Hospital admission with non-alcoholic fatty liver disease is associated with increased all-cause mortality independent of cardiovascular risk factors.

Non-alcoholic fatty liver disease (NAFLD) is common and strongly associated with the metabolic syndrome. Though NAFLD may progress to end-stage liver disease, the top cause of mortality in NAFLD is cardiovascular disease (CVD). Most of the data on liver-related mortality in NAFLD derives from specialist liver centres. It is not clear if the higher reported mortality rates in individuals with non-cirrhotic NAFLD are entirely accounted for by complications of atherosclerosis and diabetes. Therefore, we aimed to describe the CVD burden and mortality in NAFLD when adjusting for metabolic risk factors using a 'real world' cohort. We performed a retrospective study of patients followed-up after an admission to non-specialist hospitals with a NAFLD-spectrum diagnosis. Non-cirrhotic NAFLD and NAFLD-cirrhosis patients were defined by ICD-10 codes. Cases were age-/sex-matched with non-NAFLD hospitalised patients. All-cause mortality over 14-years follow-up after discharge was compared between groups using Cox proportional hazard models adjusted for demographics, CVD, and metabolic syndrome components. We identified 1,802 patients with NAFLD-diagnoses: 1,091 with non-cirrhotic NAFLD and 711 with NAFLD-cirrhosis, matched to 24,737 controls. There was an increasing burden of CVD with progression of NAFLD: for congestive heart failure 3.5% control, 4.2% non-cirrhotic NAFLD, 6.6% NAFLD-cirrhosis; and for atrial fibrillation 4.7% control, 5.9% non-cirrhotic NAFLD, 12.1% NAFLD-cirrhosis. Over 14-years follow-up, crude mortality rates were 14.7% control, 13.7% non-cirrhotic NAFLD, and 40.5% NAFLD-cirrhosis. However, after adjusting for demographics, non-cirrhotic NAFLD (HR 1.3 (95% CI 1.1-1.5)) as well as NAFLD-cirrhosis (HR 3.7 (95% CI 3.0-4.5)) patients had higher mortality compared to controls. These differences remained after adjusting for CVD and metabolic syndrome components: non-cirrhotic NAFLD (HR 1.2 (95% CI 1.0-1.4)) and NAFLD-cirrhosis (HR 3.4 (95% CI 2.8-4.2)). In conclusion, from a large non-specialist registry of hospitalised patients, those with non-cirrhotic NAFLD had increased overall mortality compared to controls even after adjusting for CVD.

Introduction

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in Europe [1] and is strongly associated with all features of the metabolic syndrome [2]. The majority of patients with NAFLD have simple steatosis (non-alcoholic fatty liver, NAFL) and only a minority have non-alcoholic steatohepatitis (NASH), with or without fibrosis. However a small, but significant proportion do progress to end-stage liver disease [3].

NAFLD is thought to be associated with increased all-cause and cardiovascular mortality [4-6]. It has been established that fibrosis is the main predictor of long-term liver-related morbidity in NAFLD [3, 7–9] and that patients with NASH but no fibrosis have a similar outcome to those with NAFL and no fibrosis. However, these studies included biopsy-proven patients in specialist clinics and therefore there is likely significant ascertainment bias in estimating rates of hepatic complications. Recent data also suggests that fibrosis stage is a critical predictor of cardiovascular events in NAFLD [10]. The natural history of NAFLD and its impact upon clinical services is an important topic that divides expert opinion [11, 12].

Cardiovascular disease (CVD) is the commonest cause of mortality in patients with NAFLD [3]. A recent large-scale analysis strongly suggests that this is due to prevalence of classical CVD risk factors such as type 2 diabetes and dyslipidaemia [13]. Insulin resistance is understood to be the primary driver linking all these features of the metabolic syndrome. In response to the positive energy balance of obesity, subcutaneous adipose becomes dysfunctional and there is expansion of visceral white adipocytes, which are less insulin sensitive and have a higher basal rate of lipolysis [14]. Elevated insulin and increased substrate delivery to the liver promotes hepatic steatosis by driving increased de novo lipogenesis without increasing glucose uptake [15]. Cumulatively, this results in a rise in circulating triglycerides, impaired low-density lipoprotein clearance, and higher serum glucose. Hepatic steatosis is also thought to alter the composition of secreted lipoparticles [16].

A further increasingly important consideration is the burden of cardiovascular co-morbidity in patients with end-stage NAFLD for whom transplantation is an option [17]. There is currently comparatively limited data on the prevalence of CVD in patients with NAFLD cirrhosis [18]. One study of 133 patients with cryptogenic cirrhosis found nearly half to have diabetes and 18% had experienced a major cardiovascular or cerebrovascular event [19]. CVD events are common post-transplant sequelae and chronic kidney disease is linked to reduced graft survival [20].

Whilst several previous natural history studies have included comparison to age- and gender-matched control populations, they have been unable to control for CVD [21-23]. Therefore, it remains unclear whether NAFLD is associated with increased all-cause mortality after correction for cardiac and metabolic disease risk factors.

We aimed to first describe the burden of CVD across the NAFLD disease spectrum: non-cirrhotic-NAFLD and NAFLD-cirrhosis (end-stage fibrosis). Then to assess whether they are associated with increased all-cause mortality in a real life cohort of hospitalised UK patients from the ACALM (Algorithm for Comorbidities, Associations, Length of stay and Mortality) registry, after correction for CVD and metabolic risk factors.

Materials and methods

Study design

The study was conducted as a retrospective cohort study of adult patients in England during 2000–2013 who were admitted to 7 different hospitals with naturalistic follow-up. All available data was included. Tracing of anonymised patients was performed using the ACALM (Algorithm for Comorbidities, Associations, Length of stay and Mortality) study protocol to develop the ACALM registry and has been previously described by our group [24-29]. Briefly, medical records were obtained from local health authority computerized Hospital Activity Analysis register, which is routinely collected by all NHS hospitals. This provides fully anonymized data on hospital admissions and allows for the long-term tracing of patients at an individual hospital. The ACALM protocol uses the International Classification of Disease, 10th edition (ICD-10) and Office of Population Censuses and Surveys Classification of Interventions and Procedures (OPCS-4) coding systems to trace patients. This data was obtained separately for the seven included hospitals. Similar data could be obtained through national Hospital Episode Statistics or from any local Hospital Activity Analysis register.

Participant identification

ICD-10 codes were used to identify patients with NAFL (non-alcoholic fatty liver, K76.0), NASH (non-alcoholic steatohepatitis, K75.8), and NAFLD-cirrhosis (cryptogenic cirrhosis, K74.6). Where a patient was coded with both NAFL and NASH, they were included in the NASH group. Patients coded with both NAFL and NAFLD-cirrhosis, or NASH and NAFLD-cirrhosis, were included in the cirrhosis group. NAFL and NASH groups were combined to create a non-cirrhotic-NAFLD group. As per UK practice, the diagnosis of NAFL, NASH, or NAFLD-cirrhosis were made according to clinical judgement and the latest guidelines but the results of the investigations used to derive the diagnoses were not available. An age- and sex-matched control group (with no liver-related diagnoses) was identified from the same ACALM registry and matched 10:1 to patients with NAFLD diagnoses. In line with recent recommendations [30], patients with a history of alcohol excess (F10) or any other alcohol-related diagnosis were excluded. In addition, patients with other liver diseases were excluded, including: autoimmune hepatitis (K75.4), viral hepatitis (B15-B19), Wilson disease and haemochromatosis (E83), cholangitis (K83.0) and primary biliary cirrhosis (K74.3).

