Sepsis heterogeneity

Wei Wang · Chun-Feng Liu

Abstract Background Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection,with extremely high mortality.Notably,sepsis is a heterogeneous syndrome characterized by a vast,multidimensional array of clinical and biologic features,which has hindered advances in the therapeutic field beyond the current standards.Data sources We used PubMed to search the subject-related medical literature by searching for the following single and/or combination keywords: sepsis,heterogeneity,personalized treatment,host response,infection,epidemiology,mortality,incidence,age,children,sex,comorbidities,gene susceptibility,infection sites,bacteria,fungi,virus,host response,organ dysfunction and management.Results We found that host factors (age,biological sex,comorbidities,and genetics),infection etiology,host response dysregulation and multiple organ dysfunctions can all result in different disease manifestations,progression,and response to treatment,which make it difficult to effectively treat and manage sepsis patients.Conclusions Herein,we have summarized contributing factors to sepsis heterogeneity,including host factors,infection etiology,host response dysregulation,and multiple organ dysfunctions,from the key elements of pathogenesis of sepsis.An in-depth understanding of the factors that contribute to the heterogeneity of sepsis will help clinicians understand the complexity of sepsis and enable researchers to conduct more personalized clinical studies for homogenous patients.

Keywords Heterogeneity · Host response · Infection · Personalized treatment · Sepsis

Introduction

Sepsis is life-threatening organ dysfunction caused by a dysregulated host response to infection [1].An estimated 48.9 million people globally are diagnosed with sepsis each year,accounting for 11 million deaths and representing 19.7% of all global deaths [2].Notably,the percentage of global sepsis-related deaths (from any cause) in 2017 peaked in early childhood,declined through early adulthood,and peaked again in older adults [2].Therefore,not only adults but also children with sepsis should be considered.Moreover,an estimated 48 children per 100,000 population develop sepsis,22 children per 100,000 population develop severe sepsis,and 2202 neonates per 100,000 live births develop neonatal sepsis,translating into 3.0 million and 1.2 million cases of childhood sepsis in neonates and children per year globally [3].An epidemiologic survey of pediatric sepsis in regional hospitals in China revealed an estimated incidence of 181/100,000,of which 80% were in those below 5 years of age [4].Mortality in children with sepsis ranges from 4% to 50% depending on disease severity,risk factors,and geographic location [5].Therefore,sepsis has long been a public health concern given its high morbidity and mortality.

Unfortunately,despite more than 30 years of research and more than 200 randomized controlled trials,treatment for sepsis remains supportive care and antibiotic treatment without a single treatment that consistently saves lives in sepsis patients [6,7].A growing number of experts believe that the reason may be that sepsis is a heterogeneous syndrome characterized by a vast,multidimensional array of clinical and biologic features [8,9].In 2005,Ulloa et al.proposed that the definition of sepsis was too broad and encompassed heterogeneous groups of patients suffering similar,but different syndromes that were historically grouped under the general diagnosis of sepsis [10].Recently,Maslove et al.suggested that sepsis was characterized not by any particular biopsy feature,genetic mutation,microbial culture or serologic test but rather by collections of signs and symptoms that together paint the picture of a clinically recognizable entity.As a result,sepsis is heterogeneous by nature and can arise from a multitude of infections caused by many different pathogens,resulting in different patterns of organ injury[11–13].According to experts,high mortality in sepsis stems from the lack of personalized treatments for specific patients rather than because of the ineffectiveness of pharmacological therapy [14].Moreover,an increasing number of recent studies have been conducted to identify two or more homogeneous subgroups with common clinical and laboratory features or specific biologic features that could be targeted [9].These findings suggest that the heterogeneity of sepsis at the patient level,resulting in varied disease presentations,progressions,and treatment responses,has hindered advances in the therapeutic field beyond the current standards [7,15,16].Thus,an in-depth understanding of the heterogeneity of sepsis can help clinicians identify the different conditions that may develop in different patients as early as possible and researchers conduct clinical studies of different patients to carry out more personalized treatment.In this review,we have summarized contributing factors for sepsis heterogeneity,including host factors,infection,uncontrolled host response,and multiple organ dysfunctions.

