Cardiovascular disease risk in liver transplant recipients transplanted due to chronic viral hepatitis.

BACKGROUND: Cardiovascular disease (CVD) is a major cause of morbidity and mortality after liver transplantation, mostly in patients transplanted for nonalcoholic steatohepatitis, obesity and diabetes. Few data exist on cardiovascular diseases among patients transplanted for viral hepatitis.
OBJECTIVE: Our aim is to clarify the cardiovascular risk and subclinical vascular damage among liver transplant recipients for chronic viral hepatitis (i.e. hepatits C virus, hepatis B virus and hepatitis D virus infection).
METHODS: Adult patients (age ≥ 18 years) with orthotopic liver transplants (OLT) due to viral hepatitis who signed informed consent, and were admitted for a routine follow-up between June 2019 and September 2020 at the Infectious Disease outpatient clinic of the University of Campania Luigi Vanvitelli, Naples, Italy, were prospectively enrolled. An estimation of cardiovascular risk was assessed using three main risk charts, echocolor-Doppler of epiaortic vessels was performed to assess subclinical Intima-Media changes.
RESULTS: A total of 161 patients were evaluated; of these 15 were excluded because not affected by viral hepatitis. 146 patients were considered. 83 patients (56.8%) were considered at high cardiovascular risk according to Framingham, 54 patients (36.9%) to American Heart Association Arteriosclerotic Cardiovascular Disease (ASCVD) score and 19 (13.0%) to Heart Score. Only 8 patients (5.4%) showed a normal carotid ultrasound, while 52 patients (35.6%) had a carotid artery Intima-Media Thickness (IMT) and 86 (58.9%) an atherosclerotic plaque.
CONCLUSIONS: Liver transplant recipients for virus-related associated liver disease are, in light of the high percentage of carotid lesions, at high risk of CVD. Risk charts compared to subclinical carotid lesions which represent damage already established and a real localization of the disease, seem to underestimate the cardiovascular risk. A chronic inflammatory status, could play a key role. It's important to raise the awareness of cardiovascular risk in liver transplant patients to prevent cardiovascular diseases and improve the timing of early diagnosis of premature vascular lesions.

Introduction

Liver transplantation (LT) is a lifesaving procedure for patients with acute and chronic end stage liver disease, and hepatocellular carcinoma [1-3].

Hepatotrophic viruses capable of causing chronic hepatitis (i.e. hepatitis C virus, hepatitis B virus and hepatitis D virus) are among the most common causes of cirrhosis and a frequent indication for liver transplantation, in particular among individuals with co-infections who are at increased risk of end stage liver disease.

Nowadays, LT is a routine procedure with excellent outcomes in terms of quality and length of life. However, complications are frequent both in the early and long-term period, and significantly impact in terms of morbidity and mortality [4-8]. In particular, cardiovascular complications and metabolic alterations among liver transplant recipients, represent, the main early and late complications during follow-up [9].

The cumulative cardiovascular risk events are estimated between 13.6% and 42%, depending on the studies [10, 11]. The cause of this reported high risk is multifactorial, including, on one side, chronic immunosuppressive treatment [12] and, on the other side, end‐stage liver disease that leads to metabolic disturbances of lipids glucose, and blood pressure levels. As a matter of fact, it is well known that approximately half of the cirrhotic patients suffer for associated cardiomyopathy characterized by high heart rate, increased cardiac output, and decreased systemic vascular resistance [13, 14]. Moreover, cardiac dysfunction also depends from acute hemodynamic changes caused by reperfusion of the graft and the release of proinflammatory cytokines, which substantially affect early cardiovascular risk [15].

Chronic immunosuppressive therapy also represents an important and well recognized risk factor for long-term morbidity due to its influence on dyslipidemia, diabetes mellitus, ischemic heart disease and hypertension [16].

Among calcineurin inhibitors (CNIs), cyclosporine has a greater impact on metabolic and cardiovascular comorbidities than tacrolimus [9, 17–19], while the mammalian target of rapamycin (mTOR) inhibitors lower the risk of metabolic syndrome compared to CNIs, except for their impact on fat metabolism [20].

Moreover, etiology has a major impact on cardiovascular risk, being higher in patients transplanted for nonalcoholic steatohepatitis (NASH), alcoholic cirrhosis, obesity and diabetes- related cause of end-stage liver disease [21].

Although a potential association between hepatitis C virus (HCV) infection and risk of subclinical and clinical cardiovascular disease (CVD) has been described, with a 2.5–3.5% absolute risk increase of 10-year CVD in HCV-infected subjects compared to the HCV-negative population [21, 22], few data exist on CVD among stable patients transplanted for viral hepatitis due to different etiology.

In this scenario, the objective of the present study was to evaluate the risk of cardiovascular disease and the presence of subclinical vascular damage among liver transplant recipients infected with HCV and/or hepatitis B virus (HBV) and hepatitis D virus (HDV)-coinfection associated liver disease, in order to raise the awareness of cardiovascular risk in LT patients, to prevent CVD and improve the timing of early diagnosis of premature vascular lesions.

Materials and methods

Study design and patient selection

This is a cross-sectional, observational cohort study including patients with orthotopic liver transplantation (OLT) consecutively admitted for routine follow-up visit to the Infectious Disease outpatient facilities of the University of Campania Luigi Vanvitelli, Naples, Italy from June 2019 to September 2020.

