The antimicrobial polymer PHMB enters cells and selectively condenses bacterial chromosomes

To combat infection and antimicrobial resistance, it is helpful to elucidate drug mechanism(s) of action. Here we examined how the widely used antimicrobial polyhexamethylene biguanide (PHMB) kills bacteria selectively over host cells. Contrary to the accepted model of microbial membrane disruption by PHMB, we observed cell entry into a range of bacterial species, and treated bacteria displayed cell division arrest and chromosome condensation, suggesting DNA binding as an alternative antimicrobial mechanism. A DNA-level mechanism was confirmed by observations that PHMB formed nanoparticles when mixed with isolated bacterial chromosomal DNA and its effects on growth were suppressed by pairwise combination with the DNA binding ligand Hoechst 33258. PHMB also entered mammalian cells, but was trapped within endosomes and excluded from nuclei. Therefore, PHMB displays differential access to bacterial and mammalian cellular DNA and selectively binds and condenses bacterial chromosomes. Because acquired resistance to PHMB has not been reported, selective chromosome condensation provides an unanticipated paradigm for antimicrobial action that may not succumb to resistance

Determinants of recovery from post-COVID-19 dyspnoea: analysis of UK prospective cohorts of hospitalised COVID-19 patients and community-based controls

BACKGROUND: The risk factors for recovery from COVID-19 dyspnoea are poorly understood. We investigated determinants of recovery from dyspnoea in adults with COVID-19 and compared these to determinants of recovery from non-COVID-19 dyspnoea. METHODS: We used data from two prospective cohort studies: PHOSP-COVID (patients hospitalised between March 2020 and April 2021 with COVID-19) and COVIDENCE UK (community cohort studied over the same time period). PHOSP-COVID data were collected during hospitalisation and at 5-month and 1-year follow-up visits. COVIDENCE UK data were obtained through baseline and monthly online questionnaires. Dyspnoea was measured in both cohorts with the Medical Research Council Dyspnoea Scale. We used multivariable logistic regression to identify determinants associated with a reduction in dyspnoea between 5-month and 1-year follow-up. FINDINGS: We included 990 PHOSP-COVID and 3309 COVIDENCE UK participants. We observed higher odds of improvement between 5-month and 1-year follow-up among PHOSP-COVID participants who were younger (odds ratio 1.02 per year, 95% CI 1.01–1.03), male (1.54, 1.16–2.04), neither obese nor severely obese (1.82, 1.06–3.13 and 4.19, 2.14–8.19, respectively), had no pre-existing anxiety or depression (1.56, 1.09–2.22) or cardiovascular disease (1.33, 1.00–1.79), and shorter hospital admission (1.01 per day, 1.00–1.02). Similar associations were found in those recovering from non-COVID-19 dyspnoea, excluding age (and length of hospital admission). INTERPRETATION: Factors associated with dyspnoea recovery at 1-year post-discharge among patients hospitalised with COVID-19 were similar to those among community controls without COVID-19. FUNDING: PHOSP-COVID is supported by a grant from the MRC-UK Research and Innovation and the Department of Health and Social Care through the National Institute for Health Research (NIHR) rapid response panel to tackle COVID-19. The views expressed in the publication are those of the author(s) and not necessarily those of the National Health Service (NHS), the NIHR or the Department of Health and Social Care. COVIDENCE UK is supported by the UK Research and Innovation, the National Institute for Health Research, and Barts Charity. The views expressed are those of the authors and not necessarily those of the funders

Clinical characteristics with inflammation profiling of long COVID and association with 1-year recovery following hospitalisation in the UK: a prospective observational study

