosis and guiding management decisions for lung diseases such as chronic obstructive pulmonary diseaseWe attempted to develop a multidimensional prognostic AE-IPF staging system that uses commonly measured clinical and radiological variables.This retrospective study investigated data from 353 consecutive patients with IPF who were admitted to Toho University Omori Medical Center during the period from January 2008 through April 2018. A total of 106 patients who had received a first clinical diagnosis of AE-IPF satisfied the inclusion criteria. Two patients were excluded because data on 3-month survival were missing. One patient was excluded because of missing data on other variables. Ultimately, 103 AE-IPF patients were included in the analysis. The median duration of observation from the first visit to our center was 13 months (range 1–137 months). During the observation period, 48 of the 103 patients (46.7%) died within 3 months. All deaths during the first 3 months were from respiratory failure caused by AE-IPF , lactate dehydrogenase (LDH), Krebs von den Lungen-6 (KL-6), and surfactant protein D (SP-D).Clinical data were collected to determine the characteristics of underlying IPF and IPF treatment before AE. We also collected information on respiratory function during the 6-month period before AE. The analyzed covariates included PaO10. All patients had a pathological usual interstitial pneumonia (UIP) or UIP pattern on chest high-resolution computed tomography (HRCT), which was confirmed by histological examination of lung biopsy specimens, and/or satisfied the clinical diagnostic criteria for IPF. CT images and clinical records of patients with suspected IPF were subsequently reviewed by 1 thoracic radiologist (A.K.) and 5 pulmonologists , who reached a consensus diagnosis regarding clinical IPF. IPF severity before AE was assessed with the Gender-Age-Physiology (GAP) index, which is calculated by using data on gender, age, forced vital capacity (FVC)% predicted, and diffusion capacity (DLco)% predicted. Disease severity was classified as stage I–III, as previously described. Disease severity, pulmonary function, IPF treatment, and diagnostic findings on HRCT were evaluated while IPF was chronic and stable, i.e., before AE-IPF onset.IPF was diagnosed in accordance with the 2018 international IPF guideline1, with slight modifications, and was diagnosed when the following criteria were fulfilled (1) Previous or concurrent diagnosis of IPF, (2) Acute worsening or development of dyspnea , (3) Computed tomography findings showing a new bilateral ground-glass opacity and/or consolidation superimposed on a background pattern consistent with UIP pattern, (4) deterioration not fully explained by cardiac failure or fluid overload, and (5) no evidence of pulmonary infection on bronchoalveolar lavage, endotracheal aspiration, or sputum culture and negative results on blood tests for other potentially infectious pathogens .AE-IPF was defined in accordance with the International Working Group Report11, we classified the CT pattern at AE-IPF onset as diffuse or non-diffuse. HRCT images at AE-IPF onset were reviewed by evaluating 3 images, namely, those at the level of the aortic arch, carina, and 1 cm above the diaphragm. In the patients studied, new pulmonary opacities at acute exacerbation exhibited different degrees of increased opacity (mixed areas of ground-glass attenuation and consolidation). In semiquantitative analysis, ground-glass opacity and consolidation were grouped together as alveolar opacity. Each lung lobe was evaluated to determine the percentage of newly developed alveolar opacity in relation to the extent and distribution of involvement, except for honeycomb and cystic areas. The percentage for each lobe was defined as the average of the values from all investigators. A diffuse HRCT pattern was defined as an extent of involvement of newly developed alveolar opacity greater than 50%.HRCT images of all the present patients were reviewed by 3 pulmonologists  and 1 chest radiologist (A.K.) who were blinded to the clinical characteristics of the patients. Using the classification of Akira et al.18 at the time of AE-IPF diagnosis because our aim was to establish a scoring system that could be used even for patients with no previous data. The results of multivariate analysis of factors significantly associated with 3-month mortality were used to identify variables that could be used as candidates in the model. An AE-IPF clinical prediction model was developed by using retrospective data from our cohort, and logistic regression was used to evaluate the model. We retrospectively screened potential predictors of mortality in patients with AE-IPF, which yielded a model comprising 3 predictors. On the basis of these predictors, we then developed a simple point-score model and staging system (PCR index and staging system).The primary outcome was 3-month mortality. Vital status and date of death were verified by using the information in the database. Using a database of 103 cases of AE-IPF recorded at our hospital, we used multivariate analysis to identify factors associated with 3-month mortality. We selected clinical variables reported to be associated with AE-IPF outcome2/FiO2 ratio, HRCT pattern (diffuse vs non-diffuse), and antifibrotic agent use as explanatory variables, followed by stepwise variable selection (backward elimination with a threshold of P = 0.05). The values for selected variables in the multivariate analysis were categorized by using cut-off values determined by receiver operating characteristic (ROC) curve analysis. Then, multivariate logistic regression analysis with the categorized variable was again performed to validate its significance. Each selected variable was assigned an integral weight proportional to its partial regression coefficient. For all patients, the total score was defined as the sum of the values for the selected variable. The association between total score and 3-month mortality for AE-IPF was investigated by using ROC curves, and the predicted 3-month mortality cut-off value was calculated. To calibrate the PCR model, we used the Hosmer–Lemeshow test to evaluate agreement between the probability predicted by PCR score and observed probability . In addition, we performed internal validation by using the bootstrap method. Furthermore, to confirm consistency with the lifetime distribution of patients, we constructed Kaplan–Meier survival curves for each PCR index and used the log-rank test for trend to test the correspondence between the temporal trend of the curves and the PCR index. A P value of < 0.05 was considered to indicate statistical significance.Statistical analysis was performed with IBM SPSS Statistics software (version 26). Patients with missing data were excluded from the analysis. Multivariate logistic regression analysis was performed with clinical variables at AE-IPF onset, namely, age, serum KL-6, SP-D, CRP, and LDH concentrations, PaOThis retrospective study was approved by the Institutional Review Board of Toho University Omori Medical Center on June 18, 2019 . Considering the nature of the retrospective study, the informed consent was waived by the IRB because of the anonymized patient data. The study protocol was performed in accordance with the relevant guidelines.2/FiO2 ratio at AE-IPF onset. The detailed clinical characteristics of the patients before AE onset (0–6 months) are also shown in Table 2/FiO2 ratio at AE-IPF onset was significantly but weakly negatively correlated with GAP stage  and JRS classification of IPF disease severity . All patients were treated with high-dose corticosteroid pulse therapy (methylprednisolone 1000 mg/day for 3 days). Corticosteroid dose was tapered after pulse therapy (0.5–1.0 mg/kg/day). Some patients were treated with combination treatment: cyclosporin A (CsA) 2.5 mg/kg/day was administered orally, sivelestat 4.8 mg/kg/day was administered intravenously for the first 14 days, and recombinant human thrombomodulin (rhTM) 0.06 mg/kg/day was administered intravenously for the first 6 days.The characteristics of the 103 patients with AE-IPF are shown in Table 2/FiO2 ratio , diffuse HRCT pattern , serum KL-6 concentration  and serum CRP concentration  (Table P = 0.3491), antifibrotic agent use , serum LDH concentration , and serum SP-D concentration  were not associated with 3-month mortality. Neither the severity of IPF nor pulmonary function test results before AE-IPF were significantly associated with 3-month mortality. Furthermore, no AE-IPF treatment was significantly associated with 3-month mortality. Thus, these factors were not used as candidate variables in multivariate analysis.In multivariate logistic regression analysis with selected explanatory variables, 3-month mortality was significantly associated with PaO2/FiO2 ratio , diffuse HRCT pattern , and serum CRP concentration  (Table P < 0.0001). In addition, 1-month mortality was significantly associated with PaO2/FiO2 ratio  and serum CRP concentration  in multivariate analysis.In multivariate logistic regression analysis with selected explanatory variables, followed by stepwise variable selection, 3-month mortality was significantly associated with PaO2/FiO2 ratio and 5.5 for CRP concentration. The variables identified as significant risk factors for 3-month mortality were a PaO2/FiO2 ratio of < 250 , diffuse HRCT pattern , and serum CRP concentration ≥ 5.5 , all with integral weights of 1 (Table P < 0.0001). Points were assigned to variable categories to create a point-score model (PCR index), as shown in Table P < 0.0001). The optimal cut-off value for PCR score was 2, as indicated by the Youden index, and sensitivity was 0.771 and specificity was 0.655 for predicting cause-specific survival . Ultimately, a grading system was created by grouping point scores into 4 groups. Three-month mortality was 7.7% for a score of 0, 29.4% for a score of 1, 54.8% for a score of 3, and 80.0% for a score of 4 . In addition, we confirmed that values for Kaplan–Meier analysis of the 3-month mortality rate for each PCR score and the estimated 3-month mortality rate calculated by the bootstrap method exhibited similar trends . Patients were then classified as having extensive or limited disease on the basis of the 4 composite parameters. Patients with extensive disease were more likely to require mechanical ventilation and intensive therapy than were those with limited disease. In addition, 3-month mortality was higher in patients with extensive disease. The diagnostic performance in predicting 3-month mortality was examined by ROC analysis, and the area under the ROC curve significantly differed between survivors and nonsurvivors . Although this staging system accurately reflected outcomes, it includes only 