BACKGROUND:Vitamin deficiencies remain prevalent worldwide and contribute to chronic disease burden. This study evaluated serum concentrations of vitamins D, A, and E in Italian general practice populations and examined their associations with prevalent chronic diseases. METHODS:This multicenter cross-sectional study enrolled 500 adult patients (median age: 60.7 years; 40.4% male) attending 21 general practice offices across Italy between January 2021 and December 2024. Serum concentrations of 25-hydroxyvitamin D [25(OH)D], vitamin A, and vitamin E were measured alongside demographic, clinical, and biochemical parameters. Statistical analyses included chi-square tests, correlation analyses, and stepwise logistic regression. RESULTS:The median 25(OH)D concentration was 20.4 ng/mL, below optimal levels. Vitamin D deficiency was significantly associated with osteoporosis (p = 0.015), arterial hypertension (p = 0.047), and coronary artery disease (p = 0.002). The median vitamin A (654 μg/L) and vitamin E (11.3 mg/L) concentrations were within normal ranges. Notably, elevated vitamin A levels were significantly associated with arterial hypertension (p = 0.005), diabetes mellitus (p = 0.036), and cancer (p = 0.010). In the multivariate analysis, elevated vitamin A levels emerged as an independent predictor of multiple comorbidities (p < 0.001) and cancer presence (p = 0.014), alongside age, male gender, and body mass index. CONCLUSIONS:Vitamin D insufficiency is highly prevalent in Italian primary care populations. Vitamin A concentrations show independent associations with multimorbidity and cancer, suggesting a potential negative influence of an animal-based diet, warranting prospective investigation. These findings highlight the importance of comprehensive vitamin assessment in general practice settings.

Background:Adult solid organ transplant recipients (SOTRs) have decreased responsiveness to severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) vaccination and higher incidence of infection, but there are few data on the serological response in pediatric SOTR. The aim of this study was to determine serological response to SARS-CoV-2 vaccination in pediatric liver (LT) and kidney transplant (KT) recipients and compare it with adult SOTR. Methods:A European, prospective, multicenter study was performed. Samples were taken at 7 and 32 wk following COVID-19 vaccination and serological endpoints were measured by ELISA. Results:A total of 42 pediatric (16 post-LT and 26 post-KT) and 117 adult (all post-LT) were included. All pediatric participants and 94% adult participants received mRNA vaccines. Paediatric SOTR patients had significantly higher anti-Spike IgG levels than adult participants at week 7 (114 220.7 [59 285.92-220 058.55] versus 8756.7 [5643.69-13 586.71], P < 0.0001) and week 32 (46 113.2 [10 992.91-193 436.14] versus 8207.0 [3561.20-18 913.43], P = 0.0032). No significant difference in week 7 anti-Spike IgG response was found between pediatric LT and KT (129 434.4 [51 888.64-322 869.69] versus 105 304.5 [39 910.20-277 849.50], P = 0.9854). No differences were seen between children and adults in the rate of decline of anti-Spike IgG between weeks 7 and 32 (P = 0.8000). Male sex and hemolytic-uremic syndrome or postischemic kidney disease were associated with lower anti-Spike IgG levels at week 7 in pediatric SOTR. Conclusions:Paediatric SOTR demonstrate greater SARS-CoV-2 vaccine responses than comparable adult SOTR patients. These data support efficacy and safety of SARS-CoV-2 vaccination in child SOTR and may alleviate vaccine hesitancy in this patient group.