Copyright © 2022 The Author(s). Background: No effective pharmacological or non-pharmacological interventions exist for patients with long COVID. We aimed to describe recovery 1 year after hospital discharge for COVID-19, identify factors associated with patient-perceived recovery, and identify potential therapeutic targets by describing the underlying inflammatory profiles of the previously described recovery clusters at 5 months after hospital discharge. Methods: The Post-hospitalisation COVID-19 study (PHOSP-COVID) is a prospective, longitudinal cohort study recruiting adults (aged ≥18 years) discharged from hospital with COVID-19 across the UK. Recovery was assessed using patient-reported outcome measures, physical performance, and organ function at 5 months and 1 year after hospital discharge, and stratified by both patient-perceived recovery and recovery cluster. Hierarchical logistic regression modelling was performed for patient-perceived recovery at 1 year. Cluster analysis was done using the clustering large applications k-medoids approach using clinical outcomes at 5 months. Inflammatory protein profiling was analysed from plasma at the 5-month visit. This study is registered on the ISRCTN Registry, ISRCTN10980107, and recruitment is ongoing. Findings: 2320 participants discharged from hospital between March 7, 2020, and April 18, 2021, were assessed at 5 months after discharge and 807 (32·7%) participants completed both the 5-month and 1-year visits. 279 (35·6%) of these 807 patients were women and 505 (64·4%) were men, with a mean age of 58·7 (SD 12·5) years, and 224 (27·8%) had received invasive mechanical ventilation (WHO class 7–9). The proportion of patients reporting full recovery was unchanged between 5 months (501 [25·5%] of 1965) and 1 year (232 [28·9%] of 804). Factors associated with being less likely to report full recovery at 1 year were female sex (odds ratio 0·68 [95% CI 0·46–0·99]), obesity (0·50 [0·34–0·74]) and invasive mechanical ventilation (0·42 [0·23–0·76]). Cluster analysis (n=1636) corroborated the previously reported four clusters: very severe, severe, moderate with cognitive impairment, and mild, relating to the severity of physical health, mental health, and cognitive impairment at 5 months. We found increased inflammatory mediators of tissue damage and repair in both the very severe and the moderate with cognitive impairment clusters compared with the mild cluster, including IL-6 concentration, which was increased in both comparisons (n=626 participants). We found a substantial deficit in median EQ-5D-5L utility index from before COVID-19 (retrospective assessment; 0·88 [IQR 0·74–1·00]), at 5 months (0·74 [0·64–0·88]) to 1 year (0·75 [0·62–0·88]), with minimal improvements across all outcome measures at 1 year after discharge in the whole cohort and within each of the four clusters. Interpretation: The sequelae of a hospital admission with COVID-19 were substantial 1 year after discharge across a range of health domains, with the minority in our cohort feeling fully recovered. Patient-perceived health-related quality of life was reduced at 1 year compared with before hospital admission. Systematic inflammation and obesity are potential treatable traits that warrant further investigation in clinical trials. Funding: UK Research and Innovation and National Institute for Health Research.UK Research and Innovation and National Institute for Health Researc

Determinants of recovery from post-COVID-19 dyspnoea: analysis of UK prospective cohorts of hospitalised COVID-19 patients and community-based controls

Background The risk factors for recovery from COVID-19 dyspnoea are poorly understood. We investigated determinants of recovery from dyspnoea in adults with COVID-19 and compared these to determinants of recovery from non-COVID-19 dyspnoea. Methods We used data from two prospective cohort studies: PHOSP-COVID (patients hospitalised between March 2020 and April 2021 with COVID-19) and COVIDENCE UK (community cohort studied over the same time period). PHOSP-COVID data were collected during hospitalisation and at 5-month and 1-year follow-up visits. COVIDENCE UK data were obtained through baseline and monthly online questionnaires. Dyspnoea was measured in both cohorts with the Medical Research Council Dyspnoea Scale. We used multivariable logistic regression to identify determinants associated with a reduction in dyspnoea between 5-month and 1-year follow-up. Findings We included 990 PHOSP-COVID and 3309 COVIDENCE UK participants. We observed higher odds of improvement between 5-month and 1-year follow-up among PHOSP-COVID participants who were younger (odds ratio 1.02 per year, 95% CI 1.01–1.03), male (1.54, 1.16–2.04), neither obese nor severely obese (1.82, 1.06–3.13 and 4.19, 2.14–8.19, respectively), had no pre-existing anxiety or depression (1.56, 1.09–2.22) or cardiovascular disease (1.33, 1.00–1.79), and shorter hospital admission (1.01 per day, 1.00–1.02). Similar associations were found in those recovering from non-COVID-19 dyspnoea, excluding age (and length of hospital admission). Interpretation Factors associated with dyspnoea recovery at 1-year post-discharge among patients hospitalised with COVID-19 were similar to those among community controls without COVID-19. Funding PHOSP-COVID is supported by a grant from the MRC-UK Research and Innovation and the Department of Health and Social Care through the National Institute for Health Research (NIHR) rapid response panel to tackle COVID-19. The views expressed in the publication are those of the author(s) and not necessarily those of the National Health Service (NHS), the NIHR or the Department of Health and Social Care. COVIDENCE UK is supported by the UK Research and Innovation, the National Institute for Health Research, and Barts Charity. The views expressed are those of the authors and not necessarily those of the funders