2 stages and is somewhat crude. Furthermore, this grading requires complicated CT score calculations. In contrast, the PCR score does not require complicated CT score calculation, as, at diagnosis, only 3 variables have discriminatory power (AUC = 0.768), which similar to that of Kishaba’s continuous model. Moreover, PCR score can be classified into 4 stages, which enables more detailed prediction of outcome.Putative prognostic factors for AE-IPF include CRP, LDH, HRCT pattern (score), PaOWe believe that the PCR score is complimentary and has important implications for clinical practice and research. Specifically, we believe that these tools could provide clinicians and patients with a framework for discussing prognosis with a tool to investigate stage-specific management options and researchers with the means to identify at-risk study populations, which could maximize the efficiency and statistical power of clinical trials.11 classified the pattern of HRCT pulmonary opacification, such as ground-glass attenuation and consolidation, as peripheral, multifocal, and diffuse at AE-IPF onset. The diffuse HRCT pattern was associated with a poor prognosis. Because this classification is relatively straightforward for pulmonary physicians, we used it to classify HRCT findings and confirmed that a diffuse HRCT pattern was associated with poor prognosis.Akira et al.2, and HRCT score as significant predictors; however, multivariate analysis showed that only HRCT score significantly independently predicted outcome. The authors concluded that HRCT score at AE-IPF onset was independently related to outcome in patients with AE-IPF. The HRCT scoring system was adapted from the classification of Ichikado et al., which was previously shown to be an independent prognostic factor for patients with acute respiratory distress syndrome, sepsis, and acute interstitial pneumonia18. Although this scoring system is excellent for predicting prognosis, it might be too complicated for clinical use by pulmonary physicians.A previous study evaluated the pattern, distribution, and extent of HRCT findings at patient presentation and calculated the HRCT score at AE-IPF by examining areas of normal attenuation and the extent of abnormalities, including areas of ground-glass attenuation or consolidation, with or without traction bronchiectasis or bronchiolectasis, and areas of honeycombing. Univariate analysis identified serum KL-6 level, PaCO2/FiO2 ratio of < 250, CRP of ≥ 5.5, and a diffuse HRCT pattern at AE onset were identified as prognostic factors and scored. A cut-off of 2 points was used to determine risk of 3-month mortality after AE-IPF. AE-IPF is thought to be attributable to multiple interrelated risk factors, and the present risk scoring system is likely to be useful clinically.In the present analysis, a PaO9. First, the present predictors are simple to ascertain and are multidimensional, as they incorporate clinical, physiological, and radiological variables. Second, extension of the PCR index into a grading system provides a framework for grade-specific treatment and research recommendations.PCR models have important advantages over previously developed AE-IPF prediction modelsFuture studies should examine the effect of PCR models on disease management. Expanded models that incorporate more-complex baseline variables  and longitudinal measurements  should be compared with the present simple model to determine their additional prognostic value, if any, before incorporation into clinical practice.The present cohort was analyzed retrospectively, and the small sample size may affect data quality. In addition, our cohort was drawn from an academic center; thus, because of referral bias, the included patients might differ from the general population of patients with AE-IPF. In addition, the present scoring system was not validated with another cohort. Furthermore, to make the model more universally applicable, we excluded variables that are not always available to clinicians at initial or subsequent patient visits, such as serial change in lung function. Finally, although we took pains to exclude patients with clinical pulmonary infection or heart failure, the difficulty of diagnosing AE-IPF, and consequent risk of misdiagnosis, could have affected data collection.In summary, we developed a multidimensional PCR index/grading system and calculator that predict 3-month mortality in patients with AE-IPF. We believe that the PCR models are complimentary and have clinical and research utility. In addition, a future study should evaluate the present scoring system in a clinical setting. Finally, a large-scale multicenter study with a validation cohort is necessary in order to confirm the validity of the present results.


Proteobacteria and Bacteroidetes phyla. Functional potential analyses show broadly convergent patterns, contrasting with their great taxonomic variability. As-related metabolism, as well as other functional categories such as those related to the CH4 and S cycles, differs among the five communities. Particularly, we found that the distribution and abundance of As-related genes increase as the As concentration rises. Approximately 75% of the detected genes for As metabolism belong to expulsion mechanisms; arsJ and arsP pumps are related to sites with higher As concentrations and are present almost exclusively in Proteobacteria. Furthermore, taxonomic diversity and functional potential are reflected in the 12 reconstructed high-quality metagenome assembled genomes (MAGs) belonging to the Bacteroidetes (5), Proteobacteria (5), Cyanobacteria (1), and Gemmatimonadetes (1) phyla. We conclude that SH microbial communities are diverse and possess a broad genetic repertoire to thrive under extreme conditions, including increasing concentrations of highly toxic As. Finally, this environment represents a reservoir of unknown and undescribed microorganisms, with great metabolic versatility, which needs further study.Microbial communities inhabiting extreme environments such as Salar de Huasco (SH) in northern Chile are adapted to thrive while exposed to several abiotic pressures and the presence of toxic elements such as arsenic (As). Hence, we aimed to uncover the role of As in shaping bacterial composition, structure, and functional potential in five different sites in this altiplanic wetland using a shotgun metagenomic approach. The sites exhibit wide gradients of As (9 to 321 mg/kg), and our results showed highly diverse communities and a clear dominance exerted by the IMPORTANCE As microbial communities inhabiting extreme environments are fundamental for maintaining ecosystems, many studies concerning composition, functionality, and interactions have been carried out. However, much is still unknown. Here, we sampled microbial communities in the Salar de Huasco, an extreme environment subjected to several abiotic stresses . We found that although microbes are taxonomically diverse, functional potential seems to have an important degree of convergence, suggesting high levels of adaptation. Particularly, arsenic metabolism showed differences associated with increasing concentrations of the metalloid throughout the area, and it effectively exerts a significant pressure over these organisms. Thus, the significance of this research is that we describe highly specialized communities thriving in little-explored environments subjected to several pressures, considered analogous of early Earth and other planets, that have the potential for unraveling technologies to face the repercussions of climate change in many areas of interest. Extreme environments such as high-altitude wetlands select for adaptations in bacterial communities that enable them to thrive. This particular and fragile environment resembles life before the oxygenation of Earth and could serve as a model for studying life on other planets. Moreover, microbial communities are critical for maintaining biogeochemical cycles, particularly in extreme environments where there is little presence of other life forms. The Salar de Huasco (SH), a high-altitude wetland located in the Chilean Altiplano  at 3,800 masl (meters above sea level), is a Ramsar protected site, considered a hot spot for microbial life 38.aioA, arrA, arsM, and arxA are less common in the environment than acr3, arsB, arsC, and arsD , such as As, is a significant selection pressure and thus one of the main drivers of the composition of microbial communities . Thus, m20–In this study, we tested different established sites in the SH regarding their taxonomic and functional heterogeneity. We focused on As-related genetic elements as well as other relevant metabolic functions. Increasingly, such analyses enable the identification of relatively small subsets of markers associated with a particular ecologically important function. Additionally, their identification and the dynamics of bacterial communities are potentially useful bioindicators for monitoring ecosystem health , 24. TheThe Salar de Huasco possesses an important level of variation within a relatively small area (the maximum separation of sampling sites is 5.9 km), including daily oscillations in a wide range of environmental parameters (temperature and humidity) and others which vary in spatial gradients, i.e., salinity and As , 26 Fig; see alsProteobacteria and Bacteroidetes are the most prevalent phyla in the sampled sediments, which are particularly enriched in the H3, H4, and H5 communities, accounting together for >60% of all observed taxa ; it also has the highest abundance of Firmicutes, Patescibacteria, and Spirochaetes phyla as well as significantly lower proportions of Actinobacteria. Moreover, the H1 community profile presents the highest abundance of Chloroflexi, Actinobacteria, Verrucomicrobia, Planctomycetes, and Acidobacteria phyla. On the other hand, H3, H4, and H5 communities are more similar to each other, despite the vast differences in low-abundance taxa, many of which are exclusively present in only one community (see Table S2).The substantial differences between the SH sampling sites are reflected by the taxonomic compositions of bacterial communities, which showed contrasting patterns and variation between sites . Overallved taxa . In turnProteobacteria genera Roseovarius and Desulfotignum in the H3, H4, and H5 communities  detected, many were not classified within any known phylum (6.5%) or genus (58.3%). Nevertheless, Halomonas and Marinobacter genera were detected, both of which are culturable and seem to be recurrent in the SH . Also, most of the highly connected nodes in the network belong to unknown or undescribed taxa.The most abundant bacteria at the lowest (available) taxonomic rank reflect the same pattern, with a great abundance of the n the SH . Of the As a whole, the