Data collection

All of these patients were then assessed for the presence of several cardiovascular co-morbidities and risk factors, including: congestive heart failure (CHF, I150.0), atrial fibrillation (I48), and non-insulin dependent diabetes mellitus (NIDDM, E11), chronic kidney disease (N18), obesity (E66.0), myocardial infarction (I21-I22), ischaemic heart disease (I20-25), ischaemic stroke (I63.9), hyperlipidaemia (E78.5), hypertension (I10), and peripheral vascular disease (I73.9). Prevalence of malignancy was collected using codes C0-C9. Patients were also assessed for liver-related events: hepatocellular carcinoma (HCC, C22.9), hepatic failure (K72), oesophageal varices (I85), portal hypertension (K76.6), splenomegaly (R16.1), and ascites (R18). A combined ‘hepatic decompensation or failure’ score was generated from the sum of all non-malignant liver-related events. Inclusion of hepatic encephalopathy (K72.9) or variceal bleeding (I98.3, I98.8, I85.9) did not identify any additional patients. Jaundice was not included due to identification of patients with obstructive (non-hepatic) jaundice.

Vital status (alive or deceased) on 31st March 2013 was determined by record linkage to the National Health Tracing Services (NHS strategic tracing service) and was received along with the raw data; this was used to calculate all-cause mortality and survival. The first admission to hospital treatment was chosen as index admission, follow-up of patients continued until 31st March 2013.

Approvals

Confidentiality of information was maintained in accordance with the UK Data Protection Act. The patient data included was fully anonymous and non-identifiable when received by the authors, and collected routinely by the hospitals. Therefore, according to local research ethics policies we were not required to seek formal ethical approval for this study.

Statistical analysis

Data analysis was performed using SPSS version 20.0 (SPSS Inc. Chicago, IL), R version 4.0.2, and GraphPad Prism version 8.0. Code used in analyses in R is available in the S1 File. Analyses were initially performed by comparing three groups: control, non-cirrhotic NAFLD (NAFL plus NASH), and NAFLD-cirrhosis. Clinical outcomes were compared between groups using chi-squared tests for categorical variables and t-tests for age. Multivariate logistic regression was used to determine adjusted odds ratios for hepatic failure/decompensation and hepatocellular carcinoma, adjusted for demographics (age, gender, ethnicity). Cox regression analysis was used to determine adjusted hazard ratios (HR) for overall mortality between groups. Cox regression was performed using four models: 1) adjusting for variations in demographics (age, gender, ethnicity); 2) adjusting for demographics plus CVD or metabolic risk factors (obesity, NIDDM, CHF, ischaemic stroke, myocardial infarction, chronic kidney disease, peripheral vascular disease, hypertension, hyperlipidaemia, ischaemic heart disease, and atrial fibrillation); 3) adjusting for demographics plus liver-related events (hepatocellular carcinoma, hepatic failure, oesophageal varices, portal hypertension, splenomegaly, and ascites); and 4) adjusting for demographics, CVD, metabolic risk factors, and liver-related events. Participants with incomplete data were excluded. Cumulative hazard survival curves were derived using Cox proportional regression models. A supplementary analysis was performed using four groups, by splitting the non-cirrhotic NAFLD group into NAFL and NASH: control, NAFL, NASH, and NAFLD-cirrhosis. All analyses from the main analyses were replicated for these four groups.

A further sub-analysis was performed by removing all participants with liver-related events and then calculating adjusted mortality hazard ratios using Cox regression adjusted for 1) demographics only, and 2) demographics, CVD, and metabolic risk factors.

Two-sided p-values were calculated for all statistical tests and then corrected for multiple testing using the Benjamini-Hochberg method. Q-values <0.05 were considered significant.

Results

1,802 patients were identified with NAFLD-spectrum diagnoses, of which 1,091 had non-cirrhotic NAFLD (994 with NAFL and 97 with NASH) and 711 with NAFLD-cirrhosis (Table 1 and S1 Table). They were matched to 24,737 hospitalised in-patient controls. The median duration of follow-up for each group was: control 5.3 years, NAFL 4.6 years, NASH 4.4 years, and cirrhosis 2.8 years (range 1 day—14 years for all groups).

Table 1

Demographics for control, non-cirrhotic-NAFLD and NAFLD-cirrhosis patients.

	Control (n = 24,737)	NAFLD (n = 1,091)	NAFLD vs. Control q-value	Cirrhosis (n = 711)	Cirrhosis vs. Control q-value	Cirrhosis vs. NAFLD q-value
Mean age (SD)	55.0 (15.4)	51.6 (15.2)	6.4E-12	63.6 (13.5)	2.0E-53	2.0E-62
Female	10,999 (44.5)	518 (47.5)	0.09	338 (47.5)	0.15	1
Caucasian	19,761 (79.9)	850 (77.9)	0.17	607 (85.4)	6.5E-04	2.10E-04
South Asian	1,652 (6.7)	140 (12.8)	4.6E-14	37 (5.2)	0.17	4.70E-07

Baseline demographics for participants included in the study. Q-values were derived from chi-squared tests (for sex and ethnicity) or t-tests (for age) with adjustment for multiple testing using the Benjamini-Hochberg method. SD, standard deviation.

The control group was older than the non-cirrhotic NAFLD group (55 years vs. 52 years, q = 6.4x10-12) but younger than the cirrhosis group (55 years vs. 64 years, q = 2.0x10-62). The non-cirrhotic NAFLD group had a higher proportion of participants self-identifying as South Asian (6.6% control vs. 12.8% non-cirrhotic NAFLD, q = 4.6x10-14).

Patients with non-cirrhotic NAFLD had a higher prevalence of metabolic risk factors (hyperlipidaemia, type 2 diabetes mellitus, obesity, and hypertension) than controls (Table 2), despite being younger. There were no differences between NAFL and NASH groups on sub-analysis (S2 Table). Compared to the NAFLD group, patients with cirrhosis were more likely to have T2DM (22% NAFLD vs. 35% cirrhosis, q = 8.7x10-10) but had a lower prevalence of hyperlipidaemia (13.3% NAFLD vs. 6.5% cirrhosis, q = 1.4x10-5).

Table 2

Cardiovascular disease burden and liver-related events across the NAFLD spectrum.