Hereditary hemochromatosis (HH) related to HFE-gene mutations is a well-known condition, yet its understanding remains complex. The BIOIRON classification emphasizes that only homozygosity for the C282Y mutation should be considered pathogenic. The penetrance of HFE-related HH is highly variable. Symptoms are often challenging to recognize at the time of presentation, and the systemic involvement may overlap with other diseases. Hyperferritinemia and elevated transferrin saturation levels are still the milestones in HH diagnosis, but they are also common findings in many other clinical conditions. Furthermore, current diagnostic and therapeutic guidelines are not always unequivocal in defining HH patients’ characteristics, as well as treatment management and goals. Our work provides a concise overview of the latest evidence regarding pathogenic mechanisms, clinical picture, differential diagnosis and diagnostic tools. Alongside this, it summarizes and compares the main recommendations from principal guidelines issued by the 2017 Hemochromatosis International Meeting, the American College of Gastroenterology, the European Association for the Study of the Liver, the European Molecular Genetics Quality Network, the DUTCH guidelines, and the British Society for Haematology. Summarizing tables for quick consultation are also provided.

Background & Aims Sodium taurocholate cotransporting peptide (NTCP) genetic polymorphisms have been described, but their role in untreated and treated patients with Chronic Hepatitis Delta (CHD) remains unknown. Virological response (VR) to NTCP inhibitor Bulevirtide (BLV) was achieved at week 48 by >70% of CHD patients, but nearly 15% experienced virological nonresponse (VNR) or partial response (PR). This study aimed to evaluate whether NTCP genetic polymorphisms affect baseline HDV RNA load and response to BLV in CHD patients. Methods Untreated and BLV treated patients were enrolled in a retrospective cross-sectional and longitudinal study. Clinical and virological characteristics were collected at baseline and up to 96 weeks in the BLV-treated patients. NTCP genetic polymorphisms were identified by Sanger sequencing. Results Of the 6 NTCP polymorphisms studied in 209 CHD untreated patients, carriers of rs17556915 TT/CC (N=142) compared to CT (N=67) genotype presented higher median HDV RNA levels (5.39 vs. 4.75 log10 IU/mL, p=0.004). 76 out of 209 patients receiving BLV monotherapy at 2 mg/day were evaluated at week 24 and 40 of them up to week 96. Higher mean baseline HDV RNA levels were confirmed in TT/CC (N=43) compared to CT (N=33) carriers (5.38 vs. 4.72 log10 IU/mL, p=0.010). Although 24-week VR was comparable between TT/CC and CT carriers (25/43 vs. 17/33, p=0.565), the former group presented VNR more often than PR (9/11 vs. 9/23, p=0.02) at week 24. 7/9 TT/CC genotype carriers remained VNR at week 48 of BLV treatment. Conclusions The NTCP rs17556915 C>T genetic polymorphisms may influence baseline HDV RNA load both in untreated and BLV treated patients with CHD and may contribute to identify patients with different early virological responses to BLV.