beta diversity analysis shows that the dispersion patterns between the communities correlate with the composition analysis. Hence, the taxonomic compositions of H3, H4, and H5 metagenomes are more similar between each other and distinctive from those of H0 and H1, producing three very well-defined groups . This coThe SH metagenomes we analyzed represent the compositions in surface sediments of 5 different sites with a great variation in As content (from 9 to 321 mg/kg). The 337.5 million quality-controlled reads were coassembled and yielded a total of 994,545 contigs  corresponding to 2.38 million genes, which were used to generate abundance/distributions and functional profiles of the 5 communities under study . The resCyanobacteria, and the Calvin Benson cycle. However, when assessing the ability of these bacterial communities to carry out necessary reactions to sustain key biogeochemical cycles, we found that S, N, and CH4 showed significant scores  for all five metagenomes, implying that the necessary metabolic pathways and machinery are present and complete in bacteria isolated from all environments . We also found representatives of several pathways, including sulfur reduction and oxidation, nitrate fixation and reduction, methanogenesis, and methane oxidation, and the utilization of a wide range of the available nutrients. Particularly, a significant difference is present in the CH4 cycle, which is much more enriched in the H1 and H0 communities; consequently, we only detected methanogens and methane oxidizer taxa in these sites. Conversely, a wide range of sulfur-associated pathways were detected in all sampled sites , suggesting its importance in environments where volcanic-derived sulfur oxidation products leach and flow into the salt flats . Particularly, the five SH communities showed specific differences in some categories such as As resistance (SEED subsystems 3), following the same tendency previously mentioned, where H0/H1 metagenomes are more similar than H3/H4/H5, with 0.21% to 0.22% and 0.26% to 0.27% of reads recruited by this category, respectively. Other significant categories in the communities were carbon, nitrogen, and phosphate metabolism, zinc, nickel, cobalt, iron, and manganese transport, osmotic stress, the circadian clock in lt flats . NonethearsR regulator, the arsC reductase and the acr3 pump were the most overrepresented genes, indicating that the As(V) reduction and subsequent As(III) expulsion strategy is the most prevalent one used by bacteria that inhabit the SH. Moreover, arsM was among the most abundant genes, particularly enriched in the H3, H4, and H5 metagenomes. Nevertheless, genes related to oxidation and respiration mechanisms were less abundant yet present an interesting distribution; those related to oxidation  are enriched in H3, H4, and H5, while those related to respiration (arrA and arxR) are enriched in H0 and H1.To gain a broader view on the communities’ functional potential related to As metabolism or resistance/tolerance, we determined the abundance of the genes belonging to the known mechanisms . Again, acr3 , was more abundant in the sites with higher As concentrations. Moreover, the higher acr3 variants and abundance could be because this pump is present in a wider number of bacteria phyla, covering most of the diversity observed  ; the same tendency was observed for the GC content (36.1% to 69.4%). On the other hand, most of the reconstructed MAGS were represented or detected in the H3, H4, and H5 metagenomes, showing differential abundance patterns in each site  to gain insights into nonculturable bacteria. This resulted in 19 metagenome-assembled genomes (MAGs) that met the completion of ≥80% and redundancy of ≤10% criteria ; togethenadetes) . We wereBacteroidetes  clustered together, as well as the Deltaproteobacteria  and Gammaproteobacteria (MAG129 and MAG144). On the other hand, the only Cyanobacteria (MAG29) is close to the Gammaproteobacteria and the only Gemmatimonadetes (MAG3) is close to the Bacteroidetes. Particularly, MAGs displayed a considerable number of genes regarding As metabolism, where the same tendency shown before was observed, with a dominant presence of genes related to As expulsion  throughout all taxa, followed by reduction (arsC), methylation (arsM), and oxidation (arsH) mechanisms , while it can also be associated with calcium carbonate (CaHAsO4) or adsorbed into Fe and Al oxides , whi, whi8), 30–l oxides . The metl oxides . It is hProteobacteria, Bacteroidetes, and Cyanobacteria  have been cultured and isolated under laboratory conditions , which correlates with the findings reported in the Argentinian Altiplano  gene was also consistently enriched. Thus, these markers could be proposed as bioindicators of As contamination, such as acr3 , improving our understanding of microbial communities  reduction and subsequent As(III) expulsion systems are the most common strategy used to detoxify the cell of As. Furthermore, regarding expulsion pumps, the most abundant was Acr3, followed by ArsB; however, in sites with high As concentrations, ArsP becomes enriched. In addition, 12 high-quality nonredundant MAGs were reconstructed from the metagenomes. These represented the dominant diversity detected across the communities as well as the metabolic variability and the presence of marker genes related to the most recurrent As resistance mechanisms . Finally, in order to further elucidate the strategies and relationships between the microbial taxa and among biotic and abiotic components within the ecosystem, further multidisciplinary studies are required as well as the use of the ever-evolving next-generation sequencing (NGS) approaches. Together with more precise database information, we will be able to better understand the evolutionary process of adaptation to the extreme conditions present in these unique ecosystems.The Altiplano array of ecological niches is a reservoir for microbial diversity, showing great richness in adapted organisms capable of facing these challenging conditions. Particularly, the Salar de Huasco is a highly diverse ecosystem, where salinity and As concentrations contribute to shaping the community composition, mainly represented by in situ  lakes with salinity and As gradients from north to south . In June260/280) ratio, and fluorescence using a Qubit 3.0 fluorometer along with the Qubit dsDNA HS assay kit . Next, paired-end (150 bp) libraries were constructed for each sample in duplicates at the Centro de Biotecnología Vegetal, Universidad Andrés Bello  using the TruSeq Nano DNA kit  according to the TruSeq Nano DNA sample preparation guide 15041110 revision D. Libraries were sent for sequencing at Macrogen Inc.  on a HiSeq 4000 platform (Illumina Inc.). Then, raw data were evaluated using FastQC v0.11.8  according to the manufacturer’s instructions. DNA integrity, quality, and quantity were verified through 1% agarose gel electrophoresis, optical density at 260 nm to optical density at 280 nm  a coassembly , (ii) its annotations , and (iii) an alignment per sample , we proceeded to assemble the quality-controlled reads from all samples using MEGAHIT v1.1.3 , with th4, and Fe biogeochemical cycles in each metagenome using the predicted proteins .We followed the anvi’o v7 pipeline . First, To estimate the abundance of genes related to As metabolism, the read counts for each predicted gene of each sample were obtained using the corresponding BAM file and the coassembly annotated GFF file, with HTSeq-Counts v0.13.5 . Also, wThe contigs in the metagenome profile were clustered through anvi’o v7  using thWe define MAGs (metagenome assembles genomes) as bins with a completion of >80% and redundancy of <10%. Then, we used CheckM v1.0.13  for a moAll MAGs were queried against the same previously constructed database (with the As metabolism genes) using CRB-BLAST v0.6.6  E value to connePRJNA573913.The whole raw data sets and the metagenome assembled genomes have been deposited at DDBJ/ENA/GenBank under the BioProject accession number


Clinical pathways (CPs) are intended to standardise and improve care but do not always produce positive outcomes, possibly because they were not adapted to suit the specific context in which they were enacted. This qualitative study aimed to explore staff perspectives of implementation of a CP for routine screening, assessment, referral and management of anxiety and depression (the ADAPT CP) for patients with cancer, focussing on perceived feasibility of the CP and negotiated adaptations made during the implementation phase.n = 88) and 6 months later, half-way through the implementation period (n = 89). Monthly meetings with lead multi-disciplinary teams at the eight enhanced sites were recorded. Data were thematically analysed.The ADAPT CP was implemented in 12 urban and regional oncology services in Australia. Services were randomised to receive core versus enhanced implementation strategies. Core sites received support until implementation commencement and could access progress reports. Enhanced sites received proactive, ongoing support during the 12-month implementation. Purposively selected staff were interviewed prior to implementation (Six overarching themes were identified: ADAPT is of high value; timing for introducing the CP and screening is difficult; online screening is challenging; a burden too much; no-one to refer patients to; and micro-logistics are key. While early screening was deemed desirable, diverse barriers meant this was complex, with adaptations made to time and screening location. Online screening prompted by email, seen as time-saving and efficient, also proved unsuccessful in some services, with adaptations made to in-clinic or phone screening, or repeated email reminders. Staff negative attitudes to ADAPT, time constraints, and perceived poor fit of ADAPT to work roles and flows, all impacted implementation, with key tasks often devolving to a few key individuals. Nevertheless, services remained committed to the ADAPT CP, and worked hard to create, review and adapt strategies to address challenges to optimise success.This study demonstrates the interactive nature of health service change, with staff actively engaging with, forming views on, and problem-solving adaptations of the ADAPT CP to overcome barriers. Obtaining staff feedback is critical to ensure health service change is sustainable, meaningful and achieves its promise of improving patient outcomes.The study was registered prospectively with the ANZCTR on 22/3/2017. Trial ID ACTRN12617000411347.The online version contains supplementary material available at 10.1186/s12913-022-07532-2. Clinical pathways (CPs) outline a sequence of standardised, evidence-based clinical management steps with timeframes and expected outcomes, for defined