**1. Introduction**

Seafood is a fundamental source of proteins and nutrients for human nutrition. Its global consumption has increased since 1960 at rates per year higher than that of all land animals, combined and individually (i.e., bovine, ovine, porcine, etc.), except for poultry [1]. However, with the increasing demand for sea products, most of the fishery resources of the world's oceans have been overfished, and many are in a condition of variable levels according to the vulnerability of the different species. In addition, the overfishing condition of continental shelf fish resources has pushed the fishing activities to move towards the exploitable living resources of the deep sea [2–4]. In this regard, deep waters (i.e., beyond the continental shelf, and deeper than approximately 200 m) have acted as a refuge for several stocks with an extensive vertical distribution, where no fishing was occurring until the first decades of the last century [5]. With the expansion of fishing to deeper waters, favoured by the development of new technologies, muddy deep bottoms and other deep-water refuges—such as soft-bottom coral gardens (CGs), cold-water coral (CWC) reefs, submarine canyons (SCs), and seamounts (SEs)—have been affected by this activity, and may no longer play their role of providing structure, food, and shelter for

**Citation:** Maiorano, P.; Capezzuto, F.; Carluccio, A.; Calculli, C.; Cipriano, G.; Carlucci, R.; Ricci, P.; Sion, L.; Tursi, A.; D'Onghia, G. Food from the Depths of the Mediterranean: The Role of Habitats, Changes in the Sea-Bottom Temperature and Fishing Pressure. *Foods* **2022**, *11*, 1420. https://doi.org/10.3390/ foods11101420

Received: 4 April 2022 Accepted: 9 May 2022 Published: 13 May 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

fishery resources. All of these deep-water ecosystems have been identified as hotspots of biodiversity (e.g., [6–8]), and since they can be valuable fishing sites, due to the occurrence of large sizes and high abundances of commercial species, they are often impacted by commercial fishing [9–20]. Fisheries for some deep-sea fish stocks have also collapsed due to heavy pressure on species whose life history traits do not make them well-suited to an intensive harvest [3,4,21].

The EU began to deal with deep-sea fisheries in 1992, as a result of assessments carried out by the International Council for the Exploration of the Sea (ICES), who stated that most of the deep-water species of commercial interest were overfished [28]. The current scientific evidence suggests that many deep-sea fish stocks are being exploited beyond sustainable levels [23,29–32], thus emphasising the need to improve the management of these species [3,32–35].

It is well-known that fishing can impact harvested populations directly by excessive removal of individuals, and indirectly by reducing habitat complex structures that guarantee their bioecological activities and provide protection from predators, as well as from adverse physical factors (e.g., [36–40]). Globally, around 40% of trawling fishing grounds are on waters deeper than the continental shelf [41], and several deep-sea habitats and ecosystems have been impacted by fishery activities, with consequent depletion of or reduction in economically important species (e.g., [3,9,21,42–45]).

According to the FAO [46], vulnerable marine ecosystems (VMEs) are groups of species, communities, or habitats that may be vulnerable to impacts from fishing activities. Corals, together with sponges, echinoderms, molluscs, and other epibenthic species, play a significant role in the formation of VMEs. Furthermore, most coral taxa are included in relevant lists of protected species, such as the Red List drawn up by the International Union for the Conservation of Nature (IUCN) [47–50]. Their vulnerability is linked to their likelihood of experiencing substantial alterations from short-term or chronic disturbances, as well as to their difficulty in recovering (for example, slow growth rate, late age of maturity, low or unpredictable recruitment, and long life expectancy) [51–55]. In addition, there is sufficient evidence that VMEs act as essential fish habitats (EFHs), defined as "those waters and substrates necessary to fish for spawning, breeding, feeding, or growth to maturity" [56–58]. Therefore, there is an international consensus in favour of the protection of VMEs in order to combine conservation and fisheries-management objectives according to the ecosystem approach to fisheries (EAF) [46,59–64].

Fish consumption has always been an important part of people's diets around the Mediterranean Sea. The annual production of about 788,000 tonnes, for a total revenue of USD 3.4 billion, offers employment opportunities to several hundred thousand people, supplies seafood products for human consumption to local and regional markets, and creates many other indirect benefits, although fisheries and related activities also produce a large amount of marine litter (including plastics) as a global increasing threat [1]. In the Mediterranean Sea the exploitation of deep-water resources only started in the first decades of the last century. In particular, the deep-water red shrimps *Aristaeomorpha foliacea* (giant red shrimp) and *Aristeus antennatus* (blue and red shrimp) were the target species for deep-water bottom trawling in the 1930s in the Ligurian Sea, where catches of these two shrimps were between 100 and 200 kg/day and, after the Second World War, they could be up to 1000 kg/day per boat [65,66]. In the 1940s, these resources began to be exploited in the Catalan and Balearic seas, and subsequently in other Mediterranean areas [67]. In the Mediterranean Basin, the development of deep-water commercial fisheries was due to the narrowness of the shelf, crossed by several submarine canyons, as well as the growth of the human population along the basin and high demand for a muchappreciated food. However, considering the multispecies nature of the Mediterranean fisheries, the deep-water red shrimps are mostly caught in bathyal muddy bottoms, between 400 and 800 m, together with many other valuable demersal species, such as the European

hake (*Merluccius merluccius*), the deep-water rose shrimp (*Parapenaeus longirostris*), the Norway lobster (*Nephrops norvegicus*), the blackspot seabream (*Pagellus bogaraveo*), the greater forkbeard (*Phycis blennoides*), and the wreckfish (*Polyprion americanus*). Other deepwater species of commercial interest, such as the blue whiting (*Micromesistius poutassou*), European conger (*Conger conger*), blackbelly rosefish (*Helicolenus dactylopterus*), angler (*Lophius piscatorius*), blackbellied angler (*Lophius budegassa*), blackmouth catshark (*Galeus melastomus*), bluntnose sixgill shark (*Hexanchus griseus*), golden shrimp (*Plesionika martia*), southern shortfin squid (*Illex coindetii*), European flying squid (*Todarodes sagittatus*), and lesser flying squid (*Todaropsis eblanae*), represent additional components of deep catches. Most of the fish species are commonly caught with trawl nets, longlines, and gillnets, while crustaceans and cephalopods are fished mainly with trawl nets, and sometime with pots.

Most of the abovementioned species are distributed across a wide depth range, between shelf and slope; some of them are typically distributed in deep waters. Moreover, past and recent investigations in deep-sea habitats with a complex and heterogeneous structure—such as coral ecosystems, submarine canyons, and seamounts (now considered VMEs)—have proven that most of the abovementioned species use these types of habitats for shelter, feeding, spawning, and nursery (e.g., [20,58,68–71]). Indeed, habitat features play an important role in determining the structure of species assemblages, and habitat loss may impact—albeit in different ways—on all life stages and critical phases of the different species [38,72]. While adult individuals may not be strictly affected by habitat as juveniles, the detrimental effects of habitat loss on juvenile survival may have longer-term impacts on adult populations [73].

The innovation and sustainability of the food (in the present case, the fishery resources represented by deep-sea fish, crustaceans, and molluscs) used by humanity are mainly based on the systems where it is produced (i.e., the deep-sea sensitive habitats and VMEs), and on the systems that allow its harvest (i.e., the fishing techniques). As part of the "*Innovations in the Food System: Exploring the Future of Food*" Special Issue, this paper aims (1) to briefly review studies that highlight a link between deep-sea fishery resources and VMEs in the Mediterranean Sea; (2) to provide new insights into commercial and experimental catches of the deep-sea fishery resources in the central Mediterranean for the past 30 years; (3) to evaluate changes in the abundance of these resources with time, sea-bottom temperature (SBT), fishing effort (FE), and depth; and (4) to reveal an effect of the Santa Maria di Leuca cold-water coral province on the abundance of the deepsea fishery resources. The implications of these findings and the presence of several geomorphological features, sensitive habitats, and VMEs in the central Mediterranean are discussed in terms of conservation of biodiversity, combined with the sustainable management of the fishery resources.

### **2. Materials and Methods**

Data on food resources from deep-sea sensitive habitats and VMEs in the whole Mediterranean basin refer to international volumes and publications, and references therein (e.g., [11,55,67,68,74–77]).

In order to define the contribution of the most important commercial deep-sea species as food resources from the depths of the Mediterranean Sea, landing data from official FAO statistics were explored (https://www.fao.org/fishery/statistics-query/en/gfcm\_capture/ gfcm\_capture\_quantity, accessed on 12 January 2022), focusing on the central part of the basin (Figure 1).

