- Academic Editors
†These authors contributed equally.
Chemotherapies have changed the prognosis of patients affected by cancer over the last 20 years, with a significant increase in survival rates. However, they can cause serious adverse effects that may limit their use. In particular, anthracyclines, widely used to treat both hematologic cancers and solid cancers, may cause cardiac toxicity, leading to the development of heart failure in some cases. This review aims to explore current evidence with regards to anthracyclines’ cardiotoxicity, with particular focus on the classifications and underlying molecular mechanisms, in order to provide an overview on the current methods of its diagnosis, treatment, and prevention. An attentive approach and a prompt management of patients undergoing treatment with anthracyclines is imperative to avoid preventable antineoplastic drug discontinuation and is conducive to improving both short-term and long-term cardiovascular morbidity and mortality.
Heart failure (HF) is a clinical syndrome consisting of typical symptoms, such as breathlessness and fatigue, and signs, like elevated jugular venous pressure, pulmonary crackles, and peripheral oedema. It is caused by a structural and/or functional abnormality of the heart that results in elevated intracardiac pressures and/or inadequate cardiac output at rest and/or during exercise . Nowadays, the incidence of HF in Europe is about 3/1000 person-years  whilst its increasing prevalence has reached about 1–2% of adults . Despite improvements in HF treatment, mortality rate is still high (67% within 5 years from the diagnosis) . The main causes of HF are coronary artery disease, hypertension, valve disease, arrhythmias, cardiomyopathies, diabetes, congenital heart disease, infectious diseases, and drugs . Drug-induced HF is emerging as a potentially preventable form, with cytostatic agents, antidepressants, and immunomodulatory agents being the most common drugs correlated with HF. First recognized in 1960 with the introduction of anthracyclines as a treatment in oncological patients, drug-induced HF remains of interest today for its impact and severity . Anthracyclines are cytostatic antibiotics derived from Streptomyces spp. and are used in the treatment of various types of cancers, as they have been the most important class of antitumor drugs available for years . Doxorubicin (DOX) (also called adriamycin) is extensively used for the treatment of several solid tumors, such as soft tissue and bone sarcomas, breast, ovary, bladder, thyroid and lung cancer . Daunorubicin and idarubicin are used for the treatment of hematologic cancers, such as leukemia [8, 9]. Epirubicin is indicated in the treatment of breast cancer both in metastatic disease and as adjuvant therapy in women with early breast cancer . Their anticancer activity depends on their ability to interact with DNA through different mechanisms, including topoisomerase II inhibition, DNA intercalation, and DNA strand breakage leading to cancer cell death. Anthracyclines may also inhibit polymerase activity, regulate gene expression, and cause damage to the DNA of cancer cells by producing reactive oxygen species (ROS) [11, 12].
In this review, we summarize the available literature on the adverse effects of anthracyclines on the heart with regards to the epidemiology and pathogenetic mechanisms of cardiac toxicity. Furthermore, we will also discuss the diagnostic workflow, the treatments available at present, and possible prevention strategies for this drug-related complication.
We comprehensively searched the literature for data on the epidemiology, molecular mechanisms, diagnostic workflow, therapies, and preventive strategies of anthracyclines-induced cardiotoxicity. We used “anthracyclines” or “doxorubicin” or “daunorubicin” or “idarubicin” or “epirubicin” and “cardiovascular prevention” or “cardiotoxicity” or “cardio-oncology” or “left ventricular dysfunction” or “heart failure” as search terms. Articles published from 1998 to 1st October 2022 in English on both PubMed and MEDLINE were included. Most recent and largest original articles and meta-analyses have been selected. Reviews, consensus papers and guidelines were included if relevant. A search across the references of selected reports helped to identify further additional relevant studies.
According to 2022 European Society of Cardiology (ESC) guidelines on
cardio-oncology , anthracyclines-induced cardiotoxicity may be either
symptomatic, when signs and symptoms of HF appear, or asymptomatic, if there is
only a reduction in systolic left ventricular (LV) function parameters in absence
of symptoms. It can be acute, early onset chronic, or late onset chronic .