Topic: 26. Sickle cell disease Background: Sickle cell disease (SCD) and thalassemia are severe genetic disorders impairing haemoglobin (Hb) function and production, resulting in significant morbidity and mortality, even in the first years of life. A recent increase in the number of people with Hb disorders (HD) has been registered in Europe due to the globalization and migration phenomena. The early detection of HD allows counselling for family members about disease management, prevention of complications and future reproductive decisions. Newborn screening (NBS) programs for HD are now recommended in the United States and Europe, although widespread implementation still needs to be achieved. Moreover, most NBS are based only on High Performance Liquid Chromatography (HPLC) method, with a clear separation and quantification of Hb fractions, and limited to at risk population. Testing with HPLC is a well-known feasible approach, but not so accurate for diagnosis, as it requires to be completed by molecular DNA confirmation analysis. This sequential workflow, in which only one gene is investigated at a time, may be time-consuming and costly, and may miss some rare causative variants, resulting in a delay in genetic counseling and unresolved cases. Next-generation sequencing (NGS) has been also recently reported as an effective method in screening HD and Hb-associated variations to facilitate diagnosis. Aims: The aim of the present study was to assess the feasibility and efficacy of a universal NBS program based on both HPLC and NGS methods, and to estimate the incidence of HD in the area of Udine (North-Eastern Italy). Methods: A public universal NBS for HD started in 2018 at the Hospital of Udine. Parents of all newborns were offered to participate to this program, and infants’ blood samples were collected 2 days after birth or at 37 gestational weeks in preterm infants. HPLC was performed as the first test on samples, then molecular analysis of the beta-globin gene completed the Hb genotype. Results: From July 2018 to December 2022 there were 7396 newborns at Udine’s Hospital. A total of 6980 neonates (94%) were tested for HD. During this period, 101 samples (1.45%) resulted positive for any Hb abnormality, in particular 1 SCD (0.014%),1 β-Thalassemia Major (0.014%), 3 HbCC (0.042%), 66 infants (0.94%) with sickle-cell trait (1 presented an S/G-Philadelphia phenotype) and 15 (0.21%) HbC carriers. Other minor Hb variants were also identified: 11 (0.16%) carriers of HbE, 2 (0.03%) HbD-Punjab, 1 (0.014%) HbG and 1 (0.014%) HbK Woolwich. Children with SCD and β-Thalassemia were immediately redirected from Neonatology to Pediatric Department for the subsequent clinical management and follow-up. All other families of Hb variant positive newborns received genetic counseling. Summary/Conclusion: This study demonstrates the feasibility of a universal NBS program for HD, giving also important information on SCD and thalassemia epidemiology in a Northern Italian area. Based on our results, universal NBS for HD appears to be useful also in a non–endemic area, supporting previous European recommendation for a universal screening. Overall, NGS has an added value in large-scale high throughput carrier screening and in the complex cases for which common molecular techniques have some inadequacies. Therefore, early detection and immediate treatment of affected newborns thanks to molecular analysis techniques may compensate the initial costs of a direct molecular approach. Keywords: Hemoglobin variants, Hemoglobinopathy, Neonate, Sickle cell disease

The treatment response criteria in autoimmune hepatitis (AIH) have been recently updated. This study aimed to assess treatment responses in 39 (16 males) patients with AIH confirmed by histology. Prednisone added to azathioprine or mycophenolate was the most frequent first-line treatment. Serum alanine aminotransferase (ALT) levels were periodically checked for a median of 45 months. Eight (20.5%) patients presented 4 weeks non-response (NR). Baseline lower multiples of ALT above the upper normal limit (UNL) (p = 0.005), Ishak liver fibrosis score > 3 (p = 0.029), and less frequent confluent necrosis > 2 (p < 0.001) were independent predictors of NR. 24 (61.5%) patients achieved complete biochemical response (CBR) at six months. Ishak liver fibrosis score ≤ 3 (p < 0.001), lobular eosinophilic infiltrate (p < 0.001), and ≥50% decrease in serum ALT levels at week 4 (p < 0.001) were independent predictors of CBR. In addition, the GLUCRE score, derived from the multiplication of serum creatinine (mg/dL) and glucose (mg/dL) levels, were identified. A baseline GLUCRE value > 100 strongly predicted CBR failure (p = 0.003) at a follow-up greater than 12 months. In conclusion, the absence of cirrhosis and a ≥50% UNL decrease in serum ALT levels were independent predictors for CBR. A baseline GLUCRE score may help identify patients maintaining longer CBR.

Background & aims: The fourth dose of anti-SARS-CoV-2 vaccine slightly improved the humoral response among previously seronegative liver transplant (LT) recipients. Mycophenolate (MMF) treatment worsens the vaccination response. This study aimed to evaluate whether temporary MMF interruption might improve the immunogenicity of the fourth anti-SARS-CoV-2 BNT16b2 vaccine dose in nonresponsive LT recipients. Methods: LT recipients negative for anti-spike glycoprotein-specific immunoglobulin G receptor-binding domain (s-RBD) antibodies after the third vaccine dose were enrolled. Anti-SARS-CoV-2 spike-specific T-cell responses were measured before and 2 months following the fourth vaccine dose, and anti-SARS-CoV-2 s-RBD antibodies also 6 months thereafter. MMF was suspended two weeks before and after vaccination. Results: Five LT recipients were enrolled. After a mean of 78 days after vaccination, all patients tested positive for anti-SARS-CoV-2 s-RBD antibodies. The mean antibody titer was 8944 UI/mL. The positive antibody response was maintained during a mean of 193 days of follow-up. Three patients developed a positive T-cell response. Two patients (one positive for T-cell response) developed a self-limited SARS-CoV-2 infection. Conclusions: Suspending MMF prior to the fourth dose of the anti-SARS-CoV-2 mRNA vaccine seems feasible and safe. This procedure could restore vaccine-induced immunogenicity in a large portion of previously nonresponsive LT recipients.