**Figure 1.** Average total landing (L) of deep-sea commercial species for each FAO Mediterranean subdivision, expressed in tonnes (t) and percentages (%), calculated for the period 1994–2019. FAO subdivisions are coded as Balearic (BAE, 1.1), Gulf of Lion (G. Lion, 1.2), Sardinia (SAR, 1.3), Adriatic (ADR, 2.1), Ionian (ION, 2.2), Aegean (AEG; 3.1), and Levant (LEV, 3.2).

The temporal trends over a period of 25 years (1994–2019) were analysed by means of Spearman's non-parametric correlation.

New observations from the central Mediterranean, the southwestern Adriatic, and the northwestern Ionian, as well as on muddy bottoms of the northwestern Ionian Sea, were derived from data collected as part of national and international study projects carried out in the last two decades by the ecology team from the Department of Biology at the University of Bari Aldo Moro. In particular, data from deep-sea sensitive habitats and VMEs were taken using different low-impact fishing techniques [12,71,78–85], while data from muddy bottoms were collected during the Mediterranean Trawl Surveys (MEDITS) programme, included in the EU Data Collection Framework to date [86]. The MEDITS surveys are carried out in the Mediterranean from late spring to summer every year, according to a standardised protocol that includes gear characteristics, haul duration, and sampling procedures, following a depth-stratified random design, from 10 to 800 m in depth [86–89].

Using MEDITS data, the abundances in weight (expressed as biomass index kg/km2) and numbers (expressed as density index N/km2) of the deep-sea species distributed on deep muddy bottoms (200–800 m) of the northwestern Ionian Sea (Figure 2) were evaluated for the period 1994–2020, and their changes over time were tested using Spearman's nonparametric correlation. Data on the sea-bottom temperature (SBT) were recorded using a probe at the start and the end of different MEDITS hauls carried out from 1998 to 2020, and an average value of SBT was computed per year. The fishing pressure of bottom trawling fleets on the resources of the northwestern Ionian Sea was analysed using fishing effort (FE) data, in terms of number of vessels and gross tonnage (GT). Data were obtained from the European Fishing Fleet Register (http://ec.europa.eu/fisheries/fleet/index.cfm, accessed on 12 January 2022) for the period 1994–2020. The relationship between abundance of the deep-sea species and SBT was evaluated using linear regression analysis. Spearman's non-parametric correlation was also applied to the abundance data in weight (kg/km2) of the deep-sea species that showed a significant trend over time versus the FE—expressed as the total annual number of vessels—throughout the study period.

**Figure 2.** Topographic features, highs, banks, canyons, sensitive habitats, and VME species and habitats along the southwestern Adriatic Sea and northwestern Ionian Sea (central Mediterranean). GS = Gondola Slide; BC FRA = Bari Canyon Fisheries Restricted Area; SML FRA = Santa Maria di Leuca Fisheries Restricted Area; TC = Tricase Canyon; GC = Gallipoli Canyon; TT = Taranto Trench; AS = Amendolara Shoal; CB = Crotone Bank; SGCS = Squillace Gulf Canyon System; PSB = Punta Stilo Bank; SCCS = South Calabria Canyon System; SMC = Strait of Messina Canyon; triangle = hard bottom corals; asterisk = hard- and soft-bottomed corals [58,81,90–97]; lined areas *= Isidella elongata* facies [98]; NA = sampling area near the SML FRA; FA = sampling area far from the SML FRA.

Furthermore, for the shrimps *A. foliacea* and *P. longirostris*, due to correlation between environmental covariates (*ρ* = −0.73, *p* < 0.001), and in order to avoid multicollinearity problems, linear regression models were estimated to investigate the dependence between log-transformed abundances and environmental drivers (i.e., SBT and FE). Transformed responses ensure, in these cases, that the model assumptions are met. For both species, *s* = {*A. foliacea*, *P. longirostris*}, and for each *yz* with *z* = {abundance in weight, abundance in number}, the linear model is specified as follows:

$$\log(y\_z^{(s)}) = \mathcal{B}\_0^{(s)} + \mathcal{B}\_1^{(s)} \mathbf{x}\_i^{(s)} + \mathbf{e}^{(s)} \tag{1}$$

where *xi* represents the independent covariate, with *i* = {FE, SBT}.

Using linear regression analysis, the relationship between length and depth was evaluated for *M. merluccius*, *P. bogaraveo*, *P. blennoides*, *H. dactylopterus*, and *Galeus melastomus*, collected both on muddy bottoms with the MEDITS trawl net and in VMEs with an experimental longline (e.g., [81]), and the boxplots of the length were represented for all of these species in both habitats.

In order to detect an effect of the presence of a VME on fishery resources, MEDITS abundance data on the weight and number of the species *A. foliacea*, *P. martia*, *M. merluccius*, *P. bogaraveo*, *P. blennoides*, and *H. dactylopterus*, for an area near the Santa Maria di Leuca (SML) cold-water coral (CWC) province (NA), were compared with those of other species far from this coral province (FA) (Figure 2). Data from 54 trawl hauls, carried out between 200 and 800 m, were used for each area. Relative boxplots of the abundances in weight and number were produced, and the differences in the abundances between the two areas were tested using the Kruskal–Wallis non-parametric test. The pressure of fishing activity in these two areas was assessed in order to exclude effects due to this activity on the results of the comparison between the two areas. Specifically, FE was calculated for NA and FA by aggregating the fleets operating close to the two areas. In particular, the fleets of Gallipoli, Leuca, and Otranto were considered for NA, while those of Corigliano Calabro and Cirò Marina for FA. The differences in the species abundances between the two areas were tested using the Kruskal–Wallis non-parametric test.

### **3. Review of the Mediterranean Studies on the Link between Deep-Sea VMEs and Fishery Resources**

In the Mediterranean Sea, the deep-water resources are mainly exploited by trawl fishing on the soft bottoms of the bathyal grounds. However, there are areas characterised by the occurrence of VME species on soft and hard bottoms, canyons, and seamounts, where the fishing is carried out using different types of gears.

### *3.1. Open Slope, Soft Bottoms*

Between the shelf break and descent to bottoms deeper than 1000 m, soft corals can be found that can form dense aggregations on soft bottoms—called sea pen fields, sea fan corals, and arborescent corals—which build up coral gardens or coral forests. Coral gardens can develop on soft or hard substrata, depending on the habitat-forming species. Both sea pen fields and coral gardens and/or coral forests contribute to making more heterogeneous and complex habitats, attracting mobile and swimming fauna [55].

The sea pen fields built up by the octocoral *Funiculina quadrangularis* are mostly distributed on the upper slope, generally at less than 400 m in depth, on soft muddy habitats characterised by noticeable bottom currents. These habitats are commonly inhabited by commercially valuable crustaceans, such as the deep-water rose shrimp (*P. longirostris*) and the Norway lobster (*N. norvegicus*). Soft-bottomed coral gardens, structured by the gorgonian *Isidella elongata* (bamboo coral), can be found from the shelf break down to 1600 m. The valuable deep-water red shrimps (*A. antennatus*, *A. foliacea*) and the golden shrimp (*P. martia*) are frequently associated with these coral gardens [11,54,66,99–101]. Bamboo coral seems to play a role in habitat formation, increasing the three-dimensional habitat complexity on flat bathyal bottoms with its candelabrum-like shape. As a passive feeder, its occurrence is often associated with plankton-rich currents which, in turn, favours a high density of prey, such as pandalid shrimps and other crustaceans [102–104]. These prey animals attract predators of different trophic levels, such as the abovementioned deep-water red shrimps, bony fishes (e.g., *M. merluccius*, *P. blennoides*, *H. dactylopterus, P. bogaraveo, L. boscii*), and sharks, such as the lesser spotted dogfish (*Scyliorhinus canicula*) and the blackmouth catshark (*G. melastomus*), along with cephalopods (e.g., *I. coindetii*, *T. sagittatus*, *T. eblanae*), all of commercial interest [70,98,105–107]. In addition to the important implications as a feeding area for bentho-pelagic species, the arborescent complexity of the colonies could further act as shelter and spawning/nursery sites for several species that can grow to greater sizes than in areas where bamboo coral does not occur [98,104,107,108]. The

commercial species associated with bamboo coral account for about 5% of all of the income of the professional fisheries in the Mediterranean [34], with increasing landings—especially in Italy and Spain—the main producers in Europe [55,109].