All adult (age ≥ 18 years) patients with liver transplantation due to viral hepatitis who signed informed consent were included; therefore, liver transplant recipients due to other etiologies were excluded.

The study was approved by the Ethics Committee of the Hospital of Campania Luigi Vanvitelli. All procedures in the study protocol were carried out in accordance with the Helsinki Declaration of 1975. Written consent was obtained.

Variables and definitions

Data collected from all patients included: demographics; etiology; presence of underlying chronic disease; immunosuppression; other chronic therapies. Data regarding independent risk factors for CVD (family history, smoke, alcohol consumption) were also collected.

Each patient underwent a complete physical examination with measurement of blood pressure, weight, height and abdominal circumference as well as routine biochemical tests.

Familial history of cardiovascular disease was defined as a first degree relative-parent, sibling or child that has history of myocardial infarction or ischemic stroke at ages younger than 55 years in men and younger than 65 years in women [23].

Definition of previous cardiovascular diseases included: ischemic heart disease (heart attack and angina pectoris), and cerebrovascular diseases (ischemic and hemorrhagic stroke).

An abdominal obesity was defined as waist circumference greater than 102 cm in male and 88 in female; hypertriglyceridemia as triglycerides values greater than150 mg/dl or when the patient was under specific therapy. Hypertension was defined as blood pressure greater than 130/85 mmHg.

Metabolic syndrome (MS) was defined by the presence (or treatment) of 3 or more of the following [24]:

Obesity (waist circumference: men >102 cm, women >88 cm)

Fasting plasma glucose ≥ 100 mg/dL (5.6 mmol/L)

Blood pressure ≥ 130/85 mm Hg

Triglycerides ≥ 150 mg/mL (1.7 mmol/L)

Low HDL cholesterol (men<40 mg/dl, women <50 mg/dl)

The three cardiovascular risk charts evaluated were: Framingham risk score [25], American Heart Association Arteriosclerotic Cardiovascular Disease (ASCVD) [26] and the Heart score [27]. We used three different risk scores to distinguish two different outcomes. In fact, Heart Score evaluates the risk of fatal major adverse cardiovascular events, while ASCVD and the Framingham risk score include non-fatal events.

As regards the echocolor-Doppler of epiaortic vessels, ultrasonography was performed by a physician specifically trained on carotid vessels images with 20 years’ experience in echocolor-Doppler ultrasound (MP), using a power echocolor-Doppler instrument with 7.5 MHz probes. An Intima-Media Thickness [IMT] ≥1 mm, of common and internal carotid for both left and right sides, was considered pathological. A carotid was classified as being affected by atherosclerotic plaque if the localized thickening was ≥1.3 mm.

A minimum of three measurements have been carried out: on the common carotid artery: 1 cm before the carotid bifurcation and at carotid bifurcation; on the internal carotid: 1 cm after the carotid bifurcation and 2 cm after the carotid bifurcation. All images were photographed and properly archived.

Statistical analysis

Continuous variables were summarized as means and standard deviations, whereas categorical variables were indicated as absolute and relative frequencies. CI 95% Clopper–Pearson and binomial test were used when appropriate.

Results

The demographic and clinical characteristics of enrolled patients are summarized in Table 1. Of the 161 patients with a history of OLT evaluated in the study period, 15 were excluded because not affected by viral hepatitis; thus, an overall of 146 patients were enrolled in the present study (median age 64 years; males 73.9%), of whom 59 (40.4%) were transplanted for HCV, 27 (18.4%) for HBV, 46 (31.5%) for dual infection HBV-HDV, 10 (6.8%) for dual infection HBV-HCV and 4 (2.7%) for triple infection HBV-HDV-HCV end-stage liver disease. For 87 patients (59.5%) the time since transplantation was greater than 10 years, while only 23 (15.6%) patients had been transplanted for less than 5 years. The most frequent immunosuppressive regimen used included tacrolimus as monotherapy (31.6%), followed by the combination of tacrolimus and mycophenolate (18.4%) and cyclosporine alone (14.5%). 36 (24.6%) were active smokers and 19 (13.0%) alcohol users. 58 patients (39.7%) had a familial history for CVD, 75 (51.3%) for diabetes mellitus and 34 (23.3%) for dyslipidemia.

Table 1

Demographics and data regarding transplant status and CVD risk n, (%).

All patients	146
Age (median, range)	64 (41–80)
Male sex, n (%)	108 (73.9)
Caucasian, n (%)	146 (100)
BMI (media)	29.1
Aetiology
HBV	27 (18.4)
HCV	59 (40.4)
HBV±HDV	46 (31.5)
HBV-HCV	10 (6.8)
HBV-HDV-HCV	4 (2.7)
Time elapsed since transplantation
≥20 years	33 (22.6)
≥10 and ≤20 years	54 (36.9)
≥5 and ≤10 years	36 (24.6)
≥1 and ≤5 years	21 (14.3)
<1 year	2 (1.3)
Immunosuppressant
Tacrolimus	46 (31.5)
Everolimus	10 (6.8)
Cyclosporine	21 (14.3)
Mycophenolate	8 (5.4)
Tacrolimus plus Mycophenolate	27 (18.4)
Cyclosporine plus Mycophenolate	15 (10.2)
Tacrolimus plus Everolimus	19 (13.0)
Instructions
Elementary schools	46 (31.5)
Middle schools	58 (39.7)
High school	35 (23.9)
University	7 (4.7)
Years of instructions, media (range)	9.05 (2–18)
Working activity
Sedentary	123 (84.2)
Light-moderate	23(15.7)
Physical movement
None	54.0 (36.9)
Usually walks	86 (58.9)
Sport activity	6 (4.1)
Familiarity
CVD	58 (39.7)
Ictus/TIA	23 (15.7)
DM	75 (51.3)
Dyslipidemia	35 (23.9)
Neoplasia	83 (56.8)
Comorbidities
CVD	8 (5.4)
Ictus/TIA	12 (8.2)
DM before LT	23 (15.7)
DM after LT	52 (35.6)
Dyslipidemia before LT	0 (0)
Dyslipidemia after LT	44 (30.1)
Neoplasia before LT	54(36.9)
Neoplasia after LT	25 (17.1)
Smoker	36 (24.6)
Alcohol users	19(13.0)
Ormonal or corticosteroid therapies	6 (4.1)
Lipid-lowering therapies	29 (19.9)
Antihypertensive therapies	92 (63.0)
Oral antidiabetics	15 (10.2)
Insulin therapy	33 (22.6)

