Experimental design

We performed a four-year retrospective study on a NAFLD cohort from January 2018 to January 2022, dividing the study period by the beginning of the lockdown social restrictions in January 2020 [Baseline (T0); intermediate (end of the pre-pandemic period–January 2020) (T1); end of the study (January 2022) (T2)]. We routinely followed up the enrolled patients with clinical, biochemical, and imaging assessments in accordance with the current CPG and presented the data as mean values of the recordings that occurred during the specific period of observation.

For the entire length of the study, we screened and eventually recorded HCC occurrence by using ultrasonography assessments following CPG [28].

The study’s experimental design is reported in Fig. 1.

Fig. 1: Study design flowchart.

ALT Alanine Aminotransferase, AST Aspartate aminotransferase, BMI Body Mass Index, CAP Controlled Attenuation Parameter, FPG Fasting Plasma Glucose, HCC Hepatocellular carcinoma, HDL High-density lipoprotein, HOMA-IR Homeostatic model assessment for insulin resistance, LDL Low-density lipoprotein, NAFLD Non-alcoholic fatty liver disease, PLT Platelet count, TG triglycerides, WHR Waist-hip ratio.

The study’s primary endpoint was to assess the impact of the SARS-CoV-2 spread-related lifestyle changes on body composition analysis and metabolic syndrome components worsening.

The secondary endpoint was to assess the impact of the pandemic on HCC occurrence, as well as, shed light on the pandemic risk factors for HCC onset.

The entire study protocol was registered on the NIH U.S. National Library of Medicine database of clinical trials (NCT05416970). The “STrengthening the Reporting of Observational Studies in Epidemiology” (“STROBE”) checklist is reported in Supplementary File 1.

Patients

This retrospective multicenter longitudinal study is in compliance with the ethical guidelines of the Declaration of Helsinki (1975) and was approved by the ethical committee of the University of Campania “L. Vanvitelli” in Naples (prot n. 15.04-20220010000, 29th Mar 2022).

Between January 2018 and January 2022, patients affected by NAFLD based on clinical, biochemical, imaging, and histology, in accordance with CPG diagnostic criteria, and continuously followed by three centers (Hepato-gastroenterology Division of the University of Campania “Luigi Vanvitelli, Internal Medicine and Hepatology Unit of the University of Salerno and Institute of Internal Medicine of the University of Foggia), were enrolled in the present study, after signing informed consent.

The inclusion criteria were age between 18 and 80 years and NAFLD diagnosis.

Exclusion criteria were the presence of chronic inflammatory diseases such as inflammatory bowel disease, acute or chronic kidney disease, rheumatoid arthritis, systemic lupus erythematosus, or other major systemic diseases or tumors, ongoing infections, alcohol or drug abuse history, other etiologies of chronic liver damage, previous HCC diagnosis, use of hepatoprotective drugs, the administration of insulin (i.e., T2DM enrolled affected patients were all insulin-naïve), and psychological/psychiatric problems that could have invalidated the informed consent.

Medical history was collected, and alcohol consumption by using the complete Alcohol Use Disorders Identification Test (AUDIT-C) questionnaire was longitudinally assessed; medications (in particular, therapies for the dysmetabolic comorbidities: T2DM, obesity, arterial hypertension, and dyslipidemia) were opportunely classified and longitudinally recorded, and drug abuse was investigated.

Blood pressure (BP) [systolic (SBP) (mmHg) and diastolic (DBP) (mmHg)] were directly measured by an expert physician according to the American Heart Association (AHA) recommendations [29]. In detail, using an aneroid sphygmomanometer (GIMA, 32725, London) with an adequate cuff size (for arm circumference of 27 to 34 cm, the “adult” size of 16 × 30 cm was considered) and stethoscope (GIMA, 32719), the auscultatory method based on the Korotkoff technique, in which peculiar sound modifications describe 5 different phases, was adopted to collect SBP and DBP. To implement the predictive power of measurements, for everyone, multiple blood pressure determinations were obtained by the same physician: two readings were taken at intervals of at least 1 min, and the average of those readings was used to represent the patient’s BP; if there was >5 mm Hg difference between the first and second readings, additional (1 or 2) readings were obtained, and then the average of these multiple readings was used [29]. Weight (kg) and height (m) were also determined, and the Body Mass Index (BMI) was calculated by dividing the weight (kg) by the square of height (m) as Quetelet’s index (kg/m2) [30].