### *3.2. Open Slope, Hard Bottoms*

Coral gardens or coral forests between the shelf break and upper slope are mainly made up of antipatharians and alcyonaceans. Among the former, the most widespread species are *Antipathes dichotoma*, *Parantipathes larix*, *Leiopathes glaberrima,* and *Antipathella subpinnata*, which form monospecific or multispecific forests [54,55]. Several commercial fish species are often associated with these antipatharians (e.g., [52,108,110]). Alcyonaceans are present on Mediterranean Sea with hard bottoms with several species, covering a wide bathymetric range. The whip-like gorgonian *Viminella flagellum* and the fan-shaped gorgonian *Callogorgia verticillata* are present in the bathyal zone at depths from 100 to 500 m and from 150 to 1000 m in depth, respectively [54,55]. Off the southwestern coasts of Sardinia, several fish species—some of commercial interest—have been observed hiding among the colonies of *L. glaberrima*, while egg capsules of the shark *S. canicula* have been found on the branches of this antipatharian at depths between 186 and 210 m [52,111]. The presence of egg capsules of this shark on *L. glablerrima* colonies had previously been observed on El Idrissi Bank (Alboran Sea) at 647 m and 452 m [112]. Cau et al. [111] suggested that the coral forest from a representative for southwestern Sardinia represent nursery grounds for *S. canicula*.

In the eastern Ionian Sea, the shark *G. melastomus* and the teleost fish *H. dactylopterus* were the most common fish species caught in the area, characterised by the presence of black coral (*L. glaberrima*) and bamboo coral (*Isidella elongata*) [108]. The shark seems to use the its habitat as a feeding area, and the teleost as a refuge area [108]; *H. dactylopterus* was also found together with other commercial fish species, such as the silver scabbardfish (*Lepidopus caudatus*), and the wreckfish (*P. americanus*) in a coral forest dominated by *L. glaberrima* on the Malta Escarpment (310–315 m) [113].

The fishes that coral zooxanthellate reefs produce in tropical waters account for 17% of animal protein consumed [114]. On the open slope of the Mediterranean Sea, there are still hard bottoms characterised by cold-water coral (CWC) communities, including solitary and colonial zooxanthellae cnidarians [94]. Colonial species have a complex branching morphology, and are habitat formers. The main species, known as white corals, are the colonial species *Madrepora oculata* and *Lophelia pertusa* (recently renamed as *Desmophyllum pertusum*), as well as the solitary coral *Desmophyllum dianthus*. These species have a broad frame-building ability, being able to deposit calcium carbonate and build up durable biogenic substrata. CWCs, as passive suspension feeders, depend on the supply of current-transported particulate organic matter and zooplankton for their trophic requirements. They are preferentially distributed on topographic irregularities on the slope, in canyons and on seamounts, where there are strong currents and the sedimentation rate is low [53,91,115–117].

CWC habitats are impacted by fishing due to the occurrence of large sizes and high abundances of commercial species [9,10,13,16,18,58,70,118,119] and references therein]. The presence of corals is generally known to the local fishers, who experience gear damage and losses, although they often fish close to these areas with the aim of obtaining a greater catch and larger specimens of valuable commercial species, such as the deep-water red shrimps (*A. antennatus* and *A. foliacea*) and the European hake (*M. merluccius*). In fact, side-scan sonar and underwater video images show the characteristic seabed scars of otter trawls ploughing through the coral banks [12,82]. Longline is also used in these areas of complex bottom topography, and is not accessible to trawling, so as to catch wreckfish, greater forkbeard, blackbelly rosefish, blackspot seabream, and bluntnose sixgill shark [71,78,81].

CWC habitats provide a suitable ground for larval settlement and juvenile growth of benthic species. They are spawning and nursery areas for vagile and swimming

fauna, acting as an EFH for several commercial and non-commercial fish and invertebrate species [12,17,57,71,78–81,83,84,111].


**About the Editor**

## **Jinghua Wang**

Dr. Jing-Hua Wang is an accomplished researcher and educator, currently serving as an assistant professor and principal investigator at the College of Korean Medicine and the Institute of Bioscience & Integrative Medicine (IBIM) at Daejeon University in South Korea. Dr. Wang earned his bachelor's degree in the College of Clinical Chinese Medicine from Anhui University of Chinese Medicine (AUCM), China, and his master's and Ph.D. in the College of Korean Medicine from Daejeon University (DJU), South Korea. He also served as a visiting professor at the Institute of Korean Medicine at Dongguk University from 2013 to 2018. Dr. Wang has nearly two decades of experience in the fields of hepatology, herbal medicine, and the gut microbiome, with his research primarily focused on exploring natural products to alleviate various hepatobiliary diseases. In recognition of his contributions to the field, Dr. Wang was granted a bursary from the European Association for the Study of the Liver (EASL) in 2011. He has also authored 52 SCI papers, holds two patents, and completed twenty-three research projects funded by the Korean government, such as the National Research Foundation of Korea (NRF). Dr. Wang's expertise and contributions to the field make him a valuable member of the academic community, and his work inspires his colleagues and students alike.

## *Editorial* **Advances in Pathogenesis and Therapeutics of Hepatobiliary Diseases**

**Jing-Hua Wang**

Liver and Immunology Research Center, Institute of Bioscience & Integrative Medicine, Daejeon University, 75, Daedeok-daero 176 Beon-Gil, Seo-gu, Dunsan-dong, Daejeon 35235, Republic of Korea<EMAIL_ADDRESS>Tel.: +82-42-257-6397; Fax: +82-42-257-6398

The hepatobiliary system, comprising the liver, gallbladder, and bile ducts, performs a diverse array of functions that are essential to maintaining homeostasis [1], including digestion, nutrient metabolism, detoxification, coagulation, and immune modulation [2]. Alterations in the normal function of the hepatobiliary system can have serious implications for human health, resulting in a broad range of pathologies spanning from liver disease to bile system disorders.

The Special Issue of *Biomedicines*, entitled "Advances in Pathogenesis and Therapeutics of Hepatobiliary Diseases", presents cutting-edge research findings, innovations, and insights into a wide range of topics related to the hepatobiliary system, such as the molecular mechanisms underlying disease pathogenesis, diagnostic advancements, novel therapeutic approaches, and prevention strategies. The Special Issue comprises seven original articles and four review articles regarding NAFLD, primary biliary cholangitis (PBC), hepatitis B and C, and hepatocellular carcinoma (HCC).

NAFLD is currently a major global health issue. It affects approximately 25% of the global population [3], with the highest prevalence in Western countries (25–30%) [4]. It affects 80–100 million people in the United States, making it the most common type of chronic liver disease [3]. NAFLD is also prevalent in Asia, particularly in Korea (affecting up to 30.3% of adults; men: 41.1%, women: 20.3%) [5]. As a result, many researchers are working to identify methods to prevent and treat NAFLD [6–9]. However, the lack of FDAapproved therapeutic options for NAFLD remains a significant impediment, emphasizing the critical importance of timely and accurate diagnosis [10]. Herein, Cazac et al. offer a comprehensive review of ultrasound-based hepatic elastography as a non-invasive method for diagnosing NAFLD in patients with type 2 diabetes mellitus (T2DM) [11]. They also highlight the limitations of liver biopsy and the need for non-invasive diagnostic tools to assess NAFLD in high-risk patients [11]. Bertran et al. utilized systems biology tools and public databases to identify the potential molecular mechanisms linking RUNX1 and NAFLD, resulting in a promising therapeutic strategy and a novel approach to treating NAFLD [12]. Furthermore, Abu-Freha et al. shed light on an important and relatively under-researched field of study: sex-based differences in NAFLD [13]. They used clinical big data from Clalit Health Services (CHS) in Israel. This discovery would improve our understanding of sex-based differences in NAFLD by highlighting disparities in comorbidities, outcomes, and mortality rates between females and males [13].

Furthermore, cholestatic diseases are significant because they have the potential to cause severe liver damage and long-term complications, emphasizing the importance of early diagnosis and treatment. One review provided a comprehensive overview of cholestatic diseases, including subtypes and causes, as well as the most up-to-date treatment options for cholestatic diseases, including pharmacotherapeutic agents and gene therapy [14]. It is worth noting that the discussion of gene therapy for inherited and acquired cholestasis is insightful and demonstrates the future promise of using gene therapy to address this medical issue [14]. Another review summarized current knowledge

**Citation:** Wang, J.-H. Advances in Pathogenesis and Therapeutics of Hepatobiliary Diseases. *Biomedicines* **2023**, *11*, 1140. https://doi.org/ 10.3390/biomedicines11041140

Received: 6 April 2023 Accepted: 7 April 2023 Published: 10 April 2023

**Copyright:** © 2023 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

regarding pharmacological interventions for the treatment of primary biliary cholangitis (PBC) [15]. As is known, ursodeoxycholic acid (UDCA) is the first-line therapy for PBC and has been proven to normalize liver markers, delay disease progression, and prolong transplant-free survival. Obeticholic acid (OCA) is the only approved second-line treatment for individuals with PBC who do not respond to UDCA. Floreani et al. provided a comprehensive review of the pharmacological properties of OCA, including its mechanism of action, as well as its tolerability and effectiveness in treating PBC patients [16]. Moreover, one cohort study conducted by Bauer and colleagues, comprising 138 PBC patients and 90 non-PBC patients, revealed that the presence of antibodies against the kelch-like 12 (KLHL12) protein is a highly specific marker for diagnosing PBC [17]. When used in conjunction with other markers, it has the potential to significantly enhance the accuracy of PBC diagnosis.