BMI: Body massa index, TIA: Transient ischaemic attack, DM: Diabetes mellitus, LT: Liver transplantation.

51 (34.9%) patients had diabetes arose in the post-transplant, 44 (30.1%) suffer from dyslipidemia and 11 (7.5%) patients had already experienced an acute cardiovascular event in the form of stroke/transient ischemic attack (TIA).

More than half of the patients (63.1%) were taking antihypertensive therapy, 19.7% a lipid-lowering drug and 32.9% a hypoglycemic therapy in the form of oral antidiabetic drugs or insulin.

Metabolic status and biochemical values are shown in Table 2. At biochemical tests 71 (48.6%) and 52 (35.6%) patients showed, respectively, pathological glycemia and triglycerides values. More than half of the patients (65.8%) showed poor blood pressure control at the visit with values higher than normal. Furthermore, 50 (34.2%) patients had 3 or more criteria for metabolic syndrome.

Table 2

Metabolic status.

Variables	N, (%)
Abdominal Obesity	25 (17.1)
Hypertriglyceridemia	52 (35.6)
Low HDL cholesterol	67 (45.8)
Hypertension	90 (65.8)
Hyperglycemia	71 (46.8)
Metabolic syndrome	50 (34.2)

As regards the cardiovascular risk charts (Table 3), an overall of 83 patients (56.8%) were considered at high cardiovascular risk according to Framingham, 54 patients (36.9%) to ASCVD and 19 (13.0%) to Heart Score.

Table 3

Cardiovascular risk charts.

Cardiovascular Risk	Framingham	ASCVD	Heart-SCORE
Low risk	35 (23.8)	15 (10.3)	8(5.5)
Borderline	-	13 (9.0)	-
Intermediate	29 (19.7)	63(43.5)	-
Moderate Risk	-	-	117 (80.1)
High risk	83 (56.5)	54(37.2)	19 (13.0)
Very high risk	-	-	2 (1.4)

Analyzing the echocolor-Doppler features of the patients enrolled (Table 4), only 8 patients (5.4%) showed a normal carotid ultrasound. 138 (94.5%) patients showed pathological ultrasonographic findings; of these, 86 (58.9%) had atherosclerotic plaque and 52 (35.6%) had abnormal IMT (CI 95% Clopper–Pearson = 29,58%–46,32%; binomial test: p = 0.0048).

Table 4

Echocolor-Doppler features.

Normal n,%	IMT (≥1) n,%	Plaque (≥1,3) n,%
8 (5.4)	52 (35.6)	86 (58.9)

51.1% of patients who had a plaque had been transplanted for HCV, 22.2% for co-infection HBV±HDV and 17% for HBV. IMT was more frequently found in patients transplanted for co-infection HBV±HDV (44.4%), followed by HCV (25.9%) and HBV (18.5%) monoinfection.

Regarding the correlation between immunosuppressive regimen and echocolor-Doppler features, 31% of patients showing plaques were treated with tacrolimus as monotherapy, followed by the association of tacrolimus plus mycophenolate (15.6%), tacrolimus plus everolimus (15.6%), cyclosporine as monotherapy (15.6%) or combined with mycophenolate (15.6%) and everolimus as monotherapy (6.6%).

Discussion

Cardiovascular disease has emerged as the leading cause of non-graft-related deaths [28, 29]. In fact, LT represents a major stress which can affect and worsen preexisting cardiovascular dysfunctions [9, 12], mostly in early post-transplant follow-up. Studies concerning long-term cardiovascular complications are fewer than those focused on early morbidity.

The risk of CVD in liver transplant recipients is due to several mechanisms: aging, systemic hemodynamic changes due to advanced liver disease, chronic inflammation and immunosuppressive treatment.

A large number of studies suggest an increased risk of cardiovascular disease events in patients transplanted for nonalcoholic steatohepatitis (NASH), alcoholic cirrhosis, obesity and diabetes-related causes of end-stage liver disease [21]. In fact, a large multicenter cohort study including 32,810 patients with LT showed that major cardiovascular events at 30 and 90 days were independently predicted by age (more than 65 years), pre-transplant creatinine, pretransplant comorbidities such as atrial fibrillation and stroke, and non-viral liver disease such as alcoholic cirrhosis, and non-alcoholic steatohepatitis [21].