Factors affecting sepsis heterogeneity

Heterogeneity is present in patients with sepsis.Diversity in clinical manifestations and prognoses has been observed in patients with sepsis,such as disease severity and complications due to differences in patient age,biological sex,gene susceptibility,underlying conditions,site of infection,source of infection,timing,method of treatment and intervention,etc.The following sections will discuss the heterogeneous manifestations of sepsis regarding host factors,infection,disease progression,and prognosis (Fig.1).

Fig.1 Heterogeneous manifestations of sepsis.Various factors contribute to sepsis heterogeneity,including host factors,infection,uncontrolled host response and multiple organ dysfunctions.Top left: people with different ages,sexes,comorbidities,and genetics have different sepsis characteristics.Among them,tumors,heart disease,lung disease,and bone marrow transplantation are common comorbidities.Top right: a variety of bacteria,viruses,and fungi infect different parts of the human body,including the CNS,respiratory tract,abdomen,cardiovascular system,bloodstream,skin,and genitourinary system,and will have different host responses.Middle:When a pathogen invades the human body,immune cells recognize the pathogen,secrete cytokines or phagocytose the pathogen.Vascular endothelium supports protective immune responses by increasing the expression of adhesion molecules and widening gap junctions.Neutrophils release extracellular traps (NETs) for platelet activation.Platelets,endothelial cells and leukocytes release micro-particles.Tissue factor from monocytes released into the blood,together with NETs and micro-particles,constitutes an “immune thrombus” that traps pathogens in the thrombus.Bottom: However,sepsis is a state of dysregulated responses,such as immune response,endothelial,and coagulation disorders,which ultimately lead to different organ dysfunctions,including cardiovascular,respiratory,neurological,liver,kidney,and blood dysfunction.Different patients are in different dysregulated states,resulting in the heterogeneity of sepsis.It should be noted that sepsis also has other systemic host response disorders,such as neurohumoral and metabolic disorders.CNS: central nervous system.The figure was created with BioRender.com and we have the reprint copyright transfer of the figure.

Host factors

Age

Sepsis incidence is disproportionate in elderly adults,but age is an independent predictor of mortality in adults.Compared with younger patients with sepsis,elderly patients die earlier during hospitalization,while elderly survivors require skilled nursing or rehabilitative care more frequently after being discharged [17].An epidemiological study in the United States showed that the incidence increased >100-fold with age (20/100,000 in children to 2620/100,000 in those >85 years old),and mortality also increased with age,from 10% in children to 38.4% in those >85 years old [18].In addition,a comparison study showed that the incidence of sepsis in the oldest old (≥ 85 years old) was 9414 cases per 100,000 population in 2012,which was 31 folds greater than the adult incidence (18–64 years old) (303 cases per 100,000 population) and three-fold greater than the elderly incidence (65–84 years old) (2908 cases per 100,000 population) in Taiwan of China [19].Furthermore,a global epidemiological study has shown that there were an estimated 20.3 million incident sepsis cases worldwide among children younger than five years,4.9 million incident sepsis cases among children and adolescents aged 5–19 years,and 23.7 million incident sepsis cases among adults 20 years and older in 2017 globally [2].These studies have shown a high incidence of sepsis in elderly individuals (especially those older than 85 years old) and children (especially those younger than 5 years old).A systematic review suggested that mortality rate ranged from 1% to 5% for sepsis and 9% to 20% for severe sepsis in children,while mortality varied between 11% and 19% for neonatal sepsis [3].In addition,a global epidemiological study suggested that the percentage of global sepsis-related deaths (from any cause)in 2017 peaked in early childhood,declined through early adulthood,and rose among older adults [2].Furthermore,a meta-analysis showed that younger age was a risk factor for severe sepsis in children [20].These studies indicate that peak sepsis incidence and mortality occur in extreme age groups,with newborn babies,young children,and elderly people at highest risk.

Moreover,sepsis clinical manifestations in adults differ from those in children,such as a higher prevalence of hyper-dynamic and hypodynamic septic shock in adults and children,respectively [21],and an increased sepsis-induced heart dysfunction in the latter [22].Differences in manifestations and host responses in children and adults may be partly due to different developmental states or underlying diseases [23].

