The aims of this study were to assess prescription patterns, dosages, discontinuation rates, and association with prognosis of conventional heart failure medications in patients with transthyretin cardiac amyloidosis (ATTR-CA).P < .001] and in a pre-specified subgroup of patients with a left ventricular ejection fraction (LVEF) >40% ; and treatment with low-dose beta-blockers was independently associated with a reduced risk of mortality in a pre-specified subgroup of patients with a LVEF ≤40% . No convincing differences were found for treatment with ACEi/ARBs.A retrospective analysis of all consecutive patients diagnosed with ATTR-CA at the National Amyloidosis Centre between 2000 and 2022 identified 2371 patients with ATTR-CA. Prescription of heart failure medications was greater among patients with a more severe cardiac phenotype, comprising beta-blockers in 55.4%, angiotensin-converting enzyme inhibitors (ACEis)/angiotensin II receptor blockers (ARBs) in 57.4%, and mineralocorticoid receptor antagonists (MRAs) in 39.0% of cases. During a median follow-up of 27.8 months (interquartile range 10.6–51.3), 21.7% had beta-blockers discontinued, and 32.9% had ACEi/ARBs discontinued. In contrast, only 7.5% had MRAs discontinued. A propensity score-matched analysis demonstrated that treatment with MRAs was independently associated with a reduced risk of mortality in the overall population [hazard ratio (HR) 0.77  0.66–0.89), Conventional heart failure medications are currently not widely prescribed in ATTR-CA, and those that received medication had more severe cardiac disease. Beta-blockers and ACEi/ARBs were often discontinued, but low-dose beta-blockers were associated with reduced risk of mortality in patients with a LVEF ≤40%. In contrast, MRAs were rarely discontinued and were associated with reduced risk of mortality in the overall population; but these findings require confirmation in prospective randomized controlled trials.  Cheng and S.A.M. Cuddy, 2 Until recently, ATTR-CA was considered a rare, untreatable disease. However, improvements in diagnostics coupled with emerging high-cost therapies are challenging these long-held beliefs. The ATTR-CA is far more common than previously suspected, and there is a potential for successful therapeutic intervention.3Transthyretin cardiac amyloidosis (ATTR-CA) causes progressive, fatal, heart failure (HF), due to misfolding of transthyretin (TTR), forming insoluble amyloid fibrils, which are deposited within the myocardial extracellular space.4 However, unfortunately, the high cost associated with tafamidis has resulted in restricted use, and tafamidis has not been approved for the treatment of ATTR-CA in many countries.5The only drug proved to be associated with prognostic benefit in ATTR-CA is tafamidis, which is a highly specific drug that targets the circulating TTR protein and stabilizes the TTR tetramer to prevent dissociation into amyloidogenic monomers that deposit in the myocardium, causing an infiltrative and restrictive cardiomyopathy. Tafamidis was shown in a phase 3 placebo-controlled trial (ATTR-ACT) to reduce the combined primary endpoint of cardiovascular hospitalizations and mortality.6–13 Hence, the value of conventional HF medications in patients with ATTR-CA is still debated. Small-scale studies have yielded contrasting results, with some suggesting that low doses of conventional HF medications are well tolerated,15 while others reported that not only are these medications poorly tolerated, but they may result in worse outcomes.17 The lack of large-scale clinical trials has resulted in a significant knowledge gap, although a position statement from the European Society of Cardiology (ESC) working group on myocardial and pericardial diseases regarding HF medications in ATTR-CA recommends stopping beta-blockers, and avoiding angiotensin-converting enzyme inhibitors (ACEis) and angiotensin II receptor blockers (ARBs), and are silent about mineralocorticoid receptor antagonists (MRAs).18At present, it is unknown whether conventional HF medications that have substantial benefits in patients with HF of other aetiologies may also benefit those with ATTR-CA, as patients with known ATTR-CA have been excluded from previous HF trials.The aims of this study were to: (i) assess the prescription pattern of conventional HF medications in patients with ATTR-CA; (ii) assess the dosages and discontinuation rates of HF medications in patients with ATTR-CA; and (iii) assess the association between treatment with HF medications and survival in patients with ATTR-CA.Consecutive patients in whom a diagnosis of ATTR-CA was confirmed at the National Amyloidosis Centre (NAC), between January 2000 and September 2022, were included. Patients with evidence of ATTR-polyneuropathy were excluded, as many have autonomic neuropathy and are not treated with HF medications due to concomitant postural hypotension.99mTechnetium labelled 3,3-diphosphono-1,2-propanodicarboxylic acid (99mTc-DPD) scintigraphy was utilized, and diagnosis established based on ATTR-amyloid in an extra-cardiac biopsy with cardiac uptake on 99mTc-DPD scintigraphy; or grade 2–3 cardiac uptake on 99mTc-DPD scintigraphy in the absence of biochemical evidence of a plasma cell dyscrasia. All patients underwent genetic sequencing of the TTR gene and provided written consent for their data to be retrospectively analysed and published, in line with the Declaration of Helsinki and approval from the Royal Free Hospital ethics committee (REC 21/PR/0620).Between 2000 and 2005 the diagnosis of ATTR-CA was established based on HF symptoms together with a characteristic CA echocardiogram and either direct endomyocardial biopsy proof of ATTR-amyloid or ATTR-amyloid in an extra-cardiac biopsy. From 2006 onwards cardiac magnetic resonance was added to the assessment if there was diagnostic doubt. From 2010 onwards, 19 Management decisions utilized a combined decision-making process involving local clinicians and the NAC team. Considering the knowledge gap, decisions concerning the initiation or discontinuation of HF medications were made following each clinical assessment on a case-by-case basis.All patients are enrolled into a protocolized follow-up program that consists of 6–12 monthly consultations. Data regarding whether HF medications were initiated, continued, or stopped, and medication dosages were all recorded. Medication classes were defined based on the ESC HF guidelines and comprised beta-blockers, ACEi/ARBs and MRAs. Target doses from the guidelines enabled comparisons by converting the daily dose to a percentage of the target dose. Medication classes were recorded regardless of whether the specific drug had been used in previous HF trials.t-test was used to compare means if the data were normally distributed in each treatment group, or its non-parametric equivalent was used to compare the distributions of the two treatment groups. One-way analysis of variance if the data were normally distributed in each treatment group was used to compare means in more than two groups; or its non-parametric equivalent was used to compare the distributions of multiple groups. A significant result was followed by post hoc Bonferroni corrected pairwise comparisons to establish where differences lay. Categorical data are presented as absolute numbers and frequencies (%) and compared using the χ2 test.Statistical analysis was performed using Stata . All continuous variables were tested for normality (Shapiro–Wilk test) and presented as mean ± standard deviation if the distribution was normal or median [interquartile range (IQR)] otherwise, other than N-terminal pro-B-type natriuretic peptide (NT-proBNP) which was log-transformed for bivariate testing. The independent sample All mortality data were obtained via the UK Office of National Statistics, which is the formal government registry for all deaths throughout the UK. The mortality endpoint was defined as time to death from date of diagnosis for all deceased patients and time to censor date (25 October 2022) from date of diagnosis among the remainder. Follow-up was restricted to ≤60 months, after which patients were censored due to the majority of events occurring in the first 60 months, and a low number of patients at risk after 60 months. To account for amyloid-specific disease-modifying therapy or clinical trials, patients were censored at their start date.a priori based on clinical relevance, association with HF medication treatment and association with survival .Survival was evaluated using Cox proportional hazards regression analysis, providing estimated hazard ratios (HRs) with 95% confidence intervals (CIs). The proportional hazards assumption was checked and confirmed using weighted Schoenfeld residuals. With regard to the survival analysis, patients were classed as being treated with HF medications if they were treated continuously for at least 6 months following their initial assessment, or an event occurred within the first 6 months while patients were continuously treated. If the medication was stopped during the first 6 months, then patients were classed as not taking the medication. The initial survival analysis was performed on the whole study population using a multivariable Cox proportional hazards regression adjusting for covariates selected a priori were carried out in the subgroup of patients with a left ventricular ejection fraction (LVEF) ≤40% and the subgroup of patients with a LVEF >40% (based on the guideline definition for HF with reduced ejection fraction being a LVEF ≤40%).19 Kaplan–Meier curves were constructed with statistical significance being assessed with a log-rank test. Significant results were followed by sensitivity analyses to assess whether these results could be replicated; firstly using an ‘intention to treat’ approach whereby patients were classed as treated, or not treated based on their treatment status at diagnosis (rather than over the first 6 months), secondly without censoring patients for the start date of clinical trials or disease-modifying therapy, and lastly analysing the medication use as a time-varying exposure. Statistical significance was defined as P < .05.Propensity score (PS) matching is widely used to reduce confounding biases in observational studies. The PS is a score between 0 and 1 that reflects the likelihood of the patient receiving one of the HF medications of interest conditional on a set of variables, so that those with similar PSs are independent of these variables. Prior to PS matching, missing data were replaced using single imputation, whereby missing values of numerical variables were replaced by the relevant median, and missing values of categorical variables were replaced by the relevant mode, to overcome potential bias introduced by excluding patients with missing data. In order to compare two particular HF medications, a PS for each individual was determined using all the aforementioned variables, apart from the HF medications being assessed. After finding the area of common support (in which the histograms of the PSs overlapped), the patients were then matched on the basis of their PSs in the two medication groups in a 1:1 ratio using the nearest neighbour approach without replacement and calliper width equal to 0.20 times the standard deviation of the logit of the PSs. The adequacy of matching was verified by ensuring that the standardized differences between groups were <0.10 for all