The association between CVD and HCV infection in subjects without LT was already known. In fact, a meta-analysis of aggregate data from 22 studies showed that, compared with uninfected controls, HCV-infected patients are at increased risks of CVD-related mortality, carotid plaques and cerebrocardiovascular events [30]. However, despite these data, few studies explore cardiovascular risk and subclinical damage among stable patients transplanted for viral hepatitis.

In the present study, we evaluated the cardiovascular risk and the subclinical damage of the carotid vessels among liver transplant recipients for HCV, HBV±HDV associated liver disease. Until now, cardiovascular assessment among these patients has been poorly investigated, also because they are not perceived at increased risk, differently from patients transplanted for nonalcoholic steatohepatitis (NASH), alcoholic cirrhosis, obesity and diabetes. Unexpectedly, our data suggested that this category of patients are at high cardiovascular risk. In fact, according to Framingham risk score, patients at high cardiovascular risk were more than 50%. Analyzing the echocolor-Doppler images, it is noteworthy that 94.7% of the patients showed pathological ultrasonographic findings (IMT or plaques). This shows that Framingham risk score in these subjects performs better than ASCVD and Heart Score, but underestimates the presence of subclinical vascular damage. We hypothesize that, in this phenomenon, inflammatory mechanisms, not explored by the risk scores at present validated, could play a role. Also immunosuppressive therapy could contribute to the possible interplay between immune suppression and immune activation. In this, patients in treatment with tacrolimus seem to have a similar pattern of cardiovascular risk and vessel damage development with respect to the other immunosuppressive regimens, even if the number of patients does not allow to infer definitive conclusions.

Possible limits of our study are the number of enrolled subjects, and the absence of a control group. An evaluation of inflammatory biomarkers is warranted to better explore the role of chronic endothelial inflammation in this phenomenon.

In conclusion, this is the first study among long term liver transplant recipients for viral hepatitis, revealing an unexpectedly high presence of patients at increased cardiovascular risk, and an even higher number of patients with subclinical epi-aortic vessel lesions. We suggest that echocolor-Doppler investigation of carotid vessels could be introduced among periodic routine controls of liver transplant recipients for HCV, HBV± HDV-associated disease.

16 Jul 2021

PONE-D-21-16062

Cardiovascular Disease (CVD) risk in liver transplant recipients for HCV, HBV± HDV-associated liver disease

PLOS ONE

Dear Dr. Maggi,

Thank you for submitting your manuscript to PLOS ONE. 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.

The Editor reviewed the manuscript. This work requires more relevant information to make suggested conclusions.

The Editor's comments:

The title needs to be modified. What is “HCV, HBV+/- HDV-associated liver disease”? This is not obvious or even clear for a professional and especially for a lay reader. The title needs to be better expressing the entire idea.

In the Abstract, the authors use multiple abbreviations (ASCVD, IMT, CVD) without an explanation; all abbreviations need to be spelled out when used the first time.

The conclusion sentences in the Abstract need to be re-written as “Liver transplant recipients for HCV, HBV±HDV-associated liver disease are at high risk of CVD. Comparing the high percentage of subclinical carotid lesions with data of the risk charts, the latter seem to underestimate the real extent of the endothelial damage. A chronic inflammatory status could play a key role. It’s important to raise the awareness of cardiovascular risk in liver transplant patients to prevent cardiovascular diseases and improve the timing of early diagnosis of premature vascular lesions” is not clear. The first sentence does not clearly state the main findings. The second sentence also needs to be better expressed.

It is confusing the use of CV as cumulative cardiovascular risk as well as CVD as clinical cardiovascular disease. What is the difference? It is not convincing why the authors use these two similar terms. Please explain better each term or use one universal term. The risk of “a 10-year CVD risk 2.5-3.5% higher in HCV-infected subjects compared to the HCV-negative population” is not very impressive. If the risk is for example 20% and an increase is to 22.5% such increase may be biologically not significant. The argument is not convincing. Please explain better this argument.

The last paragraph in the Introduction section needs to be re-written. For example it should be: “The objective of this study was to evaluate the risk of cardiovascular disease or at least subclinical vascular damage among liver transplant recipients infected with HCV and/or HBV.”

The authors fail to explain the principal idea for their work. After reading the Abstract and the Introduction, the reader still does not understand what is “HBV ± HDV-associated liver disease”. The abbreviations need to be explained and the combination of HBV +/- HDV should be clearly spelled out and explained.

The best description of this study would be: “This an observational study of patients with orthotopic liver transplants (OLT) admitted for a routine follow-up between June 2019 and September 2020 at the Infectious Disease outpatient clinic of the University of Campania Luigi Vanvitelli, Naples, Italy. All adult (age ≥ 18 years) patients with liver transplants due to viral hepatitis who signed informative consent were included.” Some form of this description should be in the Abstract.

The authors need to provide the list of all abbreviations with spell out text.

All abbreviations used in the Results section needs to be explained “were transplanted for HCV, 27 (18.4%) for HBV, 46 (31.5%) for dual infection HBV-HDV, 10 (6.8%) for dual infection HBV-HCV and 4 (2.7%) for triple infection HBV-HDV-HCV. For 87 patients (59.5%)”.

The Introduction section must explain very clearly about different types of hepatitis. There is no any information about what is hepatitis. Only after looking at a website, one can learn “Hepatitis is most commonly caused by the viruses hepatitis A, B, C, D and E. The authors need to write a section with an explanation about hepatitis with explained abbreviations. What are the differences among patients with hepatitis B, C and D versus combination of two different types?