When acute, it occurs usually after a single dose. This presentation accounts for
|Dose of drug (mg/m
||Incidence of left ventricular dysfunction (%)|
Cardiomyocytes are vulnerable to anthracycline-induced toxicity and as such, LV
systolic dysfunction is the most common cardiac adverse effect of anthracyclines
. Several mechanisms have been implicated in the pathophysiology of
cardiotoxicity, including oxidative stress, inflammation, mitochondrial injury,
apoptosis, calcium (Ca
It has long been known that anthracyclines can cause a dose-dependent redox cycling with increased level of intracellular ROS . The oxidative stress caused by the production of both ROS and reactive nitrogen species (RNS), via induction of nitric oxide synthase, seems to play a crucial role in the development of cardiotoxicity . Indeed, DOX has a quinone moiety which facilitates electron transfer to oxygen molecules and other cellular redox enzymes (e.g., cytochrome P450 reductase, NADH dehydrogenase). Reduction of DOX produces the semiquinone radical, which re-oxidizes in the presence of O2 generating ROS that is associated with protein oxidation lipid peroxidation and DNA damage . RNS can damage cardiomyocytes through nitration and inactivation of key enzymes in the heart, such as myofibrillar creatine kinase [21, 22]. Free iron also contributes to DOX-mediated oxidative stress due to the propagation of ROS formation .
There is a strong interplay between inflammation and oxidative stress, with both
causing myocardial injury. Indeed, oxidative stress may stimulate an inflammatory
response through activating nuclear factor kappa B (NF-
Mitochondrial injury is also a hallmark of exposure to anthracyclines. Indeed,
electrostatic binding between mitochondrial cardiolipin and DOX leads to
disruption of the activity of complexes I, III, and IV in the electron transport
chain (ETC). DOX accumulation in mitochondria is associated with enhanced
production of ROS and RNS  followed by peroxidation of lipids and oxidative
damage to DNA and proteins, resulting in mitochondrial DNA damage, loss of
adenosine triphosphate (ATP) levels, peroxidation of cardiolipin and
mitochondrial permeability transition . The subsequent release of
cytochrome C may trigger apoptosis of cardiac cells. In this setting, the role of
nicotinamide adenine dinucleotide phosphate (NADPH) oxidase/ROS-mediated NF-
Calcium dysregulation is another well-known and established mechanism
contributing to anthracycline-induced cardiotoxicity . Anthracyclines might
modulate the sarco/endoplasmic reticulum Ca21 ATPase (SERCA) present on
sarcoplasmic reticulum (SR) and the sodium/potassium exchanger on sarcolemma
[37, 38] while mitochondrial ROS generated from the exposure of cardiac cells to
DOX might lead to an increase in cytosolic calcium levels. Increased levels of
calcium is correlated with calcineurin-dependent activation of the nuclear factor
of activated T-lymphocytes, which promotes cardiac cell death. In addition,
anthracyclines may also alter adrenergic and adenylate cyclase function to
trigger abnormalities in Ca
Recently, Wang et al.  found that the DOX-activated TRPA1 channel in cardiomyocytes could also cause cardiotoxicity by promoting endoplasmic reticle stress (ER) stress.
Narikawa et al.  demonstrated that DOX could increase the
expression of metalloproteases, transforming growth factor-
Evidence about the effect of anthracyclines on autophagy regulation in cardiomyocytes is controversial . It has been shown that DOX could stimulate autophagy through increased ratio of microtubule-associated proteins 1A/1B light chain 3-II and upregulated expression of p62, Beclin-1, by stimulating the expression of c-Jun N-terminal kinases and p70S6 kinase . Furthermore, the inhibition of mechanistic target of rapamycin by DOX promotes autophagy .
Finally, anthracyclines may suppress protein synthesis by directly binding to DNA and may also induce sarcomere disruption, with the ensuing cardiac “sarcopenia” being typically associated to anthracycline-induced HF . Cardiomyocytes are not the unique target of anthracycline toxicity, indeed endothelial cells, progenitor cells and fibroblasts in the heart, are also targets, contributing to a multifaced pathogenesis of anthracycline-induced cardiotoxicity. Increased arterial stiffness due to endothelial vascular damage caused by the alteration of the vascular extracellular matrix and by the interference with the endothelial regulation of vascular tone due to reduction of nitric oxide synthesis is also associated with anthracyclines. They may also increase the expression of cytokines leading to inflammation and vascular damage . The main proposed pathogenetic mechanisms of anthracyclines-induced cardiotoxicity are summarized in Fig. 1.
