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Introduction Loss of blood-brain barrier (BBB) integrity is hypothesised to be one of the earliest microvascular signs of Alzheimer’s disease (AD). Existing BBB integrity imaging methods involve contrast agents or ionising radiation, and pose limitations in terms of cost and logistics. Arterial spin labelling (ASL) perfusion MRI has been recently adapted to map the BBB permeability non-invasively. The DEveloping BBB-ASL as a non-Invasive Early biomarker (DEBBIE) consortium aims to develop this modified ASL-MRI technique for patient-specific and robust BBB permeability assessments. This article outlines the study design of the DEBBIE cohorts focused on investigating the potential of BBB-ASL as an early biomarker for AD (DEBBIE-AD).Methods and analysis DEBBIE-AD consists of a multicohort study enrolling participants with subjective cognitive decline, mild cognitive impairment and AD, as well as age-matched healthy controls, from 13 cohorts. The precision and accuracy of BBB-ASL will be evaluated in healthy participants. The clinical value of BBB-ASL will be evaluated by comparing results with both established and novel AD biomarkers. The DEBBIE-AD study aims to provide evidence of the ability of BBB-ASL to measure BBB permeability and demonstrate its utility in AD and AD-related pathologies.Ethics and dissemination Ethics approval was obtained for 10 cohorts, and is pending for 3 cohorts. The results of the main trial and each of the secondary endpoints will be submitted for publication in a peer-reviewed journal.

Nearly 300 million individuals live with chronic hepatitis B virus (HBV) infection (CHB), for which no curative therapy is available. As viral diversity is associated with pathogenesis and immunological control of infection, improved methods to characterize this diversity could aid drug development efforts. Conventionally, viral sequencing data are mapped/aligned to a reference genome, and only the aligned sequences are retained for analysis. Thus, reference selection is critical, yet selecting the most representative reference a priori remains difficult. We investigate an alternative pangenome approach which can combine multiple reference sequences into a graph which can be used during alignment. Using simulated short-read sequencing data generated from publicly available HBV genomes and real sequencing data from an individual living with CHB, we demonstrate alignment to a phylogenetically representative 'genome graph' can improve alignment, avoid issues of reference ambiguity, and facilitate the construction of sample-specific consensus sequences more genetically similar to the individual's infection. Graph-based methods can, therefore, improve efforts to characterize the genetics of viral pathogens, including HBV, and have broader implications in host-pathogen research.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has emphasized the importance and challenges of correctly interpreting antibody test results. Identification of positive and negative samples requires a classification strategy with low error rates, which is hard to achieve when the corresponding measurement values overlap. Additional uncertainty arises when classification schemes fail to account for complicated structure in data. We address these problems through a mathematical framework that combines high dimensional data modeling and optimal decision theory. Specifically, we show that appropriately increasing the dimension of data better separates positive and negative populations and reveals nuanced structure that can be described in terms of mathematical models. We combine these models with optimal decision theory to yield a classification scheme that better separates positive and negative samples relative to traditional methods such as confidence intervals (CIs) and receiver operating characteristics. We validate the usefulness of this approach in the context of a multiplex salivary SARS-CoV-2 immunoglobulin G assay dataset. This example illustrates how our analysis: (i) improves the assay accuracy, (e.g. lowers classification errors by up to 42% compared to CI methods); (ii) reduces the number of indeterminate samples when an inconclusive class is permissible, (e.g. by 40% compared to the original analysis of the example multiplex dataset) and (iii) decreases the number of antigens needed to classify samples. Our work showcases the power of mathematical modeling in diagnostic classification and highlights a method that can be adopted broadly in public health and clinical settings.