The title of this study is about cardiovascular disease risk in liver recipients with hepatitis. There is no convincing argument that these patients are indeed at higher risk than any other group of patients. Please provide more data and the comparison with non-infected patients for their risk for cardiovascular diseases. The statement at the end of the Discussion section is not satisfactory “Possible limits of our study are the number of enrolled subjects, and the absence of a control group.” In addition, the authors state “However, this is the first study among long term liver transplant recipients for viral hepatitis, revealing an unexpectedly high presence of patients at high CVD risk, and an even higher number of patients with subclinical epiaortic vessel lesions.” The argument needs to be supported by the comparison with other groups of patients.

Reviewer # 1:

The manuscript presents the results of a observational study which assessed cardiovascular risk in long-term liver transplant recipients (most >5 years) transplanted for viral hepatitis using different risk charts and subclinical intima-media damage via carotid ultrasound. Many recipients had high CV risk and the majority had either IMT or a carotid plaque on CD carotid ultrasound.

The study lacks control groups as was observed in the discussion, however the authors did not draw any exaggerated conclusions Only proportions were reported, with no formal statistical testing, which is in line with the study design.

Table 1 stated that 39,7% of patients had previous cardiovascular disease, how was it defined?

As the present cross-sectional study was not designed to record adverse events at all, carotid ultrasound was used as surrogate. Even though it was previously established that IMT correlates with major adverse cardiovascular events, the risk scores were not originally designed to asses endothelial damage. The risk scores used in the study also differ in outcomes; Heart Score only evaluates the risk of fatal major adverse cardiovascular events, while ASCVD and the Framingham risk score include non-fatal events. Limitations regarding differences in outcomes should be noted in the discussion.

Another observation is that the authors stated in their discussion that patients on tacrolimus monotherapy seemed more prone to developing vessel lesions although the proportions reported were similar to those of the total study population (around 31%).

Reviwer # 2:

The paper by Dr. Magii et al. is a description of a brief observational study of a group of liver transplant recipients whose initial liver failure was due to viral etiology. Some questions should be addressed before publication.

1.     What was the medical history of patients before liver transplantation? Please make sure to include summarized and statistically analyzed information on parameters relevant to CVD risk, such as CVD diagnosis, BMI, diet and lifestyle, lipid profiles etc. Please comment on the change in the CVD risk factors before and after transplantation. This is one way to address the lack of a healthy control group in the study.

2.     The statement about correlation between echo-Doppler features and immunosuppression regimen has to be accompanied with statistical confirmation (probably Chi-squared test).

3.     Please make sure percentages in Table 3 add up to 100 (may require checking rounding).

4.     The manuscript will benefit from proofreading to fix language errors.

==============================

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Additional Editor Comments:

The Editor reviewed the manuscript. This work requires more relevant information to make suggested conclusions.

The Editors comments:

The title needs to be modified. What is “VCV, HBV+/- HDV-associated liver disease”? This is not obvious or even clear for a professional and especially for a lay reader. The title needs to be better expressing the entire idea.

In the Abstract, the authors use multiple abbreviations (ASCVD, IMT, CVD) without an explanation; all abbreviations need to be spelled out when used the first time.

The conclusion sentences in the Abstract need to be re-written as “Liver transplant recipients for HCV, HBV±HDV-associated liver disease are at high risk of CVD. Comparing the high percentage of subclinical carotid lesions with data of the risk charts, the latter seem to underestimate the real extent of the endothelial damage. A chronic inflammatory status could play a key role. It’s important to raise the awareness of cardiovascular risk in liver transplant patients to prevent cardiovascular diseases and improve the timing of early diagnosis of premature vascular lesions” is not clear. The first sentence does not clearly state the main findings. The second sentence also needs to be better expressed.

It is confusing the use of CV as cumulative cardiovascular risk as well as CVD as clinical cardiovascular disease. What is the difference? It is not convincing why the authors use these two similar terms. Please explain better each term or use one universal term. The risk of “a 10-year CVD risk 2.5-3.5% higher in HCV-infected subjects compared to the HCV-negative population” is not very impressive. If the risk is for example 20% and an increase is to 22.5% such increase may be biologically not significant. The argument is not convincing. Please explain better this argument.

The last paragraph in the Introduction section needs to be re-written. For example it should be: “The objective of this study was to evaluate the risk of cardiovascular disease or at least subclinical vascular damage among liver transplant recipients infected with HCV and/or HBV.”

The authors fail to explain the principal idea for their work. After reading the Abstract and the Introduction, the reader still does not understand what is “HBV ± HDV-associated liver disease”. The abbreviations need to be explained and the combination of HBV +/- HDV should be clearly spelled out and explained.

The best description of this study would be: “This an observational study of patients with orthotopic liver transplants (OLT) admitted for a routine follow-up between June 2019 and September 2020 at the Infectious Disease outpatient clinic of the University of Campania Luigi Vanvitelli, Naples, Italy. All adult (age ≥ 18 years) patients with liver transplants due to viral hepatitis who signed informative consent were included.” Some form of this description should be in the Abstract.

The authors need to provide the list of all abbreviations with spell out text.

All abbreviations used in the Results section needs to be explained “were transplanted for HCV, 27 (18.4%) for HBV, 46 (31.5%) for dual infection HBV-HDV, 10 (6.8%) for dual infection HBV-HCV and 4 (2.7%) for triple infection HBV-HDV-HCV. For 87 patients (59.5%)”.