patient groups . They arWithin the literature, there is significant debate regarding if, and to what extent, CPs should be adapted during implementation. Some advocate for strict adherence to the published CP , 14; othOur group developed a CP for screening, assessment, referral, and management of anxiety and depression in adult cancer patients (ADAPT CP) . The eviWe developed implementation strategies  to addreThe ADAPT CP was implemented for 12 months, in a cluster randomised controlled trial (CRCT) evaluating two different implementation strategies (core versus enhanced) in 12 Oncology services in New South Wales (NSW) representing approximately 25% of cancer centres in the state, which were set in 7 of the 15 Local Health Districts of NSW, the most populous state in Australia . The stuWe have earlier described how the CP was tailored during the initial engagement with services prior to implementation . This pan = 12) were purposively selected to provide diversity in urban versus regional settings and size of patient load, and randomised to core or enhanced implementation support arms.Participating sites were public or private oncology services providing cancer care for ≥ or < 100 patients per year (to examine how the ADAPT CP was used in sites with more or less patient throughput). Exclusion criteria included inability to commit to required study processes, and insufficient technology to support use of the ADAPT Portal. Potential sites , a subset of staff at each service, purposively selected to include members and non-members of the lead team and multi-disciplinary representation, were invited to participate in a semi-structured phone interview exploring attitudes to and expectations of the ADAPT CP. We aimed for a sample sufficient to ensure saturation of themes within different staff groups and different services.Non-members of the lead team were included to ensure representation of views of frontline staff enacting the ADAPT CP. Interviews were conducted by three female researchers trained in qualitative methods with no involvement in preparing services to deliver the ADAPT CP. Interviews were conducted again 6 months later (T1) to obtain feedback regarding how the ADAPT CP was working in practice, any CP adaptations made and their rationale. The interview guide was developed based on a recent systematic review and implementation outcomes , 24 and Research staff minuted monthly meetings to capture discussion and decisions made.Interviews were audio recorded, transcribed verbatim and coded in NVivo12 . Data wen = 10), private (n = 1) and public-private partnership (n = 1) services were included.Seven metropolitan and five regional cancer services participated in the ADAPT CRCT  and 89 were interviewed at T1. Due to staff changes, leave or unavailability, different individual staff members were approached at T1; 64 staff were interviewed at both T0 and T1, 24 only at T0 and 25 only at T1. Average interview duration was 22 mins and 24 min at T0 and T1 respectively. Participants had on average 6 years of experience in their role (range 0 to 33). See Table (M = 10.50). Average meeting duration was 35 min. Forty-one meetings (49%) were delivered in-person, 35 (42%) online (web-conference), and eight (10%) via teleconference. There were 366 attendances across all meetings (M = 4.21 per meeting); the majority of attendees  were lead team members  participant ID (P), and assessment point (T) are identified for each interview quote e.g., NURS_S01P05T1. See Table Attitudes towards ADAPT CP were positive throughout implementation. Participants believed the ADAPT CP would ensure all patients who needed psychosocial help would be identified, including those with non-visible distress, facilitate early identification of morbidity to enable timely and appropriate care, and keep patients positively connected to the service.“I think it [ADAPT CP] is needed and useful, I think everyone knows it’s common for cancer patients to have anxiety and depression, we all recognise that treatment is useful.” (MED_S11P05T0).“It’s seen as a … positive way to stay engaged with the team even after discharge.” (PSYCH_ S07P05T0).“I’ve had a couple of patients actually thank me about it, … just reaching out … I think … they’ve found that quite nice.” (NURS_S01P04T1).“The staff have said that, because of ADAPT, we have picked up patients who needed help even though they didn’t appear to need help.” (MED_S01P09T1).“somewhat redundant”  and duplicated existing services, “re-inventing the wheel” (PSYCH_S12P07T1).However, some felt ADAPT was “To be honest, I think we had a pretty good way of doing it before ADAPT … but it’s good to have a bigger version and it’s online so people can do it self-directed” (NURS_S07P04T0).Despite positive attitudes, challenges to implementing ADAPT CP were identified by staff, including timing. Some sites initially chose to introduce ADAPT and the rationale for regular screening at the patient’s first oncologist appointment. However, patients were sometimes hard to identify at this time (due to multiple entry points into the system) and too overwhelmed. Staff were focused on co-ordinating treatment plans and yet to develop a relationship with the patient. If some time elapsed before the patient was screened, some patients forgot or lost motivation.“I think it’s given at the wrong time … they have a thousand things to think about when they’re first seeing the doctors in that situation. So … I think that’s very inappropriate” (NURS_S04P03T1).“Team says if the patient does not know them at this stage and if it is introduced over the phone, it could be difficult to sell.” .tough” for nursing staff on top of existing clinical responsibilities, and time consuming.Screening in clinic immediately prior to or during treatment also presented issues, as anxiety and depression scores could be transiently and contextually high. Staff also found introducing ADAPT during clinics was “… we had a few people that have screened while they’re having treatment, … they can screen quite high because they’re quite anxious just about how it’s going to go, and what’s happening and it’s all new... Whereas if they did it at home or after their treatment, then they might actually have screened differently.” (PSYCH_S02P01T1).“After discussion with the ADAPT team, several services changed to later registration which was perceived to work better:“We were doing the distress thermometer on first presentation and so, there was an awful lot of people that I was contacting. So now … they’re screened more appropriately and at a better time. because everyone’s going to flag high on initial presentation … it’s culled a bit of work out.” (PSYCH_ S05P01T1).One service shifted from introducing ADAPT at the surgical clinic to discharge planning, although even this was not deemed optimal:“ emailed staff regards inclusion of ADAPT in discharge planning. She feels this has had no impact on ADAPT activity, mainly because patients are very pre-occupied with other issues at discharge.” .more feasible” as patients were there much longer (PSYCH_S03P08T1). Another said:One nurse noted that conducting screening in the chemotherapy suite was ““So I’ve spoken to all my colleagues … we feel to introduce them to the ADAPT program is best when the nurse does drug education … and then when they come in to Cycle 1 in the chair … we then say, would you like to register? And if they do, we register there and then.” (NURS_S02P09T1).One site organised a back-up approach by the Nursing Unit Manager if ADAPT was not introduced; other sites simply accepted that some patients would be missed.While initially thought to offer considerable benefit in saving time and providing greater convenience to patients, online screening prompted by email had a low uptake, and even when patient-preferred, low adherence . Staff cited barriers such as older age and lower socio-economic background contributing to patients not having an email address, lacking access to devices at home or lacking the technological literacy to complete email screening independently. Furthermore, without staff present to prompt screening, it was quickly forgotten by patients.“We’d imagined that people would register and then they would screen via emails and electronically, but that hasn’t happened.” (PSYCH_S10P01T1).“We have quite an older population here, so a lot of people don’t have email addresses.” (AH_S06P03T1).“A few people have said, oh, yeah, yeah, I remember that being in my inbox and, um, they just don’t want to address it really.”(NURS_S12P01T1).However, some staff believed that the way ADAPT was communicated to patients was critical in influencing adherence, as opposed to the medium or format of screening.“I think … that we have to push it so that it’s almost not optional … this is what we do; how do you want to receive this? Do you want to do it here, or do you want to do it at home?” (NURS_S02P08T1).One site identified two instances where family members had completed email screening without patients being aware this had taken place , which raised concerns. Another site became concerned that email screening may lead to delays in offering support to patients expressing high distress, violating their duty of care.“[Nursing staff] was questioned … about patients screening highly distressed/suicidal on a Friday night and then this not being acted on until a Monday and conveyed it would not be acceptable to leave them 3 days without contact.” .In the case above, it was ultimately decided that since patients are automatically referred by the online portal to resources and helplines if their distress score is high, and since a direct suicide question is not asked, then online screening by email could continue. One site developed an email reminder system for patients if they did not screen immediately:“Team reconfirmed that email is preferred as finding people in clinic and getting hold of them over the phone can be logistically difficult and time-consuming … Email reminders sent by  to remind patients to screen.” .Other sites reverted to in-clinic screening or telephone follow-ups with patients to prompt screening.not my role,” that they lacked required skills, or just could not fit them into their already stretched workload, and that ADAPT caused friction. A social worker said:Despite initially reporting that the ADAPT CP fulfilled service and personal mission, many staff felt ADAPT tasks were ““Nurses are run off their feet … Nurses cannot and will not prioritise this work”. .“They’re like, that’s not my thing, that’s not my job …” (NURS_S01P06T1).