	Control (n = 24,737)	NAFLD (n = 1,091)	NAFLD vs. Control q-value	Cirrhosis (n = 711)	Cirrhosis vs. Control q-value	Cirrhosis vs. NAFLD q-value
Obesity	307 (1.2)	92 (8.4)	3.6E-77	27 (3.8)	2.0E-08	2.8E-04
Type 2 Diabetes	2328 (9.4)	235 (21.5)	4.2E-38	250 (35.2)	3.3E-110	8.7E-10
Hyperlipidaemia	2015 (8.1)	145 (13.3)	7.6E-09	46 (6.5)	0.16	1.4E-05
Hypertension	5655 (22.9)	343 (31.4)	2.2E-10	225 (31.6)	1.1E-07	1
Ischaemic heart disease	2951 (11.9)	115 (10.5)	0.24	112 (15.8)	4.0E-03	2.2E-03
Myocardial infarction	940 (3.8)	24 (2.2)	0.02	21 (3.0)	0.33	0.48
Atrial fibrillation	1174 (4.7)	64 (5.9)	0.16	86 (12.1)	3.3E-18	1.1E-05
Congestive Heart Failure	865 (3.5)	44 (4.0)	0.45	63 (8.9)	2.7E-13	7.4E-05
Ischaemic stroke	498 (2.0)	12 (1.1)	0.08	24 (3.4)	0.03	2.2E-03
Peripheral vascular disease	381 (1.5)	13 (1.2)	0.47	14 (2.0)	0.49	0.33
Chronic Kidney Disease	309 (1.2)	23 (2.1)	0.04	33 (4.6)	8.8E-14	5.5E-03
Any malignancy	1876 (7.6)	103 (9.4)	0.05	176 (24.8)	1.2E-60	1.1E-17
GI malignancy	395 (1.6)	38 (3.5)	9.4E-06	111 (15.6)	3.4E-151	6.6E-19
Hepatic failure/decompensation	116 (0.5)	61 (5.6)	1.3E-86	300 (42.2)	<1E-300	1.0E-78
Hepatocellular carcinoma	45 (0.2)	14 (1.3)	4.5E-12	93 (13.1)	<1E-300	6.5E-24
All-cause mortality	3,635 (14.7)	149 (13.7)	0.44	288 (40.5)	1.9E-225	5.9E-91

Crude rates of mortality, metabolic, cardiovascular, and liver-related outcomes for control, non-cirrhotic-NAFLD, and NAFLD-cirrhosis, patients during a 14-year study period. Q-values were derived from chi-squared tests with adjustment for multiple testing using the Benjamini-Hochberg method.

There was an increasing burden of cardiovascular co-morbidity with more advanced liver disease. Compared to the control group, patients with NASH had a higher prevalence of heart failure (10.5% vs. 3.5%, q = 4.5x10-3, S2 Table). The cirrhosis group showed higher prevalence of atrial fibrillation and CKD compared to the NAFLD group (Table 2). No differences in prevalence of ischaemic stroke were observed.

The prevalence of hepatic events (hepatic failure or decompensation and development of hepatocellular carcinoma (HCC)) was higher in all groups of patients with NAFLD (Table 2) and was similar between NAFL and NASH groups (S2 Table). The increased prevalence of liver-related events remained after adjustment for age, sex, and ethnicity (Table 3).

Table 3

Adjusted odds ratios for liver-related outcomes and adjusted mortality hazard ratios.

	NAFLD vs. Control		Cirrhosis vs. Control		Cirrhosis vs. NAFLD
	Adj. OR (95% CI)	q-value	Adj. OR (95% CI)	q-value	Adj. OR (95% CI)	q-value
Hepatic failure/decompensation	2.6 (2.3–2.9)	4.00E-57	4.9 (4.7–5.2)	<1E-300	2.4 (2.1–2.7)	3.60E-49
Hepatocellular carcinoma	2.2 (1.5–2.7)	1.10E-11	4.2 (3.8–4.6)	8.30E-107	1.9 (1.4–2.6)	7.60E-11
	HR (95% CI)	q-value	HR (95% CI)	q-value	HR (95% CI)	q-value
Mortality adjusted for demographic characteristics	1.3 (1.1–1.5)	3.70E-03	3.8 (3.4–4.2)	8.30E-146	3.7 (3.0–4.5)	1.30E-38
Mortality adjusted for demographics, metabolic risk factors, and CVD	1.2 (1.0–1.4)	0.04	3.2 (2.9–3.6)	8.30E-107	3.4 (2.8–4.2)	1.10E-33
Mortality adjusted for demographics and liver-related events	1.1 (0.9–1.3)	0.61	2.4 (2.1–2.8)	2.80E-31	3.0 (2.4–3.7)	4.80E-24
Mortality adjusted for demographics, metabolic risk factors, CVD, and liver-related events	1.0 (0.9–1.2)	0.8	2.1 (1.8–2.4)	9.00E-23	2.8 (2.3–3.5)	2.00E-21

Odds ratios for liver-related events (hepatic failure/decompensation and hepatocellular carcinoma) were calculated using multivariable logistic regression adjusted for age, sex, and ethnicity. Adjusted hazard ratios of overall mortality were calculated between control, non-cirrhotic NAFLD, and NAFLD-cirrhosis groups using Cox proportional regression. Adjustment for demographic characteristics were gender, age and ethnicity. Adjustment for metabolic risk factors and CVD included: obesity, type 2 diabetes mellitus, CHF, ischaemic stroke, myocardial infarction, chronic kidney disease, peripheral vascular disease, hypertension, hyperlipidaemia, ischaemic heart disease, and atrial fibrillation. Adjustment for liver-related events included: hepatocellular carcinoma, hepatic failure, oesophageal varices, portal hypertension, splenomegaly, and ascites. Control n = 25,780; NAFLD n = 1,343; and Cirrhosis n = 1,235. Q-values were calculated from p-values using the Benjamini-Hochberg method. Adj. OR, adjusted odds ratio; CI, confidence interval; CVD, cardiovascular disease; HR, hazard ratio.

Unadjusted 14-year all-cause mortality was 14.7% for patients in the control group, 13.7% for patients with non-cirrhotic NAFLD, and 40.5% for those with NAFLD-cirrhosis (Table 2). However, the control group were significantly older than those with non-cirrhotic NAFLD, therefore after adjustment for age, gender and ethnicity, all-cause mortality hazard ratio was higher in the non-cirrhotic NAFLD group compared to the control group (HR 1.3 (95% CI 1.1–1.5), q = 3.7x10-3, Table 3 and Fig 1A). After adjustment for cardiovascular factors, all-cause mortality was still elevated compared to the control group: non-cirrhotic NAFLD HR 1.2 (95% CI 1.0–1.4, q = 0.04, Fig 1B) and NAFLD-cirrhosis HR 3.4 (95% CI 2.8–4.2, q = 1.1x10-33). However, after adjusting for liver-related events there was no difference in mortality between controls and non-cirrhotic NAFLD patients (HR 1.1 (95% CI 0.9–1.3), q = 0.61, Fig 1C) but elevated mortality remained for those with NAFLD-cirrhosis (HR 3.0 (95% CI 2.4–3.7), q = 4.8x10-24). No differences in adjusted mortality ratios were observed between NAFL and NASH groups on sub-analysis (S1 Fig and S3 Table).