Background: Medications that modify the renin–angiotensin system may reduce Alzheimer’s disease pathology and reduce the rate of disease progression. Objective: This study investigated whether taking the antihypertensive drug losartan, in addition to normal care, would slow the progression of Alzheimer’s disease when compared with a placebo. Design: A double-blind multicentre randomised controlled trial, after a 4-week open-label phase, with follow-up at 14 days and at 3, 6, 9 and 12 months. The primary outcome was based on measured imaging differences in brain volume between baseline and 12 months. Setting: Twenty-three NHS hospital trusts across England, Scotland and Northern Ireland. Participants: Patients diagnosed with mild-to-moderate Alzheimer’s disease were eligible to participate if they met the following criteria: (1) aged ≥ 55 years; (2) a Mini Mental State Examination score of 15–28; (3) a modified Hachinski Ischaemic Score of ≤ 5; (4) a previous computerised tomography, single-photon emission computed tomography or magnetic resonance imaging scan consistent with a diagnosis of Alzheimer’s disease; (5) a study companion who was willing to participate in the study; and (6) capacity to consent for themselves. Patients were ineligible if they were (1) taking or intolerant to renin–angiotensin system-related medications, (2) unlikely to undergo magnetic resonance imaging or (3) unlikely to complete the trial protocol. People who had blood pressure outside the normal ranges, defined cardiovascular issues, impaired liver or renal function, or a primary neurodegenerative disease that was not Alzheimer’s disease were also excluded, as were women who had not reached menopause and were unwilling to take relevant protocol-specific safety precautions. Intervention: The intervention was either 100 mg of overencapsulated losartan (Teva Pharmaceuticals Industries Ltd, Petah Tikva, Israel) daily or a matched placebo for 12 months. Main outcome measures: Difference in brain atrophy, represented by measurement of whole-brain volume before and following 12 months of treatment post randomisation, was measured using volumetric MRI and determined by boundary shift interval analysis. Secondary outcomes included changes in rates of Alzheimer’s disease progression (as assessed using the ADAS-Cog, Mini Mental State Examination and Neuropsychiatric Inventory), the volume of white matter hyperintensities, cerebral blood flow (assessed by magnetic resonance imaging), blood pressure, magnetic resonance imaging measures of atrophy and association with measures of cognitive decline, and drug compliance and tolerability. Results: A total of 261 participants entered the open-label phase, of whom 211 were randomised to the intervention (n = 105) or placebo (n = 106) arms. Of the 197 people (93%) who completed the study, 81% (n = 171) had a valid primary outcome. The difference in brain volume between arms was consistent with chance (–2.79 ml, 95% confidence interval –6.46 to 0.89 ml; p = 0.19), and there was no evidence of benefit for any of the secondary outcome measures. Limitations: Our study had 82% power to detect treatment-based changes and, as a result, may have been underpowered or, more likely, the intervention, which may not have crossed the blood–brain barrier as much as expected, may have been given too late or for an insufficient amount of time in the disease process to influence the outcomes. Conclusions: Losartan administered over 12 months did not alter brain atrophy in Alzheimer’s disease. Future work: Other related ‘sartans’ could be tested in patient groups with mild cognitive impairment and for longer to fully test this hypothesis. Trial registration: Current Controlled Trials ISRCTN93682878 and EudraCT 2012-003641-15. Funding: This project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a Medical Research Council and National Institute for Health Research (NIHR) partnership. This will be published in full in Efficacy and Mechanism Evaluation; Vol. 8, No. 19. See the NIHR Journals Library website for further project information.

In severe viral pneumonia, including Coronavirus disease 2019 (COVID-19), the viral replication phase is often followed by hyperinflammation, which can lead to acute respiratory distress syndrome, multi-organ failure, and death. We previously demonstrated that alpha-1 adrenergic receptor (⍺1-AR) antagonists can prevent hyperinflammation and death in mice. Here, we conducted retrospective analyses in two cohorts of patients with acute respiratory distress (ARD, n = 18,547) and three cohorts with pneumonia (n = 400,907). Federated across two ARD cohorts, we find that patients exposed to ⍺1-AR antagonists, as compared to unexposed patients, had a 34% relative risk reduction for mechanical ventilation and death (OR = 0.70, p = 0.021). We replicated these methods on three pneumonia cohorts, all with similar effects on both outcomes. All results were robust to sensitivity analyses. These results highlight the urgent need for prospective trials testing whether prophylactic use of ⍺1-AR antagonists ameliorates lower respiratory tract infection-associated hyperinflammation and death, as observed in COVID-19.