The Introduction section must explain very clearly about different types of hepatitis. There is no any information about what is hepatitis. Only after looking at a website, one can learn “Hepatitis is most commonly caused by the viruses hepatitis A, B, C, D and E. The authors need to write a section with an explanation about hepatitis with explained abbreviations. What are the differences among patients with hepatitis B, C and D versus combination of two different types?

The title of this study is about cardiovascular disease risk in liver recipients with hepatitis. There is no convincing argument that these patients are indeed at higher risk than any other group of patients. Please provide more data and the comparison with non-infected patients for their risk for cardiovascular diseases. The statement at the end of the Discussion section is not satisfactory “Possible limits of our study are the number of enrolled subjects, and the absence of a control group.” In addition, the authors state “However, this is the first study among long term liver transplant recipients for viral hepatitis, revealing an unexpectedly high presence of patients at high CVD risk, and an even higher number of patients with subclinical epiaortic vessel lesions.” The argument needs to be supported by the comparison with other groups of patients.

Reviewer # 1:

The manuscript presents the results of a observational study which assessed cardiovascular risk in long-term liver transplant recipients (most >5 years) transplanted for viral hepatitis using different risk charts and subclinical intima-media damage via carotid ultrasound. Many recipients had high CV risk and the majority had either IMT or a carotid plaque on CD carotid ultrasound.

The study lacks control groups as was observed in the discussion, however the authors did not draw any exaggerated conclusions Only proportions were reported, with no formal statistical testing, which is in line with the study design.

Table 1 stated that 39,7% of patients had previous cardiovascular disease, how was it defined?

As the present cross-sectional study was not designed to record adverse events at all, carotid ultrasound was used as surrogate. Even though it was previously established that IMT correlates with major adverse cardiovascular events, the risk scores were not originally designed to asses endothelial damage. The risk scores used in the study also differ in outcomes; Heart Score only evaluates the risk of fatal major adverse cardiovascular events, while ASCVD and the Framingham risk score include non-fatal events. Limitations regarding differences in outcomes should be noted in the discussion.

Another observation is that the authors stated in their discussion that patients on tacrolimus monotherapy seemed more prone to developing vessel lesions although the proportions reported were similar to those of the total study population (around 31%).

Reviwer # 2:

The paper by Dr. Magii et al. is a description of a brief observational study of a group of liver transplant recipients whose initial liver failure was due to viral etiology. Some questions should be addressed before publication.

1. What was the medical history of patients before liver transplantation? Please make sure to include summarized and statistically analyzed information on parameters relevant to CVD risk, such as CVD diagnosis, BMI, diet and lifestyle, lipid profiles etc. Please comment on the change in the CVD risk factors before and after transplantation. This is one way to address the lack of a healthy control group in the study.

2. The statement about correlation between echo-Doppler features and immunosuppression regimen has to be accompanied with statistical confirmation (probably Chi-squared test).

3. Please make sure percentages in Table 3 add up to 100 (may require checking rounding).

4. The manuscript will benefit from proofreading to fix language errors.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

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: No

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

Reviewer #1: Yes

Reviewer #2: 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

**********

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

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 manuscript presents the results of a observational study which assessed cardiovascular risk in long-term liver transplant recipients (most >5 years) transplanted for viral hepatitis using different risk charts and subclinical intima-media damage via carotid ultrasound. Many recipients had high CV risk and the majority had either IMT or a carotid plaque on CD carotid ultrasound.

The study lacks control groups as was observed in the discussion, however the authors did not draw any exaggerated conclusions Only proportions were reported, with no formal statistical testing, which is in line with the study design.

Table 1 stated that 39,7% of patients had previous cardiovascular disease, how was it defined?

As the present cross-sectional study was not designed to record adverse events at all, carotid ultrasound was used as surrogate. Even though it was previously established that IMT correlates with major adverse cardiovascular events, the risk scores were not originally designed to asses endothelial damage. The risk scores used in the study also differ in outcomes; Heart Score only evaluates the risk of fatal major adverse cardiovascular events, while ASCVD and the Framingham risk score include non-fatal events. Limitations regarding differences in outcomes should be noted in the discussion.

Another observation is that the authors stated in their discussion that patients on tacrolimus monotherapy seemed more prone to developing vessel lesions although the proportions reported were similar to those of the total study population (around 31%).

Reviewer #2: The paper by Dr. Magii et al. is a description of a brief observational study of a group of liver transplant recipients whose initial liver failure was due to viral etiology. Some questions should be addressed before publication.

1. What was the medical history of patients before liver transplantation? Please make sure to include summarized and statistically analyzed information on parameters relevant to CVD risk, such as CVD diagnosis, BMI, diet and lifestyle, lipid profiles etc. Please comment on the change in the CVD risk factors before and after transplantation. This is one way to address the lack of a healthy control group in the study.

2. The statement about correlation between echo-Doppler features and immunosuppression regimen has to be accompanied with statistical confirmation (probably Chi-squared test).

3. Please make sure percentages in Table 3 add up to 100 (may require checking rounding).

4. The manuscript will benefit from proofreading to fix language errors.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Dulat Bekbolsynov

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25 Nov 2021

Dear Editor,

We re-submit our paper “Cardiovascular Disease (CVD) risk in liver transplant recipients for HCV, HBV± HDV-associated liver disease” modified according to the suggestions of the editor and of the reviewers.