variables used to create the PS. A Cox proportional hazards regression model was then applied using the matched groups to compare the effect on survival of the two medications of interest. Additional PS-matched analyses specified 2. Overall, 1955 patients (82.4%) were treated with a diuretic. In most cases (76.8% patients) a loop diuretic was prescribed either alone or in combination (Table 1). A total of 467 (19.7%) patients were enrolled into clinical trials, or treated with disease-modifying therapy . These patients were younger and had a milder cardiac phenotype at diagnosis than the rest of the study population (see Table S1).We identified 2371 patients diagnosed with ATTR-CA. The population compromised 1840 (77.6%) with wild-type ATTR-CA and 531 (22.4%) with hereditary ATTR-CA. The mean age of patients was 77.5 ± 7.3 years, and 90.0% were men. About two-thirds of patients were in New York Heart Association (NYHA) classes I and II, the median NT-proBNP was 2925 ng/L, and the mean LVEF was 48.2% . The median NT-proBNP among patients treated with beta-blockers was significantly higher, while median eGFR was significantly lower than patients not receiving beta-blockers. Patients treated with beta-blockers had a larger bi-atrial size, lower LVEF, lower tricuspid annular plane systolic excursion (TAPSE), and worse longitudinal strain than those not receiving this type of treatment.A total of 1362 (57.4%) patients were treated with an ACEi or ARB (60.5% in patients with a LVEF ≤40%) at diagnosis. As for beta-blockers, those treated with ACEi/ARBs had a higher prevalence of IHD, diabetes mellitus, and atrial fibrillation compared to patients not receiving this type of treatment. In addition, patients treated with an ACEi/ARB were more likely to have hypertension than patients not receiving this type of treatment. Those treated with ACEi/ARBs had a severe cardiac phenotype, with a higher NYHA class and NAC disease stage, and a higher proportion of patients having chronic kidney disease stages 3–5 than patients not receiving ACEi/ARBs. Patients treated with ACEi/ARBs had a larger bi-atrial size, lower LVEF, and worse longitudinal strain than those not receiving this type of treatment.Table 2).A total of 925 (39.0%) patients were treated with an MRA (47.5% in patients with a LVEF ≤40%) at diagnosis. Those treated with MRAs had a higher prevalence of diabetes mellitus and atrial fibrillation but, unlike beta-blocker and ACEi/ARB treatment, patients treated with an MRA did not have more IHD. Those treated with MRAs had a more severe cardiac phenotype, with a worse functional capacity as evidenced by NYHA class and 6MWT, and a higher NAC disease. The median NT-proBNP among patients treated with MRAs was significantly higher, while median eGFR was significantly lower than patients not receiving MRAs. Patients treated with MRAs had a larger right atrial area, lower stroke volume, lower LVEF, lower TAPSE, higher E/e′, and worse longitudinal strain than those not receiving this type of treatment (Table 3).A total of 417 (17.6%) patients were treated with all three classes of HF medications  at diagnosis, 804 (33.9%) were treated with a combination of two classes of HF medications, 741 (31.3%) were treated with one of the three classes of HF medications, and 409 (17.2%) were not treated with any prognostic HF medications. The most frequent combination of two HF medications was a beta-blocker and ACEi/ARB in 454 (56.5%) patients, followed by a beta-blocker and MRA in 180 (22.4%) patients, and an ACEi/ARB and MRA in 170 (21.1%) patients. Those treated with more HF medications had a higher prevalence of IHD, diabetes mellitus, and atrial fibrillation. They had more severe HF, with a worse functional status, as evidenced by NYHA class and 6MWT, and a higher NAC disease stage, and a higher proportion of patients having chronic kidney disease stages 3–5. Patients treated with more HF medications had a larger left ventricular wall thickness, larger bi-atrial size, and worse biventricular systolic function , and there was a greater use of HF medications in patients with a LVEF ≤40%.19 The most commonly prescribed beta-blocker was bisoprolol , with the majority of patients treated with ≤2.5 mg per day . Only 75 (5.7%) patients had the target beta-blocker dose prescribed, most of which had atrial fibrillation . The overwhelming majority of the study population  and all patients with a LVEF ≤40%  were treated with beta-blockers approved for HF with reduced ejection fraction. During follow-up 285 (21.7%) patients had their beta-blocker discontinued [median duration to discontinuation: 14.1 (6.8–28.9) months], and 117 (8.9%) had their beta-blocker dose reduced [median duration to reduction: 15.7 (7.4–34.5) months]. Patients who discontinued beta-blocker treatment had a lower blood pressure and heart rate than those who continued treatment. Only 63 (4.8%) patients had their beta-blocker dose increased, of which only 8 patients eventually had the target dose prescribed. During follow-up, 55 patients were initiated on beta-blockers, and the majority were treated with ≤25% of the target dose , of which 4 (7.2%) had their beta-blocker subsequently discontinued.Of the 1313 patients treated with beta-blockers, over half were treated with ≤25% of the target dose for HF .19 The most commonly prescribed ACEi/ARB was ramipril , with the majority of patients treated with ≤2.5 mg per day . Only 158 (11.6%) patients were treated with the target ACEi/ARB dose. During follow-up 448 (32.9%) patients had their ACEi/ARB discontinued [median duration to discontinuation: 14.4 (6.9–26.8) months], and 77 (5.7%) had their ACEi/ARB dose reduced [median duration to reduction: 14.2 (7.4–26.6) months]. Patients who discontinued ACEi/ARB treatment had a lower blood pressure than those who continued treatment (see Table S2). Only 35 (2.6%) patients had their ACEi/ARB dose increased, of which only 3 patients were treated with the target dose. During follow-up, 41 patients were initiated on ACEi/ARBs, and the majority were treated with ≤37.5% of the target dose  of which 8 (19.5%) had their ACEi/ARB subsequently discontinued.Of the 1362 patients treated with ACEi/ARBs, over half were treated with ≤37.5% of the target dose , followed by 50 mg . During follow-up 69 (7.5%) patients had their MRAs discontinued [median duration to discontinuation: 12.5 (7.9–24.9) months], and 31 (3.4%) had their MRA dose reduced [median duration to reduction: 14.1 (7.9–24.9) months]. Only 77 (8.3%) patients had the dose of their MRA increased, of which 53 were prescribed with 50 mg. During follow-up, 158 patients were initiated on MRAs, and the majority were treated with ≥25 mg , of which only 5 (3.2%) had their MRA subsequently discontinued.Of the 925 patients treated with MRAs, 742 (80.2%) were treated with spironolactone, and 183 (19.3%) were treated with eplerenone. The most commonly prescribed dose of both drugs was 25 mg , and the death rate was 14.9 deaths per 100 patient-years (95% CI 13.9–15.9). There were 1274 patients classed as being treated with beta-blockers for the survival analysis, and the death rate was 14.8 deaths per 100 patient-years (95% CI 13.5–16.2). There were 1306 patients classed as being treated with ACEi/ARBs for the survival analysis, and the death rate was 15.0 deaths per 100 patient-years (95% CI 13.8–16.4). There were 915 patients classed as being treated with MRAs for the survival analysis, and the death rate was 14.6 deaths per 100 patient-years (95% CI 13.1–16.1).P = .004] was convincingly associated with a lower risk of mortality (see Table S3).In a multivariable Cox regression analysis with covariates age, sex, IHD, diabetes mellitus, hypertension, atrial fibrillation, NAC disease stage, wild-type or hereditary ATTR-CA, IVSd, longitudinal strain, beta-blocker, ACEi/ARB, and MRA, only 4 covariates  were associated with a higher risk of mortality; and only one treatment , although the 95% CI of the estimate was wide and did not exclude clinically important effects (see Table S4). A second PS-matched cohort was constructed to assess the association between treatment with beta-blockers and risk of mortality in patients with a LVEF ≤40%. This comprised of 338 patients (169 treated with beta-blockers vs. 169 not treated with beta-blockers), and demonstrated a 39% lower risk of mortality in patients treated with beta-blockers  (see Table S5). These findings were confirmed with sensitivity analysis, utilizing an ‘intention to treat’ approach , and whereby patients were no longer censored for the start date of clinical trials and disease modifying therapy , and where beta-blocker treatment was analysed as a time-varying exposure . Following exclusion of patients with coexistent IHD and their corresponding pairs, repeat analysis confirmed a lower risk of mortality in patients with a LVEF ≤40% treated with beta-blockers . A third PS-matched cohort was constructed to assess the association between treatment with beta-blockers and risk of mortality in patients with a LVEF >40%. This comprised of 1378 patients (689 treated with beta-blockers vs. 689 not treated with beta-blockers) and did not provide convincing evidence for a difference in the risk of mortality between the two groups , although the estimate was imprecise .To minimize the potential selection bias inherent with the baseline treatment of HF medications we also performed PS-matched cohort analyses to assess the association between treatment with each HF medication and survival. Missing data were imputed for NAC stage in 37 patients, IVSd in 115 patients, and longitudinal strain in 296 patients. The remaining variables did not have any missing data. The PS-matched cohort constructed to assess the association between treatment with beta-blockers and risk of mortality comprised of 1756 patients (878 treated with beta-blockers vs. 878 not treated with beta-blockers) and did not provide convincing evidence for a difference in the risk of mortality between the two groups  (see Table S7). A second PS-matched analysis was constructed to assess the association between treatment with ACEi/ARBs and the risk of mortality in patients with a LVEF ≤40%. This comprised of 368 patients (184 treated with ACEi/ARBs vs. 184 not treated with ACEi/ARBs) and did not provide convincing evidence for a difference in the risk of mortality between the two groups , although the estimates were imprecise (Table S8). A third PS-matched analysis was constructed to assess the association between treatment with ACEi/ARBs and the risk of mortality in patients with a LVEF >40%. This comprised of 1390 patients (695 treated with ACEi/ARBs vs. 695 not treated with ACEi/ARBs) and did not provide convincing evidence for a difference in the risk of mortality between the two groups  .The PS-matched cohort constructed to assess the association between treatment with ACEi/ARBs and the risk of mortality comprised of 1782 patients (891 treated with ACEi/ARBs vs. 891 not treated with ACEi/ARBs) and did not provide convincing evidence for a difference in the risk of mortality between the two groups  (see Table S10). These findings were confirmed with sensitivity analysis, utilizing an ‘intention to treat’ approach ; and whereby patients were no longer censored for the start date of clinical trials and disease modifying therapy , and where MRA treatment was analysed as a time-varying exposure . A second PS-matched analysis was constructed