Fig 1

Adjusted cumulative hazard of mortality for hospitalised controls, patients with non-cirrhotic NAFLD, and NAFLD-cirrhosis.

Survival curves showing cumulative hazard of mortality derived from four models of adjustment using Cox proportional regression. Data shows 95% CI for control n = 24,737; NAFLD n = 1,091; and Cirrhosis n = 711. (A) Adjustment for demographic characteristics only (gender, age and ethnicity). (B) Adjustment for demographics plus CVD and metabolic risk factors (obesity, type 2 diabetes mellitus, CHF, ischaemic stroke, myocardial infarction, chronic kidney disease, peripheral vascular disease, hypertension, hyperlipidaemia, ischaemic heart disease, and atrial fibrillation). (C) Adjustment for demographics and liver-related events (hepatocellular carcinoma, hepatic failure, oesophageal varices, portal hypertension, splenomegaly, and ascites). (D) Adjustment for demographics, CVD, metabolic risk factors, and liver-related events.

After removing patients who had experienced liver-related events (S4 Table), there was no difference in hazard ratio of mortality for patients with non-cirrhotic NAFLD compared to control when adjusting for demographics (HR 1.2 (95% CI 1.0–1.4), q = 0.08).

Discussion

This study provides important non-specialist “real life” data amongst hospitalised patients demonstrating an increased mortality for patients with non-cirrhotic NAFLD, even after adjustment for CVD. This difference appeared to be due to liver-related events (hepatic failure/decompensation and HCC) over a 14 year follow-up period. The size of the cohort and lack of link to specialist liver centres reduces likelihood of bias. These data will help inform healthcare demand for this cohort of patients, complementing modelling estimates [31, 32]. In addition, we have highlighted the burden of CVD in patients with NAFLD-cirrhosis, which poses a particular issue for transplantation. Whilst all participants in our cohort were hospitalised, which may increase their risk of future clinical events, so were the controls, thus the comparisons between groups remain valid within this setting.

The strong association between NAFLD and CVD has been well established [33-35]. Relationships have been identified between NAFLD and heart failure [36], atrial fibrillation [37], hypertension [38], stroke [39], chronic kidney disease [40], and coronary artery disease. NAFLD has even been linked to increased mortality in acute heart failure [41]. However strong observational data from a large European meta-analysis suggests that NAFLD is not causal in acceleration of CVD [13]. Our data highlights the particularly increased prevalence in patients with cirrhosis and is broadly consistent with a previously reported cohort from Italy [19]. Indeed, hypertension has been highlighted as an independent risk factor for advanced fibrosis in NAFLD [42]. A further consideration is whether heart failure contributes to accelerated fibrosis in NAFLD [43], though causality is difficult to establish.

Some previous studies with biopsy-defined cohorts have been smaller, did not adjust for cardiovascular diagnoses [3, 22], and found no difference in mortality between participants with NAFLD and no fibrosis, and controls. Kim et al. used NHANES data to stratify patients by non-invasive fibrosis scores, and again found no increase in mortality in patients with ultrasound-defined NAFLD, after correction for diabetes and hypertension [21]. This may be accounted for by differences in the ethnicity of the cohort and also the general, rather than specialist, nature of our population. A more recent Italian study in a prospective, consecutively recruited cohort found that after adjusting for metabolic risk factors patients with NAFLD had higher rates of cardiovascular events [10]. Moreover, they used non-invasive fibrosis scores to demonstrate that higher fibrosis stage was associated with increased risk of cardiovascular events, which is generally consistent with our results from the NAFLD-cirrhosis group. Similar results were observed in a further biopsy-proven NAFLD cohort of 285 patients [44].

NAFLD may itself be a marker of sub-clinical CVD. For example, increased carotid intima media thickness has been found in adolescents with NAFLD [45]. This is mechanistically plausible as hepatic steatosis occurs (in part) secondary to peripheral insulin resistance and elevated substrate delivery from lipolysis of adipose tissue. Steatosis itself then contributes to systemic insulin resistance [14, 46]. Therefore, in this analysis, despite adjusting for metabolic covariates and cardiovascular risk factors, elevated mortality may reflect the sub-clinical nature of atherosclerosis associated with NAFLD, even at an early stage.

Given the shared disease mechanisms and clinical outcomes for NAFLD and CVD, these data suggest a common framework for treatment. Weight loss is the only established treatment strategy for NAFLD [47] and there is data suggesting that specific dietary regimens (including the Mediterranean diet) are beneficial [48]. The same lifestyle interventions and aggressive risk factor modification will have dual impact on reducing cardiovascular [49, 50] and hepatic events. Statins are likely to be recommended for many patients with NAFLD due to their CVD risk factors, often in combination with treatment for type 2 diabetes. Given the relative risk of incident morbidity from CVD compared to liver-related events, these treatments are likely to primarily influence cardiac events [51, 52]. We were unable to determine drug therapy in the participants included in this study, which has the potential to influence disease outcomes [53, 54] and therefore should be considered as a limitation of the work.

Whilst we were not able to determine cause of death or admission in our cohort, we were able to determine that liver decompensation events were increased in all groups relative to the control group. In addition, excluding participants who had experienced liver-related events, removed the mortality difference between control and non-cirrhotic NAFLD patients.

This study is limited by its retrospective design and the use of generic coding, which did not provide information on how the diagnosis was obtained i.e. imaging, liver function tests, or liver biopsy and we were unable to identify whether NAFLD was the cause of admission or an existing co-morbidity in cases. Therefore, the sub-analysis for NAFL and NASH groups should be interpreted with this in mind. However, coding improvements along with standardised diagnosis of NAFLD in the UK means that the impact of inaccurate coding may be low.

ICD-10 coding is likely to be a poorly sensitive method to exclude NAFLD from the control group, due to the asymptomatic nature of the condition. However this bias would likely result in an under-estimation of the hazard ratios comparing control and NAFLD groups. The use of ICD-10 codes for the exclusion of other causes of liver dysfunction (for example, viral hepatitis) and patients with a history of alcohol consumption may have also contributed to inaccurate coding. However, such biases have been limited from our previous study looking at the association between cardiovascular and respiratory conditions [29]. There is likely to be a degree of under coding of NAFLD, especially as clinical awareness of NAFLD was not optimal at the beginning of the data capture [55]. It should be noted that the follow-up in this study ended in 2013, and since there has been a progressive increase in prevalence of NAFLD as well as development in non-invasive diagnostic techniques that were not widely available in 2000–2013 (e.g. elastography)

Our analysis has illustrated that a substantial proportion of participants with cirrhosis have risk factors for vascular events. Cirrhosis is a state of disordered clotting with patients at increased risk for thrombosis yet also exhibit hyperfibrinolysis [56]. In this study we were unable to further examine the characteristics of participants experiencing vascular events due to a lack of detailed participant-level data.