“I also get a sense that there might be some hesitation to actually discuss anxiety and depression … they [nursing staff] are worried that if they open a can of worms, the patient will … almost decompensate, and what do they do?” (PSYCH_S06P13T1).In one site, the view was expressed that ADAPT would never be accepted unless it was made mandatory:“ noted that the only way people will engage with it is if this is mandated …. if [Med Onc] and [Director of Cancer Services] make them do it.” .Conversely, other staff felt that ADAPT had reduced their burden.If anything, it’s probably made things a little bit easier for us, [because] we can pick the patients up earlier …. rather than waiting until things fall down and then, we’re trying to pick them back up.” (NURS_S01P04T1).“Some services put significant thought into role responsibilities to maximise efficiency and efficacy, and make use of existing staff skills, both initially, and over time.“We’ve got the girls out the front. We’ve got it set up that they’ll be able to help them out once … the patient has been educated about the program and the screening …” (ADMIN_S02P05T0).“We were mindful during the planning stage of … who’d have the clinical confidence to go asking questions or running through surveys with patients, so admin were delegated tasks which were specific to their role, and nursing staff and the new [psychosocial staff] were allocated roles which were according to their competencies.” (NURS_S05P06T0).Some sites tried to reduce burden by involving more staff to share the load.“ADAPT is ‘everybody’s business’ such that all people need to be involved rather than this being the responsibility of a single person” ."… there’s five of us. So we each just take one day and then if … an alert comes on in that day then it’s our responsibility to do the triage conversation” (PSYCH_S02P01T1).In some sites, however, back-up staff were unwilling to help out, particularly to pick up patients not seen by the designated screener. In other sites, roles devolved to only a few, often the champion or lead team, to ensure tasks were completed."This theme has been raised consistently throughout the implementation, but attempts to engage medical staff, ward staff, step down facility staff, outpatient staff have failed.” .“So we have a lead team … we were meant to share the workload between us …. But it’s ended up being … just between three of us … and I think that’s what’s been so difficult” (NURS_S10P02T1).“invisible on the ward.”However, restricting ADAPT roles to the lead team could mean that most clinical staff lost awareness of ADAPT, which became “They’re heavily involved in it and they’re managing it quite well. So it’s … almost to the point, non-existent for everyone else on the ward.” (NURS_S07P06T1).Finally, if the person responsible for screening went on leave, or was sick, the system broke down:“Registrations: have been lower than expected over the last 2 months and it became apparent that in the absence of the administrator who had been placing pamphlets and registration slips, this task had not been completed by anyone else which led to a breakdown in the workflow.” .One site decided to make adaptations by devolving some of the work to patients themselves, by prompting patients to screen via email and complete their own registration:“If all patients screen via email and patients fill out mandatory fields themselves, it could be possible to eliminate the registration slip step...” .Some services had no, or insufficient, inhouse psychosocial staff, and were concerned that having identified psychological morbidity, they could not facilitate appropriate and timely support. Some services relied on referral to a local GP, private providers or a psychiatrist.“… at the moment, … we have only got one clinical psychologist … and so, that is quite difficult … for our patients to get into that, so … some people that potentially could, afford to … pay to go to see somebody privately...”(NURS_ S01P04T0).“We don’t have much psychosocial support here at all. We have a social worker.” (NURS_S03P05T0).There was also a perception by staff that external pathways were not always ideal or were difficult for patients to access. However, this was the only option for referral at some services in the absence of inhouse psychosocial staff.Some services saw this as an opportunity and took the initiative to expand referral sources when gaps were identified in planning to implement the ADAPT CP.“Pathways for these patients to go to, apart from the social worker … are starting to be developed which is great” (NURS_S03P04T1).“We’ve got access now to a Telehealth interview with an area psychiatrist who is providing psychology services one afternoon a month … for those higher-level distress patients. It’s better than it has been, because … [previously] we have to contact the GP and ask them to do a mental health plan. Then they get put on a waiting list for, um, to see a psychologist in the community. And that’s still under-resourced and hasn’t changed.”(NURS_S02P08T1).Some services discussed workflow breakdowns due to clinic processes that did not emerge until implementation was underway, for example, unclear procedures or inadequate record-keeping.Many services innovatively developed their own systems for ADAPT CP processes, such as who did what, placing ADAPT registration forms in patient files, providing a script for staff to use when introducing ADAPT, and having an agreed sequence for paperwork so that missing steps could easily be identified and rectified. These adaptations were well received."So we got the flow sheet … and tweaked it and … even during training as other things came up … with the volunteers and their concerns, things have been tweaked again … which has been really good … that has been able to work for us." (NURS_S01P04T0)."So … they’ve come up with a whole practice around that which they’ve highlighted and mapped out and done that very well so far, so, happy with the way they’re working on those." (ADMIN_S01P10T0)."Suggestion by  was the New Patient Form have an ADAPT tick-box included so specialists remember to raise it with patients" .Staff noted that the initial thought that went into developing systems could make or break the success of the CP.“And I do think all those little things add up … just make it easier when you’re in the clinic and you’re trying to think of ten things, and this one just can tell you automatically” (MED_S06P02T1).While many services adapted the ADAPT CP, services varied in the degree to which they formally notified ADAPT research staff of changes leading to the local workflow being updated. Five services notified no changes, four 1 change and three 2 changes. Examples of initial and final detailed service workflows for two services are provided in Additional file This is one of the first studies to document stakeholder experiences of implementing a CP, and the adaptations to the CP made as a result to address feasibility issues. The CP in this case was for screening and management of anxiety and depression in cancer patients (the ADAPT CP) .Despite steps taken to maximise the ADAPT CP implementation success , and a belief expressed by many stakeholders in the value of the CP, barriers to its adoption and implementation feasibility were still encountered.These included finding the right time to initiate screening when patients had formed a trusting relationship with staff, were no longer overwhelmed, yet were sufficiently early in their care to allow identification of distress before it developed into a clinically significant problem. The Australian Clinical Practice Guidelines for managing anxiety and depression in cancer patients  do not sA second issue which emerged was the virtue or otherwise of online screening. Online screening has been promoted as a way to reduce costs and increase feasibility and acceptability for patients who may be too rushed or uncomfortable to complete questionnaires in the clinic , and decAnother key barrier to implementation of the ADAPT CP identified by participants, was the perceived staff burden, on top of existing roles and responsibilities, of required tasks related to ADAPT. The degree to which this activity was felt to be a reasonable and valid part of diverse staff’s roles, core to service and individual mission, impacted the way services responded. As Damschroder et al.  has commprior to implementing a new CP.Management could also choose to strongly endorse participation in new psychosocial CPs and perform audits to ensure compliance. Furthermore, at a service level, adequate resourcing is vital to allow staff to complete required tasks without risking stress and burnout. A recent Australia survey found 38% of respondents reported lack of resources as a major barrier to implementing psychosocial care . DespiteIn the current study, many services made creative adaptations to overcome challenges, demonstrating the key importance of leadership and active engagement by a range of stakeholders (delivery partners) in implementing change, as noted by Yamey et al. . WithoutThis study is not without limitations. While we had good multidisciplinary representation, some staff did not agree to be interviewed, thus their views were not represented. While interviewers were independent of the ADAPT research team who interacted with staff during implementation, social desirability bias may nonetheless have impacted responses, leading to a more positive perspective being presented than was actually felt. Due to more intensive interaction of ADAPT Team with Enhanced arm sites during implementation , there may have been barriers/challenges and adaptations at Core services during the implementation phase to which the ADAPT Staff were not privy and thus not reported here.“Most good ideas … do not spread with such ease. They require the backing and energies of committed individuals and organizations to design and carry out strategies for expansion that are carefully tailored to the realities of their settings.” Chambers and Norton [before they are widely implemented. Furthermore, they argue it is important to recognise that interventions are adapted all the time. It is therefore critical that real world adaptation is not ignored, but rather documented and studied so that we can better understand how and why some interventions are more sustainable than others. In this study, we documented staff perspectives just before and midway through implementation of a clinical pathway to identify and manage anxiety and depression in patients with cancer, and the adaptations made to the CP to overcome feasibility and adoption challenges, adding to the scarce literature on intervention implementation in real-world studies.As noted by Simmons et al.  “Most god Norton  have argAdditional file 1. First and final ADAPT workflows for two contrasting sites. Red indicates where steps have been added to the workflow, blue indicates where adaptations to previous workflow decisions have been made.