Main molecular mechanisms of anthracyclines-induced cardiotoxicity. Each arrow pattern, refers to a different molecular pathway. For more details, see the text. AC, anthracyclines; Ca, Calcium; ETC, electron transport chain; ILs, Interleukins; NADPH, Nicotinamide Adenine Dinucleotide Phosphate; NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells; NLRP3, NLR family pyrin domain containing 3; ROS, reactive oxygen species; TLRs, Toll-like receptors.
Previous epidemiological and observational studies have shown that specific risk
factors in the clinical history of patients undergoing chemotherapy with
anthracyclines may increase the chance to develop cardiotoxicity [16, 42, 43]. It
is fundamental to recognize and, whenever possible, treat these conditions in
order to prevent and to allow an early detection of anthracyclines-induced
cardiotoxicity. A recent meta-analysis has shown that traditional cardiovascular
risk factors, such as arterial hypertension (odds ratio (OR): 1.99; 95% confidence interval (CI): 1.43–2.76),
diabetes mellitus (OR: 1.74; 95% CI: 1.11–2.74), and obesity (OR: 1.72; 95%
CI: 1.13–2.61), are associated with an increased risk of cardiotoxicity. Tobacco
smoke (OR: 1.62; 95% CI: 0.94–2.77) and hypercholesterolemia (OR: 1.48; 95%
CI: 0.99–2.20) are less associated to cardiotoxicity . Chronic kidney
disease, pre-existing LV dysfunction, and pre-existing cardiovascular diseases,
such as congestive HF, valvular heart disease, and ischemic cardiomyopathy, have
been shown to increase the risk of cardiotoxicity . Pharmacogenomics is
emerging as a potential tool to help identify patients who are at higher risk for
cardiotoxicity . For example, Aminkeng et al.  highlighted that
a nonsynonymous variant in Retinoic Acid Receptor Gamma (RARG) gene is highly
associated with anthracyclines induced cardiotoxicity. Moreover, risk factors
associated with cancer therapies, such as a previous or high dose of
|Risk factors||Risk level|
|Congestive HF or cardiomyopathy||Very High|
|Coronary artery disease||High|
|LVEF reduction (
|Previous anthracycline-based chemotherapy||High|
|Previous left chest or mediastinum radiotherapy||High|
|Borderline LVEF (50–54%)||Medium (++)|
|Age 65–79 years||Medium (++)|
|Chronic kidney disease||Medium-low (+)|
|Previous non-anthracycline-based chemotherapy||Medium-low (+)|
|Current smoker or smoking history||Medium-low (+)|
|Elevated baseline troponin||Medium-low (+)|
|Elevated baseline BNP or NT-proBNP||Medium-low (+)|
BNP, brain natriuretic peptide; HF, heart failure; LVEF,
left ventricular ejection fraction; NT-proBNP, N-terminal prohormone of brain
natriuretic peptide. “Very High Risk” patients: congestive HF or
cardiomyopathy; “High Risk” patients:
As stated previously, age is an important risk factor for anthracycline-induced
cardiotoxicity . Patients aged between 65–79 years are considered at
medium-risk whilst patients aged
Classification of Anthracycline-associated Cardiotoxicity
Chemotherapy-associated cardiotoxicity can be divided into five main types:
Type 1: Cardiac dysfunction/cardiomyopathy/HF (cancer therapy related cardiac dysfunction CTRCD)
Type 2: Myocarditis
Type 3: Vascular toxicity
Type 4: Hypertension
Type 5: Arrhythmias and QTc prolongation .
Anthracyclines are primarily associated with cardiac dysfunction (type 1
cardiotoxicity). According with the ICOS consensus statement, cardiac dysfunction
is divided into symptomatic and asymptomatic . Symptomatic systolic
dysfunction is characterized by symptoms and signs of HF due to structural or
functional heart damage. It is classified into very severe, severe, moderate, and
mild based on the intensity of symptoms and the need for hospitalization.
Asymptomatic cardiac dysfunction is defined as LV ejection fraction (LVEF)
Classification of Cancer Therapy Related Cardiac Dysfunction. LVEF, Left Ventricular Ejection Fraction; GLS, Global Longitudinal Strain; HF, heart failure.