We have made significant revisions to the original manuscript and we are sure that the comments of the reviewers have improved significantly the final version of the paper. We are convinced that it would be of interest to PLOS ONE.

Kind regards,

Paolo Maggi

On behalf of all authors

POINT-BY-POINT ANSWER TO THE COMMENTS OF THE EDITOR

Point 1: The title needs to be modified. What is “HCV, HBV+/- HDV-associated liver disease”? This is not obvious or even clear for a professional and especially for a lay reader. The title needs to be better expressing the entire idea.

Answer to point 1: We thank the Editor for this suggestion. We have accordingly modified the title.

Point 2: In the Abstract, the authors use multiple abbreviations (ASCVD, IMT, CVD) without an explanation; all abbreviations need to be spelled out when used the first time.

Answer to point 2: We thank the Editor for this suggestion. We accordingly modified the text in the abstract spelled out all the abbreviations.

Point 3: The conclusion sentences in the Abstract need to be re-written as “Liver transplant recipients for HCV, HBV±HDV-associated liver disease are at high risk of CVD. Comparing the high percentage of subclinical carotid lesions with data of the risk charts, the latter seem to underestimate the real extent of the endothelial damage. A chronic inflammatory status could play a key role. It’s important to raise the awareness of cardiovascular risk in liver transplant patients to prevent cardiovascular diseases and improve the timing of early diagnosis of premature vascular lesions” is not clear. The first sentence does not clearly state the main findings. The second sentence also needs to be better expressed.

Answer to point 3: We thank the Editor for this comment. We accordingly modified the text, and clarified some unclear points by providing better explanations.

Point 4: It is confusing the use of CV as cumulative cardiovascular risk as well as CVD as clinical cardiovascular disease. What is the difference? It is not convincing why the authors use these two similar terms. Please explain better each term or use one universal term.

Answer to point 4: We thank the Editor for this comment. We have modified the text accordingly and, when appropriate, use a universal term.

Point 5: The risk of “a 10-year CVD risk 2.5-3.5% higher in HCV-infected subjects compared to the HCV-negative population” is not very impressive. If the risk is for example 20% and an increase is to 22.5% such increase may be biologically not significant. The argument is not convincing. Please explain better this argument.

Answer to point 5: We thank the Editor for this comment. In the study of Badawi et al. the authors using a multivariable linear regression model found as HCV infection was significantly associated with a 2.5–3.5% absolute risk increase of 10-year CVD compared to HCV negative population. We have accordingly modified the text.

Point 6: The last paragraph in the Introduction section needs to be re-written. For example it should be: “The objective of this study was to evaluate the risk of cardiovascular disease or at least subclinical vascular damage among liver transplant recipients infected with HCV and/or HBV.”

Answer to point 6: We thank the Editor for this suggestion. We have accordingly modified the last paragraph in the Introduction.

Point 7: The authors fail to explain the principal idea for their work. After reading the Abstract and the Introduction, the reader still does not understand what is “HBV ± HDV-associated liver disease”. The abbreviations need to be explained and the combination of HBV +/- HDV should be clearly spelled out and explained.

Answer to point 7: We thank the Editor for this comment. We have explained all the abbreviations and we have spelled out the combination of HBV +/- HDV as coinfection.

Point 8: The best description of this study would be: “This an observational study of patients with orthotopic liver transplants (OLT) admitted for a routine follow-up between June 2019 and September 2020 at the Infectious Disease outpatient clinic of the University of Campania Luigi Vanvitelli, Naples, Italy. All adult (age ≥ 18 years) patients with liver transplants due to viral hepatitis who signed informative consent were included.” Some form of this description should be in the Abstract.

Answer to point 8: We thank the Editor for this suggestion. We have accordingly modified the section of methods in the abstract.

Point 9: The authors need to provide the list of all abbreviations with spell out text. All abbreviations used in the Results section needs to be explained “were transplanted for HCV, 27 (18.4%) for HBV, 46 (31.5%) for dual infection HBV-HDV, 10 (6.8%) for dual infection HBV-HCV and 4 (2.7%) for triple infection HBV-HDV-HCV. For 87 patients (59.5%)”.

Answer to point 9: We thank the Editor for this comment. We have already explained all the abbreviations in the Introduction.

Point 10: The Introduction section must explain very clearly about different types of hepatitis. There is no any information about what is hepatitis. Only after looking at a website, one can learn “Hepatitis is most commonly caused by the viruses hepatitis A, B, C, D and E. The authors need to write a section with an explanation about hepatitis with explained abbreviations. What are the differences among patients with hepatitis B, C and D versus combination of two different types?

Answer to point 10: We thank the Editor for this comment. We have added this information to lines 66-69 of the new manuscript.

Point 11: The title of this study is about cardiovascular disease risk in liver recipients with hepatitis. There is no convincing argument that these patients are indeed at higher risk than any other group of patients. Please provide more data and the comparison with non-infected patients for their risk for cardiovascular diseases. The statement at the end of the Discussion section is not satisfactory “Possible limits of our study are the number of enrolled subjects, and the absence of a control group.” In addition, the authors state “However, this is the first study among long term liver transplant recipients for viral hepatitis, revealing an unexpectedly high presence of patients at high CVD risk, and an even higher number of patients with subclinical epiaortic vessel lesions.” The argument needs to be supported by the comparison with other groups of patients.