to assess the association between treatment with MRAs and the risk of mortality in patients with a LVEF ≤40%. This comprised of 416 patients (208 patients treated with MRAs vs. 208 patients not treated with MRAs) and did not provide convincing evidence for a difference in the risk of mortality between the two groups , although the 95% CI of the estimate was wide and did not exclude clinically important effects (see Table S11). A third PS-matched analysis was constructed to assess the association between treatment with MRAs and the risk of mortality in patients with a LVEF >40%. This comprised of 1334 patients (667 treated with MRAs vs. 667 not treated with MRAs) and demonstrated that there was a 25% lower risk of mortality in patients treated with MRAs  . These findings were confirmed with sensitivity analysis, utilizing an ‘intention to treat’ approach ; and whereby patients were no longer censored for the start date of clinical trials and disease modifying therapy , and where MRA treatment was analysed as a time-varying exposure .The PS-matched cohort constructed to assess the association between treatment with MRAs and the risk of mortality comprised of 1788 patients (894 patients treated with MRAs vs. 894 patients not treated with MRAs) and demonstrated that there was a 23% lower risk of mortality in patients treated with MRAs  (see Table S13). These findings were confirmed with sensitivity analysis, utilizing an ‘intention to treat’ approach ; and whereby patients were no longer censored for the start date of clinical trials and disease modifying therapy , and where treatment was analysed as a time-varying exposure .In the overall population, a PS-matched cohort was constructed to compare the association with risk of mortality, between treatment with all three classes of HF medications  and treatment with two classes of HF medications (beta-blockers and ACEi/ARBs). This comprised of 680 patients  and demonstrated that there was a 37% lower risk of mortality in patients treated with all three HF medications , although the estimates were imprecise . Data on reasons for medication discontinuation and the association between survival and both medication dosage and medication discontinuation are presented in Appendix S1.A PS-matched cohort was used to compare the association with risk of mortality, between treatment with two classes of HF medications (beta-blockers and ACEi/ARBs) and treatment with just ACEi/ARBs. This comprised of 558 patients (279 treated with beta-blockers and ACEi/ARBs vs. 279 treated with just ACEi/ARBs) and did not provide convincing evidence for a difference in the risk of mortality between the two groups [HR 1.06 (95% CI 0.81–1.39), Structured Graphical Abstract).In this study we comprehensively evaluated the prescription pattern and discontinuation rates of HF medications in >2000 patients with ATTR-CA, and assessed the association between treatment with HF medications and the risk of mortality in these individuals. Our study demonstrated that: (i) patients with ATTR-CA and a severe cardiac phenotype were more commonly treated with HF medications; (ii) beta-blockers and ACEi/ARBs were generally prescribed in low doses and often discontinued, whereas in contrast, MRAs were rarely discontinued; and (iii) MRAs were independently associated with a lower risk of mortality in the overall population, and in patients with LVEF >40%; and low-dose beta-blockers were independently associated with a lower risk of mortality in patients with a LVEF ≤40% , ACEi/ARBs (57.4%), and MRAs (39.0%). Treatment with HF medications in patients with ATTR-CA appears to be driven by the presence of comorbidities and the severity of their cardiac disease. Heart failure medications were more commonly prescribed in patients with atrial fibrillation, diabetes mellitus, and chronic kidney disease. Beta-blockers and ACEi/ARBs are also more commonly prescribed in patients with IHD.21 In contrast, MRAs were rarely discontinued, with less than one-tenth having their MRA discontinued. This is probably related to the limited effect on blood pressure, compared with beta-blockers and ACEi/ARB, and their possible diuretic effect. The mainstay of symptom management in ATTR-CA has long been meticulous volume control, facilitated by high-dose loop diuretics. The MRAs may have a synergistic effect when utilized alongside loop diuretics and also increase potassium reabsorption, which is often needed when high doses of loop diuretics are utilized.22Beta-blockers and ACEi/ARBs were commonly discontinued, with over one-fifth of patients having their beta-blocker discontinued, and nearly one-third having their ACEi/ARB discontinued during follow-up. Beta-blocker intolerance may be exacerbated the underlying pathophysiology of ATTR-CA. In the context of a fixed stroke volume, caused by restrictive physiology, a higher heart rate is required to maintain cardiac output. The inability to augment stroke volume in response to the vasodilation may also contribute to the intolerance of ACEi/ARBs.12In the current study, which represents the largest analysis of HF medications in patients with ATTR-CA to date, both regression-based and PS-matched analyses demonstrated that treatment with MRAs was independently associated with a lower risk of mortality in the overall ATTR-CA population; and PS-matched analysis demonstrated that low-dose beta-blockers were associated with a lower risk of mortality in patients with a LVEF ≤40%. MRAs were associated with a lower risk of mortality in patients with a LVEF >40%, but not in patients with a LVEF ≤40%. The point estimates for these analyses were similar, hence a greater sample size may have increased power sufficiently to demonstrate a benefit in patients with a LVEF ≤40%. Another possibility is that the benefit derived from MRAs is greater earlier in the disease process, and therefore increased survival benefit occurs in patients with a LVEF >40%. The reduced risk of mortality associated with low-dose beta-blockers in patients with a LVEF ≤40% was maintained when excluding patients with concomitant IHD, suggesting that the benefit is related to treating ATTR-CA rather than treating comorbidities, and this is consistent with previous HF trials that demonstrated that improved outcomes were confined to patients with a reduced ejection fraction.23 It is therefore plausible that patients with ATTR-CA would derive prognostic benefit from neurohormonal modulation. However, a recent position statement by the ESC on the treatment of ATTR-CA recommended the withdrawal of beta-blockers, avoiding ACEi/ARBs, and did not discuss the use of MRAs in patients with ATTR-CA, reflecting the perceived poor tolerability of these agents and lack of trial evidence to support their use .18 Several small observational studies have contributed to these recommendations. However, differences in methodology and patient selection could explain our contrasting results. Previous studies have not matched patients, and therefore the worse outcomes in patients treated with HF medications were confounded by disease severity. Our study excluded patients with concomitant polyneuropathy, who often have autonomic disease and hypotension, resulting in a poor tolerance of HF medications.17 Importantly, our results are supported by a retrospective analysis of the TOPCAT trial, whereby an enriched cohort with echocardiographic characteristics of CA derived benefit from MRA therapy.22 This analysis is featured in a recent American College of Cardiology (ACC) consensus document that recommends MRA therapy alongside loop diuretics to augment diuresis.24 Our study is the first to sub-categorize ATTR-CA patients by LVEF. The majority of HF patients with a LVEF ≤40% experience chronic adrenergic overstimulation, and higher serum noradrenaline levels than their counterparts with preserved systolic function. A similar pathophysiological mechanism may exist in ATTR-CA, and therefore patients with a LVEF ≤40% could derive benefit from beta-blockade.23 Lastly, the majority were treated with bisoprolol (a cardio-selective beta-blocker), which potentially has a different haemodynamic profile to beta-blockers used in previous studies, while still providing neurohormonal modulation, and therefore, the observed benefit could potentially be confined to cardio-selective beta-blockers.It has been well established that patients with ATTR-CA have a similar and possibly greater neurohormonal activation than is observed in patients with HF of different aetiologies. Furthermore, elevated neurohormone levels  have been associated with a worse prognosis.22 While clinical trials are clearly needed, we believe that the data presented in this study call into question the consensus recommendations to discontinue beta-blockers and that neglect to mention MRAs.18While the observational analyses reported here have limitations in their ability to provide causal estimates of treatments in individuals with ATTR-CA, they do raise the question as to whether there could be benefit from some neurohumoral therapies in such patients and support testing this hypothesis in prospective randomized controlled trials.There is an unavoidable prescription bias, with comorbid patients with more advanced cardiac disease being treated with more HF medications; but it is also possible that clinicians may have avoided using HF medications in some higher risk patients. Treatment decisions were made on a case-by-case basis, and therefore clinical decisions must factor in each individual’s tolerance of HF medications. It is possible that patients may have discontinued HF medications prior to their first NAC assessment, and this could not be factored into the analysis. Although we performed multivariable adjustment and PS matching to account for confounders known to impact mortality in ATTR-CA, we cannot exclude the possibility of residual confounding. The present study should be considered hypothesis-generating and highlights the urgent need for randomized controlled trials. Some of the estimated HRs generated following pre-specified subgroup analysis were imprecise, and is likely to reflect the unavoidably small sample size. Lastly, a small minority were treated with angiotensin receptor-neprilysin inhibitors or sodium-glucose cotransporter 2 inhibitors and had a short duration of follow-up. Therefore, they were not included in the present study, and further studies will be required to assess these medications in patients with ATTR-CA.In summary, in this large cohort of patients with ATTR-CA, a relatively low proportion were treated with conventional HF medications, and those that had a more severe cardiac phenotype were more commonly treated with HF medications. Beta-blockers and ACEi/ARBs were often prescribed at a low dose, and frequently discontinued; in contrast to MRAs which were rarely discontinued. Both regression and PS-matched analyses demonstrated that treatment with a MRA was independently associated with a lower risk of mortality in the overall ATTR-CA population; and PS-matched analysis demonstrated that treatment with a low-dose beta-blocker was independently associated with a lower risk of mortality in patients with a LVEF ≤40%, but these findings require confirmation in prospective randomized controlled trials.European Heart Journal online.All authors declare no conflict of interest for this contribution.ehad347_Supplementary_DataClick here for additional data file.