Whilst the participants with non-cirrhotic NAFLD in this study were from non-specialist centres, they may still represent a more advanced subset of patients with NAFLD than the entire population of individuals with hepatic steatosis. We found 5.6% of patients initially diagnosed with non-cirrhotic NAFLD to experience a liver-related event during 14-years of follow-up, compared to 7.9% found in the biopsy-staged cohort from Hagström et al. [7] and 4.2% reported by Angulo et al. [3] The control cohort used in this study were also hospitalised patients, therefore comparisons using hazard ratios are valid within this setting. However our results are not generalisable to patients diagnosed with NAFLD outside of a hospital setting.

Similarly, the results comparing non-cirrhotic NAFLD with NAFLD-cirrhosis are likely to have been influenced by lead time bias. The cirrhosis group were significantly older than non-cirrhotic NAFLD group and were, by definition, at a more advanced stage in their disease. Therefore, the NAFLD-cirrhosis results illustrate a highly concentrated group of the most severe part of the NAFLD spectrum.

In conclusion, these results contribute to our understanding of co-morbidity, mortality and liver decompensation in patients hospitalised with NAFLD spectrum disease and demonstrates there is higher mortality independent of known cardiovascular risk factors.

Demographics for control, NAFL, NASH and NAFLD-cirrhosis patients.

(DOCX)

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Crude rates of mortality, metabolic, cardiovascular, and liver-related outcomes for control, non-cirrhotic-NAFLD, and NAFLD-cirrhosis, patients during a 14-year study period. Data is given a number of events (%). Q-values were derived from chi-squared tests with adjustment for multiple testing using the Benjamini-Hochberg method. GI malignancy, gastrointestinal malignancy.

(DOCX)

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Adjusted odds ratios for liver-related outcomes and adjusted mortality ratios.

Odds ratios for liver-related events (hepatic failure/decompensation and hepatocellular carcinoma) were calculated using multivariable logistic regression adjusted for age, sex, and ethnicity. Adjusted hazard ratios of overall mortality were calculated between control, non-cirrhotic NAFLD, and NAFLD-cirrhosis groups using Cox proportional regression. Adjustment for demographic characteristics were gender, age and ethnicity. Adjustment for cardiovascular risk factors and CVD included: obesity, type 2 diabetes mellitus, CHF, ischaemic stroke, myocardial infarction, chronic kidney disease, peripheral vascular disease, hypertension, hyperlipidaemia, ischaemic heart disease, and atrial fibrillation. Adjustment for liver-related events included: hepatocellular carcinoma, hepatic failure, oesophageal varices, portal hypertension, splenomegaly, and ascites. Control n = 25,780; NAFL n = 1,238; NASH n = 105; and Cirrhosis n = 1,235 Q-values were calculated from p-values using the Benjamini-Hochberg method. Adj. OR, adjusted odds ratio; CI, confidence interval; CVD, cardiovascular disease; HR, hazard ratio.

(DOCX)

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Adjusted mortality ratios for patients with no liver-related events.

Adjusted hazard ratios of overall mortality were calculated between control, non-cirrhotic NAFLD, and NAFLD-cirrhosis groups using Cox proportional regression after removal of all participants with liver-related events. Adjustment for demographic characteristics were gender, age and ethnicity. Adjustment for cardiovascular risk factors and CVD included: obesity, type 2 diabetes mellitus, CHF, ischaemic stroke, myocardial infarction, chronic kidney disease, peripheral vascular disease, hypertension, hyperlipidaemia, ischaemic heart disease, and atrial fibrillation. Control n = 25,551, NAFLD n = 1,241, and cirrhosis n = 591. Q-values were calculated from p-values using the Benjamini-Hochberg method. CI, confidence interval; CVD, cardiovascular disease; HR, hazard ratio.

(DOCX)

Click here for additional data file.

Adjusted cumulative hazard of mortality for hospitalised controls, patients with NAFL, NASH, and NAFLD-cirrhosis.

Survival curves showing cumulative hazard of mortality derived from four models of adjustment using Cox proportional regression. Data shows 95% CI for control n = 24,737; NAFL n = 994, NASH n = 97; and Cirrhosis n = 711. (A) adjustment for demographic characteristics only (gender, age and ethnicity). (B) adjustment for demographics plus CVD and metabolic risk factors (obesity, type 2 diabetes mellitus, CHF, ischaemic stroke, myocardial infarction, chronic kidney disease, peripheral vascular disease, hypertension, hyperlipidaemia, ischaemic heart disease, and atrial fibrillation). (C) adjustment for demographics and liver-related events (hepatocellular carcinoma, hepatic failure, oesophageal varices, portal hypertension, splenomegaly, and ascites). (D) adjustment for demographics, CVD, metabolic risk factors, and liver-related events.

(DOCX)

Click here for additional data file.

Code used in analyses in R 4.0.

(DOCX)

Click here for additional data file.

28 Jul 2020

PONE-D-19-32801

Hospital admission with non-alcoholic fatty liver disease is associated with increased all-cause mortality independent of cardiovascular risk factors

PLOS ONE

Dear Dr. Mann,

Thank you for submitting your manuscript to PLOS ONE. I sincerely apologise for the unusually delayed review timeframe for your manuscript. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Your manuscript has been assessed by four reviewers, whose comments are appended below. Although they are all generally quite positive about the study, they raise some concerns about the study timeframe, definition of study populations, and statistical analysis that should be discussed or addressed with the appropriate revisions. In addition, please feel free to use your discretion when deciding whether to include additional references in your revised manuscript.

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The research paper PONE-D-19-32801 entitled “Hospital admission with non-alcoholic fatty liver disease is associated with increased all-cause mortality independent of cardiovascular risk factors” has relevance to the scope and the audience of PONE.

This paper aimed to assess the mortality in NAFLD when adjusting for CVD risk factors in a ‘real world’ cohort of inpatients.

The authors suggest that there is a high burden of cardiovascular disease in NAFLD-cirrhosis patients. From a large “real-life” non-specialist registry of hospitalized patients, NAFLD patients have increased overall mortality and rate of liver-related complications compared to controls after adjusting for cardiovascular disease.

Major comments for the authors

1. This analysis was performed on an interesting issue: The effect of NAFLD on overall mortality in a real world setting regardless of other CVD risk factors.

2. It is a well written paper. The text, the figure and the tables are of appropriate extent and content as well as informative.

3. The references are up to date.

4. The results of the paper have practical implications in patients with NAFLD, especially those with cirrhosis.

Reviewer #2: This is an interesting study based on “real world” data.

1] The authors need to be more careful in using abbreviations. For example, “cardiovascular disease” is abbreviated in the Abstract but not used throughout the Abstract. Also, this term is not abbreviated the first time it appears in the Introduction.

The same for NAFLD, HCC etc.

Please check the whole text for similar errors.

2] Was p value 2-sided?

3] Useful refs to comment:

Athyros VG, Tziomalos K, Katsiki N, Doumas M, Karagiannis A, Mikhailidis DP. Cardiovascular risk across the histological spectrum and the clinical manifestations of non-alcoholic fatty liver disease: An update. World J Gastroenterol. 2015;21(22):6820‐6834.

Le MH, Devaki P, Ha NB, et al. Prevalence of non-alcoholic fatty liver disease and risk factors for advanced fibrosis and mortality in the United States. PLoS One. 2017;12(3):e0173499.