Furthermore, hepatitis B and C also represent major global health issues. According to the World Health Organization (WHO), an estimated 354 million people worldwide are living with viral hepatitis B and C [18]. Infection with HBV and HCV can increase the risk of developing liver fibrosis, cirrhosis, and even liver cancer [19]. Three reports are presented here that are associated with the diagnosis, treatment, and pathogenesis of hepatitis induced by HBV/HCV. A study conducted in Spain found variants with indels in the 3' end of HBX in most of their chronic hepatitis B (CHB) patients [20]. These variants encoded alternative versions of HBx that have the potential to play a functional role and/or alter transcriptional regulation [20]. This will provide useful insight into the genetic variability of HBV in CHB patients who do not have HCC. Another study investigated the correlation between serum miR-125b levels and liver fibrosis in CHB patients after 12 months of nucleoside analog treatment [21]. The results indicated that there is an inverse relationship between miR-125b levels and the post-treatment FIB-4 index score, but it is not a significant predictor of a higher score. Age, baseline platelet count, and ALT level were all independent predictors of a FIB-index greater than 2.9 post-treatment [21]. The last study investigated the long-term effects of direct-acting antiviral (DAA) regimens on mitochondrial DNA (mtDNA) instability in people who inject drugs (PWID) with chronic HCV [22]. The mtDNA parameters were measured nine months after treatment, and the percentage of deleted mtDNA genomes increased over time due to their replicative advantage over elimination processes [22].

In terms of clinical therapy for HCC, Tay et al. compared the clinical outcomes of trans-arterial chemoembolization (RFA) and radiofrequency ablation (TACE) as initial monotherapy for patients with early-stage HCC [23]. According to their findings, TACE could be considered a potential treatment option for patients who are unsuitable candidates for other therapies [23].

Overall, I anticipate that the collection of articles in this Special Issue will contribute significantly to ongoing efforts to improve the diagnosis, prevention, and treatment of hepatobiliary diseases, resulting in better patient outcomes and overall public health.

**Funding:** This research was funded by the National Research Foundation of Korea (NRF), grant number 2020R1F1A1074155.

**Acknowledgments:** I would like to express my sincere appreciation to all of the authors who contributed to this Special Issue. Furthermore, I extend my gratitude to Chang-gue Son at College of Korean Medicine, Daejeon University, for his support.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**

**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

## *Review* **Ultrasound-Based Hepatic Elastography in Non-Alcoholic Fatty Liver Disease: Focus on Patients with Type 2 Diabetes**

**Georgiana-Diana Cazac 1,2 , Cristina-Mihaela Lăcătus,u 1,2,\* , Cătălina Mihai 3,4, Elena-Daniela Grigorescu 1,\* , Alina Onofriescu 1,2 and Bogdan-Mircea Mihai 1,2**

**Abstract:** Non-alcoholic fatty liver disease (NAFLD) is the most prevalent liver disease and is the hepatic expression of metabolic syndrome. The development of non-invasive methods for the diagnosis of hepatic steatosis and advanced fibrosis in high-risk patients, especially those with type 2 diabetes mellitus, is highly needed to replace the invasive method of liver biopsy. Elastographic methods can bring significant added value to screening and diagnostic procedures for NAFLD in patients with diabetes, thus contributing to improved NAFLD management. Pharmacological development and forthcoming therapeutic measures that address NAFLD should also be based on new, non-invasive, and reliable tools that assess NAFLD in at-risk patients and be able to properly guide treatment in individuals with both diabetes and NAFLD. This is the first review aiming to outline and discuss recent studies on ultrasound-based hepatic elastography, focusing on NAFLD assessment in patients with diabetes.

**Keywords:** type 2 diabetes mellitus; non-alcoholic fatty liver disease; transient hepatic elastography; hepatic steatosis; liver fibrosis

#### **1. Introduction**

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disorder that is lately becoming a worldwide major public health problem in both adults and children. The high prevalence of metabolic comorbidities such as obesity, dyslipidemia, and diabetes mellitus (DM) that are frequently associated with NAFLD supports the need for increased attention from healthcare providers who should invest in screening and management [1,2].

According to the latest International Diabetes Federation (IDF) Diabetes Atlas data, an estimated 537 million adults worldwide aged 20–79 years are currently living with diabetes, representing 10.5% of all adults in this age group. In 2021, almost 240 million adults had undiagnosed diabetes [3]. Early identification of people with diabetes is key to avoiding or delaying complications and improving quality of life, thus preventing the significant burden on healthcare systems [3].

The estimative global prevalence of NAFLD is 25% of the adult population.  Approximately 10 to 15% of NAFLD patients from the United States and Europe have advanced fibrosis. Patients with NAFLD have an increased risk of liverrelated death, primarily those with histologically proven non-alcoholic steatohepatitis (NASH) [7]. T2DM doubles the risk of hepatocellular carcinoma [5]. The high prevalence

**Citation:** Cazac, G.-D.; L˘ac˘atus,u, C.-M.; Mihai, C.; Grigorescu, E.-D.; Onofriescu, A.; Mihai, B.-M. Ultrasound-Based Hepatic Elastography in Non-Alcoholic Fatty Liver Disease: Focus on Patients with Type 2 Diabetes. *Biomedicines* **2022**, *10*, 2375. https://doi.org/10.3390/ biomedicines10102375

Academic Editor: Jinghua Wang

Received: 27 August 2022 Accepted: 19 September 2022 Published: 23 September 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

of metabolic syndrome (MS) is independently associated with all-cause, liver-specific, and cardiovascular mortality. Other risk factors leading to the increased prevalence of NAFLD are represented by older age and male sex [5]. The large number of NAFLD patients that are potential candidates for progressive liver disease creates challenges in screening and management, mirroring the evolution of cardiovascular disease development on the background of T2DM, obesity, and insulin resistance [8].

NAFLD covers a spectrum of histological conditions, ranging from simple steatosis (non-alcoholic fatty liver, NAFL) to NASH, which can later progress to liver fibrosis, cirrhosis, or hepatocellular carcinoma (HCC). MS is a predictor of hepatic steatosis [9]. Fibrosis is an important prognostic factor in NAFLD. The fibrosis stage is independently associated with increased overall and liver-specific mortality and with higher rates of liverrelated complications and liver transplantation; early studies suggest a higher prevalence of NASH and advanced fibrosis stages among patients with T2DM [9,10].

Experts in the field have recently suggested the introduction of a new acronym, MAFLD (metabolic dysfunction-associated fatty liver disease), which reflects the relevant risk factors for this disease, underlining the association between insulin resistance and MS [11,12].

NAFLD and T2DM display a bidirectional relationship wherein these two pathologies have intricate effects on disease progression. On one hand, NAFLD co-existence increases the incidence of T2DM and the risk of developing micro- and macrovascular complications of diabetes [13–15]. On the other hand, T2DM is recognized as a risk factor for progressive liver disease, leading to advanced fibrosis, cirrhosis-related complications, and increased liver disease mortality [16]. An extensive meta-analysis of 33 studies carried out between 2000 and 2020, including 501,022 individuals and nearly 28,000 cases of incident diabetes, showed that patients with NAFLD had a higher risk of incident diabetes mellitus than those without NAFLD. This risk increased considerably in individuals with advanced liver fibrosis [17]. The co-existence of NAFLD and T2DM acts synergically to increase the risk for other organ complications, with the highest mortality in NAFLD attributed to a worsened cardiovascular risk profile [18]. It is thus becoming evident that the link between NAFLD and diabetes is more complex than previously believed.

Two steps are needed to diagnose NAFLD. The first step is to assess the existence of hepatic steatosis, either by imaging or biopsy, and then to exclude other causes of liver steatosis such as significant alcohol consumption, long-term use of steatogenic medication, or monogenic hereditary disorders [9]. NAFL's only feature is fatty liver infiltration that involves more than 5% of hepatocytes, whereas NASH also features inflammation and evidence of hepatocellular injury, with or without fibrosis, in the absence of alcohol consumption (daily intake of less than 20 g in women and 30 g in men) [9,14].