Results: History of HF, anterior wall myocardial infarction and Killip class II-IV (32% vs. 14% p < 0.001) were more prevalent in those with reduced compared to mildly-reduced LVEF. ACEI/ARB/ARNI and beta-blockers were used by the majority of patients in both LVEF groups, though ARNI was prescribed to only 3.9% (LVEF ≤ 40%). MRA was used by 42.9% and 12.2% of patients with LVEF ≤40% and 41–49%, respectively, and SGLT2I in about a quarter of both LVEF groups. Overall, ≥3 HF drug classes were documented in 44% of the patients. A trend towards higher rates of 90-day HF rehospitalizations, recurrent ACS or all-cause death was noted in those with reduced (7.6%) vs. mildly-reduced (3.7%) LVEF, p = 0.084. No association was observed between the number of HF drug classes or the use of ARNI and/or SGLT2I with adverse clinical outcomes. Conclusions: In current clinical practice, the majority of patients with reduced and mildly-reduced LVEF are treated by ACEI/ARB and beta-blockers early following ACS, whereas MRA is underutilized and the adoption of SGLT2I and ARNI is low. A greater number of therapeutic classes was not associated with reduced short-term rehospitalizations or mortality.Background: Guideline-directed medical therapies for heart failure (HF) may benefit patients with reduced left ventricular ejection fraction (LVEF) following acute coronary syndromes (ACS). Few real-world data are available regarding the early implementation of HF therapies in patients with ACS and reduced LVEF. Methods: Data collected from the 2021 nationwide, prospective ACS Israeli Survey (ACSIS). Drug classes included: (a) angiotensin-converting enzyme inhibitors (ACEI), angiotensin receptor blockers (ARB) or angiotensin receptor-neprilysin inhibitors (ARNI); (b) beta-blockers; (c) mineralocorticoid receptor antagonist (MRA) and (d) sodium-glucose cotransporter-2 inhibitors (SGLT2I). The utilization of HF therapies at discharge or 90 days following ACS was analyzed in relation to LVEF [reduced ≤40% ( The pharmacotherapy of heart failure (HF) with reduced left ventricular ejection fraction (LVEF) HFrEF; ≤40%) is historically based on the modulation of the renin-angiotensin-aldosterone axis and the sympathetic nervous system . Angiote0% is hisPatients with reduced LVEF following myocardial infarction (MI) are at an increased risk of adverse events such as death and recurrent hospitalizations due to exacerbation of HF . The aboThe aim of the present study was to investigate the early implementation of HF therapies in patients admitted with acute coronary syndrome (ACS) and evidence of reduced (≤40%) compared to mildly reduced (41–49%) LVEF. Furthermore, we wished to analyze the association between the utilization of evidence-based HF therapies as documented at discharge and 90-day post-hospitalization with short-term adverse cardiovascular outcomes and mortality.Data were retrieved from the Acute Coronary Syndrome Israeli Survey (ACSIS). The ACSIS is a national survey that is conducted over a 2-month period every 2–3 years. Data were collected prospectively from all consecutive patients hospitalized with a diagnosis of ACS in all 25 coronary care units and cardiology wards operating in Israel. Patient management was at the discretion of the treating physicians. Eligibility for the survey was validated before discharge. Discharge diagnoses were recorded as determined by the attending physicians based on clinical, electrocardiographic, echocardiographic, and biomarker criteria. Demographic, historical and clinical data, including medical management, were recorded on pre-specified e-forms by dedicated study personnel. The survey is governed and coordinated by the working group on acute cardiovascular care of the Israeli Heart Society in collaboration with the Israeli Center for Cardiovascular Research (ICCR). The data storage, maintenance, and processing are performed by the ICCR, which also reviews documents to ensure data validity and quality. Ninety-day clinical adverse outcomes and 1-year mortality were ascertained by hospital chart review, telephone contact, and use of the Israeli National Population Registry. The ACSIS was approved by the ethics committees of all participating centers and was conducted according to the principles expressed in the Declaration of Helsinki. All patients provided written informed consent for the collection of the data and subsequent analysis. Endpoints were prespecified by the ACSIS steering committee.In the current study, the latest ACSIS survey performed during February–March 2021 was analyzed, in order to enable the assessment of novel evidence-based HF drug classes. The survey included 1684 consecutive patients with a discharge diagnosis of ACS, including unstable angina (UA), non-ST-segment elevation myocardial infarction (NSTEMI), and ST-segment elevation myocardial infarction (STEMI). We investigated 661 of the 1684 patients that had in-hospital echocardiographic evidence of reduced LVEF of less than 50% and survived the acute hospitalization. Participants were categorized as reduced LVEF ≤ 40% or mildly reduced LVEF 41–49%. The study flowchart is presented in Data on demographics, baseline characteristics, risk factors, medical history , and medication history were prospectively collected. In addition, in-hospital information on ACS presentation, angiographic and revascularization data, use of cardiovascular procedures, in-hospital complications, laboratory values and pharmacologic management, were obtained until discharge. The drugs analyzed for the management of patients with reduced and mildly-reduced LVEF were categorized into four therapeutic groups: (a) beta-blockers, (b) ACEI, ARBs or ARNI, (c) MRA and (d) SGLT2I. In addition, the use of diuretics was documented. The utilization of medications was examined at several time points: (a) chronic use prior to the current admission, (b) medications initiated during the index hospitalization, and (c) at discharge or up to 90-day follow-up. n = 661). Referral to and participation in a cardiac rehabilitation program was also recorded. Clinical adverse outcomes within 90 days post-hospitalization (available in 547 of the 661 patients) included recurrent ACS, stent thrombosis, urgent revascularization, stroke, HF hospitalization and aborted sudden cardiac death. In addition, cardiac and all-cause mortality of patients surviving hospitalization was documented up to 1-year from discharge in all study participants  and LVEF groups . A McNemar’s test was used to compare frequencies of HF therapeutic classes between the LVEF groups in two time points (pre-admission versus discharge or 90-day follow-up). Cardiovascular outcomes and mortality were analyzed in relation to LVEF groups, the number of therapeutic classes used and the use of ARNI and/or SGLT2Is. Pairwise comparisons between 0–1, 2 and 3–4 HF therapy groups were performed with Bonferroni’s correction for the 3 comparisons in each LVEF group. Overall, data had about 10% missing values and no imputation was performed; data on HF drug therapies were complete. All tests were conducted at a two-sided overall 5% significance level (α = 0.05). All statistical analyses were performed using R statistical software .Continuous variables are reported as means and standard deviation (±SD) or medians and interquartile range (IQR), as appropriate. Categorical variables are described as numbers and percentages. Study groups were compared using the chi-square test for categorical variables and Student’s p = 0.002). Anterior location of MI on electrocardiogram was more typical in the reduced LVEF group, with the left anterior descending coronary artery the infarct-related artery in 75% of STEMI patients with an LVEF ≤ 40% compared to 45% in those with LVEF 41–49%  had an LVEF ≤ 40% and 255 (38.6%) had an LVEF of 41–49%. The mean age of the study population was 64 ± 12 years and 82% were males. Patients’ baseline characteristics according to the LVEF group (≤40% vs. 41–49%) are presented in F 41–49% . In-hospp < 0.001 .More than half of the patients presenting with ACS and reduced or mildly-reduced left ventricular systolic function were not treated by any of the HF drug classes before admission. At discharge or during 90-day follow-up, 84.3% of the patients were treated by two or more therapeutic classes, 43.6% by ≥3 drugs, and 8.5% by all four groups, with higher rates of utilization of 2–4 drug classes among patients with reduced compared to the mildly reduced LVEF group,  < 0.001 . A descrACEI, angiotensin-converting enzyme inhibitors; ARB, angiotensin receptor blockers; ARNI, angiotensin receptor neprilysin inhibitor; BBC, beta-blockers; EF, ejection fraction; MRA, mineralocorticoid receptor antagonist; SGLT2I, sodium-glucose cotransporter 2 inhibitors.p = 0.085 (p = 0.084). One-year mortality rates of patients surviving hospitalization were numerically higher in the reduced (4.2%) compared to the mildly-reduced (1.6%) LVEF group, but did not reach statistical significance, p = 0.101. No significant differences were observed in the rate of clinical adverse outcomes between 0–1, 2 and 3–4 HF therapeutic groups used at discharge or 90-day follow-up, in both LVEF groups . Overall, the four pillars of HF management were documented early following ACS in 11.3% of patients with reduced and 3.9% of mildly-reduced LVEF. The utilization of MRA, ARNI, and diuretics was significantly lower in those with mildly-reduced LVEF. Although a trend for increased rehospitalizations and mortality was noted in patients with an LVEF ≤ 40% compared to 41–49%, no significant association was seen between the number of HF drug classes used and adverse clinical outcomes, in both LVEF groups, including in participants receiving ARNI and/or SGLT2I.Among patients with acute MI, HF is a powerful predictor of death, and has important implications for medical treatment . Post-MISeveral post-MI trials of therapies with proven efficacy in patients with chronic HFrEF have demonstrated benefits also in the post-MI population with reduced LVEF. Among these therapies are beta-blockers, ACEI or ARBs and MRA . HoweverRecent ESC HF guidelines 2021) have changed the terminology of HF with mid-range LVEF to HF with mildly reduced LVEF (HFmrEF), as retrospective subgroup analysis of clinical trials and observational studies that included patients with LVEF 40–50% have shown some benefit from proven therapies for HFrEF 021 have . HoweverIn the current survey, the implementation of ARNI early following ACS was low, reaching 4% in the LVEF ≤ 40% group. Contributing to the low utilization rate is the fact that ARNI is included in the national health basket in Israel only for chronic HF patients with LVEF ≤ 35% who are currently under treatment with ACEI/ARBs and beta-blockers. In addition, ARNI may not have been widely used as there is a lack of evidence on its beneficial effects early post-MI,  althoughIn the ACSIS survey, only a small minority of the ACS patients with systolic dysfunction have received all four HF medication classes at discharge or at 90 days following hospitalization, though a significantly higher percentage (44%) had documentation of ≥3 drug classes. This is in line with recent findings from a prospective, global study on hospitalized patients with acute HF, reporting the prescription of three classes of guideline-recommended drugs , in only 37% of patients at discharge (41% in high-income countries) and 34% of survivors at 6 months follow-up . AlthougIn the current analysis, no short-term survival benefit or reduced rehospitalization rates were observed with the increase in the number of HF drug classes used or the utilization of ARNI and/or SGLT2I at discharge or 90-day follow-up, in both LVEF groups. As not all patients presenting with ACS and reduced LVEF develop a clinical syndrome of HF complicating MI, it is possible that there is a less apparent benefit from specific HF therapies in the short-term post-hospitalization phase. Additional speculations for the lack of benefit of more intensive therapy in our study population include the small sample size and observational design, residual confounding factors, and the possibility of reverse causation, such as the prescription of a higher number of therapeutic classes to higher-risk patients.This study has several limitations. First, the number of patients participating in the survey and included in the current analysis was relatively low, as was the representation of women. Nevertheless, the survey is a prospective nationwide sample, evaluating consecutive patients and representing all Cardiology departments in the country. Second, LVEF was determined according to a single echocardiogram test performed during hospitalization, and no repeat assessment was available to examine whether systolic dysfunction following MI was partially reversible or transient. Third, we did not have information on electrolyte disturbances nor excluded patients with severe kidney dysfunction. Fourth, the ACSIS survey assessed HF drug treatment at several time points but did not include data on drug dosages, and therefore, we could not assess the titration of medications to optimal doses and physiological targets. Fifth, the collected data were recorded as determined by the attending physicians in each study site, and therefore, it may be subject to confounding factors. Finally, as this is a prospective, multicenter, observational survey, the association between treatment patterns and outcomes does not determine causality.In a contemporary nationwide survey of patients admitted with an ACS, most patients with reduced or mildly-reduced LVEF were treated by ACEI/ARB and beta-blocker at discharge or 90-day post-hospitalization, though MRA was underutilized and the adoption of SGLT2I and ARNI was low. Only a minority of patients with systolic left ventricular dysfunction early following ACS were treated by all four pharmacological pillars of HF management. No association was noted between the number of HF therapeutic classes used or utilization of ARNI and/or SGLT2I with short-term rehospitalizations or mortality. The results emphasize the need for better optimization of drug therapy early following ACS in patients with reduced LVEF, and the requirement of further studies to affirm the benefit of combination HF therapies immediately post-MI in patients with systolic dysfunction that are at increased risk for progression to symptomatic HF.