Background: Vaccine hesitancy and lack of access remain major issues in disseminating COVID-19 vaccination to liver patients globally. Factors predicting poor response to vaccination and risk of breakthrough infection are important data to target booster vaccine programs. The primary aim of the current study was to measure humoral responses to 2 doses of COVID-19 vaccine. Secondary aims included the determination of factors predicting breakthrough infection. Methods: COVID-19 vaccination and Biomarkers in cirrhosis And post-Liver Transplantation is a prospective, multicenter, observational case-control study. Participants were recruited at 4–10 weeks following first and second vaccine doses in cirrhosis [n = 325; 94% messenger RNA (mRNA) and 6% viral vaccine], autoimmune liver disease (AILD) (n = 120; 77% mRNA and 23% viral vaccine), post-liver transplant (LT) (n = 146; 96% mRNA and 3% viral vaccine), and healthy controls (n = 51; 72% mRNA, 24% viral and 4% heterologous combination). Serological end points were measured, and data regarding breakthrough SARS-CoV-2 infection were collected. Results: After adjusting by age, sex, and time of sample collection, anti-Spike IgG levels were the lowest in post-LT patients compared to cirrhosis ( p < 0.0001), AILD ( p < 0.0001), and control ( p = 0.002). Factors predicting reduced responses included older age, Child-Turcotte-Pugh B/C, and elevated IL-6 in cirrhosis; non-mRNA vaccine in AILD; and coronary artery disease, use of mycophenolate and dysregulated B-call activating factor, and lymphotoxin-α levels in LT. Incident infection occurred in 6.6%, 10.6%, 7.4%, and 15.6% of cirrhosis, AILD, post-LT, and control, respectively. The only independent factor predicting infection in cirrhosis was low albumin level. Conclusions: LT patients present the lowest response to the SARS-CoV-2 vaccine. In cirrhosis, the reduced response is associated with older age, stage of liver disease and systemic inflammation, and breakthrough infection with low albumin level.

Dear Editor, We thank Dr Sookaromdee et al. for the positive comments on our paper.1 We demonstrated that a positive anti-SARS-CoV-2-s-RBD antibody response after the third BNT16b2 vaccine dose was detected in more than 90% of liver transplant patients (LT), and more than 80% of them developed a median antibody titre >100 U/ml. After a median post-vaccination follow-up of 4 months, a very low rate of SARS-CoV-2 symptomatic infections were recorded and, more importantly, all of them presented a benign clinical outcome. In the pre-vaccination era, mortality rate in LT patients due to SARS-CoV-2 infection was 19%, and 30% of patients required intensive care.2 These differences strongly confirm the indisputable clinical utility of anti-SARS-CoV-2 vaccination in LT patients. We agree with Dr. Sookaromdee et al. that some of our patients may have had an asymptomatic SARS-CoV-2 infection, since they were not periodically tested by RT-PCR on nasopharyngeal swabs. After the third dose of BNT16b2 vaccine, about two thirds of infections in immunocompetent patients are asymptomatic, but they can be transmitted to others.3 It is not known whether immunosuppressed patients may have a higher rate of asymptomatic infections or can transmit the infection more easily or for a longer period. Thus, studies focusing on this issue are urgently needed in LT patients. A further challenging topic is to evaluate the efficacy of the third vaccine dose in protecting LT patients for both the disease and the infection caused by new Omicron subvariants. In immunocompetent patients, SARS-CoV-2 BA.1, BA.2, BA.2.12.1 and XD spike variants have an increased propensity to evade neutralizing antibodies induced by three doses of BNT162b2 vaccine, and that BA.4/5 variants are much less efficiently neutralized by the vaccine.4 It can be hypothesized that the vaccine-induced immunogenicity against these emerging viral variants may be further reduced in immunosuppressed patients. Recent data indicate that the administration of a fourth BNT162b2 vaccine dose in immunocompetent patients was associated with high protection against hospitalizations and deaths during a surge associated with the Omicron variants.5 This suggests that LT patients should be prioritized in receiving the fourth anti SARS-CoV-2 vaccination dose, waiting for the efficacy and safety data of the updated versions of the mRNA vaccines. We can conclude that the first half of the match ended in our favour, but the opponent is very dangerous and there is still a lot of time to play to be sure we have won the match.