Biological sex

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Comorbidities

The odds ratio of progression to sepsis and death with one comorbidity or with two or more comorbidities was greater than that in the absence of comorbidities [29].In addition,a cohort study including six United States hospitals showed that the most common underlying causes of death in patients with sepsis were solid cancer,chronic heart disease,hematologic cancer,dementia,and chronic lung disease [30].Moreover,tumor status,bone marrow transplantation,chronic neurological disease,and markers of severity were found to be significantly associated with increased sepsis mortality in a cohort study including children [31].Consequently,it can be inferred that differences in existing comorbidities result in various host responses,leading to diverse outcomes.

Several epidemiological studies have shown that the incidence of sepsis is higher in men than in women [18,24,25].Interestingly,a recent meta-analysis showed that the independent prognostic effect of biological sex on all-cause hospital,28-day all-cause,and all-cause intensive care unit(ICU) mortalities in critically ill adults with sepsis was inconclusive.However,female sex may be associated with decreased 1-year all-cause mortality [26].Notably,various experimental and clinical studies have demonstrated beneficial effects of estrogen on the cardiopulmonary,immune and central nervous system (CNS),liver,kidneys,and overall survival of the host [27].In addition,other mechanisms,such as the immunosuppressive role of testosterone and X-linked mosaicism,were thought to contribute to the observed biological sex-dependent differences in sepsis [28].Therefore,biological sex is partly associated with sepsis heterogeneity.

Gene susceptibility

Individual heterogeneity of sepsis may be modulated by genetic variation,and such regulatory variants might only functionally modulate gene expression in the disease context of sepsis,which is referred to as gene susceptibility.For example,a recent genome-wide association study (GWAS)comprising four cohorts identified common variants inFERthat were associated with a reduced risk of death from pneumonia-induced sepsis [32].In addition,Davenport et al.identified cis-and trans-acting expression quantitative trait loci (eQTL) for key immune and metabolic response genes and the sepsis response network [33].To define the genes modulated by genetic variation and to identify specific functional regulatory polymorphisms,associations between genetic variants [usually single-nucleotide polymorphisms(SNPs)] and differences in gene expression between individuals can be mapped as eQTL.Therefore,genetic susceptibility may be important in sepsis heterogeneity.

Infection

Infection sites

Sepsis infection sites can be the abdomen,respiratory tract,bloodstream,skin,CNS,genitourinary and cardiovascular system.An international study of the prevalence and outcomes of infection in ICUs showed that among the 7000 patients diagnosed with infection,the most common site was the lungs (64%),followed by the abdominal cavity (20%),bloodstream infections (15%),and the kidneys or genitourinary system (14%) [34].For children,a 2015 global pediatric severe sepsis survey found that the most common primary site of infection was the lungs (40%),

Sepsis can cause dysfunction of multiple systems throughout the body,including the circulatory,respiratory,nervous,urinary,and digestive systems [54].A study of the global epidemiology of pediatric severe sepsis showed patients with respiratory (82.7%),cardiovascular (70.2%),hematologic (30.9%),hepatic (25.2%),neurologic (21%),and renal (16.4%) dysfunctions at the screening stage [22].Moreover,67% of patients had multi-organ dysfunction at sepsis recognition,with subsequent development of new or progression of multi-organ dysfunction in 30% of pediatric patients with severe sepsis [22].An epidemiological study found that the majority of children with sepsis who had multiple organ dysfunction resulting in death (84% overall) had organ dysfunction on the day of death as follows: cardiovascular (72%),respiratory (95%),renal (38%),hepatic (44%),hematologic (40%),and neurologic (58%)[57].Therefore,temporal heterogeneity in organ systems involved can be observed in pediatric patients with sepsis.Notably,organ failure contributed cumulatively to mortality,with temporal improvements in survival among pediatric patients with fewer than three failing organs [24].However,the organs that failed most frequently in adult patients with sepsis were the lungs (18%) and kidneys (15%);less frequent were cardiovascular (7%),hematologic (6%),metabolic (4%),and neurologic (2%) failures [24].In addition to age,sepsis-induced multiple organ dysfunctions have enormous heterogeneity in the severity of organ failure and the number of organ failures.For example,a patient may have severe,even refractory,shock but remain on room air with only mild dysfunction of the kidneys and brain.Conversely,some patients develop severe acute kidney injury or acute respiratory distress syndrome but maintain appropriate hemodynamics [58].These results suggest that sepsisinduced multiple organ dysfunctions also have individual heterogeneity.Therefore,temporal heterogeneity and individual heterogeneity are challenges in the clinical treatment of sepsis-induced multiple organ dysfunction.