4] The median duration of follow-up differed between groups. Does this translate to the severity of the disease in each group? For example, do patients with cirrhosis died earlier? Please comment.

5] Although lifestyle interventions do remain the first-line therapeutic option for NAFLD/NASH, other drugs may be helpful and especially statins and antidiabetic drugs. A brief comment should be added. Useful refs

Athyros VG, Alexandrides TK, Bilianou H, et al. The use of statins alone, or in combination with pioglitazone and other drugs, for the treatment of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis and related cardiovascular risk. An Expert Panel Statement. Metabolism. 2017;71:17‐32.

Stahl EP, Dhindsa DS, Lee SK, Sandesara PB, Chalasani NP, Sperling LS. Nonalcoholic Fatty Liver Disease and the Heart: JACC State-of-the-Art Review. J Am Coll Cardiol. 2019;73(8):948‐963.

6] Drug therapy may affect clinical outcomes. The inability to evaluate drug effects in this study should be mentioned in the limitations.

Katsiki N, Perakakis N, Mantzoros C. Effects of sodium-glucose co-transporter-2 (SGLT2) inhibitors on non-alcoholic fatty liver disease/non-alcoholic steatohepatitis: Ex quo et quo vadimus?. Metabolism. 2019;98:iii‐ix.

Athyros VG, Polyzos SA, Kountouras J, et al. Non-Alcoholic Fatty Liver Disease Treatment in Patients with Type 2 Diabetes Mellitus; New Kids on the Block. Curr Vasc Pharmacol. 2020;18(2):172‐181.

Reviewer #3: This is a retrospective study based on a registry data. I have some comments and suggestions.

Why the inclusion period was only until to 2013 (and not until 2018-2019) and why now do you choose to publish these data?

The diagnosis of this pathology have suffered modification between 2000 and 2013. Therefore, the codification is not sufficiently to be sure that the patients was appropriately included. What where the modalities of diagnosis for these pathologies? I think that this is the main cornerstone of this study (the inhomogeneity of diagnosis for these pathologies).

In table 1, I do not see the statistical significance (p). It would be important. All-cause mortality has the same percentage in controls and NAFL.

In table 2 why do you not put the sixth column for p? It would be easier to follow these data.

``The cirrhosis group showed higher prevalence of heart failure, atrial fibrillation, CKD, and ischaemic heart disease, compared to the NAFL group.`` I suggest to analyze separately this subgroup of patients. It might offers very interestingly data about the relationship between thrombosis risks in patients with cirrhosis.

Reviewer #4: In “Hospital admission with non-alcoholic fatty liver disease is associated with increased all-cause mortality independent of cardiovascular risk factors” Mann and collegues tried to define whether NAFL and NASH are associated with increased all-cause mortality in hospitalised patients. The study was conducted as a retrospective cohort study using ICD-10 coding to define the presence of NAFL, NASH and NAFLD related cirrhosis.

Output of the study were: overall mortality and hepatic outcomes (liver decompensation and HCC). In addition, authors characterized metabolic and cardiovascular comorbidities of NAFLD patients recording specific ICD code, listed in methods section.

Major:

I have some concerns about the populations definition, the statistical methods and the strength of the results.

Populations definition:

1. As authors reported in study limitation section, the use of ICD code to define the presence or the absence of NAFL/NASH, is highly defective. However, some bias could be reduced considering NAFL and NASH as a unique non cirrhotic NAFLD group. Often, asymptomatic NAFLD patients classified as NAFL patients hide NASH patients with normal liver function tests and non-invasive markers of fibrosis.

2. The absence of an ICD10 which refers to NAFLD spectrum does not allow to exclude the presence of nafld among controls (as stated in study limitation section). However, the authors should stress that this bias could underestimated the HR in the analysis and not conversely.

Statistical methods:

3. Since the authors talk about prevalence of liver decompensation/failure or HCC, I suppose that were recorded ad admission in hospital (follow up start time). In statistical methods authors properly say they used Multivariate logistic regression to define factors associated with this outcome. However, they improperly use the “hazard ratio” to define the statistical outcome. In this case, being a no time-corrected method, the statistical outcome is “odds ratio”, please amend properly

4. Figure 1. I think that the Kaplan Mayer curves, being the graphic representation of an univariate model, are not the best graphic model to represent survival in this study. At follow up start time patients and controls had different prevalence of cardiovascular (and liver-related?) conditions that may affect survival. Please consider using multiple survival curve (controls vs NAFLD (as suggested in point 1) and control vs patients with cirrhosis) derived from cox-regression analyses.

5. Why liver decompensation/failure/HCC was not included in cox multivariate regression? The differences in survival may partially been explained by different prevalence of liver complications. An interesting way to test this hypothesis could be an additional cox regression analysis performed after excluding patients whit liver complications

Strength of results:

6. Concern reported in point 5 and the inability to define with certain the cause of hospital admission (follow up start time) raise some doubts that NAFLD patients included in this cohort could represents a cluster of most severe patients in which NAFLD plays a key role.

Minor:

1. Lines 124: another study described the CV burden in patients with NAFLD-cirrhosis, please discuss it (https://doi.org/10.1016/j.atherosclerosis.2017.03.038)

2. Table 1: Adjusted and unadjusted HRs for overall mortality and ORs (?) for Liver failure/decompensation and HCC should be reported in a separate table.

3. The sentence “increased mortality for patients with NAFLD, irrespective of fibrosis” (lines 277-278) is not supported by results. In this study, the only differentiation in the severity of fibrosis could be made comparing cirrhotic with non-cirrhotic patients and, among them there is a wide difference in survival (HR 3.8 vs 2.6 vs. 65.4!)

4. Lines 298-304: a recent study proved higher incidence of CV disease in patients with NAFLD compared to metabolic controls. In addition, in the same study, non-invasive markers of fibrosis identify patients at higher risk for CV events among those with NAFLD. The same study could be discussed in introduction section (lines 101-107) to affirm that fibrosis, in NAFLD patients, is the major predictors of CVE also. doi: 10.1016/j.cgh.2019.12.026

5. Please check through the paper the correct use of NAFLD/NAFL terms. Eg: in figure 1 “NAFLD” was incorrectly used instead of NAFL

**********

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Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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14 Sep 2020

Thank you for your review of our manuscript. We have carefully responded to the Reviewers’ comments and feel that we have been able to address them all adequately, such that our manuscript is substantially improved. The major changes we have made are:

– Combining NAFL and NASH groups into a single ‘non-cirrhotic NAFLD’ group

– Inclusion of liver-related events as co-variates in Cox regression analyses

– Sub-analysis by removing patients with liver-related events

– Re-formatting and re-arranging our results tables as suggested by the Reviewers

– Reviewed the inclusion/exclusion criteria for all patients in light of recent guidelines and comments from the Reviewers

– Changing our figure to a cumulative hazard survival curve derived from the Cox proportional regression models

Below is a point-by-point responses to the Reviewers’ comments.