Waist (cm) and hip circumference (cm) were directly measured by using a tape measure (Xrten, B08DTG7RQ8), and the waist-to-hip ratio (WHR) was calculated. Waist circumference was recorded at the minimum circumference between the iliac crest and the rib cage, whereas hip circumference was recorded at the maximum circumference over the buttocks [30]. For each patient, these measurements were obtained in duplicate with subjects standing dressed in underwear, and both were rounded to the nearest 0.5 cm [30].

The following biochemical variables were recorded during the entire study period: insulin, fasting plasma glucose (FPG), the homeostatic model assessment for IR (HOMA-IR), aspartate aminotransferase (AST), alanine aminotransferase (ALT), platelet count (PLT) and plasma albumin (PA), total cholesterol, triglycerides (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL). Insulin levels (μU/mL) were measured enzymatically using commercially available kits (R&D Systems, Minneapolis, MN), whereas, AST (U/L), ALT (U/L), FPG (mmol/L), LDL and HDL, and TG using a colorimetric assay kit (AST, ALT: Amplite 13801/13803; FPG: Thermo Fisher Scientific EIAGLUC; LDL and HDL: Sigma Aldrich MAK045; TG: Sigma Aldrich MAK266). PA (g/dL) was assessed by using the BCA protein assay (Sigma Aldrich QPBCA), as a validated protein quantification method. PLT count (x103/μL) was performed with an automated hematology analyzer by using a suspension of blood cells passing through a small orifice along with an electric current of the Beckman Coulter analyzer [C11137 - DxI 9000 Analyzer, Beckman Coulter, Inc©].

HOMA-IR was calculated using the following formula: fasting insulin (μU/mL) × FPG (mmol/L)/22.5 [31].

Food intake, alcohol assumption, and physical exercise assessments

At the end of the pre-pandemic period, as well as at the end of the study, the food intake relative to a complete week, including working days and the weekend, was recorded, and evaluated by using the software WinFood, Medimatica s.r.l., Martinsicuro, Italy. Based on the quantity and quality of foods consumed, the program estimates the percentage of macronutrients and micronutrients in each food and elaborates the daily energy intake in terms of Kcal per day referring to the caloric amount as specifically related to carbohydrates, fats, and proteins dietary proportions. The complete elaboration of intakes shows the list of diet components, the ratio among components, the calories, and the subdivision into breakfast, lunch, and dinner. The availability of a list of diet components, for each patient, allowed an expert dietician to compute the dietary composition, in terms of fat types (saturated, monounsaturated, and polyunsaturated) and carbohydrate types (monosaccharides, disaccharides, and polysaccharides) proportion.

Alcohol assumption was evaluated with a standardized pre-codified questionnaire (AUDIT-C test): the quantity of daily alcohol intake was calculated based on a “drink” that corresponds to about 12 g of pure ethanol [32].

Physical exercise was assessed by a specific medical-assisted questionnaire (Supplementary File 2).

Non-invasive liver disease progression status evaluation: liver stiffness measurement, controlled attenuation parameter assessment, and calculation of NAFLD fibrosis score

Liver stiffness measurement (LSM) was obtained by transient elastography (TE), which was performed by using the FibroScan ® version 502 (Echosens, Paris, France) with M and XL probes [33]. We decided to use the XL probe when the ultrasonography measured distance between the skin to liver capsule resulted in greater than 2.5 cm and/or when BMI was >30. FibroScan ® was performed by an expert physician obtaining 10 acceptable measurements (defined as successful LSM), with the maximum number of attempts set at 20. The criteria proposed by Boursier et al. were used to consider the measurement “very reliable” (IQR/M ≤ 0.1), “reliable” [0.1 < IQR/M ≤ 0.3 or IQR/M > 0.3 with LS median < 7.1 kilopascal (kPa)], or “poorly reliable” (IQR/M > 0.3 with LS median ≥ 7.1 kPa) [33].