Background:Dementia care guidance highlights the importance of supporting people living with dementia to access engaging and meaningful activities to promote their quality of life. There is a growing evidence base for the efficacy of heritage settings and arts-based interventions to provide social prescribing opportunities to help support wellbeing in this population. This study extended previous research and explored the potential processes underlying this effect in multiple small group object handling sessions in a museum setting.   Methods:A mixed-methods design was used comprising a measure of subjective wellbeing and thematic analysis to explore in-the-moment session content across multiple sessions. Four people with dementia participated in three, one-hour group object handling sessions led by two facilitators.Results:Pre-post wellbeing scores showed increases after each session though this was largely not significant. Qualitative findings provided more compelling results, however, and identified four key themes: facilitating, interest in exploring objects, active participation, and group collaboration; interpretations were made around the dynamic interaction of themes and subthemes over the course of three sessions.Conclusions: This is the first study we are aware of that has taken an in-depth look at multiple museum-based group object handling sessions for people living with dementia. Findings offer ways to optimise object handling sessions for people with dementia by providing in-depth information about the processes involved across multiple object handling sessions facilitated by museum/heritage professionals in a museum setting. This has useful implications for community-based activities as part of dementia care planning and public health programming. The study contributes to a deeper understanding and elucidates the processes that enhance wellbeing for this population who participate in such sessions. It also helps to develop further theoretical understanding about why these types of activities are helpful in community-based dementia care. Limitations and implications for future research are discussed. Although there are a range of symptoms, dementia is a syndrome often characterised by progressive decline in cognitive functioning, motivation, affective control and social behaviour . It can In the interest of supporting people to live well with dementia, it is important to explore opportunities for interventions that can increase quality of life and wellbeing in the wider community. Participative arts interventions  have become a growing area of interest due to their potential for positive outcomes for PLWD and carers , includiResearching art gallery and museum-based interventions for this population has suggested positive outcomes related to cognitive functioning . The research adhered to theTo help further situate the sample and act as a screening tool, the brief version of the mini mental state examination  that were part of the museum’s collection. Olfactory experiences in dementia care have been shown to be potentially engaging experiences that can evoke personal responses and reflection. Three sessions were chosen based on the design of a previous arts intervention study bySessions were guided by a protocol , providing an uninterrupted 360-degree recording of group interaction, were used to record the verbal and visual content of sessions. About the size of a tennis ball, this device is unobtrusive and did not appear to distract from the objects or group interaction. An additional audio recording device was also used as a backup. Sessions were led by two Wellcome Collection facilitators who were trained in working with people living with dementia. LD, PC and HZ observed all sessions unobtrusively from the back of the room but did not take part. A handout was provided after each session  and post-intervention (post-session three), session-by-session and baseline to post-intervention repeated measures pairwise t-tests were completed usingNVivo 12. FollowingAudio content from the entirety of the three object handling sessions was transcribed and subsequently coded using software packageQuality assurance.Data were gathered from four white British participants diagnosed with a dementia , all of M = 11.25;p = .037). However, although some pre-to-post changes approached significance, the majority of pre-to-post change scores were not significant. This is likely a Type II error due to the very small sample (N = 4) and with a larger sample size, may have been significant.Mean pre-post CWS scores for each of the five subscales  and composite scores of all subscales were calculated for each object handling session . The greatest subscale change score at session 1 was for the Well subscale, in session 2 the Interested subscale and at session 3 the Happy subsale. For the composite score, average change scores ranged from an increase of 30 points at session 1 to 15 at session 3 (out of a possible score of 500). These change scores are in the same direction as those reported byData were analysed using an inductive thematic approach to identify themes in order to address three research questions: Q1: What is the process of facilitation? Q2: What are the roles of material objects? Q3: What is the process of person-to-person interaction within the group? The final thematic map  and the Facilitating conditions created by the contributions of the facilitators, led to groupmembers actively participating in expressing themselves and interacting in a variety of ways,exploring objects from a range of perspectives. This led to a sense of group cohesion andgroup collaboration (including both participants and facilitators) in the further exploration of objects. Objects appeared to provide a shared focus, acting as a vehicle through which these processes took place.The thematic map displays the themes and subthemes identified within and across the group sessions. Arrows and lines depict the dynamic interactions between themes, with thicker lines representing a stronger relationship.Facilitatingguiding and pacing, validating and sharing.This theme relates to the process of facilitation during the object handing sessions and comprises three subthemes:Guiding and pacing reflects the direct guidance facilitators expressed to encourage exploration and learning. This included direct questions to participants to prompt or further exploration , relating to participants own experiences or knowledge , eliciting opinions or preferences  and instructions or changes in topic, which also appeared to help structure the sessions . This also included information provided by facilitators around objects  or to pace sessions by prompting further group discussion before sharing knowledge about an object .Validating describes the action taken by facilitators to acknowledge what participants have said, responding to and encouraging their contributions . This also included efforts to reach out directly to participants who may have been quieter in the session to support their participation, valuing all members of the group (F1: “Would you like to pass it on to [name] and see what he thinks”).Sharing referred to facilitators self-disclosing within the sessions, such as expressing not knowing , sharing personal information and their opinions around objects (F2: “It’s got a great fragrance.”) and using humour (F2: “So it wouldn’t have gin in it (laughs)?”).facilitating may have contributed to a sense of equal status in the group between facilitators and participants and enabled safe and respecting conditions for participation.Overall,Active participation. This theme relates to the contributions of participants within the object handing sessions and is made up of the subthemes:curiosity, connecting, voicing difference andsharing. Independence and the confidence to actively participate within the sessions may have been enabled by the conditions created by the process of facilitation.Curiosity relates to the participants’ stance in the sessions around the objects and associated group discussion in asking questions  and guessing and hypothesising . The latter formed the most prominent part of the sessions towards exploring and discovering more about objects. This may reflect the sense of safety created in the group that allowed participants to take risks and guess without the fear that may be associated with giving an incorrect response.Connecting describes the ways participants communicated with other participants and facilitators within the sessions. This was often in the form of asking questions, which may have been directed at other participants, facilitators or the wider group (P3: “Which country was that in then? Where was that?”), to clarify what had been said , or responding more generally. This is inclusive of all participants and their own personal patterns of communication. For example, one group member generally communicated using shorter phrases  than other participants, however the frequency of their contributions suggested they were engaged. Another group member more frequently took on the role of changing topic or moving the group on . This may reflect participants’ idiosyncratic personalities and the roles they take up in groups, or possibly the impact of dementia on communication skills.Voicing difference demonstrates participants’ ability to express differing opinions, disagree with and challenge others in the group . This occurred frequently during discussion as hypotheses were generated and appeared to be tolerated by and even drive the group in making further hypotheses.Sharing refers to participants self-disclosing within the session. This may relate to the sharing by facilitators, which may have been enabling for participants to feel able to share. Participants expressed not knowing (P2: “I don’t know what you’d use it for”), shared personal opinions (P1: “I think this is fantastic.”), personal stories and experiences  and humour (P3: “Get that out and hope it doesn’t mess up the rest of the stew”. (All laugh)).properties and features, meaning-making andassociations, refers to how objects were explored through discussion in the OH sessions by both participants and facilitators. The number of these reflects the “multifaceted” (P3) nature of many of the objects. The fact that many of the objects were items participants, and in some cases the facilitators, did not know much about, appeared to allow them to be explored from many different angels. As such, the objects may have acted as a vehicle for a wide range of interaction within the group.This theme, comprising three subthemes:Properties and features of objects refer to discussion about the physical objects themselves. For example, their weight or material , smell , and decorative features (P4: “It’s got a lovely pattern on the bottom”).Meaning-making describes how group members responded to and made sense of objects beyond their physical properties. There was much discussion around the potential identity and function of the objects  but I don’t think that’s what it was for”) and their age (P1: “I mean to me it looks 20th century”). This subtheme also included discussion around whether objects were authentic , their origin (P3: “It’s from China is it?”), and what they may represent .Associations captures the links that were made beyond the objects. This included likening objects to other things  and conversations that led on from the discussion of objects. For example, during a conversation about an iron fish, discussion led to the role of iron in diet  and when exploring a glass furniture leg protector . This also links to personal stories that were shared, for example when using spices in the session .Group collaboration This theme relates to the process of the group coming together as a whole within the object handing sessions and comprises the subthemes:sharing responsibility and process of discovery.Sharing responsibility describes the finding that both facilitators and group members came to share, as reflected within the themes offacilitating andactive participation. This participation of sharing stories, using humour and moving the group on as previously reported, as well as recapping what had been discussed (F1: “I think you said cinnamon.”) appeared to reflect a shared responsibility for group participation. This may have contributed to a sense of equal status in the group and group cohesion.Process of discovery reflects the process through which members built on each other’s ideas. This described the learning of new information , sharing different ideas and problem solving around objects  and the co-curation of a display case in the final session .exploring objects (including both participants and facilitators and in particular around identity and function) and codes relating tofacilitating andactive participation: asking questions (both facilitators and members), participants guessing and hypothesising, participants sharing opinions and preferences and facilitators providing information.Researchers  looked at the final frequency of each code across each of the three sessions as well as the identified themes, to explore whether any clear changes or patterns could be identified across the sessions. Some fluctuation in the frequency of codes was observed with the varying topics