A large number of studies have found that sepsis caused by different infection sources has different clinical characteristics,such as patient characteristics,organs affected,cultured pathogens,supportive treatments,and length of hospital stay [35,37,38].For example,a multicenter,prospective cohort study in Japan showed that septic shock was more frequent among patients with intra-abdominal(72.2%) and urinary tract (70.2%) infections than other sites,and the in-hospital mortality rate due to severe sepsis and septic shock was 23.4% [range 11.9% (urinary tract infection) to 47.6% (CNS infection))][37].Another multicenter study in European countries compared the differences in infection between respiratory and abdominal origins,suggesting that septic shock was more common in patients with abdominal infections who were also more likely to have early coagulation failure and acute renal failure,while patients with respiratory infection were more likely to have early neurological failure.In addition,the median length of hospital stay was longer in patients with abdominal infection than in those with respiratory infection [38].These studies indicate that different sites of infection tend to present various clinical manifestations in sepsis.

Sepsis heterogeneity could be partly explained by sourcespecific host response dysregulations.A phenotypic heterogeneity analysis by site of infection in surgical sepsis showed that abdominal infections caused more prolonged pro-inflammation,immunosuppression,and persistent organ dysfunction,whereas pulmonary infections caused similar protracted pro-inflammation and organ dysfunction but normalized immunosuppression [39].Skin/soft tissue and genitourinary infections occurred in younger patients with fewer comorbidities,less perturbed immune responses,and faster resolution of organ dysfunction with better longterm outcomes.Vascular sepsis patients were older males with more comorbidities.Pro-inflammation was blunted with baseline immunosuppression and organ dysfunction that persisted with the worst long-term outcomes [39].In addition,Peters-Sengers et al.conducted blood leukocyte transcriptomes of diverse sources and found stronger inflammatory and cytokine responses,loss of vascular integrity,and coagulation activation in abdominal sepsis than in respiratory sepsis.Endothelial cell activation was prominent in urinary,cardiovascular and skin infections,while CNS infection was associated with the fewest host response aberrations [40].Therefore,it is necessary to actively understand the differences in host responses at different infection sites and explore more personalized therapeutic regimens.

Pathogenic organisms

Strikingly,ensuring greater awareness on the part of both the public and health care workers to recognize sepsis and to understand it as a true time-critical medical emergency is a crucial step in reducing the global burden of sepsis.Furthermore,attention to bolstering public health initiatives to prevent sepsis,surveillance systems for detecting outbreaks early,and provision of simple early treatment can help to counterbalance the effects of a lack of critical care facilities in many LMICs [66].Consequently,sepsis as a major threat to patient safety and global health requires coordinated efforts by politicians,policymakers,health care administrators,researchers,and clinicians working with people of all ages in all health care settings and in the community.

The differences in host responses caused by diverse pathogens may be due to various virulence factors.For example,RNAs or DNAs of viruses that infiltrate cells activateretinoic acid-inducible gene I(RIG-I)-like receptors (RLRs),melanoma differentiation-associated gene 5(MDA-5),cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) receptors,or several specific toll-like receptors (TLRs) to promote downstream signals and promote anti-activation of viral type 1 interferon signaling.In addition,C-type lectin receptors (CLRs) and TLRs recognize fungal cell wall components,such as O-linked mannose,glycolipid component phospholipomannan,β-(1,3)-glucan,and α-mannose,producing downstream signals against fungi,whereas endotoxins produced by Gram-negative bacteria,such as lipopolysaccharides and exotoxins produced by Gram-positive bacteria,are recognized by various TLRs and nucleotide-binding leucine-rich repeat receptors(NLRs),causing activation of downstream effector pathways[48].Thus,different virulence factors are recognized by different receptors and generate different downstream signals.Furthermore,finding biomarkers to distinguish infections is critical in sepsis treatment,facilitating the early initiation of specific antibiotic therapy for bacterial infections and avoiding unnecessary antibiotic administration for non-bacterial infections.Interestingly,some studies have analyzed transcriptomics to identify gene signatures that can differentiate between viral and bacterial infections [49–51].For example,Sweeny et al.used multi-cohort analysis to derive a set of seven genes for robust discrimination of bacterial and viral infections.Of the seven genes in the bacterial/viral metascore,six have previously been linked to infections or leukocyte activation.BothIFI27andJUPwere shown in singlecohort genome-wide expression studies to be induced in response to viral infection,whereasTNIP1andCTSBwere important in modulating the nuclear factor kB and necrotic responses to bacterial infection.Finally,LAX1(upregulated in viral infections) is involved in the activation of T and B cells,andHK3is instrumental in the neutrophil differentiation pathway [49].From this point of view,sepsis caused by different pathogenic infections is the key to heterogeneity and makes clinical diagnosis and treatment more difficult.