Over time, multiple surveillance protocols have been proposed, according to patients’ baseline risk of toxicity, to promptly diagnose anthracyclines-induced cardiotoxicity as to avoid the progression to HF. Most these protocols use repeated echocardiography and blood tests. The surveillance protocol recently proposed by the ESC is shown in Fig. 3 . Due to its reproducibility, versatility, and availability, echocardiography appears to be the cornerstone method for the evaluation of patients affected by neoplasms who are candidates to chemotherapy . Modified biplane Simpson’s technique  2D echocardiography (2DE) has been the most widely used tool for the evaluation of ventricular contractility . Despite this, it suffers from a series of limitations:
Compared to the 2D method, 3D echocardiography (3DE) allows more accurate volume measurements as it is not affected by geometric approximations and suffers less temporal variability and has a better intra-interobserver and test-retest variability . For that reason, in agreement with the latest guidelines of the European Society of Cardiology, 3DE appears to be the method of choice for measuring the volumes and systolic function of the left ventricle . 2DE also fails to detect small changes in LV contractility, underestimating the rate of mild asymptomatic cardiac dysfunction. The scientific interest has therefore focused on other parameters, such as those estimating myocardial deformation (strain and strain rate).
Surveillance protocol during anthracycline treatment. TX, Treatment; TTE, Trans Thoracic Echocardiography.
Strain refers to the patterns of myocardial contraction and relaxation that occur during each cardiac cycle. It encompasses radial, circumferential, and longitudinal strain. While evidence for the use of radial and circumferential strain is scarce, GLS is a parameter with high reproducibility and accuracy for early detection of subtle alterations in myocardial function that precede changes in LVEF .
Negishi et al.  evaluated women with breast cancer who underwent
treatment with trastuzumab (46% of whom received anthracycline sequentially with
trastuzumab) and found that a relative decrease of 11% in GLS was strongly
associated with CTRCD. Similarly, Wang et al.  found that,
in patients affected by diffuse large B-cell lymphomas and undergoing
anthracycline treatment, a relative GLS decrease of 13.8% at the third month of
chemotherapy was the best predictor of CTRCD, with a sensitivity of 75% and
specificity of 91% (Table 3, Ref. [67, 68, 69, 70]). In accordance, the American Society of
Echocardiography (ASE) and the European Association for Cardiovascular Imaging
(EACVI) suggest that a relative decline in GLS
|Primary end point||Definition of CTRCD||INDEX||AUC||Sensitivity and Specificity|
|Wang et al. ||Early detection of CTRCD||LVEF reduction
||Relative GLS reduction of 13.8% at the third cycle of chemotherapy||0.826||75% and 91%|
|Negishi et al. ||Early detection of CTRCD||Symptomatic LVEF reduction of 5% or an asymptomatic 10% reduction to an LVEF of 55%||Relative GLS reduction of 11% at 6 months after the start of chemotherapy||0.84||65% and 94%|
|Gripp et al. ||Early detection of CTRCD||Symptomatic LVEF reduction of 5% or an asymptomatic 10% reduction to an LVEF of 55%||Relative GLS reduction of 14% at 3 months after the start of chemotherapy.||0.97||80% and 99%|
|Sawaya et al. ||Early detection of CTRCD||Reduction of LVEF
||Relative GLS reduction of 11% at 3 months after the start of chemotherapy.||78% and 79%|
AUC, area under the curve; CTRCD, cancer therapy related cardiac dysfunction; GLS, Global longitudinal strain HF, heart failure; LVEF, left ventricular ejection fraction.
Despite its reproducibility and better accuracy than echocardiography in the evaluation of cardiac volumes and function, cardiac magnetic resonance (MRI) is currently not routinely used . It is particularly useful for the evaluation of cardiac masses and in case of technical difficulties in performing echocardiography . Similarly, multigated acquisition (MUGA) has also a limited role, with it being recommended only when echocardiography is equivocal and MRI is not available .