of conversation around different objects. However, no clear changes or patterns were found, suggesting the frequency of the codes and the overall themes were relatively stable across each of the three sessions. Across all three sessions, the most frequently recorded codes were those relating toPre-post CWS scores may suggest an overall increase in participants’ self-reported wellbeing after object handling sessions.guiding and pacing,validating andsharing to encourage participation), which may have empowered participants to haveactively participate in expressing themselves . This led togroup collaboration, between participants and facilitators,sharing responsibility for the group discussion and in building on each other’s ideas to come todiscover more about an object together. The objects appeared to provide a shared focus within the group through which these processes took place, demonstrated by the many perspectives from which they were explored . It is possible these processes impacted on participants’ experiences of feeling happy, well, interested, confident and optimistic, as reflected in the CWS.The identified themes generated from the verbal content of sessions suggest that wellbeing may have been increased through the process of facilitation , and also linked to anactive participation . These subthemes interacted to contribute to the theme ofgroup collaboration, illustrating a dynamic interaction of subthemes and themes in forming the in-the-moment processes within the group.active participation.The field notes and observations of the three researchers who were present across all sessions, revealed that these dynamic processes appeared to take place more quickly in sessions 2 and 3; this observation was confirmed after viewing the video data. This may have been linked to the familiarity of the setting and as the group, including the facilitators, became more adept at creating an atmosphere that promotedThat there were no clear changes found in the frequency of codes or in the identified themes across the three sessions may be a limitation of the methodology used, or a reflection of the high level of engagement across the sessions, perhaps due to the different objects used. It is also possible that benefits were limited to in-the-moment changes and were not maintained or built on across sessions due to the range of impairments associated with the types of dementia the participants in this study were living with. Future research could explore this further by focussing on a greater number of sessions to see effects over time.This study offers a tentative understanding of the processes through which group object handling sessions may promote wellbeing in people living with dementia. Although this was a small-scale exploratory study, it nevertheless highlights key components of sessions that can inform future training of facilitators to optimise sessions for this population. This also has important implications for the role of museums in public health , and socExpanding training beyond those who work in museum and heritage settings would raise awareness of dementia in the wider community. This could also enable people working in creative settings to adapt their ways of working to be more accessible for PLWD in line with The Prime Minister’s Challenge on Dementia . For exaThe small sample size can be viewed as both a strength and a limitation of the research. The participants were not representative of the demographic diversity of the population of PLWD across characteristics such as ethnicity, socioeconomic status and type and stage of dementia, thus limiting the applicability of the present study’s findings. Further, due to the sample size, the statistical analysis lacked power and the general lack of significant findings is possibly due to a Type II error. More longitudinal data collected over several months would be helpful to understand the ongoing effect of the sessions on subjective wellbeing. Input from care partners  about their observations would further illuminate if the sessions were impactful in day-to-day life.active participation and its implications for understanding the agency of people with dementia  to help explain the possible processes present in the facilitation of sessions, the roles of material objects, and person-to-person interactions. Facilitators’ guidance created conditions within the group that led to group members to actively participate in expressing themselves (including voicing different opinions), leading to group cohesion and collaboration between participants and facilitators in sharing responsibility for the group and building on each other’s ideas to discover more about objects. Objects were explored from many different perspectives and provided a shared focus within the group through which these processes took place. These findings should be viewed tentatively due to the small sample size; however, they offer important insights concerning how to optimise sessions for this population. Future research using multiple groups and a more diverse sample can extend the present study’s findings but perhaps more importantly, other qualitative methodologies may provide additional information about the narrative and discourse that occurs in museum object handling groups. And finally, as a freely available measure that is non-obtrusive to use and easy to score, the Canterbury Wellbeing Scales, should be considered in community-based programmes for early to middles stages of different dementias.This was the first study to explore the process of facilitated small group object handling sessions involving people living with a dementia, in a museum setting, across multiple sessions. Findings suggest a positive influence of object handling on the subjective wellbeing in people living with dementia, and identified four key themes  describing the nature of their interest and their qualifications, the intended use of the data, plans for obtaining ethical approval at their respective institution, and signing a confidentiality agreement to assure protection of data whilst in their possession. When ethical approval has been provided as evidenced by a signed letter from the ethics panel, transcript data will be transferred via an encrypted and password protected file. Access to transcripts is only permitted by the requesting researcher(s) and cannot be shared with others.Quantitative data. Zenodo: Subjective wellbeing in people living with dementia: exploring processes of multiple object handling sessions in a museum setting underlying quantitative data.http://doi.org/10.5281/zenodo.4715016 - Excerpt from a coded transcript.pdf- Initial & Final codebooks.pdf .Data are available under the terms of the I appreciate Dr. Camic et al. responding so thoroughly to my inquiries. This is a well written study, although small, that contributes significantly to the area of use of museums in promoting quality of life for persons with dementia.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Dementia, palliative care, museum studiesI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This is a small mixed method study looking at the effect of object handling for persons living with dementia. It constituted four individuals diagnosed with dementia, participating in three exploratory sessions of one hour each. Sessions were led by a facilitator, audio and video recorded. The Canterburry Wellbing Scales were used pre and post experience.  A thematic map was developed from qualitative analysis of the sessions. Comments: First of all I would like to thank the authors for their contribution on expanding the effects of museum based interventions for persons with dementia. Current literature and the framework in which this study sits were well presented in the paper. My comments are mostly addressing clarifications. I would like to know more details regarding the process itself. How many objects were presented per session? How were they chosen? Page 6 what is the purpose behind “novel, diverse”, etc. Was one object discussed at a time among all participants as it was passed around? I even wonder how close participants were sitting to each other and the facilitator? (Pre Covid I am assuming). I would like to know more about the questions asked. Was there a process or script in which the questions were created? The second session with smell adds a little complexity, why were smells chosen? Was there also a tactile component such as touching the cinnamon sticks? A table with a list of the objects and attributes might be helpful. There is little data, to my knowledge, about how persons with dementia respond to the types of artwork presented in museum programs for dementia/care partners 9abstract, sculpture, everyday objects in new settings etc).  It appears this study could contribute to that literature with more description of participants specific reactions? I would like to see a little more written about the “museum setting”.  A conference room was used within the museum. Is there an aspect of one coming to a museum, a special place, that is specifically helpful to this process? Was it the use of museum objects that were special? I wasn’t sure if there was a connection to current displays that might tie in with which objects were presented? Minor comments: P4 What was the intervention in the Ander study? P4 Paddon, what objects were used? P4 The questions at the end of the paragraph referencing Keady seem confusing. Is the inference meant to be that these questions are less relevant? P5 Stakeholder involvement – not sure this paragraph is needed. With the thematic map I wondering about the overlap/connection with socialization and group collaboration as socialization tends to be a key factors in activities such as yours. Facilitators sharing, sharing responsibility and participants sharing, are these components of socialization? This is touched upon in summary of findings “shared focus” but again is this at least in part socialization? P14 discussion of the olfactory experience. I wonder if this and description of the objects could be earlier in the paper – methods. With the pandemic occurring and impacting in person gatherings and in person events for persons with dementia (even post Covid) I wonder if it would be helpful to postulate how this process may be done via video with participants having the same object in hand to discuss? Thank you for allowing to review this interesting paper.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Dementia, palliative care, museum studiesI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Responding to Reviewer 2, Dr. Lee Burnside  Thank you for your helpful and insightful comments and for your close read of our paper. Our responses to each of your comments follow. 1.    I would like to know more details regarding the process itself. How many objects were presented per session? How were they chosen? Page 6 what is the purpose behind “novel, diverse”, etc. Was one object discussed at a time among all participants as it was passed around? I even wonder how close participants were sitting to each other and the facilitator? (Pre Covid I am assuming). I would like to know more about the questions asked. Was there a process or script in which the questions were created? Response: Thank you for these comments and questions. We have added to and revised the following (subsection Object handling sessions): “Facilitators welcomed participants at the beginning of each session passing around one object at a time and encouraged touching and generating discussion through asking a range of sample prompts and questions to encourage participation and exploration before sharing information about each object. Questions were used flexibly, in an open dialogic way, within the sessions based on the interaction of participants. A total of 20 objects were used across the sessions; 14 objects came from the museum’s collections including 4 spices, while others were contributed by PC. Six to nine objects were used in each session. Objects were jointly decided upon by the facilitators and researchers and selected to be novel and diverse in their cultural, historical and sensory qualities in order to maximize variety, interest and opportunities for exploration that did not rely on previous memories or knowledge. The study’s protocol, including sample questions, photos and descriptions of the objects used in each session are available as extended open access files .2.    The second session with smell adds a little complexity, why were smells chosen? Was there also a tactile component such as touching the cinnamon sticks? A table with a list of the objects and attributes might be helpful. esponse: We chose to include smells based on (a) previous research about olfactory responses in dementia care , and (b) the museum had a collection of spices that were frequently refreshed and part of the interactive visitor experience to relevant collections. The museum facilitators and researchers agreed that this would both highlight objects in the collection and provide an olfactory and tactile experience. Spices were contained in small glass bottles and passed around, one at a time, as were the other objects. This generated a good deal of discussion about how these spices might be used .  