Uncontrolled host response

The pathogenesis of sepsis is complex,involving multiple networks of multiple systems,including the immune response,metabolic reprogramming,endothelial cell damage,coagulation disorders,neurohumoral dysregulation,etc.[52,53].Among them,immune responses,endothelial cell damage,and activation of the coagulation system interact with each other,promote and accelerate each other,and jointly enhance the occurrence and development of sepsis.

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When pathogens invade the body,the pattern recognition receptors of innate immune cells recognize pathogen-associated molecular patterns,causing the release of pro-inflammatory and anti-inflammatory mediators.Vascular endothelium supports protective immune responses by increasing the expression of adhesion molecules and widening gap junctions,allowing immune cells to adhere and migrate to sites of infection.Pro-inflammatory cytokines induce neutrophils to release extracellular traps (NETs),a network of pro-coagulant aggregates including DNA,antimicrobial proteins,and enzymes that together form a scaffold for platelet activation.These activated platelets,endothelial cells and leukocytes release micro-particles,which are vesicles generated from the plasma membrane and contain inflammatory factors,pro-oxidants,pro-coagulant lipids,and various proteins such as tissue factor,angiopoietin-2,and vWF multimers.Thereafter,tissue factor expression in circulating monocytes is upregulated.Together with the previously released NETs and micro-particles,NETs constitute an “immune thrombus” that traps pathogens and further activates white blood cells [52].However,homeostasis is disrupted when the body is unable to clear the pathogen,and the pathogen continues to stimulate the host cell production of immune thromboses.

Regarding the immune response,the balance between pro-inflammatory and immunosuppressive responses would be disrupted,with the intensity of both responses depending on multiple factors of both the host—such as genetics and comorbidities—and the pathogen—such as type,virulence,and burden [54].An extreme manifestation of an excessive inflammatory storm is macrophage activation syndrome(MAS),with a frequency ranging between 3% and 4%,and is associated with early mortality after 10 days of sepsis [55].Similarly,an increasing number of studies have shown that immunosuppression in sepsis is closely related to mortality[53].When endothelial cells are over-activated,increased leukocyte adhesion,hypercoagulability,vasodilation,and barrier function loss occur,resulting in diffuse tissue edema,multiple organ dysfunction,and even septic shock [52].In addition,some patients may develop thrombocytopeniaassociated multiple organ failure (TAMOF).The mechanism may involve decreased levels of ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin motifs)and increased von Willebrand factor activity [56].The most severe manifestation of TAMOF is disseminated intravascular coagulation (DIC) [53].Uncontrolled host responses can cause sepsis phenotypes of varying severities,such as septic shock and diverse organ dysfunctions,which may depend on the host factors and pathogen.The future direction of sepsis treatment is to adopt different treatment strategies according to different phenotypes.

Organ dysfunction

followed by the bloodstream (19%),abdominal cavity(8%),CNS (4%),and genitourinary system (4%)[22].The highest fatalities belonged to cases with multiple infection sites,and the lowest fatalities were in the case of genitourinary infection sources [35,36].

Conclusion and prospects

AcknowledgementsThanks to Biorender Website where the figure was created.

Notably,while early recognition and improved management of acute episodes are important steps in reducing death and disability from sepsis,a substantial reduction in the burden of sepsis-related disease requires action across the entire healthcare system [62].For example,the majority of sepsis cases,approximately 70%–80%,are community acquired,making emergency departments and primary care key targets to improve recognition and early management,especially for low-and middle-income countries (LMICs),which bear the greatest burden of sepsis [63].Recently,the Indian government announced the Ayushman Bharat Program with two components,Health and Wellness Centers (HWCs),to deliver comprehensive primary health care (PHC) services to the entire population [64],which could be new opportunities and challenges to manage sepsis in India.HWCs pay more attention to preventive and promotive health services and strengthen the referral system to ensure continuity of care,representing advantages in managing acute and severe diseases such as sepsis [65].In addition,post-discharge mortality after sepsis is about the same as sepsis-related mortality in the hospital of LMICs [66].“Continuum of care”through coordination between PHCs and secondary and tertiary care services tends to be an effective measure [65].