The controlled attenuation parameter (CAP) measures ultrasonic attenuation in the liver at 3.5 MHz using signals acquired by the FibroScan® M and XL probes based on physical principles described elsewhere [34]. The CAP was measured only on validated measurements according to the same criteria used for LSM [33, 34].

NAFLD fibrosis score (NFS) was determined by using the formula: -1.675 + 0.037 × age (years) + 0.094 × BMI (kg/m2) + 1.13 × T2DM (yes = 1, no = 0) + 0.99 × AST/ALT ratio−0.013 × PLT (×109/L)−0.66 × PA (g/dL) [35].

Multicompartment bioimpedance body composition analysis assessment

A multifrequency bioelectrical impedance analysis (BIA) system (InBody, Seoul, Korea) was used to perform the body composition assessment. For the analysis, two electrodes on the right foot and hand were placed. The patients assumed the supine position and were relaxed for at least 15 minutes before the assessment that was performed in duplicate.

The mean values of the two different measurements were used for the analysis. For this purpose, a gentle voltage waveform at 50 kHz was passed through the body of each enrolled patient from the hand electrodes to the foot ones. Using the reactance (Xc), resistance (R), and phase angle [arctangent (Xc/R) × (180/π)] the BIA system, thanks to a series of types of machinery algorithms elaborated the total body water (TBW), the intracellular and extracellular body water (ICW/ECW), the FFM, the FM, body cell mass (BCM) expressed both in percentage and kilograms (Kg). Skeletal Muscle Mass Index (SMMI) was calculated by dividing the Skeletal Muscle Mass (SMM) by the square of the height (m2).

Recording of HCC occurrence and definition of HCC overall and Milan-out criteria

The screening for HCC was adequately performed by using ultrasonography assessments, as well as HCC diagnosis was opportunely achieved respecting the European Association for Study of the Liver (EASL) CPG: contrast-enhanced ultrasound (CEUS) showing HCC imaging hallmarks (LiRADS 5) defined this neoplasm [28]. On the diagnosis, to stage HCC occurrence, Milan criteria were adopted [36]: patients presenting 1 lesion ≥ 2 cm and ≤ 5 cm or up to 3 lesions, each ≥1 cm and ≤ 3 cm, without evidence of vascular invasion or extra-hepatic metastases, were defined as “HCC Milan-in criteria”, contrariwise individuals not respecting the abovementioned criteria were considered “HCC Milan-out criteria”. Hence, “HCC overall” represented the sum of “HCC Milan-in criteria” and “HCC Milan-out criteria”.

Statistical analysis

Baseline demographics were summarized using descriptive statistics. Continuous data were described as mean and standard deviations, while categorical variables were summarized as n (%).

The Kolmogorov-Smirnov test for normality was performed to evaluate if parametric or non-parametric analysis should be applied. Wilcoxon signed ranks test and t-test for dependent groups were performed to compare continuous variables between two times of observation.

The Kruskal-Wallis test or ANOVA test with post-hoc Tukey analysis, in the case of non-normal or normal distribution respectively, were performed to compare the continuous variables among three times of observation.

Time-to-event analyses on HCC occurrence and Milano-out staging at the diagnosis were performed by using the Kaplan-Meier method and the Log-rank test for the curve comparison considering a p-value < 0.05 as statistically significant. The proportional hazard assumption was verified using the Schoenfeld residuals test.

The odds ratios (OR) of the study variables on the just mentioned events were calculated considering the confounding variables (age, sex, BMI, T2DM, SARS-CoV-2 infection, and LSM) by using logistic regression models. Sensitivity analyses were performed stratifying the overall sample by age, sex, BMI, type 2 diabetes, SarsCoV2, and LSM.

Statistical significance was defined as p < 0.05 in a two-tailed test with a 95% confidence interval. Statistical Program for Social Sciences (SPSS®) vs.18.0 was used to perform the analysis.