[Line numbers refer to the ‘clean’ manuscript version.]

Reviewer #1:

� Thank you for your comments. We have updated our manuscript in response to some suggestions from the other Reviewers.

Reviewer #2: This is an interesting study based on “real world” data.

1] The authors need to be more careful in using abbreviations. For example, “cardiovascular disease” is abbreviated in the Abstract but not used throughout the Abstract. Also, this term is not abbreviated the first time it appears in the Introduction.

The same for NAFLD, HCC etc.

Please check the whole text for similar errors.

� Apologies for these inconsistencies. We have carefully corrected our abbreviations throughout.

2] Was p value 2-sided?

� Yes, we have now added this into the Methods (line 213).

3] Useful refs to comment:

Athyros VG, Tziomalos K, Katsiki N, Doumas M, Karagiannis A, Mikhailidis DP. Cardiovascular risk across the histological spectrum and the clinical manifestations of non-alcoholic fatty liver disease: An update. World J Gastroenterol. 2015;21(22):6820‐6834.

Le MH, Devaki P, Ha NB, et al. Prevalence of non-alcoholic fatty liver disease and risk factors for advanced fibrosis and mortality in the United States. PLoS One. 2017;12(3):e0173499.

� Thank you for these suggestions. We have added them.

4] The median duration of follow-up differed between groups. Does this translate to the severity of the disease in each group? For example, do patients with cirrhosis died earlier? Please comment.

� This is an important point to consider. We agree that the results of the cirrhosis group can be attributed to lead time bias. They were older at the start of follow-up and are, by definition, at a more advanced stage of disease. We have added a section in the Discussion to specifically comment on this limitation (lines 378-382).

� We do not feel that this limitation applies to NAFL/NASH groups. They are of similar ages and earlier stage disease is generally relatively asymptomatic.

5] Although lifestyle interventions do remain the first-line therapeutic option for NAFLD/NASH, other drugs may be helpful and especially statins and antidiabetic drugs. A brief comment should be added. Useful refs

Athyros VG, Alexandrides TK, Bilianou H, et al. The use of statins alone, or in combination with pioglitazone and other drugs, for the treatment of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis and related cardiovascular risk. An Expert Panel Statement. Metabolism. 2017;71:17‐32.

Stahl EP, Dhindsa DS, Lee SK, Sandesara PB, Chalasani NP, Sperling LS. Nonalcoholic Fatty Liver Disease and the Heart: JACC State-of-the-Art Review. J Am Coll Cardiol. 2019;73(8):948‐963

� Thank you for highlighting this. We have added a comment in the Discussion as well as the suggested references (lines 331-335).

6] Drug therapy may affect clinical outcomes. The inability to evaluate drug effects in this study should be mentioned in the limitations.

Katsiki N, Perakakis N, Mantzoros C. Effects of sodium-glucose co-transporter-2 (SGLT2) inhibitors on non-alcoholic fatty liver disease/non-alcoholic steatohepatitis: Ex quo et quo vadimus?. Metabolism. 2019;98:iii‐ix.

Athyros VG, Polyzos SA, Kountouras J, et al. Non-Alcoholic Fatty Liver Disease Treatment in Patients with Type 2 Diabetes Mellitus; New Kids on the Block. Curr Vasc Pharmacol. 2020;18(2):172‐181.

� This is an important consideration. We have added a comment in the Discussion as well as the suggested references (lines 335-338).

Reviewer #3:

Why the inclusion period was only until to 2013 (and not until 2018-2019) and why now do you choose to publish these data?

� We appreciate that the end of follow-up was several years ago however the process of data extraction to build our database for took two years to complete after acquiring the data in 2014.

� We unfortunately do not have the ability to update our data on these individuals due the logistical process of extracting from the NHS systems. We acknowledge the limitation of this and have now commented on it in the Discussion (lines 359-361).

� We had initially aimed to publish this data in 2016 and have updated our methodology substantially since then in light of new evidence and further reports on this subject.

� In addition, completion of this work has proved challenging for a variety of reasons, including personal/family circumstances.

� We appreciate that this delay detracts from the novelty of the results but trust that it still provides a marginal contribution to the literature of on this topic.

The diagnosis of this pathology have suffered modification between 2000 and 2013. Therefore, the codification is not sufficiently to be sure that the patients was appropriately included. What where the modalities of diagnosis for these pathologies? I think that this is the main cornerstone of this study (the inhomogeneity of diagnosis for these pathologies).

� We agree that the principal limitation of this work is that we cannot know how each coded diagnosis was identified. Also in response to Comment 1 from Reviewer #4, we have merged the NAFL and NASH groups into a non-cirrhotic NAFLD group. We have now presented all our results in the main text for: Control, non-cirrhotic NAFLD, and NAFLD-cirrhosis. We have included data as NAFL/NASH groups in the Supplementary Material.

� In light of your comments, and recent guidance on retrospective identification of NAFLD diagnoses (Noureddin et al., Gastroenterology 2020 159(2):422-427.e1), we have added more stringent exclusion criteria into our methodology. This has led to the exclusion of some patients but reassuringly the findings of our study remains unchanged. This has increased our confidence that the patients described are those with NAFLD and the effect observed are true.

� The data comes from comparatively small, district hospitals in the North of England and therefore it is unlikely that many patients will have had advanced imaging (e.g. magnetic resonance spectroscopy or Fibroscan).

� We speculate that the majority of participants will have been diagnosed with NAFLD through ultrasound or incidentally on computed tomography (CT), whereas those with NASH will have been diagnosed using liver biopsy.

In table 1, I do not see the statistical significance (p). It would be important.

� Thank you for this suggestion. We have added q-values (false-discovery rate-corrected p-values) into all our tables to help describe all the comparisons.

All-cause mortality has the same percentage in controls and NAFL.

� Crude all-cause mortality does not differ between controls and NAFL (or NAFLD) however, after adjusting for age, sex, and ethnicity they have increased mortality. The control group is older than the NAFLD cohort, therefore adjustment for age is important in this analysis. Apologies for not having been clear on this initially.

� We have added a comment in the Results to highlight this difference and the importance of the adjusted hazard ratio for interpretation of the mortality data (line 263).

In table 2 why do you not put the sixth column for p? It would be easier to follow these data.

� We have also added q-values into table 2.

``The cirrhosis group showed higher prevalence of heart failure, atrial fibrillation, CKD, and ischaemic heart disease, compared to the NAFL group.`` I suggest to analyze separately this subgroup of patients. It might offer very interestingly data about the relationship between thrombosis risks in patients with cirrhosis.

� We agree that this is an interesting sub-group however, we are unfortunately limited by the depth of data we have available on each participant. If we had further data (e.g. biochemistry, anthropometry, platelet count, coagulation profile), then we would interrogate the characteristics of participants with cirrhosis who did and did not suffer events. The drawback of our real world routinely-collected data is that it doesn’t allow detail for such an analysis.

� We have added a short paragraph in the Discussion to acknowledge this as an important topic worthy of further investigation (lines 363-367).