Ethiopia has made significant progress in reducing malnutrition in the past two decades. Despite such improvements, a substantial segment of the country’s population remains chronically undernourished and suffers from micronutrient deficiencies and from increasing diet-related non-communicable diseases such as diabetes, hypertension and cancer. This survey aims to assess anthropometric status, dietary intake and micronutrient status of Ethiopian children, women and adolescent girls. The study will also assess coverage of direct and indirect nutrition-related interventions and map agricultural soil nutrients. The survey will serve as a baseline for the recently developed Ethiopian Food System Transformation Plan and will inform the implementation of the National Food and Nutrition Strategy.As a population-based, cross-sectional survey, the study will collect data from the 10 regions and 2 city administrations of Ethiopia. The study population will be women of reproductive age, children aged 0–59 months, school-aged children and adolescent girls. A total of 16 596 households will be surveyed, allowing the generation of national and regional estimates. A two-stage stratified cluster sampling procedure will be used to select households. In the first stage, 639 enumeration areas (EAs) will be selected using probability-proportional-to-size allocation. In the second stage, 26 eligible households will be selected within each EA using systematic random selection. Primary outcomes include coverage of direct and indirect nutrition interventions, infant and young child feeding (IYCF) practices, food insecurity, dietary intakes, mental health, anthropometric status, micronutrient status and soil nutrient status.The protocol was fully reviewed and approved by the Institutional Review Board of the Ethiopian Public Health Institute (protocol no: EPHI-IRB-317–2020). The study is based on voluntary participation and written informed consent is required from study participants. The findings will be disseminated via forums and conferences and will be submitted for publication in peer-reviewed journals. The survey covers a large geographical area, collecting data on anthropometric status, 24 hours recall quantitative dietary intakes and the determination of micronutrient status in the same participants or household, while also capturing data on the food system in Ethiopia.The study aims to improve understanding of nutritional problems across multiple facets—from agricultural soil to people to the environment in Ethiopia.Inherent to the cross-sectional design of the study, the findings of this study cannot be used to establish cause and effect.The study design prevents us from considering seasonal differences in nutritional outcomes and determinants.Globally, one in every three persons is affected by one of more forms of malnutrition.2).Despite significant progress over the past two decades, the burden of malnutrition in Ethiopia remains high.The Sustainable Development Goals (SDGs) recognise the importance of nutrition, primarily driven by the need to mitigate its detrimental consequences. Further, the 2012 World Health Assembly identified global targets to be achieved by 2025 that aim to reduce stunting, anaemia, low birth weight and childhood obesity. These targets are used to track progress in SDG 2: zero hunger.Multiple factors operating at the immediate, underlying and basic levels contribute to malnutrition.Therefore, this study aims to provide the first ever comprehensive information on the nutritional status of different populations in Ethiopia to support evidence-based implementation of the National Food and Nutrition Strategy.The overall goal of this study will be to produce nationally and regionally representative estimates on anthropometric status, coverage of nutrition interventions, dietary intakes, and micronutrient status for children, adolescent girls and WRA in Ethiopia.Specific objectives include:Assess the coverage of direct and indirect nutrition interventions.Assess food consumption patterns and nutrient intake of children aged 6–59 months and WRA.12)Assess the micronutrient status of children , adolescent girls and WRA , adolescent girls and WRA.Assess the geographical distribution of soil micronutrient status in the Ethiopian agricultural soil.This study is a nationally and subnationally  representative cross-sectional survey that will characterise dietary intake, micronutrient status and access to nutrition-related services for different target populations. Given that soil nutrient content can influence micronutrient content of foods and hence affect nutrient intake, the soil nutrient composition will also be analysed. The study will have four main components. The first component will assess nutrition-specific and nutrition-sensitive indicators (NSS) for all target groups  using semistructured questionnaires. The second component will measure quantitative dietary intake for children aged 6–59 months and WRA (15–49 years). The third component of the survey will collect biomarker samples from all children (6–59 months), school-age children (6–12 years), adolescent girls (10–19 years) and WRA (15–49 years). The final component of the study will measure micronutrients in agricultural soils. The study data will be collected from July 2021 to December 2023.Ethiopia has an estimated population size of 120 million and is the second most populous country in Africa.The target population of this study are (1) WRA aged 15–49 years, (2) Children aged 0–59 months, (3) School-age children aged 6–12 years, (4) Adolescent girls aged 10–19 years and (5) Household heads.Sample size was estimated to guarantee adequate precision to generate national and regional estimates for selected indicators for each study target group. Indicators used for each target group are shown in 10.1136/bmjopen-2022-067641.supp14Supplementary data10.1136/bmjopen-2022-067641.supp15Supplementary dataFor WRA, dietary and biomarker data will be collected in half of the selected households within each enumeration area (EA). This selection will yield a total sample size of 7386 WRA (50% of the expected 14 772 WRA). The sample size needed to assess dietary intakes and micronutrient status of WRA was calculated using the prevalence of inadequate zinc intake, which yielded the largest sample size.8 19A two-stage stratified cluster sampling procedure will be used to select households. In the first stage, 639 EAs, 257 urban and 382 rural, will be selected using probability-proportional-to-size allocation. We will use the 2018 Ethiopia Population and Housing Census EAs sampling frame to select EAs (the primary sampling units). The Central Statistical Agency prepared the EAs sampling frame. An EA typically contains 100–150 households. EA maps will be used to delineate the boundaries of the selected EA. In the second stage of sampling to identify eligible households, all households with the EA will be listed. A household will be eligible for selection if at least one of the study target groups are residents (de jure) or stayed at the household the night before the interview (de factor).Twenty-six (26) eligible households will be selected within each EA using systematic random selection. All target groups will be eligible for the NSS interview in the selected households. All children aged 6–59 months will also be eligible for dietary assessment. Women residing in 13 households (out of 26 households) who will be selected randomly will be eligible for dietary assessment. Biomarker samples will be collected for all children under 5 years of age, school-age children and adolescents in the selected households. Similar to dietary assessment, biomarker samples will be collected for women residing in half of the selected households .A structured questionnaire will be used to determine the coverage of direct and indirect nutrition interventions provided to children aged 6–59 months, WRA and adolescent girls. Direct nutrition interventions included vitamin A supplementation, iron supplementation, zinc supplementation, growth monitoring and promotion, nutrition counselling services, and food fortification. Water, sanitation and hygiene, coverage of food or cash assistance programme, women empowerment, and mental health will be some nutrition-sensitive indicators considered in this study . We willUsing standardised procedures, anthropometric measurements, including weight, height/length and mid-upper arm circumference, will be taken for all study target populations.IYCF practices will be assessed using the new WHO and UNICEF recommended 17 indicators to evaluate IYCF practices.21The Food Insecurity Experience Scale (FIES) will be used to assess household food security.In addition to these outcome indicators, information on the sociodemographic characteristic of households, child health, maternal health, employment status and household agricultural practices will be collected using structured questionnaires.Common mental health disorders will be assessed using the WHO Self-reporting Questionnaire which consists of 20 questions. Women will be classified as having a common mental health disorder if the row score will be greater or equal to 6 out of 20.23We will measure dietary intake for children aged 6–59 months and WRA. A 1 day quantitative multiple-pass 24-hour recall will be conducted to assess dietary intakes. The interactive multiple-pass 24-hour recall interview consists of four steps designed to enhance memory.We will use 15 food groups to assess the dietary intakes of women (15–49 months) and children aged 24–59 months. These food groups were: (1) Cereals and their products, (2) Starchy roots and tubers, and their products, (3) Pulses and their products, (4) Vegetables and their products, (5) Fruits and their products, (6) Meat and poultry their products, (7) Eggs and their products, (8) Fish, shellfish and their products, (9) Milk and milk products, (10) Fats and oils, (11) Nuts and seeds, (12) Sugar and sweetened