Progressive diseases tend to be heterogeneous in their underlying aetiology mechanism, disease manifestation, and disease time course. Here, Young and colleagues devise a computational method to account for both phenotypic heterogeneity and temporal heterogeneity, and demonstrate it using two neurodegenerative disease cohorts.

BackgroundThe human hippocampus comprises a number of interconnected histologically and functionally distinct subfields, which may be differentially influenced by cerebral pathology. Automated techniques are now available that estimate hippocampal subfield volumes using in vivo structural MRI data. To date, research investigating the influence of cerebral β-amyloid deposition-one of the earliest hypothesised changes in the pathophysiological continuum of Alzheimer's disease-on hippocampal subfield volumes in cognitively normal older individuals, has been limited.MethodsUsing cross-sectional data from 408 cognitively normal individuals born in mainland Britain (age range at time of assessment = 69.2-71.9 years) who underwent cognitive assessment, 18F-Florbetapir PET and structural MRI on the same 3 Tesla PET/MR unit (spatial resolution 1.1 x 1.1 x 1.1. mm), we investigated the influences of β-amyloid status, age at scan, and global white matter hyperintensity volume on: CA1, CA2/3, CA4, dentate gyrus, presubiculum and subiculum volumes, adjusting for sex and total intracranial volume.ResultsCompared to β-amyloid negative participants (n = 334), β-amyloid positive participants (n = 74) had lower volume of the presubiculum (3.4% smaller, p = 0.012). Despite an age range at scanning of just 2.7 years, older age at time of scanning was associated with lower CA1 (p = 0.007), CA4 (p = 0.004), dentate gyrus (p = 0.002), and subiculum (p = 0.035) volumes. There was no evidence that white matter hyperintensity volume was associated with any subfield volumes.ConclusionThese data provide evidence of differential associations in cognitively normal older adults between hippocampal subfield volumes and β-amyloid deposition and, increasing age at time of scan. The relatively selective effect of lower presubiculum volume in the β-amyloid positive group potentially suggest that the presubiculum may be an area of early and relatively specific volume loss in the pathophysiological continuum of Alzheimer's disease. Future work using higher resolution imaging will be key to exploring these findings further.

IntroductionPrescription drug abuse is a major public health problem in rural and suburban areas of the United States, however its emergence in large urban settings with endemic injection drug use remains understudied. We examined temporal trends in injection drug use initiation and mortality among people who inject drugs (PWID) in Baltimore, Maryland.MethodsData were derived from the baseline assessment of PWID enrolled in a community-based cohort study with longitudinal follow-up for mortality assessment. PWID were recruited from 2005-2008 (N = 1,008) and 2015-2018 (N = 737). We compared characteristics by birth cohort (before/after 1980) and type of drug initiated (prescription opioids, prescription non-opioids, non-injection illicit drugs, or injection drugs). We calculated standardized mortality ratios (SMR) using the US general population as the reference.ResultsPWID born after 1980 were more likely to initiate drug use with prescription opioids and non-opioids and had higher levels of polysubstance prior to injection initiation, compared to individuals born before 1980. Overall mortality was high: 2.59 per 100 person-years (95% CI: 2.27-2.95 per 100 person-years). Compared to the US population, the highest SMRs were observed among participants between 40-44 years of age, with especially high mortality among women in this age group (SMR:29.89, 95% CI: 15.24-44.54).ConclusionsMirroring national trends, the profile of PWID in Baltimore has changed with increased prescription drug abuse and high levels of polysubstance use among younger PWID. Interventions need to reach those using prescription drugs early after initiation of use in order to reduce transition to injecting. Urgent attention is warranted to address premature mortality, particularly among middle-aged and female PWID.