There is still a great debate on the use of biomarkers in the setting of CTRCD . Even though, they can identify subclinical LV dysfunction, the evidence in favor of their routine use in the follow up of patients undergoing chemotherapy is scarce and mostly based on expert opinions . Most of the available evidence involves the use of cardiac troponins (cTnT/I) and natriuretic peptides (NP), such as brain natriuretic peptide (BNP) and N-terminal prohormone of brain natriuretic peptide (NT-Pro BNP). cTnT/I are markers of myocardial injury and their role in the context of cardiac ischemic disease is well established . In a study conducted on 703 patients with breast cancer undergoing anthracyclines-based chemotherapy, it was shown that an increase in Troponin I levels at 3 and 6 months was associated with an increased risk of LV systolic dysfunction . Recently a meta-analysis conducted on 61 trials with 5691 patients investigated the predictive values of both cTnT/I and NP. They found that cTnT/I, but not NP, might be a useful screening marker for systolic dysfunction (negative predictive value of 93%) . Furthermore, a combined diagnostic approach with cTnT/I and imaging (such as GLS) could increase its ability to predict systolic dysfunction . Nevertheless, there is no conclusive evidence regarding the association between a rise in cTnT/I levels and the development of cardiotoxicity-related HF or cardiotoxicity-related mortality. While NP are a cornerstone in the diagnosis of HF , their role as a predictive tool for cardiotoxicity is less clear. Since NP are strongly related to a patient’s fluid volume status, their diagnostic power could be limited . Rug̈er et al.  have shown that levels of NT-pro-BNP measured at week 6 of anthracycline-regimen in 853 patients with breast cancer was significantly associated with the development of cardiotoxicity (OR: 1.03; 95% CI: 1.008–1.055; p = 0.01). Most guidelines currently recommend measuring both biomarkers at baseline and repeatedly during a chemotherapy regimen, in relation to the baseline risk of cardiovascular toxicity. However, uncertainties about the correct timing still persist .
Recommendations regarding preventive measures are based on the baseline risk of
anthracycline-related cardiotoxicity. In patients at high- and very high-risk pf
cardiotoxicity or in those who undergo high doses of anthracyclines (i.e., DOX
Alternatively, liposomal preparations of DOX are used to reduce anthracycline toxicity  as they block their entry into cardiac cells, thus limiting their cardiotoxic effect.
Current guidelines recommend starting a preventive therapeutic strategy with beta blockers (BB), angiotensin converting enzyme inhibitors (ACEI) and statins in patients with high-/very high-risk of developing cardiotoxicity and in patients with mild/moderate asymptomatic systolic dysfunction. Neurohormonal therapy may play a crucial role in preventing cardiotoxicity. Data pointing to a positive effect of ACEi and BB in preventing the decrease in LVEF are summarized in Table 4 (Ref. [87, 88, 89, 90, 91]). These findings are consistent with a recent meta-analysis of 17 trials with a total of 1984 patients with a follow-up ranging from 4 months to 2 years . However, it is currently debated whether the beneficial effect of neurohormonal therapy might translate into improved clinical outcomes. It is also interesting to note that in this large meta-analysis the absolute improvement in terms of LVEF assessed by 2DE was only 5%, i.e., that is within the range of interest variability of the measurement.
|Drugs used||Type of cancer||Inclusion criteria||Primary endpoint||Results vs controls|
|Janbabai et al. ||Enalapril 5 mg bid||Breast Cancer||Normal LVEF; Normal troponin level||6 months LVEF change from baseline||59.61% |
|Bosch et al. ||Enalapril 2.5 bid + Carvedilol 6.25 bid||Hematological||Normal LVEF + Normal troponin level||6 months LVEF change from baseline||–0.17 (–2.41 to 3.13) vs –3.04 (–6.01 to 0.11) (p = 0.04)|
|Kalay et al. ||Carvedilol 12.5 mg od||Breast cancer and Lymphoma||Normal LVEF; Normal troponin level||6 months LVEF change from baseline||68.9% vs 52.3% (p |
|Cardinale et al. ||Enalapril 20 mg od||Breast and Hematological malignancies||Increased Troponin level; Normal LVEF||Occurrence of cardiotoxicity||0 (0%) vs 25 (43%) (p |
|Avila et al. ||Carvedilol from 3.125 mg bid to 25 mg bid||Breast cancer||Normal LVEF||Prevention of a 10% reduction in LVEF||14 (14.5%) vs 13 (13.5%), p = 1|
LVEF, Left Ventricular Ejection Fraction.
Statins, among their pleiotropic effects, can also reduce ROS generation and can inhibit topoisomerase II. Since both these mechanisms are involved in anthracycline-related cardiotoxicity, a beneficial effect of statins has been hypothesized . Nabati et al.  evaluated the effect of rosuvastatin 20 mg od in the prevention of anthracycline-related cardiotoxicity during a 6 months follow-up, with rosuvastatin having prevented a 2DE estimated drop in LVEF in the intervention group. However, there was no difference between the two groups of patients with regards to GLS. The available evidence investigating the role of statins in preventing anthracycline-related cardiotoxicity is summarized in Table 5 (Ref. [94, 95]).
|Drugs used||Type of cancer||Inclusion criteria||Primary endpoint||Results: Intervention vs control|
|Nabati et al. ||Rosuvastatin 20 mg od||Breast cancer||Normal LVEF||Changes in the LVEF||53.54% vs 49.95% (p = 0.015)|
|Acar et al. ||Atorvastatin 40 mg od||Hematologic disorders||Normal LVEF||Patients with LVEF
||1 vs 5 (p = 0.18)|
LVEF, Left Ventricular Ejection Fraction.