products, (13) Beverages, (14) Spices and condiments, and (15) Miscellaneous. For children aged 6–23 months, we will use the updated WHO, UNICEF food groups: (1) Breast milk, (2) Grains, roots and tubers, (3) Pulses, nuts and seeds, (4) Dairy products, (5) Flesh foods , (6) Eggs, (7) Vitamin-A rich fruits and vegetables, and (8) Other fruits and vegetables. These food groups were adapted from the FAO/WHO Global Individual Food consumption data Tool food groups.25We carried out presurvey work to aid dietary data collection following recommendations set by the Intake: Centre for Dietary Assessment.12, red blood cell (RBC) folate and 25-hydroxyvitamin D. Additionally, markers of inflammation, alpha(1)-acid glycoprotein (AGP), high-sensitivity C reactive protein (hsCRP) will also be measured. We will also analyse parasites from stool specimens. All laboratory analyses will be performed at the EPHI Clinical chemistry, and Food Science and Nutrition Laboratories. Both laboratories participate in an external quality assessment scheme and are accredited by the Ethiopian National Accreditation Office. Collection, storage and analytical procedures for blood, urine, stool and salt samples are described below. The details of each biomarker analysis are described in Blood specimens will be collected from the study population to determine serum retinol, ferritin, soluble transferrin receptor (sTfR), zinc, folate, vitamin B10.1136/bmjopen-2022-067641.supp1Supplementary data10.1136/bmjopen-2022-067641.supp2Supplementary data10.1136/bmjopen-2022-067641.supp3Supplementary data10.1136/bmjopen-2022-067641.supp4Supplementary data10.1136/bmjopen-2022-067641.supp5Supplementary data10.1136/bmjopen-2022-067641.supp6Supplementary data10.1136/bmjopen-2022-067641.supp7Supplementary data10.1136/bmjopen-2022-067641.supp8Supplementary data10.1136/bmjopen-2022-067641.supp9Supplementary data10.1136/bmjopen-2022-067641.supp10Supplementary data10.1136/bmjopen-2022-067641.supp11Supplementary dataPlasmodium.12 and ferritin will be measured using Cobas 6000 analyzer . Serum retinol will be measured using the high-performance liquid chromatography method,Venous blood samples (5–7 mL) will be collected using vacutainer tubes following standard operating procedures.31Stool samples will be collected using stool cups and stored in 10% formalin to preserve the parasite until analysis.36Salt samples will be collected from households with WRA for whom dietary data will be collected. At least 25 grams (one coffee cup) of salt will be collected to determine iodine content using the iodometric titration method.37Soil samples will be collected from three households in each EA. Zigzag or cross-sampling method will be used to collect 10–20 subsamples (0–30 cm depth) constituting one composite sample. Subsamples will be collected at a separation distance of 5 m. After thoroughly mixing the composite samples, 1 kg soil sample will be transferred to polyethylene bags. The collected soil samples will be air-dried in wooden trays and disaggregated using a ceramic mortar and pestle (soil grinder) at the EPHI soil laboratory. Samples will then pass through a 6 mm sieve of stainless steel screens to remove debris and homogenise the soil sample. The sieved fraction will be further pulverised to pass through a 1 mm sieve for the micronutrient analysis. Soil zinc, iron, copper and manganese will be determined following standard procedures.Training of trainers on components of the survey will be held before training the data collectors and supervisors. After 15 days of training on methodological procedures, questionnaires and quality assurance, the questionnaires will be tested in a pilot group , and adapted based on the received feedback from the survey team. The questionnaires (including the food list) were translated into local languages  and back-translated to English to ensure the quality of the translation. The data collectors’ measurements will be standardised to ensure that the interobserver variability is within tolerable limits. Supervisors received additional training on teamwork and on monitoring and supervising the data collection process. All data collection tools are programmed using open-source software (ODK) . Data qu10.1136/bmjopen-2022-067641.supp12Supplementary dataThe primary data analysis will focus on computing frequencies and percentages for categorical variables and summary statistics  for summarising continuous variables. Sample weights will be constructed based on the selection probabilities of EAs, eligible households and non-response rates. All analyses will also be adjusted for the survey design. Additional subgroup analysis will be computed for variables with adequate sample sizes for each category. The Biomarkers Reflecting Inflammation and Nutrition Determinants of Anemia working group’s regression correction approach will be used to account for inflammation in the study of all micronutrients status using the biomarkers CRP and AGP. Geostatistical analyses will be employed to determine the spatial patterns of micronutrient distribution in the soil and blood samples. The wealth index will be constructed using principal component analysis.None.The study protocol is approved by the Institutional Review Board of the EPHI (protocol no: EPHI-IRB-317–2020). Written informed consent will be obtained from each respondent and participants may withdraw at any time . Confide10.1136/bmjopen-2022-067641.supp13Supplementary dataThe study’s findings will be disseminated through several communication channels, including stakeholder workshops, various local and international conferences, and technical reports. Additionally, the findings will be submitted for publication in peer-reviewed journals.This comprehensive, nationally representative survey will for the first time characterise simultaneously the dietary intake and micronutrient status of Ethiopian children, adolescent girls and WRA. Besides, the study assesses key drivers of malnutrition including soil nutrient composition, as well as coverage of direct and indirect nutrition interventions. The survey will provide key insights informing the implementation of Ethiopia’s National Food and Nutrition Strategy.High-quality and timely data are critical to assess the burden of nutritional problems, identify vulnerable populations and priority actions, track the implementation of nutrition programmes, and assess impact.This study will allow us to evaluate progress relative to previous food consumption and micronutrient surveys, but, more importantly, will serve as a baseline against which future progress related to the implementation of the National Food and Nutrition Strategy will be evaluated. Furthermore, the current survey will also serve as a baseline for the Ethiopian Food System Transformation Plan by capturing the majority of indicators used for monitoring food systems-related progress, thus filling information gaps that could have impeded successful implementation of the National Food and Nutrition Strategy. By establishing 13 strategic objectives, the National Food and Nutrition Strategy is intended to be aligned with the strategic directions of the Food and Nutrition Policy. Each strategy direction includes initiatives, actions and key performance indicators, as well as leading and collaborating sectors. The key performance indicators should be evaluated to determine the progress of each implementing sector’s achievement. The current survey will provide up-to-date national and subnational information on the current food and nutrition situation in Ethiopia for different target populations as well as provide a comprehensive list of indicators that are pertinent to the implementation of the policy.Additionally, effective multisectoral interventions that address the immediate and underlying determinants of malnutrition must be implemented in order to accelerate the reduction of malnutrition in all its forms.12 that have not been included in previous studies. Finally, the inclusion of adolescent girls and school-age children will provide vital information on nutritional indicators for these target groups, which are often not included in other nationally representative surveys. This survey will also provide information on the coverage of direct interventions implemented in the health sectors and indirect interventions implemented in the agriculture, Water, Sanitation and Hygiene (WASH), education and social protection sectors for whom scant data exist. Hence, this study will provide valuable information that will guide the implementation of strategic actions for the reduction of malnutrition in Ethiopia.Although this study will provide regionally and nationally representative estimates for key indicators and critical life stages, it has several limitations. Inherent to the cross-sectional design of the study, the findings of this study cannot be used to establish cause and effect. Additionally, the design prevents us from considering seasonal differences in nutritional outcomes and determinants. This study also relies on self-reported data, which are subject to recall bias. Notwithstanding the abovementioned limitations, this study is uniquely designed to combine the assessment of anthropometric status, 24-hour recall quantitative dietary intakes and the determination of micronutrient status in the same participants, while at the same time capturing data on the food system. Additionally, the study will be evaluating micronutrients in agricultural soil, which will expand our understanding of factors that influence nutrition. To the best of our knowledge, this will be among—if not—the first study to simultaneously collect these variables from the same household. This could contribute to a better understanding of nutritional problems across multiple facets—from soil to people to the environment. In the past, nutrition programmes implemented in Ethiopia have relied on information provided from small-scale studies and population-based surveys such as the Ethiopia Demographic and Health Surveys.