R In the subsection, Subjective wellbeing  (in the Discussion section), we have added the following: “Olfactory experiences in dementia care  have generally revealed positive responses across different stimuli  and should be considered when planning object handling sessions.”3.    There is little data, to my knowledge, about how persons with dementia respond to the types of artwork presented in museum programs for dementia/care partners . It appears this study could contribute to that literature with more description of participants specific reactions? Response: We definitely agree, there is a paucity of research that looks specifically at how people with dementia respond to different types of art works. The Meet Me at MoMA programme was the first study that we are aware of that looked at figurative and abstract modern art, and although not designed as a comparative study, did find abstract art often produced more engaged and longer discussions.      A cross-sectional observational study, undertaken in The Netherlands, addresses this issue: Hendriks, I., Meiland, F., Slotwinska, K., Kroeze, R., Weinstein, H., Gerritsen, D., & Dröes, R. (2019). How do people with dementia respond to different types of art? An explorative study into interactive museum programs. International Psychogeriatrics, 31(6), 857-868. doi:10.1017/S1041610218001266. An earlier, smaller scale, mixed-methods study in the UK, also looked at art works in two different museums , Camic, P. M., Tischler, V. and Pearman, C. H. (2014). Viewing and making art together: a multi-session art-gallery-based intervention for people with dementia and their carers. Aging & Mental Health, 18, 161–168. doi: 10.1080/ 13607863.2013.818101, and there may be others.      Because the present study used different types of material objects, most of which would not be considered “art”, we are not confident that describing individual responses in more detail than we have already done, would make a meaningful contribution. However, future researchers could access our audio and video files (access instructions are provided at the end of the article) and explore this interesting issue.4.    I would like to see a little more written about the “museum setting”. A conference room was used within the museum. Is there an aspect of one coming to a museum, a special place, that is specifically helpful to this process? Was it the use of museum objects that were special? I wasn’t sure if there was a connection to current displays that might tie in with which objects were presented? Response: In the Methods section (Design and setting subsection) we have made clear this is a “specialist” museum and added the following: “The museum’s focus is to connect science, medicine, health and the arts and this is reflected in its collections, special exhibitions, public engagement, and lecture programme. The object handing sessions took place in a well-lit, comfortable conference room with windows on a high floor. Facilitators and participants were seated around a rectangular table and close enough to allow for the easy passing of objects. The physical environment of the room was considered and the decision to hold the sessions in a private space rather than a gallery room was made to allow quiet for listening, fewer distractions to support engagement and confidentiality for audio recording.”  5.    P4 What was the intervention in the Ander study?Response: They used museum objects from London and Oxford museums in inpatient neurological rehabilitation (London), outpatient neurological rehabilitation (Oxford), and inpatient mental health care (Reading). Objects were brought to the respective location and occupational therapy clinicians engaged one-on-one with participants. Forty-two museum objects comprising specimens from anthropology, archaeology, art, geology and zoology collections were compiled and brought in boxes, each box contained 6 different objects. 6.    P4 Paddon, what objects were used?Response:  Objects comprised archaeological and ethnographic artefacts, etchings and printing plates, fossils, mineral samples and zoology specimens that varied in their tactile, visual and kinaesthetic properties .7.    P4 The questions at the end of the paragraph referencing Keady seem confusing. Is the inference meant to be that these questions are less relevant? Response: Apologies if we were not clear. We meant to imply that these questions are more relevant and have added the following (underlined): “More relevant questions for a dementia population include, How long do these moments last? What comes before and after them? Can different moments be linked together? .”8.    P5 Stakeholder involvement – not sure this paragraph is needed. Response: We feel it is relevant for the following reasons: Very few psychometric questionnaires have involved people with dementia in their development. Most often, researchers decide which questions are the most important to ask with the assumption that they know best. The Canterbury Wellbeing Scales (CWS) were developed with direct input from people with dementia and caregivers, along with older adult specialist psychologists and Alzheimer’s Society staff. Secondly, the length and structure of the sessions in the present study were based on feedback from participants (people living with a dementia and caregivers) in previous studies, consulting with members of the Rare Dementia Support charity (https://www.raredementiasupport.org/) and talking to museum staff at multiple musuems. In addition to these reasons, we concur with the (UK) Health Research Authority that states, “Public involvement in research means research that is done ‘with’ or ‘by’ the public, not 'to', 'about' or 'for' them. It means that patients or other people with relevant experience contribute to how research is designed, conducted and disseminated” (https://www.hra.nhs.uk/planning-and-improving-research/best-practice/public-involvement/ ). 9.    With the thematic map I wondering about the overlap/connection with socialization and group collaboration as socialization tends to be a key factors in activities such as yours. Facilitators sharing, sharing responsibility and participants sharing, are these components of socialization? This is touched upon in summary of findings “shared focus” but again is this at least in part socialization? Response: This is an insightful comment, and we agree socialisation is potentially part of what is described in the thematic map in that the sessions were formed by people socially interacting with others around a specific task and may have adapted to behave in a way acceptable to the group. How to socialise in a group setting is something most people likely learn in school at a young age. However, the thematic map describes and attempts to tease apart some of the processes associated with what is specific to object handling, and socialisation, for us, is an overly broad construct that would need to be further broken down and measured , in our opinion, to be useful. Considering that two people had a type of dementia that can make socialising quite challenging (FTD), the responses and interactions of participants across the three sessions provide support that object handling—as a focal activity—has some potential in dementia care.10.    P14 discussion of the olfactory experience. I wonder if this and description of the objects could be earlier in the paper – methods. Response: Thank you for this suggestion. We have added the following sentence to the Methods section in the Object handing sessions, subsection: “The second session also involved smelling four spices contained in small glass bottles (passed one at a time to participants) that were part of the museum’s collection. Olfactory experiences in dementia care have been shown to be potentially engaging experiences that can evoke personal responses an reflection.”11.    With the pandemic occurring and impacting in person gatherings and in person events for persons with dementia (even post Covid) I wonder if it would be helpful to postulate how this process may be done via video with participants having the same object in hand to discuss? Response: Thank you for this. The first author is currently engaged with other colleagues as part of a funded study which examines personal objects within the context of on-line support group sessions with dementia caregivers who are caring for someone with a rare form of dementia. We feel the information we have to date, while promising, requires additional development. The study reported here was completed before the pandemic but it is interesting to ponder, however, about how object handling sessions may be done via video. It is could be important to explore this as a way to increase accessibility, but this would forgo important aspects of the museum setting .         Facilitators could video from a carefully selected space in the museum (interesting but not too busy background that conveys the presence of a museum), orienting people to the room they can see on the screen and to each other, allowing for pauses after each person speaks for any delays and some time at the start for any technical issues to be problem solved. Facilitators might consider presenting visually some objects and generate discussion  but may limit who can participate (sight/hearing/staying focused when on a screen and other things going on in the setting they are joining from) and may require the support of someone at home to navigate the technology. Participants could share objects from home or all be asked to bring something such as a spice so all have the same object to touch/smell/discuss. Items could be posted out beforehand but unsure how this would work in practice. Just some thoughts. The article “Subjective wellbeing in people living with dementia: exploring processes of multiple object handling sessions in a museum setting. Version 1” describes a study that expands on previous research and explores the potential processes underlying the effect of multiple small group sessions on handling objects, carried out in a museum. A mixed design was used to assess the subjective wellbeing of participants with dementia, using a quantitative self-report measurement, the Canterbury Wellbeing Scales (CWS). A thematic analysis of the transcripts of the previously recorded sessions and the observers' field-notes was also carried out. The article begins with a good contextualization of the research on the role of art-based interventions to improve the quality of life of people with dementia. The most relevant and recent research on the subject is mentioned and, after commenting on it, the basic hypothesis of the study is presented: that the sessions of handling objects in the museum will increase the wellbeing of participants with dementia. The method section describes the aspects relating both to the quantitative measures used and the qualitative approaches. The results of this study indicate an average increase in participants' wellbeing when comparing the measures taken before the sessions with those carried out afterwards, although this increase does not reach statistical significance. The topics and sub-topics identified through the analysis of the session transcriptions and the field-notes suggest that wellbeing may have increased through facilitation processes that could have empowered the participants by facilitating their self-expression. Among the most important aspects of the work, its authors highlight that it provides a greater understanding of the processes through which handling object activities in a museum can promote the wellbeing of people with dementia. The main limitation of the work, also acknowledged by the authors, is the small sample size of people with dementia participating in this study, four people, which considerably reduces the applicability possibilities of the study's findings. It is true that the small sample size allows an in-depth analysis of issues that would not be possible with a larger sample. Still, given the social relevance of the issue of caring for and improving the quality of life of people with dementia, the study lacks more people, which would provide the authors' findings with a more solid basis. Despite this limitation, the adequate conceptual and methodological basis of the work inclines me to recommend its publication. Subsequent studies may refer to this research and use a larger sample of participants with dementia.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Art Education for people with dementiaI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. possibly due to other circumstances. These include the required commitment to come to three sessions, difficulty arranging transport from home to the museum in a busy part of central London, and any uncertainty people may have had about coming to a new place (the museum). The study also coincided with a dramatic reduction of funding for a charity we had partnered with. They had to close their local day and drop in facility, which was a close walk from the museum.We would like to thank Dr Anna Ullán for taking the time to review our manuscript. We appreciate her comments and agree, a larger number of participants would have strengthened the study. Unfortunately, recruitment was challenging, perhaps due to the time of year (winter) but