Reviewer #4:

Major:

Populations definition:

1. As authors reported in study limitation section, the use of ICD code to define the presence or the absence of NAFL/NASH, is highly defective. However, some bias could be reduced considering NAFL and NASH as a unique non cirrhotic NAFLD group. Often, asymptomatic NAFLD patients classified as NAFL patients hide NASH patients with normal liver function tests and non-invasive markers of fibrosis.

� We agree that this is a key limitation of attempting to separate NAFL and NASH groups, therefore we have now merged the two into a single (non-cirrhotic) NAFLD group. We have updated all our figures and tables accordingly.

� We have moved our data on NAFL and NASH groups into the Supplementary material, in order to provide the raw data for readers to review.

2. The absence of an ICD10 which refers to NAFLD spectrum does not allow to exclude the presence of nafld among controls (as stated in study limitation section). However, the authors should stress that this bias could underestimated the HR in the analysis and not conversely.

� Thank you for this suggestion we have now added a couple of sentences in the discussion to reflect this (lines 351-353).

Statistical methods:

3. Since the authors talk about prevalence of liver decompensation/failure or HCC, I suppose that were recorded ad admission in hospital (follow up start time). In statistical methods authors properly say they used Multivariate logistic regression to define factors associated with this outcome. However, they improperly use the “hazard ratio” to define the statistical outcome. In this case, being a no time-corrected method, the statistical outcome is “odds ratio”, please amend properly

� Apologies for this inaccuracy. We have now corrected all the tables and data in the text appropriately.

4. Figure 1. I think that the Kaplan Mayer curves, being the graphic representation of an univariate model, are not the best graphic model to represent survival in this study. At follow up start time patients and controls had different prevalence of cardiovascular (and liver-related?) conditions that may affect survival. Please consider using multiple survival curve (controls vs NAFLD (as suggested in point 1) and control vs patients with cirrhosis) derived from cox-regression analyses.

� Thank you for the suggestion. We have exchanged our Kaplan-Meier curve for cumulative hazard survival curves derived from the Cox proportional regression models, which more accurately reflect the differences between groups.

5. Why liver decompensation/failure/HCC was not included in cox multivariate regression? The differences in survival may partially been explained by different prevalence of liver complications. An interesting way to test this hypothesis could be an additional cox regression analysis performed after excluding patients whit liver complications.

� We have now performed four models for assessment of mortality, with adjustment for: demographics only, demographics + CVD, demographics + liver-related events, and demographics + CVD + liver-related events. This found that adjusting for liver-related events attenuated the difference in mortality between control and non-cirrhotic NAFLD group (HR 1.1 (0.9-1.3)). Apologies for not having included this before now; thank you for the suggestion.

� In addition, we have performed the sub-analysis as the Reviewer suggested (Table S4), which similarly found that after removal of these participants there was no difference in mortality between the non-cirrhotic NAFLD and control groups.

Strength of results:

6. Concern reported in point 5 and the inability to define with certain the cause of hospital admission (follow up start time) raise some doubts that NAFLD patients included in this cohort could represents a cluster of most severe patients in which NAFLD plays a key role.

� We appreciate that our results are likely to represent a more severe part of all patients with NAFLD. Comparing the rates of liver-related events in our cohort with previously reported data suggests that they are similar to other biopsy-cohorts. However our control population were also hospitalised individuals therefore had some other form of substantial morbidity, so we believe our comparisons within this study are valid. Therefore, we suggest that our results are accurate for the analyses performed but are not generalisable to other patients with NAFLD (e.g. outpatients, asymptomatic individuals in the general population).

� We have added a paragraph in our Discussion (lines 369-376) about this limitation of the generalisability of our results and have also edited our Conclusion.

Minor:

1. Lines 124: another study described the CV burden in patients with NAFLD-cirrhosis, please discuss it (https://doi.org/10.1016/j.atherosclerosis.2017.03.038)

� Thank you for highlighting this important study to us. We have now commented on it in the Introduction and Discussion (lines 116-118).

2. Table 1: Adjusted and unadjusted HRs for overall mortality and ORs (?) for Liver failure/decompensation and HCC should be reported in a separate table.

� We have now re-arranged our tables into three: demographics, crude disease/event rates, and adjusted HR/ORs.

3. The sentence “increased mortality for patients with NAFLD, irrespective of fibrosis” (lines 277-278) is not supported by results. In this study, the only differentiation in the severity of fibrosis could be made comparing cirrhotic with non-cirrhotic patients and, among them there is a wide difference in survival (HR 3.8 vs 2.6 vs. 65.4!)

� Apologies for this inaccuracy. We had intended to say that our non-cirrhotic NAFLD group also had increased mortality compared to controls. We have edited this section (line 288) to make it more accurate.

4. Lines 298-304: a recent study proved higher incidence of CV disease in patients with NAFLD compared to metabolic controls. In addition, in the same study, non-invasive markers of fibrosis identify patients at higher risk for CV events among those with NAFLD. The same study could be discussed in introduction section (lines 101-107) to affirm that fibrosis, in NAFLD patients, is the major predictors of CVE also. doi: 10.1016/j.cgh.2019.12.026

� Thank you for highlighting this important study, which has come out since the original version of our paper was submitted. We have now commented on it in both the Introduction and Discussion (lines 312-317).

5. Please check through the paper the correct use of NAFLD/NAFL terms. Eg: in figure 1 “NAFLD” was incorrectly used instead of NAFL

� Apologies for these typographical errors. We have carefully checked the paper and made corrections.

Submitted filename: Mann_response_v5.docx

Click here for additional data file.

14 Oct 2020

Hospital admission with non-alcoholic fatty liver disease is associated with increased all-cause mortality independent of cardiovascular risk factors

PONE-D-19-32801R1

Dear Dr. Mann,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Michael W Greene, Ph.D.

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is a well written paper with results that have clinical implications for the treatment of patients with NAFLD-cirrhosis.

The research paper PONE-D-19-32801 entitled “Hospital admission with non-alcoholic fatty liver disease is associated with increased all-cause mortality independent of cardiovascular risk factors” has relevance to the scope and the audience of PONE.

This paper aimed to assess the mortality in NAFLD when adjusting for CVD risk factors in a ‘real world’ cohort of inpatients.

The authors suggest that there is a high burden of cardiovascular disease in NAFLD-cirrhosis patients. From a large “real-life” non-specialist registry of hospitalized patients, NAFLD patients have increased overall mortality and rate of liver-related complications compared to controls after adjusting for cardiovascular disease.

Major comments for the authors

1. This analysis was performed on an interesting issue: The effect of NAFLD on overall mortality in a real world setting regardless of other CVD risk factors.

2. It is a well written paper. The text, the figure and the tables are of appropriate extent and content as well as informative.

3. The references are up to date.

4. The results of the paper have practical implications in patients with NAFLD, especially those with cirrhosis.

5. Any issues with the paper in the original submission were addressed in a proper way in the revision

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

16 Oct 2020

PONE-D-19-32801R1

Hospital admission with non-alcoholic fatty liver disease is associated with increased all-cause mortality independent of cardiovascular risk factors

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