Long COVID research: an update from the PHOSP-COVID Scientific Summit

The severity of acute SARS-CoV-2 infection has decreased with the introduction of public health policies, vaccination, improved management of acute disease, and a degree of protective immunity in those who have survived past infection. However, in the wake of the pandemic, post-acute sequelae of COVID-19—referred to as long COVID—have emerged. The UK National Institute for Health and Care Excellence (NICE) describes long COVID as a condition in which signs and symptoms continue or develop after acute COVID-19 (>4 weeks), including ongoing symptomatic COVID-19 and post-COVID-19 syndrome (≥12 weeks).PHOSP-COVID is jointly funded by a grant from the Medical Research Council UK Research and Innovation and the Department of Health and Social Care through the National Institute for Health Research (NIHR) rapid response panel to tackle COVID-19 (MR/V027859/1 and COV0319)

Accelerated immune ageing is associated with COVID-19 disease severity

Availability of data and materials: All data generated or analysed during this study are included in this published article and its supplementary information files.Supplementary Information is available online at: https://immunityageing.biomedcentral.com/articles/10.1186/s12979-023-00406-z#Sec23 .Background: The striking increase in COVID-19 severity in older adults provides a clear example of immunesenescence, the age-related remodelling of the immune system. To better characterise the association between convalescent immunesenescence and acute disease severity, we determined the immune phenotype of COVID-19 survivors and non-infected controls. Results: We performed detailed immune phenotyping of peripheral blood mononuclear cells isolated from 103 COVID-19 survivors 3–5 months post recovery who were classified as having had severe (n = 56; age 53.12 ± 11.30 years), moderate (n = 32; age 52.28 ± 11.43 years) or mild (n = 15; age 49.67 ± 7.30 years) disease and compared with age and sex-matched healthy adults (n = 59; age 50.49 ± 10.68 years). We assessed a broad range of immune cell phenotypes to generate a composite score, IMM-AGE, to determine the degree of immune senescence. We found increased immunesenescence features in severe COVID-19 survivors compared to controls including: a reduced frequency and number of naïve CD4 and CD8 T cells (p < 0.0001); increased frequency of EMRA CD4 (p < 0.003) and CD8 T cells (p < 0.001); a higher frequency (p < 0.0001) and absolute numbers (p < 0.001) of CD28−ve CD57+ve senescent CD4 and CD8 T cells; higher frequency (p < 0.003) and absolute numbers (p < 0.02) of PD-1 expressing exhausted CD8 T cells; a two-fold increase in Th17 polarisation (p < 0.0001); higher frequency of memory B cells (p < 0.001) and increased frequency (p < 0.0001) and numbers (p < 0.001) of CD57+ve senescent NK cells. As a result, the IMM-AGE score was significantly higher in severe COVID-19 survivors than in controls (p < 0.001). Few differences were seen for those with moderate disease and none for mild disease. Regression analysis revealed the only pre-existing variable influencing the IMM-AGE score was South Asian ethnicity (β = 0.174, p = 0.043), with a major influence being disease severity (β = 0.188, p = 0.01). Conclusions: Our analyses reveal a state of enhanced immune ageing in survivors of severe COVID-19 and suggest this could be related to SARS-Cov-2 infection. Our data support the rationale for trials of anti-immune ageing interventions for improving clinical outcomes in these patients with severe disease.Medical Research Council supported UK Coronavirus Immunology Consortium and the National Institute for Health Research (NIHR) supported PHOSP-COVID Collaborative study

Large-scale phenotyping of patients with long COVID post-hospitalization reveals mechanistic subtypes of disease