The glymphatics system describes a CSF-mediated clearance pathway for the removal of potentially harmful molecules, such as amyloid beta, from the brain. As such, its components may represent new therapeutic targets to alleviate aberrant protein accumulation that defines the most prevalent neurodegenerative conditions. Currently, however, the absence of any non-invasive measurement technique prohibits detailed understanding of glymphatic function in the human brain and in turn, it’s role in pathology. Here, we present the first non-invasive technique for the assessment of glymphatic inflow by using an ultra-long echo time, low b-value, multi-direction diffusion weighted MRI sequence to assess perivascular fluid movement (which represents a critical component of the glymphatic pathway) in the rat brain. This novel, quantitative and non-invasive approach may represent a valuable biomarker of CSF-mediated brain clearance, working towards the clinical need for reliable and early diagnostic indicators of neurodegenerative conditions such as Alzheimer’s disease.

BACKGROUND AND OBJECTIVE:We characterize the burden of liver disease in a cohort of PWID in Chennai, India, with a high prevalence of HCV. MATERIALS AND METHODS:1,042 PWID were sampled through community outreach in Chennai. Participants underwent fasting blood draw, questionnaire and an examination that included liver stiffness assessment using transient elastography (Fibroscan) and assessment of steatosis via ultrasound. RESULTS:The median age was 39 years, all were male, 14.8% were HIV infected and 35.6% were HCV antibody positive, of whom 78.9% were chronically infected (HCV RNA positive). Median liver stiffness was 6.2 kPA; 72.9% had no evidence of or mild stiffness, 14.5% had moderate stiffness, and 12.6% had evidence of severe stiffness/cirrhosis. Prevalence of severe stiffness/cirrhosis was significantly higher among persons who were older, had a longer duration of injecting drugs, higher body mass index, higher prevalence of insulin resistance, higher prevalence of steatosis, higher HCV RNA levels and evidence of alcohol dependence. An estimated 42.1% of severe stiffness/cirrhosis in this sample was attributable to HCV. 529 (53.0%) had some evidence of steatosis. Prevalence of steatosis was higher among those who had larger waist circumference, insulin resistance, higher HDL cholesterol and a history of antiretroviral therapy. CONCLUSIONS:We observed a high burden of liver disease in this relatively young cohort that was primarily driven by chronic HCV infection, metabolic factors (insulin resistance and steatosis) and heavy alcohol use. Interventions to improve access to HCV treatment and reduce alcohol use are needed to prevent further progression of liver disease.

Chronic liver infection by hepatitis C virus (HCV) is a major public health concern. Despite partly successful treatment options, several aspects of intrahepatic HCV infection dynamics are still poorly understood, including the preferred mode of viral propagation, as well as the proportion of infected hepatocytes. Answers to these questions have important implications for the development of therapeutic interventions. In this study, we present methods to analyze the spatial distribution of infected hepatocytes obtained by single cell laser capture microdissection from liver biopsy samples of patients chronically infected with HCV. By characterizing the internal structure of clusters of infected cells, we are able to evaluate hypotheses about intrahepatic infection dynamics. We found that individual clusters on biopsy samples range in size from 4-50 infected cells. In addition, the HCV RNA content in a cluster declines from the cell that presumably founded the cluster to cells at the maximal cluster extension. These observations support the idea that HCV infection in the liver is seeded randomly (e.g. from the blood) and then spreads locally. Assuming that the amount of intracellular HCV RNA is a proxy for how long a cell has been infected, we estimate based on models of intracellular HCV RNA replication and accumulation that cells in clusters have been infected on average for less than a week. Further, we do not find a relationship between the cluster size and the estimated cluster expansion time. Our method represents a novel approach to make inferences about infection dynamics in solid tissues from static spatial data.