Naganawa et al. Nature Communications 10.1038/s41467-023-40507-3 (2023)1. Their main concern is that the signals we detected in the ventral region represent artifacts rather than lymphatic signals2. In the first revision of our manuscript, we detailed the effects of TR, TE, and TI variation in our MR technique on signals measured in a healthy volunteer. Unfortunately, we removed these data from the manuscript in later drafts after including the results of our phantom study. Our study used a 3D-FLAIR technique with a T2-selective inversion pulse. For comparison, Dr. Naganawa presented four different 3D-FLAIR techniques: T2-selective inversion pulse, T2-preparation pulses, conventional inversion pulse similar to our techniques, and conventional inversion using longer repetition time of 8000 (TR/TE/TI: 8000/387/2370)2. We will only discuss the first 3, as the fourth technique uses parameters significantly different from those we used (TR/TE/TI: 5000/387/1800).First, we would like to thank Dr. Naganawa and his colleagues for their interest in our article. We are happy to address their concerns regarding our article2 reported that signals in the ventral region (white arrows) were similarly visible in images obtained with the T2-selective IR , and not in images obtained from their volunteer case with the conventional inversion pulse  - Interstitial Fluid (ISF) visualization in other protein-rich regions in the calvarium outside of the brain. There is a 7.2-fold greater protein concentration in perilymphatic space (PLS) versus endolymphatic area (ELS) in the guinea pig5 used the T1 SPACE DANTE MR sequence to accurately segregate the slow-flow circuits of the lymphatic vessels from the faster-flow circuits of arteries, veins, venules, and CSF. The slower-flow lymphatic circuit was concentrated in the perisinusoidal areas along the superior sagittal, straight, transverse, sigmoid, and cavernous sinus, standing apart from venous sinuses and veins and included vessel-like compartments associated with flattened vesicles. In the neck region, extracranial lymphatics connected dural lymphatic vessels with cervical lymph nodes. The exit of lymphatics from the skull was observed along blood vessels through the jugular foramen and along the carotid canal, the superior orbital fissure, the foramen rotundum, and the foramen ovale for lymphatics of the cavernous sinus. These findings corroborate our hypothesized dorsal lymphatic flow and structures and further suggest significant ventral lymphatic flow at the skull base in humans at the level of jugular foremen, sellar region, and 5th nerve pathway. Although we detected a strong signal in the anterior cranial fossa around the olfactory nerves at the level of the internal auditory canal, Jacob et al. did not detect any lymphatic signal in these regions. These differences might relate to internal marker variances . However, we cannot completely rule out possible artifacts in these regions. Therefore, we agree that prospective and well-designed live-human studies supported by histopathology are needed for clarification. Jacob et al. did not specifically report on the internal auditory canal region. Therefore, again, the dural FLAIR signal at the level of the internal auditory canal is likely a lymphatic signal similar to other dural surfaces. Our signals that extend beyond the dura into subdural or subarachnoid spaces may represent artifacts or CSF-ISF exchange in this region toward the dural lymphatics system from subdural-subarachnoid spaces. At this time, there are not ample data regarding CSF-ISF exchange at the level of the internal auditory canal in the subdural-subarachnoid spaces. Recently, subarachnoid lymphatic structures have been shown by Mezey et al.6, but more investigation is needed to confirm this finding. The possibility of artifact at the level of the internal auditory canal in the subdural and subarachnoid spaces could not be excluded. Further histopathological studies will be needed.Recently Jacob et al.Protocol Exchange7.We have also created the step-by-step protocols used in the original publication of We want to thank Dr. Naganawa and his colleagues again for their interest in our article. We hope the responses above address their concerns.Further information on research design is available in the Reporting Summary


Clinic-based recruitment for preventative interventions for child health may select for healthier populations compared with community-based outreach. Nutritional status during infancy as measured by anthropometry is predictive of mortality, growth faltering later in life, and poor cognitive development outcomes. We evaluated baseline differences in infant nutritional status among children recruited directly in their community versus clinic recruitment among infants participating in a trial of azithromycin compared with placebo for prevention of mortality in three districts of Burkina Faso. Infants between 5 and 12 weeks of age were recruited in their community of residence via vaccine outreach teams or in primary health-care clinics during vaccine clinics. Weight, height, and mid upper arm circumference were measured. We used linear and logistic regression models to compare anthropometric outcomes among community and clinic recruited infants, adjusting for age at enrollment, gender, and season. Among 32,877 infants enrolled in the trial, 21,273 (64.7%) were recruited via community outreach. Mean weight-for-age z-score (WAZ) was −0.60 ± 1.2 (SD), weight-for-length z-score (WLZ) was −0.16 ± 1.5, and length-for-age z-score was−0.53 ± 1.3. Infants enrolled in the community had lower WAZ  and WLZ . Community-recruited infants were more often underweight  and wasted . There was no evidence of a difference in height-based measures. Community and clinic recruitment likely reach different populations of children. For child health, examples of these interventions include distribution of seasonal malaria chemoprevention during the high malaria transmission season, community vaccine campaigns, and community health worker-led screening for acute malnutrition.–Twice-yearly community-based distribution of azithromycin has been shown to reduce all-cause childhood mortality significantly among children 1 to 59 months of age.Burkina Faso is a landlocked country in West Africa that experiences seasonal malaria transmission and has a relatively high burden of childhood undernutrition. In a population-based sample of infants in Nouna District, Burkina Faso, approximately 20% of infants 1 to 11 months of age were underweight.–Here, we used data from an individually randomized trial of azithromycin for prevention of infant mortalityData for this secondary analysis arose from an individually randomized trial of single-dose oral azithromycin compared with placebo administered at age 5 to 12 weeks in conjunction with routine vaccination for prevention of infant mortality by 6 months of age.This study was conducted in Kossi, Banfora, and Karankasso-Vigue provinces of Burkina Faso between September 2019 and September 2022. Burkina Faso is located in the Sahel, and experiences highly seasonal rainfall from approximately July through October. In the study area, regular outreach is made to each community for childhood vaccination. Each month, a vaccination team consisting of nurses from primary health-care facilities performs outreach directly in communities to offer childhood vaccinations.Infants were eligible for the parent trial if they were between 5 and 12 weeks of age at the time of enrollment, had no documented allergy to macrolides, and their family was planning to stay in the study area for the duration of the study (6 months). Participants were recruited either directly in their communities as part of vaccine outreach visits or in the primary health-care clinic during clinic vaccinations. The location of recruitment and enrollment of each infant enrolled in the trial was recorded in the study’s mobile data collection application. Infants who were recruited in the community but referred to and enrolled in the clinic were considered community recruited.At baseline, weight, length, and mid upper arm circumference (MUAC) were collected on all infants. Weight was measured using a standard digital infant scale. A single weight measurement was acquired. Length was measured using a ShorrBoard . Length measurements were acquired in triplicate, with the median used for analysis. Mid upper arm circumference was measured using a standard MUAC tape. Weight-for-age z-score (WAZ), weight-for-length z-score (WLZ), and length-for-age z-score (LAZ) were calculated according to the WHO 2006 growth standards.Descriptive statistics were calculated among those recruited in the community and clinic using medians and interquartile ranges for continuous variables, and proportions for categorical variables. To assess the relationship between community versus clinic enrollment and continuous anthropometric measures , we constructed a series of linear regression models, including unadjusted; adjusted for age, gender, and month of enrollment; and adjusted for age, gender, month of enrollment, and distance from the child’s community of residence and the health-care facility. Because GPS coordinate data were only available for infants recruited in Kossi Province, models adjusting for distance from the community of residence to the clinic were restricted only to infants in Kossi Province. We evaluated the relationship between community versus clinic recruitment and dichotomized measures of anthropometric status, including underweight (WAZ < −2), wasted (WLZ < −2), and stunted (LAZ < −2) using a similar series of logistic regression models. The SEs of all models were clustered by the community of recruitment. Because missing data were extremely rare (< 0.1%), complete case analysis was used. All analyses were done using Stata 17.0 .Among 32,877 infants enrolled in the trial, 21,273 (64.7%) were recruited directly in their community and 11,604 (35.3%) were recruited in clinics. Infants recruited in the community tended to be younger than those recruited in clinics . As expected, infants recruited in the community lived considerably farther from the clinic than those recruited in clinics  and wasting (WLZ < −2) was significantly greater in infants recruited in the community compared with the clinic, and the relationship between recruitment location was stronger with wasting  compared with underweight  are likely healthier on average than the general population. Although randomized controlled trials of azithromycin for mortality have been underpowered for subgroup analysis, there has been some evidence that nutritional status may modify the effect of azithromycin on infant mortality.These results must be considered in the context of several limitations. All infants enrolled in the trial were reached by vaccine outreach teams or in clinics. It is possible that infants who are not reached at all by vaccination teams are substantively different than those who are. The number of “zero-dose” children, or those who have had no doses of a given vaccine series, varies substantially by country and is low in Burkina Faso.In this study of trial-enrolled infants, infants recruited via community-based vaccine outreach had lower anthropometric scores for most weight-related measures, reflecting a worse overall nutritional status. We posit that these children may be more vulnerable to poor child health outcomes, including increased risk of mortality, chronic undernutrition, and poor child development outcomes. Studies recruiting children exclusively from clinics will likely miss important subgroups that are more vulnerable. As a result, such studies may not be generalizable to populations that have reduced access to health-care facilities.


Cardiotoxicity linked with hematopoietic stem cell transplantation (HSCT) is a well-described phenomenon associated with an increased mortality risk; however, the majority of cardiac events present over 100 days following transfusion and are often attributed to graft-versus-host disease or pre-treatment conditioning by chemotherapy with or without radiation therapy. Here, we present the case of a 60-year-old female with a medical history of chronic lymphocytic leukemia complicated by a myelodysplastic syndrome that progressed to acute myeloid leukemia who developed chest pain immediately following an allogeneic HSCT. Electrocardiogram showed dynamic ST-depressions in leads V3-5 without evidence of reciprocal changes. Transthoracic echocardiography revealed pericardial effusion without signs of tamponade. The patient was thought to have acute pericarditis and was subsequently treated with high-dose intravenous methylprednisolone with a taper for two weeks. Her symptoms promptly subsided, and the pericardial effusion resolved on repeat echocardiography, which confirmed the diagnosis. Acute pericarditis is a rarely described complication of HSCT that is fatal if left untreated and prompts urgent management. This atypical case of acute pericarditis in the early post-transplant phase highlights the importance of cardiac stratification in patients with active malignancy undergoing treatment. It would suggest a potential benefit in closely monitoring high-risk individuals who have a history of coronary artery disease, smoking, or pericarditis in the pre-engraftment phase of transplantation. Allogeneic hematopoietic stem cell transplantation (HSCT) is an increasingly used treatment of choice for a variety of hematological malignancies and other non-malignant hematological disorders due to its curative potential and improved overall survival ,2. The HLong-term adverse effects associated with HSCTs are established and can be life-threatening. HSCT-associated cardiotoxicity is estimated to account for 9.8% of cardiac events, including cardiac arrhythmias, acute heart failure, and hypertension with an increased one-year mortality risk ,8. CardiA 60-year-old female with a history of secondary myelodysplastic syndrome complicated by advanced acute myeloid leukemia presented for a scheduled allogeneic stem cell transplant. Her conditioning regimen consisted of weight-based dosing of fludarabine and melphalan with total body irradiation totaling 4 Gray in two fractions. Shortly after the transplant, she developed chest pain in the sternum area described as sharp, non-radiating, and exacerbated with deep breath or position changes. In addition, she noted shortness of breath with exertion, intermittent nausea, and non-bloody, bilious emesis. She denied any vision changes, jaw pain, or epigastric or abdominal pain. The chest pain persisted overnight and the patient was seen by cardiology the following morning.The patient had no prior history of coronary artery disease, radiation therapy, heart failure, hypertension, diabetes, hyperlipidemia, or thyroid disease. Family history was negative for prior cardiac conditions. Social history was significant for a 13-pack-year smoking history quit 20 years ago. Vital signs showed the patient was normotensive and afebrile with a regular heart rate. On physical exam, heart sounds were normal with no appreciable murmur. Laboratory values revealed a peak troponin of 0.21 ng/mL (0-0.04 ng/mL). Significant anemia was not present and therefore hemolytic markers were not obtained. Electrocardiogram showed dynamic ST depressions in precordial leads V3-5 without evidence of reciprocal changes  was obtained and revealed normal left ventricular wall motion and contractility with resolution of the pericardial effusion. She was also given metoprolol 12.5 mg orally twice a day for suspected myocardial ischemia, with plans to evaluate for coronary artery disease in the outpatient setting. The patient was unfortunately lost to follow-up.Cardio-oncology is an emerging field dedicated to describing cancer therapy-related cardiac dysfunction and has thoroughly outlined intermediate- and long-term cardiac risks associated with cancer therapy . DespiteAdditionally, the pathophysiology of acute pericarditis in the setting of HSCTs remains unclear; however, it can include conditioning-related cardiotoxicity, infectious causes, or complications of graft-versus-host disease. Further, the literature review suggests that polyserositis can be an early complication of endothelial origin, related to complications occurring within the first 100 days of transfusion . This asAcute pericarditis is an uncommon complication of stem cell transplantation that can be fatal if left untreated, prompting urgent management and emphasizing the importance of early recognition and intervention. Within 24 hours of symptom onset, the patient was successfully treated with intravenous high-dose steroids. Although non-steroidal anti-inflammatory drugs and colchicine are often first-line therapies , aggressThis case is unique in the temporal association of pericarditis shortly following the infusion and unusual presentation with non-specific EKG findings. The acuity of acute pericarditis in the early post-transplant phase highlights the importance of cardiac stratification in patients with active malignancy undergoing treatment. It would thus be reasonable to maintain a high clinical suspicion for cardiac events with close monitoring of individuals who have a history of coronary artery disease, smoking, or pericarditis in the pre-engraftment phase of transplantation.