Data availability: This is an open access article under the CC BY 4.0 license. The PHOSP-COVID protocol, consent form, definition and derivation of clinical characteristics and outcomes, training materials, regulatory documents, information about requests for data access, and other relevant study materials are available online at ref. 76. Access to these materials can be granted by contacting [email protected] and [email protected]. The ISARIC4C protocol, data sharing and publication policy are available at https://isaric4c.net. ISARIC4C’s Independent Data and Material Access Committee welcomes applications for access to data and materials (https://isaric4c.net). The datasets used in the study contain extensive clinical information at an individual level that prevent them from being deposited in an public depository due to data protection policies of the study. Study data can only be accessed via the ODAP, a protected research environment. All data used in this study are available within ODAP and accessible under reasonable request. Data access criteria and information about how to request access is available online at ref. 76. If criteria are met and a request is made, access can be gained by signing the eDRIS user agreement.Code availability: Code was written within the ODAP, using R v4.2.0 and publicly available packages (‘data.table v1.14.2’, ‘EnvStats v2.7.0’, ‘tidyverse v1.3.2’, ‘lme4 v1.1-32’, ‘caret v6.0-93’, ‘glmnet v4.1-6’, ‘mdatools v0.14.0’, ‘ggpubbr v0.4.0’, ‘ggplot2 v3.3.6’, ‘bootnet v1.5.6’ and ‘qgraph v1.9.8’ packages). No new algorithms or functions were created and code used in-built functions in listed packages available on CRAN. The code used to generate data and to analyze data is publicly available at https://github.com/isaric4c/wiki/wiki/ISARIC; https://github.com/SurgicalInformatics/cocin_cc and https://github.com/ClaudiaEfstath/PHOSP_Olink_NatImm.One in ten severe acute respiratory syndrome coronavirus 2 infections result in prolonged symptoms termed long coronavirus disease (COVID), yet disease phenotypes and mechanisms are poorly understood1. Here we profiled 368 plasma proteins in 657 participants ≥3 months following hospitalization. Of these, 426 had at least one long COVID symptom and 233 had fully recovered. Elevated markers of myeloid inflammation and complement activation were associated with long COVID. IL-1R2, MATN2 and COLEC12 were associated with cardiorespiratory symptoms, fatigue and anxiety/depression; MATN2, CSF3 and C1QA were elevated in gastrointestinal symptoms and C1QA was elevated in cognitive impairment. Additional markers of alterations in nerve tissue repair (SPON-1 and NFASC) were elevated in those with cognitive impairment and SCG3, suggestive of brain–gut axis disturbance, was elevated in gastrointestinal symptoms. Severe acute respiratory syndrome coronavirus 2-specific immunoglobulin G (IgG) was persistently elevated in some individuals with long COVID, but virus was not detected in sputum. Analysis of inflammatory markers in nasal fluids showed no association with symptoms. Our study aimed to understand inflammatory processes that underlie long COVID and was not designed for biomarker discovery. Our findings suggest that specific inflammatory pathways related to tissue damage are implicated in subtypes of long COVID, which might be targeted in future therapeutic trials.This research used data assets made available by ODAP as part of the Data and Connectivity National Core Study, led by Health Data Research UK in partnership with the Office for National Statistics and funded by UK Research and Innovation (grant ref. MC_PC_20058). This work is supported by the following grants: the PHOSP-COVD study is jointly funded by UK Research and Innovation and National Institute of Health and Care Research (NIHR; grant references MR/V027859/1 and COV0319). ISARIC4C is supported by grants from the National Institute for Health and Care Research (award CO-CIN-01) and the MRC (grant MC_PC_19059) Liverpool Experimental Cancer Medicine Centre provided infrastructure support for this research (grant reference C18616/A25153). Other grants that have supported this work include the UK Coronavirus Immunology Consortium (funder reference 1257927), the Imperial Biomedical Research Centre (NIHR Imperial BRC, grant IS-BRC-1215-20013), the Health Protection Research Unit in Respiratory Infections at Imperial College London and NIHR Health Protection Research Unit in Emerging and Zoonotic