Microbial translocation (MT) is thought to be a major contributor to the pathogenesis of HIV-related immune activation, and circulating lipopolysaccharide (LPS) from gram-negative bacteria is the principle measurement of this process. However, related research has been impeded by inconsistent LPS test results.Specimens were obtained from HIV-infected adults enrolled in the PEARLS study (ACTG A5175) and HIV-HCV co-infected participants enrolled in a study of liver disease staging using MRI elastography. Pig-tailed macaque specimens were obtained from SIV-infected and -uninfected animals. Samples were tested for LPS using the LAL assay with diazo-coupling modifications to improve sensitive detection.When exogenous LPS was added to macaque plasma, >25% inhibition of LPS detection was found in 10/10 (100%) samples at 20% plasma concentration compared to control; in contrast 5/10 (50%) samples at 2% plasma concentration (p = 0.07) and 0/10 (0%) at 0.1% plasma concentration (p = 0.004) showed >25% inhibition of LPS detection. Similarly, when LPS was added to human serum, >25% inhibition of LPS detection was found in 5/12 (42%) of samples at 2% serum concentration compared to control, while 0/12 (0%) of samples in 0.1% serum showed >25% inhibition of LPS detection (p = 0.07). Likewise, LPS detection in human sera without exogenous LPS was improved by dilution: LPS was detected in 2/12 (17%) human samples in 2% serum, ranging from 3,436-4,736 pg/mL, compared to 9/12 (75%) samples in 0.1% serum, ranging from 123 pg/mL -60,131 pg/mL (p = 0.016). In a separate validation cohort of HIV-HCV co-infected participants sampled at two different times on the same day, LPS measured in 0.2% plasma and with diazo-coupling was closely correlated between the first and second samples (R = 0.66, p

Traditional herbal medicines are commonly used in sub-Saharan Africa and some herbs are known to be hepatotoxic. However little is known about the effect of herbal medicines on liver disease in sub-Saharan Africa.500 HIV-infected participants in a rural HIV care program in Rakai, Uganda, were frequency matched to 500 HIV-uninfected participants. Participants were asked about traditional herbal medicine use and assessed for other potential risk factors for liver disease. All participants underwent transient elastography (FibroScan®) to quantify liver fibrosis. The association between herb use and significant liver fibrosis was measured with adjusted prevalence risk ratios (adjPRR) and 95% confidence intervals (CI) using modified Poisson multivariable logistic regression.19 unique herbs from 13 plant families were used by 42/1000 of all participants, including 9/500 HIV-infected participants. The three most-used plant families were Asteraceae, Fabaceae, and Lamiaceae. Among all participants, use of any herb (adjPRR = 2.2, 95% CI 1.3-3.5, p = 0.002), herbs from the Asteraceae family (adjPRR = 5.0, 95% CI 2.9-8.7, p

Fibrosis stages from liver biopsies reflect liver damage from hepatitis C infection, but analysis is challenging due to their ordered but non-numeric nature, infrequent measurement, misclassification, and unknown infection times.We used a non-Markov multistate model, accounting for misclassification, with multiple imputation of unknown infection times, applied to 1062 participants of whom 159 had multiple biopsies. Odds ratios (OR) quantified the estimated effects of covariates on progression risk at any given time.Models estimated that progression risk decreased the more time participants had already spent in the current stage, African American race was protective (OR 0.75, 95% confidence interval 0.60 to 0.95, p = 0.018), and older current age increased risk (OR 1.33 per decade, 95% confidence interval 1.15 to 1.54, p = 0.0002). When controlled for current age, older age at infection did not appear to increase risk (OR 0.92 per decade, 95% confidence interval 0.47 to 1.79, p = 0.80). There was a suggestion that co-infection with human immunodeficiency virus increased risk of progression in the era of highly active antiretroviral treatment beginning in 1996 (OR 2.1, 95% confidence interval 0.97 to 4.4, p = 0.059). Other examined risk factors may influence progression risk, but evidence for or against this was weak due to wide confidence intervals. The main results were essentially unchanged using different assumed misclassification rates or imputation of age of infection.The analysis avoided problems inherent in simpler methods, supported the previously suspected protective effect of African American race, and suggested that current age rather than age of infection increases risk. Decreasing risk of progression with longer time already spent in a stage was also previously found for post-transplant progression. This could reflect varying disease activity, with recent progression indicating active disease and high risk, while longer time already spent in a stage indicates quiescent disease and low risk.