Answer to point 11: The Editor is right but unfortunately, we have not explored the impact of cardiovascular disease risk in other groups of patients. This limit was stated in the Discussion section; surely, it can be interesting to investigate it for a further development of the research. However, it should be noted that there is an important literature supporting the fact that patients with viral hepatitis are at high cardiovascular risk and that subclinical carotid atherosclerosis are asymptomatic stepping stones to clinical CVD. Therefore, considering the consolidated data, our goal was to investigate the cardiovascular risk in this particular category of patients, i.e. patients undergoing liver transplantation for viral hepatitis, and we found a impressively high presence of patients with subclinical epiaortic vessel lesions and high cardiovascular risk according to risk charts. Fe feel this is a relevant datum worth to be communicated and furtherly studied.

Reviewer # 1:

Table 1 stated that 39,7% of patients had previous cardiovascular disease, how was it defined?

RE: Thank you for this suggestion We specified this definition in the method section.

As the present cross-sectional study was not designed to record adverse events at all, carotid ultrasound was used as surrogate. Even though it was previously established that IMT correlates with major adverse cardiovascular events, the risk scores were not originally designed to asses endothelial damage. The risk scores used in the study also differ in outcomes; Heart Score only evaluates the risk of fatal major adverse cardiovascular events, while ASCVD and the Framingham risk score include non-fatal events. Limitations regarding differences in outcomes should be noted in the discussion.

RE: Thank you for these observations. In the discussion we have argued them as limit of the study.

Another observation is that the authors stated in their discussion that patients on tacrolimus monotherapy seemed more prone to developing vessel lesions although the proportions reported were similar to those of the total study population (around 31%).

Re: Thank you for this comment. We corrected this discrepancy in the text.

Reviwer # 2:

1. What was the medical history of patients before liver transplantation? Please make sure to include summarized and statistically analyzed information on parameters relevant to CVD risk, such as CVD diagnosis, BMI, diet and lifestyle, lipid profiles etc. Please comment on the change in the CVD risk factors before and after transplantation. This is one way to address the lack of a healthy control group in the study.

Re: thank you for this observation. We agree that these data can provide a further starting point for discussion and enrichment of the manuscript. However, most patients are long term transplantation recipients. For this reason, variables such as BMI, lipid, diet style and others, have changed over time (20 years and more, in most cases!) and cannot be useful for a direct comparison between pre and post transplantation

2. The statement about correlation between echo-Doppler features and immunosuppression regimen has to be accompanied with statistical confirmation (probably Chi-squared test).

Re: Thank you for this comment. The Chi-squared test was done. The Method section was corrected accordingly.

3. Please make sure percentages in Table 3 add up to 100 (may require checking rounding).

Re: We apologize for the mistake. We corrected it.

4. The manuscript will benefit from proofreading to fix language errors.

Re: Thank you for the suggestion. The manuscript was revised by a native English speaker.

We thank the Editor and the reviewers for helping us to improve our paper.

We hope that the paper is now worthy of publication in PLOS ONE

Best regards,

Prof Paolo Maggi

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

28 Feb 2022

Cardiovascular Disease risk in liver transplant recipients for chronic viral hepatitis

PONE-D-21-16062R1

Dear Dr. Maggi,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Vanessa Carels

Staff Editor

PLOS ONE

Additional Editor Comments (optional):

Please modify the title to ensure that it is meeting PLOS’ guidelines (https://journals.plos.org/plosone/s/submission-guidelines#loc-title). In particular, the title should be "specific, descriptive, concise, and comprehensible to readers outside the field" and in this case we recommend the reviewer's suggestion "Cardiovascular Disease risk in liver transplant recipients transplanted due to chronic viral hepatitis"

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

**********

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

**********

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

Reviewer #1: Yes

Reviewer #2: 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

**********

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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: (No Response)

Reviewer #2: This is an important study that provides new results about cardiovascular disease risk in patients with liver transplants who had different types of liver infections. I do not have new comments to add on this manuscript, as it appears solid and important to me, and resort to providing my opinion about how the comments by previous reviewers were addressed.

For points addressing the title of the manuscript, I feel like its updated version is still imperfect in terms of grammar. I would recommend changing the title to something like "Cardiovascular Disease risk in liver transplant recipients transplanted due to chronic viral hepatitis".

Regarding point 11 from the Editor, I agree with the authors that measuring cardiovascular disease risk in non-infected patients was beyond the scope of this manuscript. Going back and revising the study to include comparison to non-infected patients would equate to rejecting the manuscript that otherwise has a valid and important message to deliver. Expanding this study to statistically measure the relative risk of cardiovascular complications in infected and non-infected liver transplant patients can be viewed as a follow-up project to this study.

Reviewer 1 - I feel like their comment about the difference between cardiovascular and Framingham and ASCVD risk scores could be addressed better. The Discussion section does contain a few sentences on this matter (rows 231-234), so it can be argued that the manuscript does not need further revision due to this comment.

Reviewer 2 - while the point 1 about previous medical history is valid, I feel like information provided in Table 1 in the context of the authors' note about long follow-up in most of patients is a satisfactory response.

Overall, I believe this manuscript is in the form suitable for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Dulat Bekbolsynov

2 Mar 2022

PONE-D-21-16062R1

Cardiovascular Disease risk in liver transplant recipients transplanted due to chronic viral hepatitis

Dear Dr. Maggi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Vanessa Carels

Staff Editor

PLOS ONE