The European Association for the Study of the Liver (EASL), the European Association for the Study of Diabetes (EASD), and the European Association for the Study of Obesity (EASO) recommend ultrasonography as a first-line screening test for NAFL, whilst liver biopsy represents the essential tool for the diagnosis of NASH [14]. However, the limits of invasive biopsy procedures are acknowledged, and no distinct screening information in the existing guidelines refers to the identification of this metabolic liver disease amongst patients with diabetes.

Therefore, this is the first review aiming to identify and analyze the current elastographybased imaging strategy for NAFLD screening and diagnosis, focusing on its applicability in patients with T2DM. This category of patients has become more and more clinically significant, as the increased prevalence of diabetes and obesity have become important public health issues in recent decades. Among them, non-invasive diagnostic tests such as ultrasound-based hepatic elastography are highly needed to replace liver biopsy, to develop a new protocol for screening patients at risk for NAFLD or those with a history of steatosis diagnosed by hepatic imaging/biopsy, and to non-invasively monitor patients with NAFLD and diabetes and their response to treatment.

#### **2. Common Approaches in NAFLD Assessment**

Measures to limit disease progression must be based on the identification of metabolic risk factors (obesity, dyslipidemia, hypertension, and diabetes), on the assessment of anthropometric indices and laboratory tests (fasting blood glucose, oral glucose tolerance test, HbA1c, complete lipid profile, uric acid, and thyroid markers), on the calculation of related biomarkers (homeostatic model assessment for insulin resistance index, estimated glomerular filtration rate, urinary albumin-creatinine ratio), and on imaging techniques [9,19,20].

Liver-related outcomes are influenced by the advanced stage of fibrosis and not steatosis. Commonly used non-invasive tests (NIT) widely available in clinical practice to estimate fibrosis are represented by the fibrosis-4 (FIB-4) index, NAFLD fibrosis score (NFS), and aspartate aminotransferase (AST) [21,22]. By using such NIT in a large cohort of patients with T2DM, Singh et al. identified a high prevalence of fibrosis [23]. In fact, in line with the European guidelines, these scores should be calculated for every patient with NAFLD [14]. Their use can exclude the presence of advanced fibrosis in 50–67% of patients with diabetes [24]. A new prediction model, diabetes liver fibrosis score (DLFS) was recently developed to help identify patients with diabetes at significant risk for liverrelated morbidity: DLFS values over 68.9 maximize specificity (98%) and positive predictive value (86%), while values less than 14.5 maximize sensitivity (95%) and negative predictive value (92%) [25]. Other NIT such as AST to platelet ratio index (APRI) or Hepascore had less accuracy predicting cirrhosis in patients with NAFLD and diabetes [26].

Ultrasonography stands at the forefront as a non-invasive method of screening and diagnosing steatosis in patients with diabetes. The lipid deposits in the liver can be detected by ultrasound when steatosis exceeds 30% of the liver parenchyma, which is visualized as a bright liver echotexture (hyperechoic) blurring the deeper structures [16,27]. An ultrasonography-based study showed that 127 out of 204 patients with T2DM had hepatic steatosis on ultrasound, and 87% of those having consented to a liver biopsy had NAFLD confirmed by histology [28]. The Edinburgh Type 2 Diabetes Study, the first study using ultrasound grading compared with magnetic resonance spectroscopy to determine NAFLD prevalence in a population of patients with T2DM, showed that the disadvantage of ultrasound is its inability to differentiate grade 1 or 2 of steatosis [29]. Therefore, the most accurate method to quantify fat is magnetic resonance imaging (MRI); however, MRI is limited by its high costs and lesser availability and is mainly used in clinical trials [21].

The "gold standard" in diagnosing NAFLD is liver biopsy and histologic examination. However, liver biopsy is limited by its invasive nature, potentially prone to complications such as pain, bleeding, or sampling errors [30]. The difficulties in repeating biopsies to assess changes in hepatic steatosis and fibrosis and in performing them on individuals with high abdominal circumferences require alternative non-invasive assessment tools [9,31].

The main purposes of following-up patients with diabetes are to identify patients with MS and risk of NAFLD, to detect individuals with a worsening prognosis, and to monitor them once the therapeutic strategy is implemented [32,33]. While the evidence for novel and innovative therapy approaches for NAFLD in subjects with T2DM is rising, elastography techniques might have a reliable role in monitoring patients with NAFLD and diabetes and their response to treatment [34]. Liver imaging plays an important role in NAFLD assessment in patients with T2DM because no clinical manifestations exist in the early stages of disease and functional tests may be within normal limits [35].

The recent recommendations by the EASL, EASD, and EASO designate clinical scores, serum biomarkers, and the elastographic evaluation of the liver as NIT accepted for the diagnosis and staging of hepatic fibrosis [21]. The American Diabetes Association (ADA) guidelines also suggest the use of transient elastography (TE) and non-invasive biomarkers for risk stratification [36]. The elastographic method evaluates the presence and severity of liver fibrosis according to the etiology of the liver disease and has already been tested in many liver-related conditions [37,38].

#### **3. Ultrasound-Based Hepatic Shear Wave Elastography**

Ultrasound-based elastography has found its place among NIT used to screen and assess the severity of NAFLD and is represented by TE, point shear wave elastography (pSWE), and two-dimensional shear wave elastography (2D-SWE) [16]. Within this category, TE is extensively available and can be used as a point-of-care test to estimate liver fibrosis by measuring liver stiffness and hepatic steatosis using controlled attenuation parameter (CAP) measurement [39,40]. The practice guidelines of the Brazilian Society of Hepatology and Brazilian College of Radiology have recently supported the use of elastography, among others, as a tool to assess fibrosis and steatosis in various chronic liver diseases, including NAFLD; due to its accuracy, elastography seems to be a non-invasive and cost-effective alternative to liver biopsy [41].

The screening for undiagnosed non-alcoholic fatty liver disease and non-alcoholic steatohepatitis (SUNN) study suggests that even asymptomatic high-risk individuals should, nevertheless, be screened for NAFLD. Using TE and CAP, Eskridge et al. found that 57% of the study population had steatosis without fibrosis and 16% of them had both steatosis and fibrosis [42]. However, the results of this study likely overestimate the presence of steatosis by using a cut-off value of ≥238 dB/m [43]. Even though specialists have not yet reached a consensus on cut-off values, the EASL guidelines suggest that a CAP value >275 dB/m might be used to diagnose hepatic steatosis [44]. A meta-analysis by Petroff et al. found that the optimal cut-offs when using the XL probe are 297, 317, and 333 dB/m for >S0, >S1, and S2, respectively [43]. Another study showed that the cut-off for S ≥ S2 of 331 dB/m is accurate for the identification of moderate steatosis [39]. Obesity, diabetes, and arterial hypertension proved to be statistically significant risk factors for NAFLD and NASH development [42]. In line with the results of this study, applying such efficient screening strategies to high-risk individuals may help to properly implement therapy and, over time, reduce the burden of NAFLD.

#### *3.1. Elastography-Based Imaging Techniques to Assess Hepatic Fibrosis*

Ultrasound-based shear wave elastographic methods for the assessment of advanced fibrosis in NAFLD are represented by [37,45]:

	- pSWE (point shear wave elastography)
	- 2D-SWE (two-dimensional shear wave elastography), or 3D-SWE (three-dimensional shear wave elastography) [46].

#### 3.1.1. Transient Elastography

TE is a non-invasive imagistic technique able to stage liver fibrosis by LSM. The use of TE to estimate liver fibrosis severity was first described by Sandrin et al. in 2003 [47]. Besides being recommended as a clinical diagnostic method in many liver-related conditions such as chronic viral hepatitis, cholestatic diseases, alcoholic liver disease, and autoimmune hepatitis, an accumulating body of evidence supports the use of TE for the diagnosis and staging of liver fibrosis in NAFLD [44,48].

TE is a method non-integrated into standard ultrasound-based systems and performed using the Fibroscan® device (Echosens, Paris, France) that is well correlated with histologically diagnosed liver fibrosis in NAFLD. LSM quantification of liver fibrosis is expressed in kilopascals (kPa) [33,49]. TE can use an M probe for normal-weight patients and an XL probe for patients with obesity [50]. The feasibility of TE using both M and XL probes is 93.5% [51].

According to the Baveno VI consensus, TE has enabled the identification of asymptomatic patients with advanced fibrosis (stage F3–F4) at risk for clinical complications. This consensus has proposed the term "compensated advanced chronic liver disease" (cACLD) as an alternative to chronic liver disease in asymptomatic F3–F4 patients, who are at risk of

developing severe portal hypertension. Values between 10 and 15 kPa need confirmation of cACLD, and a value >15 kPa is suggestive of cACLD in the absence of clinical signs [52].