Sepsis can be caused by a broad range of pathogens,the most common being bacteria,fungi,and viruses.A 2015 global pediatric severe sepsis survey found that 65% of patients tested positive for pathogens,of which 26.5% were Gram-positive bacteria,27.9% were Gram-negative bacteria,13.4% were fungi,and 21% were viruses.Sources of positive isolates include blood,urine,cerebrospinal fluid,respiratory system,stool,wound,and other normally sterile body fluids,and some infections are poly-microbial [22].Notably,different proportions occurred between adults and children infected with different pathogens.A cross-sectional study in Southeast Asia showed that among sepsis patients infected with identified pathogens,viruses were identified alone compared to bacteria identified alone (36% vs.12%)in children and (12% vs.31%) in adults [41].In addition,fungal infections are generally caused by opportunistic pathogens,and immuno-compromised patients,such as those with malignant tumors and bone marrow transplantation,are more susceptible to this type of infection [42].Among fungal infections,those caused byCandidaspecies are by far the predominant cause of fungal sepsis,accounting for 5% of all sepsis cases [43].Furthermore,some studies have shown that the mortality rate of sepsis caused by different microbial infections is different.For example,patients with identified viral infections had a lower risk of death than those with identified bacterial infections,and these were also observed in sepsis patients infected with only one pathogen[41].Moreover,a retrospective analysis of an international study suggested that compared to patients with bacteremia,patients with candidemia had higher ICU mortality (43% vs.25% to 29%) and longer hospital stays [44].Additionally,different types of bacterial infections have different fatality rates.For example,gram-negative infections tend to have a higher mortality rate than gram-positive infections [45].In addition,a survey about the epidemiology and microbiology of severe sepsis and septic shock in children showed that the mortality was the highest for methicillin-resistantStaphylococcus aureus(14.42%) among Gram-positive organisms and forPseudomonas(21.49%) among Gram-negative organisms [46].Notably,methicillin-resistantStaphylococcus aureusinfection is an independent risk factor for inhospital mortality,and the mortality rate is higher than 50% in patients with this type of infection [47].

In summary,differences in host factors (genetics,biological sex,age,comorbidities) (Fig.1),infection etiology,host response dysregulation,and multiple organ dysfunctions all contribute to sepsis heterogeneity,which make it difficult to effectively treat and manage sepsis patients.Strikingly,risk stratification and sub-classification have become promising approaches to address sepsis heterogeneity.Using biomarkers for patient stratification according to prognosis and severity of illness can help clinicians identify patients with poor prognosis early.In addition,sub-classification enables us to find similar populations in sepsis patients,enabling the discovery of more personalized drug targets and contributing to the effective and precise implementation of clinical research.At present,an increasing number of researchers have used omics technology and bioinformatics methods to classify sepsis patients,which has good application prospects [59–61].We believe that in future,joint efforts in multiple fields and industries of medicine,scientific research,bioengineering and pharmaceutical research are needed to develop sepsis classification standards applicable to different ages,countries and regions to solve the problem of sepsis heterogeneity.

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FundingThis work was supported by the National Natural Science Foundation of China (81971810 to Chun-Feng Liu),Liaoning Province Science and Technology Major Special Project (No.2020JH1/10300001 to Chun-Feng Liu),Shenyang Science and Technology Plan Project(20–205-4–002 to Chun-Feng Liu).

Author contributionsLCF: performed study concept,design and revision of the paper (Conceptualization,Writing—review &editing);WW: performed development of writing the paper (Writing—original draft).

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Data availabilityAll relevant raw data can be freely available to any researcher wishing to use them for non-commercial purposes from the corresponding author.

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Declarations

Conflict of interestThe authors declare no conflicts of interest.No financial or non-financial benefits have been received or will be received from any party related directly or indirectly to the subject of this article.

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