Infections at University of Liverpool, both in partnership with Public Health England, (NIHR award 200907), Wellcome Trust and Department for International Development (215091/Z/18/Z), Health Data Research UK (grant code 2021.0155), MRC (grant code MC_UU_12014/12) and NIHR Clinical Research Network for providing infrastructure support for this research. We also acknowledge the support of the MRC EMINENT Network (MR/R502121/1), which is cofunded by GSK, the Comprehensive Local Research Networks, the MRC HIC-Vac network (MR/R005982/1) and the RSV Consortium in Europe Horizon 2020 Framework Grant 116019. F.L. is supported by an MRC clinical training fellowship (award MR/W000970/1). C.E. is funded by NIHR (grant P91258-4). L.-P.H. is supported by Oxford NIHR Biomedical Research Centre. A.A.R.T. is supported by a British Heart Foundation (BHF) Intermediate Clinical Fellowship (FS/18/13/33281). S.L.R.-J. receives support from UK Research and Innovation (UKRI), Global Challenges Research Fund (GCRF), Rosetrees Trust, British HIV association (BHIVA), European & Developing Countries Clinical Trials Partnership (EDCTP) and Globvac. J.D.C. has grants from AstraZeneca, Boehringer Ingelheim, GSK, Gilead Sciences, Grifols, Novartis and Insmed. R.A.E. holds a NIHR Clinician Scientist Fellowship (CS-2016-16-020). A. Horsley is currently supported by UK Research and Innovation, NIHR and NIHR Manchester BRC. B.R. receives support from BHF Oxford Centre of Research Excellence, NIHR Oxford BRC and MRC. D.G.W. is supported by an NIHR Advanced Fellowship. A. Ho has received support from MRC and for the Coronavirus Immunology Consortium (MR/V028448/1). L.T. is supported by the US Food and Drug Administration Medical Countermeasures Initiative contract 75F40120C00085 and the National Institute for Health Research Health Protection Research Unit in Emerging and Zoonotic Infections (NIHR200907) at the University of Liverpool in partnership with UK Health Security Agency (UK-HSA), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford. L.V.W. has received support from UKRI, GSK/Asthma and Lung UK and NIHR for this study. M.G.S. has received support from NIHR UK, MRC UK and Health Protection Research Unit in Emerging and Zoonotic Infections, University of Liverpool. J.K.B. is supported by the Wellcome Trust (223164/Z/21/Z) and UKRI (MC_PC_20004, MC_PC_19025, MC_PC_1905, MRNO2995X/1 and MC_PC_20029). The funders were not involved in the study design, interpretation of data or writing of this manuscript. The views expressed are those of the authors and not necessarily those of the Department of Health and Social Care (DHSC), the Department for International Development (DID), NIHR, MRC, the Wellcome Trust, UK-HSA, the National Health Service or the Department of Health. P.J.M.O. is supported by a NIHR Senior Investigator Award (award 201385). We thank all the participants and their families. We thank the many research administrators, health-care and social-care professionals who contributed to setting up and delivering the PHOSP-COVID study at all of the 65 NHS trusts/health boards and 25 research institutions across the United Kingdom, as well as those who contributed to setting up and delivering the ISARIC4C study at 305 NHS trusts/health boards. We also thank all the supporting staff at the NIHR Clinical Research Network, Health Research Authority, Research Ethics Committee, Department of Health and Social Care, Public Health Scotland and Public Health England. We thank K. Holmes at the NIHR Office for Clinical Research Infrastructure for her support in coordinating the charities group. The PHOSP-COVID industry framework was formed to provide advice and support in commercial discussions, and we thank the Association of the British Pharmaceutical Industry as well the NIHR Office for Clinical Research Infrastructure for coordinating this. We are very grateful to all the charities that have provided insight to the study: Action Pulmonary Fibrosis, Alzheimer’s Research UK, Asthma and Lung UK, British Heart Foundation, Diabetes UK, Cystic Fibrosis Trust, Kidney Research UK, MQ Mental Health, Muscular Dystrophy UK, Stroke Association Blood Cancer UK, McPin Foundations and Versus Arthritis. We thank the NIHR Leicester Biomedical Research Centre patient and public involvement group and Long Covid Support. We also thank G. Khandaker and D. C. Newcomb who provided valuable feedback on this work. Extended Data Fig. 10 was created using Biorender