The TE technique has acquired widespread use in clinical studies and daily medical activities, ranging from the screening and diagnosis of hepatic steatosis and fibrosis in patients with suspected NAFLD to the assessment of T2DM prevalence among patients with NAFLD and the follow-up protocols searching for improvements after the initiation of pharmacologic and non-pharmacologic treatment [53].

The Rotterdam Study found the highest probabilities of fibrosis among participants with diabetes and steatosis [54]. It is noteworthy that most studies having investigated the prevalence of NAFLD and its risk factors by the TE tool resorted to non-diabetic cohorts for validating their results, so further studies are needed to stratify the diabetesassociated risk, as optimal cut-offs may be influenced by diabetes mellitus or body mass index (BMI) [55]. On the other hand, the risk of developing diabetes may be influenced by a NAFLD-associated status that evolves over time [56]. A cross-sectional study using TE to evaluate fibrosis among various chronic liver disease populations in a tertiary center in Lebanon appreciated that more than 58% of subjects had NAFLD; also, almost 50% of patients had at least one metabolic risk factor and 20% had T2DM [57].

The performance of NAFLD diagnostic tools among patients with T2DM varies according to the assessment methods. In healthy people, TE measurements of Young's modulus range from 4.4 to 5.5 kPa [37]. Ahn et al. found a significantly higher LSM in the diabetes group (11.22 ± 10.51 kPa) than in the non-diabetes group (8.07 ± 7.29 kPa), and a higher prevalence of diabetes in patients with NAFLD than in those with chronic viral hepatitis [58]. A cohort study on 283 patients performed by Patel and colleagues revealed 82.5% of them were diagnosed with T2DM and one-fifth with severe obesity; the cut-off values applied for LSM were 8.2 kPa for significant fibrosis, ≥9.5 kPa for advanced fibrosis, and >13 kPa for cirrhosis. In this study, 76.5% of patients with BMI values greater than 40 kg/m<sup>2</sup> required the use of the XL probe [59]. XL probes are designed for obese patients to improve the measurability of liver stiffness [60]. According to Garg et al., TE using the XL probe has a lower rate of failure than the M probe in patients with obesity, being able to evaluate hepatic steatosis and fibrosis in almost 60% of the obese persons with a BMI <sup>≤</sup> 45 kg/m<sup>2</sup> [30].

In a cross-sectional trial conducted in Vietnam, assessing diabetic patients by TE, a 73.3% prevalence of NAFLD was found among patients with T2DM. The LSM values in patients with F2 (significant fibrosis), F3 (advanced fibrosis), and F4 (cirrhosis) were ≥7 kPa, ≥8.7 kPa, and 11.5 kPa, respectively. After applying multivariable logistic regression, the investigators found AST and platelets as predictors of advanced fibrosis in patients with T2DM [61]. Therefore, patients with diabetes and increased AST values may be predisposed to increased liver stiffness [62].

The heterogeneity of study results may be influenced by specific BMI and waist circumference cut-off values depending on the country and ethnic origin of patients. The rising rates of obesity, dyslipidemia, hypertension, and MS in people with NAFLD support the need for evaluating MS components in patients with fatty liver, but also for NAFLD screening among patients with metabolic risk factors [63].

Several studies compared the use of TE alone with combined NIT for the detection of fibrosis. The STELLAR study demonstrated that the combined use of two NIT among patients with enhanced liver fibrosis (ELF), NFS, FIB-4, and liver stiffness by TE improved the diagnostic performance by reducing the proportion of patients with advanced fibrosis due to NASH and indeterminate results [64].

Combining clinical scores and serum markers with LSM by TE may facilitate and improve the diagnosis of advanced fibrosis and steatosis [65–67]. The Fibroscan-AST (FAST) score combines LSM and CAP measured by TE with aspartate aminotransferase, having already been validated in large global cohorts [68]. Comparison of NIT to accurately identify advanced fibrosis due to NASH subsequently reduces the need for liver biopsy to

assess the fibrosis stage [64]. The implementation of such a strategy may be particularly beneficial in high-risk patients such as those with T2DM.

The development of novel therapeutic strategies to improve NAFLD-related outcomes also requires high-value evaluation methods such as LSM. Unlike liver biopsy, this tool is widely available and reproducible, avoids patient reluctance, and can be repeated to monitor the results of pharmacological treatment [69].

As LSM by TE has become the most investigated and embraced method for evaluating NAFLD, forthcoming years will show whether it may be designated as a future "gold standard" among non-invasive assessment tools. Studies focusing on the estimation of liver stiffness with TE in patients with diabetes and NAFLD are described in Table 1.

*Biomedicines* **2022**, *10*, 2375

14

#### 3.1.2. Point Shear Wave Elastography (pSWE)

This technique, based on ARFI, is integrated into conventional ultrasound systems [101,102]. A significant advantage of pSWE is that it can assess liver fibrosis and evaluate the liver parenchyma on the same examination [102].

Shear wave elastography was also used in measuring liver stiffness in a case series of ten patients with diabetes and dyslipidemia in which the safety and effectiveness of saroglitazar in improving NAFLD, a dual PPAR α/γ agonist approved for diabetes in India, were assessed [103].

When the accuracies of LSM by TE, ARFI, and supersonic shears wave (SSI) for the staging of fibrosis were compared on a cohort of patients with NAFLD using liver biopsy as a reference, ARFI performance was found to be better for severe fibrosis and cirrhosis than for mild to moderate fibrosis. As more than half of the selected population had T2DM, variables such as BMI <sup>≥</sup>30 kg/m<sup>2</sup> , waist circumference ≥102 cm, or increased intercostal wall thickness may have interfered with and provided unreliable results when ARFI was used, compared with other imaging techniques [104].

However, mixed results are reported in this area. A meta-analysis assessing the diagnostic performance of pSWE vs. TE for staging liver fibrosis found a higher rate of failure in TE measurements using the M probe, more than in pSWE estimations, and obesity appeared to have a lesser influence on the results (11.3% vs. 0.8%) [105]. Giuffrè et al. screened several subjects with obesity having undergone bariatric surgery and reported that LSM is machine-dependent when taking into consideration the skin-to-liver distance (SLD) effect and not just the BMI [106].

While the Ultrasound Liver Elastography Consensus Statement, of the Society of Radiologists, recommends the "rule of four" (5, 9, 13, and 17 kPa) for liver stiffness cut-off values obtained using pSWE or 2D-SWE in NAFLD, no cut-off values specific to the T2DM population exist [107].

There are limited studies on the diagnostic ability of pSWE in patients with NAFLD and diabetes, and some of them included a limited number of patients with diabetes in the selected population. However, intriguing results reported by Meyer et al. using pSWE revealed a relatively high prevalence of liver fibrosis associated with NAFLD, even in patients with type 1 diabetes mellitus (T1DM), with a rate of 16% vs. 31% in T2DM subjects [108].

Existing studies have predominantly involved populations with obesity (adults and children), of which some underwent bariatric surgery [109–111]. Studies focusing on the estimation of liver fibrosis with pSWE in patients with T2DM and NAFLD are described in Table 2.

*Biomedicines* **2022**, *10*, 2375

*Abbreviations:* NAFLD, non-alcoholic fatty liver disease; T1DM, type 1 diabetes mellitus; T2DM, type 2 diabetes mellitus; BMI, body mass index; F, fibrosis; N, normal.

#### 3.1.3. Two-dimensional Shear Wave Elastography (2D-SWE)

Two-dimensional shear wave elastography, or SSI, is also an ARFI-based technique that seems to be a rapid and reproducible technique that adapts ultrasound imaging to measure liver stiffness [102]. In healthy populations, liver stiffness values found by 2D-SWE range between 4.4 and 4.9 kPa [46].

In the two-center study by Cassinotto et al., the relevant covariates influencing the results of the 2D-SWE method were increased waist circumference, higher BMI values, thicker intercostal wall, and, in some cases, diabetes [104].

MS is associated with high liver stiffness [115]. Moreover, a cross-sectional, onecenter Japanese study in people with abdominal obesity (Japanese diagnostic criteria for MS include waist circumference values of ≥85 cm for men and ≥90 cm for women) showed that waist circumference was significantly and independently correlated with liver stiffness measured by 2D-SWE [116]. The advantages of 2D-SWE have become evident in individuals with NAFLD and severe obesity, where its findings showed a higher success rate in comparison with TE, one of the most validated tools available [117]. In patients with clinically severe obesity that were evaluated before and after metabolic surgery by 2D-SWE-based LSM, improved characteristics were seen [118].