Current paradigms of anti-tumor therapies are not qualified to evacuate the malignancy ascribing to cancer stroma’s functions in accelerating tumor relapse and therapeutic resistance. Cancer-associated fibroblasts (CAFs) has been identified significantly correlated with tumor progression and therapy resistance. Thus, we aimed to probe into the CAFs characteristics in esophageal squamous cancer (ESCC) and construct a risk signature based on CAFs to predict the prognosis of ESCC patients.The GEO database provided the single-cell RNA sequencing (scRNA-seq) data. The GEO and TCGA databases were used to obtain bulk RNA-seq data and microarray data of ESCC, respectively. CAF clusters were identified from the scRNA-seq data using the Seurat R package. CAF-related prognostic genes were subsequently identified using univariate Cox regression analysis. A risk signature based on CAF-related prognostic genes was constructed using Lasso regression. Then, a nomogram model based on clinicopathological characteristics and the risk signature was developed. Consensus clustering was conducted to explore the heterogeneity of ESCC. Finally, PCR was utilized to validate the functions that hub genes play on ESCC.Six CAF clusters were identified in ESCC based on scRNA-seq data, three of which had prognostic associations. A total of 642 genes were found to be significantly correlated with CAF clusters from a pool of 17080 DEGs, and 9 genes were selected to generate a risk signature, which were mainly involved in 10 pathways such as NRF1, MYC, and TGF-Beta. The risk signature was significantly correlated with stromal and immune scores, as well as some immune cells. Multivariate analysis demonstrated that the risk signature was an independent prognostic factor for ESCC, and its potential in predicting immunotherapeutic outcomes was confirmed. A novel nomogram integrating the CAF-based risk signature and clinical stage was developed, which exhibited favorable predictability and reliability for ESCC prognosis prediction. The consensus clustering analysis further confirmed the heterogeneity of ESCC.The prognosis of ESCC can be effectively predicted by CAF-based risk signatures, and a comprehensive characterization of the CAF signature of ESCC may aid in interpreting the response of ESCC to immunotherapy and offer new strategies for cancer treatment. It primTargeted therapies have made remarkable strides in the management of diverse neoplastic conditions, such as esophageal squamous cell carcinoma (ESCC). Immunotherapy, encompassing the utilization of immune checkpoint inhibitors (ICIs)/immunomodulators, therapeutic vaccines, monoclonal antibodies, and adoptive cellular immunotherapy, constitutes a novel approach in the management of esophageal cancer (EC). Of noteworthy importance, ICIs have demonstrated efficacy in the management of melanoma and non-small cell lung cancer, and have exhibited encouraging outcomes in the treatment of advanced ESCC . An overRecent progresses in single-cell sequencing has shed new light on exploring biological systems with revolutionary solutions . DiffereNumerous studies have been conducted on CAFs in esophageal squamous cancer (ESCC), but the systematic characteristics of CAFs and their correlation with ESCC prognosis and immunotherapy response are not yet fully comprehended. In this study, we obtained single-cell RNA sequencing (scRNA-seq) data and transcriptome data from accessible databases to differentiate CAF subclusters and establish a CAF-based risk signature for ESCC. We evaluated the clinical significance of the CAF-based signature and further analyzed the immune landscape and responsiveness to immunotherapy associated with it. Finally, we developed a novel nomogram that combines the CAF-based risk signature with clinicopathological features to facilitate the clinical use of CAF features in the prognosis of ESCC. Our findings could provide novel insights into the pathophysiology of ESCC, bringing about personalized treatments and improved outcomes for patients with ESCC.22.1ESCC scRNA-seq data was acquired in Gene Expression Omnibus (GEO) database (accession number GSE191756). We screened out single cells with any gene expressed in fewer than three cells or those expressing fewer than 250 genes. The percentage of rRNA and mitochondria was then calculated with the PercentageFeatureSet function in the Seurat R package . ConsequWe further collected transcript data, single-nucleotide variants (SNV), copy number variants (CNV), and corresponding clinical data of ESCC from The Cancer Genome Atlas (TCGA) database. Samples lacking outcome status or survival data were excluded and 94 ESCC samples were obtained, which were utilized for external validation. GSE53624 data with 119 tumor samples and 119 normal ones was used as the training cohort after abandoning samples without follow-up acquired from Gene Expression Omnibus (GEO) database . Based on the literature, ten cancer-associated pathways  were identified and analyzed about their gene expression profiles in our dataset.2.2The scRNA-seq data of ESCC was re-analyzed using the Seurat package, with the aim of providing a systematic characterization of the CAF signature. To start the data preprocessing, cells with less than 250 or more than 6000 expressed genes were removed, and the remaining expressed genes were log-normalized. Next, the FindIntegrationAnchors function was utilized. To reduce the dimensionality of the data, the t-distributed Stochastic Neighbor Embedding (tSNE) method was applied, utilizing a resolution of 0.1 and selecting 30 principal components. The tSNE method employed was non-linear in nature. To classify the single cells into various subgroups, we utilized the FindNeighbors and FindClusters functions (dim = 30 and resolution = 0.1). Additionally, we performed tSNE dimensional reduction using the RuntSNE function. Fibroblasts were annotated according to four marker genes, including PDGFRB, ACTA2, FAP, and NOTCH3. Subsequently, the fibroblasts were re-clustered using the FindClusters and FindNeighbors functions. To define the marker genes for each CAF cluster, we used the FindAllMarkers function with a comparison between different clusters . We utilized the CopyKAT R package to analyze the CNV characteristics of the CAF clusters and distinguish them from tumor cells and normal ones. Finally, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was conducted on the marker genes using the clusterProfiler package .2.3Differentially expressed genes (DEGs) between normal and tumor tissue were identified using the limma package , 25, bas2.4After conducting univariate and multivariate Cox regression analyses based on the risk signature and clinicopathological features , 28, a n2.5The correlation between the risk signature and the tumor immune microenvironment (TIME) was comprehensively assessed using several algorithms, including CIBERSORT, EPIC, MCPCOUNTER, and TIMER . Stromal2.6Anti-PD-1 or anti-PD-L1 checkpoint inhibition therapy has gained increasing attention as a crucial component of immunotherapy. Transcriptomic data as well as corresponding clinical data from patients who received anti-PD-L1 therapy from the IMvigor210 cohort were collected to evaluate the performance of the risk signature in predicting responsiveness to immunotherapy (immune checkpoint blocks). Additionally, transcriptomic data from the GSE78220 cohort, which included melanoma patients who received anti-PD-1 checkpoint inhibition therapy before treatment, were downloaded.2.7To further probe into the heterogeneity of ESCC, all ESCC patients were separated into different clusters according to the expression of CAF-related genes with the R package ‘ConsensusClusterPlus’ . Differe2.8Total RNA was isolated from ESCC cells or tissues using TRIzol reagent . The complementary DNA (cDNA) was synthesized as per the manufacturer’s instructions, utilizing the RevertAid™ First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). qRT-PCR was performed with SYBR Green PCR kit  on a StepOne Real-Time PCR system (Thermo Fisher Scientific). The relative gene expression levels were quantified by employing the 2-△△CT method.2.9All statistical analyses were performed using R software (version 4.1.0). The Wilcoxon test was used for comparing two groups, while Spearman or Pearson correlation was used for correlation matrices. Survival differences through K-M curves were assessed using the Log-rank test, where statistical significance was defined as p-value < 0.05.33.1The flow-process diagram of our study was depicted in 3.2To probe into the correlations between tumor progression and the CAF clusters, we explored the features of ten tumor-related pathways based on the six CAF clusters. GSVA scores of these divergent pathways were estimated based on various CAF clusters, and the results were depicted in Furthermore, the ssGSEA score of the marker genes  and Gene Ontology (GO) analysis were conducted. As shown in 3.6After conducting an investigation into the landscape of immune and stromal cell infiltrations in both low- and high-risk groups, Additionally, the relationship between risk genes and immunity was probed into. On the one hand, 3.7Under the circumstances that T-cell immunotherapy has gained great achievements in recent years, we performed the assessment of prognostic value of our signature for immune-checkpoint therapy in GSE78220 and IMvigor210 cohorts. Divergent degrees of responsiveness of anti-PD-L1 receptor blockers were identified in the 348 patients from the IMvigor210 cohort, including partial response (PR), complete response (CR), progressive disease (PD), and stable disease (SD). As depicted in 3.8Moreover, unsupervised consensus clustering was conducted to explore molecular subtypes based on the expression of CAF-related genes comprising the risk signature. With k = 3 deemed as the optimal clustering stability, patients in GSE53624 cohort were grouped into three clusters  exhibited elevated IC50 values for chemotherapeutic medications such as Bosutinib, Gefitinib, and AICAR. Additionally, patients in cluster 1 (C1) were observed to be more receptive to AMG.706, IPA.3, and the like, while those in cluster 3 (C3) demonstrated poorer response rates to the majority of chemotherapeutic treatments  can be found within the article/All human experiments in this study have been approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University .QR, PZ, HL and YY contributed conception and design of the study. XZ finished the dada collection. XZ and YF performed the statistical analysis. QR wrote the first draft of the manuscript. HL and YY revised the manuscript. LL, HL and YY gave the final approval of the version to be submitted. All authors contributed to the article and approved the submitted version.