Controlled evaLuation of Angiotensin Receptor Blockers for COVID-19 respIraTorY disease (CLARITY): Statistical analysis plan for a randomised controlled Bayesian adaptive sample size trial (preprint)

The CLARITY trial (Controlled evaLuation of Angiotensin Receptor Blockers for COVID-19 respIraTorY Disease) investigates the effectiveness of angiotensin receptor blockers in addition to standard care compared to placebo (in Indian sites) with standard care in reducing the duration and severity of lung failure in patients with COVID-19. The CLARITY trial is a multi-centre, randomised controlled Bayesian adaptive trial with regular planned analyses where pre-specified decision rules will be assessed to determine whether the trial should be stopped due to sufficient evidence of treatment effectiveness or futility. Here we describe the statistical analysis plan for the trial, and define the pre-specified decision rules, including those that could lead to the trial being halted. The primary outcome is clinical status on a 7-point ordinal scale adapted from the WHO Clinical Progression scale assessed at Day 14. The primary analysis will follow the intention-to-treat principle. A Bayesian adaptive trial design was selected because there is considerable uncertainty about the extent of potential benefit of this treatment. Trial registrationClinicalTrials.gov, NCT04394117. Registered on 19 May 2020. https://clinicaltrials.gov/ct2/show/NCT04394117 Clinical Trial Registry of India: CTRI/2020/07/026831 Version and revisionsVersion 1.0. No revisions.

Immunological dysfunction persists for 8 months following initial mild-moderate SARS-CoV-2 infection (preprint)

A proportion of patients surviving acute COVID-19 infection develop post-COVID syndrome (long COVID) encompassing physical and neuropsychiatric symptoms lasting longer than 12 weeks. Here we studied a prospective cohort of individuals with long COVID (the ADAPT study) compared to age/gender matched subjects without long COVID, healthy donors and individuals infected with other non-SARS CoV2 human coronaviruses (the ADAPT-C study). We found an elevated diffuse serum inflammatory cytokine profile in symptomatic long COVID subjects that was maintained at 8 months post-infection and was not observed in asymptomatic COVID-19 survivors. This inflammatory profile consisted of 15 cytokines that positively correlated; revealing an apparent diffuse, potentially coordinated, low level up regulation of a spectrum of immune and inflammatory mediators. In addition, we found an absence of subsets of un-activated naive T and B cells in peripheral blood of long COVID subjects, that did not reconstitute over time. In contrast, individual serum cytokines from the interferon I and III classes, T cell activation markers and plasma ACE2, while elevated in the serum of people previously infected with SARS-CoV-2 were not further elevated in subjects with long COVID symptoms. This work defines immunological parameters associated with long COVID and suggests future opportunities to prevention and treatment.

Plasma ACE2 activity is persistently elevated following SARS-CoV-2 infection: implications for COVID-19 pathogenesis and consequences (preprint)

COVID-19 causes persistent endothelial inflammation, lung and cardiovascular complications. SARS-CoV-2 utilises the catalytic site of full-length membrane-bound angiotensin converting enzyme 2 (ACE2) for cell entry causing downregulation of tissue ACE2. We reported downregulation of cardiac ACE2 is associated with increased plasma ACE2 activity. In this prospective observational study in recovered COVID-19 patients, we hypothesised that SARS-CoV-2 infection would be associated with shedding of ACE2 from cell membranes and increased plasma ACE2 activity. MethodsWe measured plasma ACE2 catalytic activity using a validated, sensitive quenched fluorescent substrate-based assay in a cohort of Australians aged [≥]18 years (n=66) who had recovered from mild, moderate or severe SARS-CoV-2 infection (positive result by PCR testing) and age and gender matched uninfected controls (n=70). Serial samples were available in 23 recovered SARS-CoV-2 patients. ResultsPlasma ACE2 activity at a median of 35 days post-infection [interquartile range 30-38 days] was 97-fold higher in recovered SARS-CoV-2 patients compared to controls (5.8 [2-11.3] vs. 0.06 [0.02-2.2] pmol/min/ml, p<0.0001). There was a significant difference in plasma ACE2 activity according to disease severity (p=0.033), with severe COVID-19 associated with higher ACE2 activity compared to mild disease (p=0.027). Men (n=39) who were SARS-CoV-2 positive had higher median plasma ACE2 levels compared to women (n=27) (p<0.0001). We next analysed whether an elevated plasma ACE2 activity level persisted following SARS-CoV-2 infection in subjects with blood samples at 63 [56-65] and 114 [111-125] days post infection. Plasma ACE2 activity remained persistently elevated in almost all subjects, with no significant differences between timepoints in post-hoc comparisons (p>0.05). DiscussionThis is the first description that plasma ACE2 activity is elevated after COVID-19 infection, and the first with longitudinal data indicating plasma ACE2 activity remains elevated out to a median of 114 days post-infection. Larger studies are now needed to determine if persistent elevated plasma ACE2 activity identifies people at risk of prolonged illness following COVID-19.

Hypertension and renin-angiotensin system blockers are not associated with expression of Angiotensin Converting Enzyme 2 (ACE2) in the kidney (preprint)

Angiotensin converting enzyme 2 (ACE2) is the cellular entry point for severe acute respiratory syndrome coronavirus (SARS-CoV-2) - the cause of COVID-19 disease. It has been hypothesized that use of renin-angiotensin system (RAS) inhibiting medications in patients with hypertension, increases the expression of ACE2 and thereby increases the risk of COVID-19 infection and severe outcomes or death. However, the effect of RAS-inhibition on ACE2 expression in human tissues of key relevance to blood pressure regulation and COVID-19 infection has not previously been reported. We examined how hypertension, its major metabolic co-phenotypes and antihypertensive medications relate to ACE2 renal expression using information from up to 436 patients whose kidney transcriptomes were characterised by RNA-sequencing. We further validated some of the key observations in other human tissues and/or a controlled experimental model. Our data reveal increasing expression of ACE2 with age in both human lungs and the kidney. We show no association between renal expression of ACE2 and either hypertension or common types of RAS inhibiting drugs. We demonstrate that renal abundance of ACE2 is positively associated with a biochemical index of kidney function and show a strong enrichment for genes responsible for kidney health and disease in ACE2 co-expression analysis. Collectively, our data indicate that neither hypertension nor antihypertensive treatment are likely to alter individual risk of SARS-CoV-2 infection or influence clinical outcomes in COVID-19 through changes of ACE2 expression. Our data further suggest that in the absence of SARS-CoV-2 infection, kidney ACE2 is most likely nephro-protective but the age-related increase in its expression within lungs and kidneys may be relevant to the risk of SARS-CoV-2 infection.