A comparison of TE, 2D-SWE, and magnetic resonance elastography (MRE) methods found them to be viable alternatives to liver biopsy for examining hepatic stiffness in 231 NAFLD patients. No differences were found between these techniques in the ability to diagnose the F1–3 stages, but MRE was superior to TE and 2D-SWE in detecting the F4 stage. Patients in this study included more than 60% subjects with diabetes, but no other information about this category was available [119].

In another study, obesity, T2DM, and arterial hypertension were independent predictors of a 2D-SWE value ≥ 8 kPa; patients with T2DM and hypertension exhibited a double risk for a hepatologist referral, while patients with obesity had a threefold risk. Therefore, focusing on patients with these medical conditions may improve NAFLD-related risk stratification [120].

Even though pSWE and 2D-SWE are less available in tertiary hepatology clinics and current evidence in patients with T2DM is limited, they may become a forthcoming routine tool for the screening, diagnostic, and therapeutic follow-up of patients with both NAFLD and diabetes [121].

Studies using the 2D-SWE method to assess liver fibrosis in patients with T2DM and NAFLD are described in Table 3.

**Table 3.** Diagnostic performance and comparison of results for different fibrosis stages using 2D-SWE in patients with NAFLD and T2DM.

*Abbreviations:* NAFLD, non-alcoholic fatty liver disease; BMI, body mass index; kPa, kilopascals; SWE, shear wave elastography.

#### *3.2. Additional Results Obtained by Imaging Methods Complemented with Elastography*

CAP uses ultrasound waves to detect and quantify liver fat by measuring the degree of ultrasound attenuation by hepatic steatosis after the initial attenuation in the adipose tissue within the abdominal wall [87,122]. It is an affordable method that can identify and monitor persons at risk for NAFLD. Fibroscan® software added CAP in 2010, so it can assess both fibrosis by LSM and steatosis by CAP at the same time [123]. CAP is derived from the attenuation of the same ultrasound data used to track the shear wave speed [124]. Several measurement algorithms based on the same principle as CAP are available on other ultrasound systems [124] but are less used in studies on patients with NAFLD.

CAP qualifies today as a standardized non-invasive measure of liver steatosis. The clinical use of CAP is limited due to difficulties in establishing optimal cut-offs for every steatosis grade and to the influence of other conditions such as diabetes. It appears that the steatosis prevalence in a specific population, its etiology, BMI values, and the co-existence of diabetes must be taken into consideration when interpreting CAP [125].

Several studies using LSM and CAP support the use of CAP to screen for NAFLD in patients with T2DM [87,89,99]. However, current guidelines do not yet recommend it as a standard routine method to identify NAFLD among asymptomatic, even though high-risk, populations [9]. It is noteworthy that a large number of studies overestimated the grade of hepatic fat by using lower, inappropriate CAP cut-offs, as described in Table 4 [75,76,83,89,90].

*Biomedicines* **2022**, *10*, 2375

*Biomedicines* **2022**, *10*, 2375

In the Vietnamese study previously mentioned in the TE section of this paper, steatosis severity was graded using the following CAP cut-off values: S0 (26.7% steatosis) for CAP ≤ 233 dB/m, S1 (20.5% steatosis) for CAP 234–269 dB/m, S2 (21.8% steatosis) for CAP 270–300 dB/m, and S3 (31% steatosis) for CAP > 301 dB/m [61]. This is another example of a study that used an inappropriate CAP cut-off and overestimated the results.

The simultaneous use of LSM and CAP to assess liver fibrosis and steatosis was brought into the spotlight by their implementation in patients with severe obesity that were candidates for bariatric surgery [126]. Only 60% of subjects were eligible for the use of the XL Fibroscan® probe. The results suggested that TE could estimate significant fibrosis (an LSM cut-off value ≥ 9 kPa) and significant hepatic steatosis (CAP ≥ 305 dB/m). The histological findings of patients who underwent liver biopsies appeared to correlate with LSM and CAP results [127].

As previously mentioned, higher estimates of hepatic tissue stiffness are associated with elevated BMI and waist circumference values. Moreover, Sporea et al. found supplementary associations between waist circumference, BMI, elevated AST, HbA1c, severe steatosis, higher CAP values, and advanced fibrosis [87].

The usefulness of CAP in monitoring therapeutic effects is the objective of several studies [34]. Liraglutide was able to reduce CAP-measured hepatic steatosis in addition to its well-known effects on body weight and plasma glucose control [128]. A study investigating, by TE, the effects of the GLP-1 receptor agonist dulaglutide in patients with T2DM was not able to show a reduction of intrahepatic fat, probably due to the short 12-week period of treatment [129]. Another study with a novel thiazolidinedione (lobeglitazone), using CAP by TE and having a primary endpoint of hepatic fat reduction, found improvements in NAFLD in patients with T2DM [130]: a 65% improvement in steatosis, comparable to the PIVENS trial where 69% of NAFLD patients responded to pioglitazone treatment [130,131]. Shimizu et al. assessed the impact of dapagliflozin, an SGLT-2 inhibitor, on liver steatosis and fibrosis: after 24 weeks of therapy, LSM decreased from 9.49 ± 6.05 kPa to 8.01 ± 5.78 kPa and CAP reduced from 314 ± 61 to 290 ± 73 dB/m in the dapagliflozin group [132].

As most studies did not have CAP available when evaluating liver stiffness, and some other studies used only the CAP software to assess NAFLD, we have chosen to address LSM and CAP separately in this paper. Studies using the CAP method to assess liver steatosis in patients with T2DM and NAFLD are described in Table 4.

#### **4. The Place of Elastography-Based Techniques in the Screening Algorithm for NAFLD**

As previously mentioned, an accumulating body of evidence supports the systematic use of ultrasound-based elastography for assessing hepatic steatosis and advanced fibrosis in high-risk patients. The appropriate management of patients with NAFLD must rely on accurate identification of fibrosis and steatosis severity [133].

Whether a screening strategy using NIT such as TE for the diagnosis of liver fibrosis is cost-effective is still a matter of debate [134]. Future results of the LiverScreen project, which aims to screen for liver fibrosis in the general population in European countries, will probably answer this question after 2025. If the results of this study help identify groups at high risk for chronic liver disease in the general or high-risk population, particularly in patients with obesity and diabetes, improved prevention of liver complications will perhaps become possible, thus ameliorating the burden on healthcare systems [135].

Until then, alternative non-invasive scores such as NFS or FIB-4 are recommended to rule out advanced fibrosis when TE is unavailable, thus minimizing the costs [136]. Other methods, such as MRE, have better sensitivity and specificity but are limited by cost and availability [44,137]. The selection of NIT for the diagnostic algorithm in low-prevalence populations must be performed by consulting a liver specialist [44].

Preliminary results of an ongoing cross-sectional trial reported that less than 2% of patients with diabetes are screened for liver fibrosis in primary and secondary care. A high proportion of cases in which liver fibrosis was confirmed (80.6%) were identified using serum fibrosis markers associated with TE or liver biopsy [138]. On the other hand, in the cross-sectional study by Park et al., the patients with diabetes benefited from fibrosis screening procedures in primary care, even in the absence of steatosis [139].

Unfortunately, the screening rate is low in this high-risk population, despite the high prevalence of significant liver fibrosis and steatosis among patients with diabetes [138]. Therefore, the systematic implementation of a routine screening algorithm is needed to improve the clinical care of patients with NAFLD and diabetes.

Current practices and guidelines have not yet adopted widespread screening because of the lack of evidence supporting the long-term benefits of screening and a favorable cost-effectiveness ratio [9,139]. This might hinder the identification of population groups at risk for NAFLD. Lomonaco et al. argue that NAFLD represents a public health problem for patients with T2DM by emphasizing the burden of the disease in a population with T2DM unaware of NAFLD that was screened with TE [77]. In line with this, Mansour et al., after demonstrating better identification of NAFLD in this category of patients, advised incorporating FIB-4 and TE as a two-tier assessment approach into the routine annual evaluation of patients with T2DM [79]. However, given the large number of people with diabetes, it is unlikely that clinicians will be able to apply TE to all T2DM patients. Therefore, it is important to identify patients at risk for fatty liver disease progression [99].

Currently, most screening suggestions for people with T2DM include non-invasive scores such as FIB-4 or NFS in association with TE [21,140]; this combination can be used to distinguish between populations at low or high risk for advanced fibrosis (Figure 1). *Biomedicines* **2022**, *10*, x FOR PEER REVIEW 20 of 30

**Figure 1.** A suggested algorithm to screen patients with T2DM for NAFLD and advanced fibrosis [2,33,63,141]. *Abbreviations:* ALT, alanine aminotransferase; WC, waist circumference; BP, blood pressure; Tg, triglycerides; HOMA-IR, homeostatic model assessment for insulin resistance; hsCRP, high-sensitive C-reactive protein. **Figure 1.** A suggested algorithm to screen patients with T2DM for NAFLD and advanced fibrosis [2,33,63,141]. *Abbreviations:* ALT, alanine aminotransferase; WC, waist circumference; BP, blood pressure; Tg, triglycerides; HOMA-IR, homeostatic model assessment for insulin resistance; hsCRP, high-sensitive C-reactive protein.