Prehospital transdermal glyceryl trinitrate in patients with ultra-acute presumed stroke (RIGHT-2): an ambulance-based, randomised, sham-controlled, blinded, phase 3 trial

Background High blood pressure is common in acute stroke and is a predictor of poor outcome; however, large trials of lowering blood pressure have given variable results, and the management of high blood pressure in ultra-acute stroke remains unclear. We investigated whether transdermal glyceryl trinitrate (GTN; also known as nitroglycerin), a nitric oxide donor, might improve outcome when administered very early after stroke onset. Methods We did a multicentre, paramedic-delivered, ambulance-based, prospective, randomised, sham-controlled, blinded-endpoint, phase 3 trial in adults with presumed stroke within 4 h of onset, face-arm-speech-time score of 2 or 3, and systolic blood pressure 120 mm Hg or higher. Participants were randomly assigned (1:1) to receive transdermal GTN (5 mg once daily for 4 days; the GTN group) or a similar sham dressing (the sham group) in UK based ambulances by paramedics, with treatment continued in hospital. Paramedics were unmasked to treatment, whereas participants were masked. The primary outcome was the 7-level modified Rankin Scale (mRS; a measure of functional outcome) at 90 days, assessed by central telephone follow-up with masking to treatment. Analysis was hierarchical, first in participants with a confirmed stroke or transient ischaemic attack (cohort 1), and then in all participants who were randomly assigned (intention to treat, cohort 2) according to the statistical analysis plan. This trial is registered with ISRCTN, number ISRCTN26986053. Findings Between Oct 22, 2015, and May 23, 2018, 516 paramedics from eight UK ambulance services recruited 1149 participants (n=568 in the GTN group, n=581 in the sham group). The median time to randomisation was 71 min (IQR 45–116). 597 (52%) patients had ischaemic stroke, 145 (13%) had intracerebral haemorrhage, 109 (9%) had transient ischaemic attack, and 297 (26%) had a non-stroke mimic at the final diagnosis of the index event. In the GTN group, participants’ systolic blood pressure was lowered by 5·8 mm Hg compared with the sham group (p<0·0001), and diastolic blood pressure was lowered by 2·6 mm Hg (p=0·0026) at hospital admission. We found no difference in mRS between the groups in participants with a final diagnosis of stroke or transient ischaemic stroke (cohort 1): 3 (IQR 2–5; n=420) in the GTN group versus 3 (2–5; n=408) in the sham group, adjusted common odds ratio for poor outcome 1·25 (95% CI 0·97–1·60; p=0·083); we also found no difference in mRS between all patients (cohort 2: 3 [2–5]; n=544, in the GTN group vs 3 [2–5]; n=558, in the sham group; 1·04 [0·84–1·29]; p=0·69). We found no difference in secondary outcomes, death (treatment-related deaths: 36 in the GTN group vs 23 in the sham group [p=0·091]), or serious adverse events (188 in the GTN group vs 170 in the sham group [p=0·16]) between treatment groups. Interpretation Prehospital treatment with transdermal GTN does not seem to improve functional outcome in patients with presumed stroke. It is feasible for UK paramedics to obtain consent and treat patients with stroke in the ultraacute prehospital setting. Funding British Heart Foundation

Long COVID and cardiovascular disease: a prospective cohort study

Background Pre-existing cardiovascular disease (CVD) or cardiovascular risk factors have been associated with an increased risk of complications following hospitalisation with COVID-19, but their impact on the rate of recovery following discharge is not known. Objectives To determine whether the rate of patient-perceived recovery following hospitalisation with COVID-19 was affected by the presence of CVD or cardiovascular risk factors. Methods In a multicentre prospective cohort study, patients were recruited following discharge from the hospital with COVID-19 undertaking two comprehensive assessments at 5 months and 12 months. Patients were stratified by the presence of either CVD or cardiovascular risk factors prior to hospitalisation with COVID-19 and compared with controls with neither. Full recovery was determined by the response to a patient-perceived evaluation of full recovery from COVID-19 in the context of physical, physiological and cognitive determinants of health. Results From a total population of 2545 patients (38.8% women), 472 (18.5%) and 1355 (53.2%) had CVD or cardiovascular risk factors, respectively. Compared with controls (n=718), patients with CVD and cardiovascular risk factors were older and more likely to have had severe COVID-19. Full recovery was significantly lower at 12 months in patients with CVD (adjusted OR (aOR) 0.62, 95% CI 0.43 to 0.89) and cardiovascular risk factors (aOR 0.66, 95% CI 0.50 to 0.86). Conclusion Patients with CVD or cardiovascular risk factors had a delayed recovery at 12 months following hospitalisation with COVID-19. Targeted interventions to reduce the impact of COVID-19 in patients with cardiovascular disease remain an unmet need