Sacubitril/Valsartan and sodium-glucose co-transporter-2 (SGLT2i) are mainstays
for the treatment of HF with reduced EF (HFrEF), with their efficacy having been
shown in different trials and in both acute and chronic settings [96, 97, 98].
However, history of chemotherapy-induced cardiomyopathy over 12 months was an
exclusion criterion in the main trials for sacubitril/valsartan, such as the
Angiotensin–Neprilysin Inhibition versus Enalapril in Heart Failure trial
(PARADIGM-HF) trial . Likewise, patients with active malignancy required
treatment were excluded in the Dapagliflozin in Patients with Heart Failure and
Reduced Ejection Fraction (DAPA-HF) trial. Therefore, solid indications regarding
the use of sacubitril/valsartan and SGLT2i for this purpose is lacking. Recently,
Garcia et al.  provided evidence on the efficacy and safety of
Sacubitril/Valsartan for CTRCD and HFrEF (LVEF
The management of patients affected by anthracycline-induced cardiotoxicity is summarized in Fig. 4, in accordance with the 2022 ESC guidelines on Cardio-Oncology . With regards to the treatment of established anthracycline-induced cardiotoxicity, it is recommended to suspend chemotherapy and start cardiovascular therapy when symptoms related to HF appear, in accordance with the 2021 ESC guidelines on the management of HF .
Management of patients with anthracycline-induced cardiotoxicity. AC, Anthracyclines; ACE-I, Angiotensin-Converting Enzyme Inhibitors; ARBs, Angiotensin Receptor Blockers; BB, Beta Blockers.
Cardiotoxicity is a potentially troublesome adverse effect of anthracycline-based chemotherapies since they may cause LV systolic dysfunction followed by HFrEF, which tends to be permanent. It is thus of great importance to assess the risk of cardiotoxicity before anthracyclines therapy, to structure a follow-up plan that is tailored on individual patient’s risk. Despite there being a general consensus on the role of echocardiography in diagnosing anthracycline-related cardiotoxicity, the optimal timeframe to perform it and the optimal parameters to be evaluated for the diagnosis are still matter of debate. Biomarkers such as cTnI/T and NP have proved to have a good negative predictive value for anthracycline-related cardiotoxicity and as such, most recent guidelines recommend their serial measurement during follow-up. However, convincing evidence about ideal cut-off values, in terms of reliability, and definitive recommendations regarding its timing are lacking. Since most cardiotoxicity is early chronic (within 1 year from the start of anthracyclines), current guidelines recommend a strict follow up during the first year for patients at high- and very high-risk of cardiotoxicity along with the introduction of an ACEi/ Angiotensin Receptor Blockers (ARB) plus BB treatment regimen. Nevertheless, recommendations differ significantly between international guidelines. Due to cardiotoxicity being usually permanent, a deeper knowledge of the molecular pathways of action of anthracyclines and their effects on the cardiovascular system is crucial. Hopefully this might help minimizing their negative impact on heart and vessels and to develop more effective preventive strategies and therapeutic options for anthracycline-related cardiotoxicity. These are essential steps that would translate in a better survival, limited life-saving chemotherapy drug discontinuation, and better prognosis for patients undergoing anthracycline-based chemotherapies.
AF, VF and AB designed the research study. AF and VF performed the research. GS, SR, GT, MV, EB provided help and advice on data searching and analysis. AF and VF analyzed the data. All authors contributed to the writing of the paper and to the editorial changes in the manuscript. All authors made substantial contributions to conception and design, to acquisition, analysis and interpretation of data. All authors have been involved in drafting the manuscript revising it critically for important intellectual content. All authors have given final approval of the version to be published. Each author have participated sufficiently in the work to take public responsibility for appropriate portions of the content and they agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript.
The authors wish to thank Dr. Melwyn Luis Muthukkattil, who has helped review the paper in order to be released.
This research received no external funding.
The authors declare no conflict of interest. Massimo Volpe is serving as one of the Editorial Board members and Guest Editors of this journal. We declare that Massimo Volpe had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Brian Tomlinson and Jerome L. Fleg.
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