#### **5. Gaps in Knowledge**

Different researchers have used various cut-offs to study where elastography is positioned in the NAFLD assessment tree. As specific LSM cut-off values to predict fibrosis stages are not yet acknowledged, the method's reliability could be impaired. The spotlight falls on the optimal LSM cut-off values used to define severe fibrosis (F3 or F4 stages). F ≥ 3 represents advanced fibrosis, while the F4 fibrosis stage usually suggests cirrhosis [142]. Supplementary difficulties arise from some studies not reporting LSM cut-offs that define different fibrosis stages [57,72,91], while others use different stage appellations that are difficult to correlate with the current standard definitions [81,83,89,116]. The age-adapted cut-offs should also be taken into account to improve the method's performance. Finally, some studies suggest that the use of lower cut-off values would optimize their negative predictive value.

As yet, patients with diabetes have not been compared directly with non-diabetic control groups in elastography-based investigation protocols. Hence, the same cut-off points were applied to stratify fibrosis and steatosis as in any other NAFLD patient. However, the diagnostic and prognostic accuracy for NAFLD of non-invasive imaging tools is significantly influenced by the presence of diabetes [143]. This category of methods needs, therefore, further investigation and validation in populations with T2DM, among which advanced fibrosis has a significantly rising prevalence.

When focusing on T2DM patients and trying to gather specific information on this NAFLD at-risk group population, researchers need to find the best methods to fill in these substantial knowledge gaps. There are only a few studies that directly targeted the diabetic population, while most research involved a larger population, among which only a subgroup of subjects had T2DM [71,96]. Because patients at risk for NAFLD may frequently have significant fibrosis, which can be overlooked on common ultrasound, especially when normal liver enzymes are associated, supplementary screening approaches should be considered, either in the general population or only in at-risk individuals represented by patients with obesity, T2DM, and MS [143]. It is, therefore, logical to presume that the utility of novel non-invasive assessment tools for NAFLD is of utmost importance, but we must acknowledge for now that their predictive ability is insufficiently demonstrated in diabetes populations [144]. At present, ADA recommends that all patients with prediabetes/T2DM and increased liver enzymes or steatosis on ultrasound should be evaluated for the presence of NAFLD, while the other guidelines have discordant approaches [36]. No guideline clearly states who should be selected for screening, who should do the screening, and which method is best to use.

Among elastography-based methods, most available evidence supports the use of TE, while pSWE and 2D-SWE, which are less available in liver clinics, feature limited data on patients with diabetes. Several studies are currently using the SWE techniques, but the available proof is not yet sufficient to generate recommendations, and the need to continue dedicated research in this at-risk population is still high.

The lack of technical information also narrows the reproducibility of data using LSM and CAP assessment. Many publications do not specify whether one or more operators were involved, if they were trained certified examiners, or if patients respected the examination protocol requiring, at least, a three-hour fast before undergoing elastography. [37,107]. The success rate depends on the operator's experience, but also other various factors such as age, BMI, visceral fat, or the presence of ascites; the probability of elastography-based methods failing increases in patients who are old, obese, or have ascites [14,37]. The number of exploratory measurements may also differ from one study to the other [37,145].

The method's applicability to NAFLD can be challenging in patients with obesity because of the high rate of failure in measurement and performance without the use of an XL probe. Some of the existing studies had limitations due to not using the XL probe to perform TE examinations on patients with obesity.

As mentioned before, liver biopsy is unsuited for large-scale applications in the diagnosis of NAFLD [143]. Moreover, the applicability of liver biopsies is limited in patients with T2DM and associated cardiovascular disease that need antiplatelet or anticoagulant therapy. However, this method is still required to confirm the results of non-invasive tools in clinical trials. Beyond designing an optimal, cost-effective algorithm for systematic risk stratification, the management of NAFLD in primary care should, therefore, include procedures to accurately estimate and minimize the need for biopsy.

At present, ultrasound-based elastography devices are not accessible in diabetes care clinics, thus requiring a strong collaboration with hepatologists to implement these new, simpler, non-invasive tools and to limit the use of invasive methods in the future. Among steps already taken in this direction, NIMBLE (non-invasive biomarkers of metabolic liver disease) [146] in the USA and LITMUS (liver investigation: testing marker utility in steatohepatitis) [147] in Europe are two projects looking to integrate non-invasive tools into clinical practice and to offer the scientific community data required to receive uniform acceptance.

#### **6. Conclusions**

To sum up, the results of recent studies show a high prevalence of NAFLD identified by TE among patients with T2DM. These findings support the need for systematic screening for NAFLD to assess the severity of hepatic steatosis and fibrosis in T2DM patients. Within the group of shear wave elastography-based methods, TE has already acquired a welldeserved place, while ARFI-based techniques have begun to collect scientific evidence supporting their value in NAFLD screening, diagnosis, and monitoring among patients with T2DM.

Priorities of this research field should include the setting of cut-off points adapted to specific situations such as the co-existence of diabetes, assessment of the cost-effectiveness and validation of quality criteria for these imaging methods, the risk stratification based on the fibrosis stage, and evaluation of elastography value in the assessment of therapeutic success. Producing a strategic algorithm to check each of these purposes could help diabetes care specialists and primary care providers. An early diagnosis in high-risk patients and the subsequent implementation of adapted interventions such as lifestyle optimization, lipid-lowering therapy, and antihyperglycemic drugs may have the chance to limit NAFLD and its extrahepatic complications, at least until further effective therapies are developed. Beyond this, supplementary research is needed to completely define all long-term benefits of these ultrasound-based elastography techniques.

**Author Contributions:** Conceptualization, G.-D.C., C.-M.L. and B.-M.M.; methodology, G.-D.C., C.M. and E.-D.G.; validation, G.-D.C., C.M. and B.-M.M.; formal analysis, C.M., E.-D.G. and A.O.; investigation, C.M., E.-D.G. and A.O.; resources, G.-D.C., C.-M.L., E.-D.G. and A.O.; writing—original draft preparation, G.-D.C., E.-D.G. and A.O.; writing—review and editing, C.-M.L., C.M. and B.-M.M.; visualization, G.-D.C. and C.-M.L.; supervision, B.-M.M.; project administration, C.-M.L. and E.-D.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


2.3.3. Cryogenic Transmission Electron Microscopy (cryo-TEM)

Cryo-TEM analyses of HIV-1 Gag-eGFP VLPs were conducted from harvested supernatants. In Figure 3A, the sample preparation procedure for VLP visualization is presented. 2–3 μL of sample were blotted onto 200 or 400 mesh Holey carbon grids (Micro to Nano, Wateringweg, the Netherlands) previously glow discharged in a PELCO easiGlow glow discharge unit. Samples were subsequently plunged into liquid ethane at −180 ◦C using a Leica EM GP cryo workstation and observed in a Jeol JEM-2011 TEM electron microscope operating at 200 kV. During imaging, samples were maintained at −173 ◦C, and pictures were taken using a CCD multiscan camera (Model No. 895, Gatan).


**2. Epidemiology**

There is a big difference in the prevalence of type LADA DM among different population groups around the world. In North America it is estimated that 5–10% of new cases of DM in adult patients could correspond to this pathology [11].

In a UK Prospective Diabetes Study (UKPDS), about 10% of adults with suspected type 2 diabetes at the time of diagnosis had evidence of islet autoimmunity in the form of circulating ICA or GAD antibodies and most progressed to dependence on insulin in 6 years [12]. See **Table 1**.

In Central America there are still no studies that describe the prevalence of LADA type Diabetes. In countries like Honduras, its diagnosis is based on the clinic; this reduces the credibility of the diagnosis, especially when there is not enough equipment to corroborate the presence or absence of the antibodies described above.

Given that an appreciable proportion of people with diabetes who do not require insulin have a high proportion of antibodies against glutamic acid decarboxylase (GAD), it has been concluded that the LADA type Diabetes is probably much more prevalent than the DM type 1 but less frequent than DM type 2 [24]. This in turn explains the reason for the use of Anti-GAD, in comparison with other antibodies, to detect type LADA DM among subjects diagnosed as DM type 2 [13, 25].

**Table 1.** Prevalence of positive glutamic acid decarboxylase antibodies (late autoimmune diabetes in adults) in subjects with type 2 diabetes among different population groups.