Background & Aims: The long-term immunogenicity of anti-SARS-CoV-2 vaccines in liver transplant (LT) recipients is unknown. We aimed to assess the long-term antibody response of the Pfizer-BioNTech (R) BNT162b2 vaccine in LT recipients compared to controls. Methods: LT recipients underwent anti-SARS-CoV-2 anti-receptor-binding domain protein IgG (anti-RBD) and anti-nucleocapsid protein IgG antibody (anti-N) measurements at the first and 1, 4 and 6 months after the second vaccination dose. Results: One hundred forty-three LT recipients and 58 controls were enrolled. At baseline, 131/143 (91.6%) LT recipients tested anti-N negative (COVID-19 naive), and 12/143 (8.4%) tested positive (COVID-19 recovered) compared to negative controls. Among COVID-19 naive, 22.1% were anti-RBD positives 1 month after the first vaccine dose, while 66.4%, 77%, and 78.8% were 1, 4 and 6 months following the second vaccine dose. In contrast, 100% of controls were positive at 4 months (p <0.001). The median anti-RBD titer 4 months after the second vaccine dose was significantly lower (32 U/ml) in COVID-19 naive than in controls (852 U/ml, p <0.0001). A higher daily dose of mycophenolate mofetil (MMF) (p <0.001), higher frequency of ascites (p = 0.012), and lower serum leukocyte count (p = 0.016) were independent predictors of anti-RBD negativity at 6 months. All COVID-19 recovered patients tested positive for anti-RBD at each time point. The median antibody titer was similar in those taking MMF (9,400 U/ml, 11,925 U/ml, 13,305 U/ml, and 10,095 U/ml) or not taking MMF (13,950 U/ml, 9,575 U/ml, 3,500 U/ml, 2,835 U/ml, p = NS) 3 weeks after the first and 1, 4 and 6 months after the second vaccine dose, respectively. Conclusions: In COVID-19-naive LT recipients, the immunogenicity of anti-SARS-CoV-2 vaccination was significantly lower than that in controls. MMF was the main determinant of vaccination failure in SARS-CoV-2-naive patients. Lay summary: The immunogenicity of anti-SARS-CoV-2 vaccination in liver transplant recipients is currently unknown. Herein, we show that liver transplant recipients who have not previously had COVID-19 are less likely to mount effective antibody responses to vaccination than a control population. The main determinant of vaccination failure was the use of the immunosuppressive drug mycophenolate mofetil. (C) 2022 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Background Patients with liver disease may be at increased risk of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection due to immune dysfunction. However, the risk of nosocomial SARS-CoV-2 infection in these patients remains unknown. This study aimed to determine whether patients with liver disease are at an increased risk of nosocomial transmission of SARS-CoV-2 infection upon admission to the hospital for diagnostic or therapeutic procedures. Methods The study prospectively enrolled 143 patients who were admitted at least once to the hepatology unit at our hospital; 95 patients (66%) were admitted at least twice during the study period. History of past symptomatic SARS-CoV-2 exposure was assessed on the day before hospital admission via an interview. Patients were evaluated for active SARS-CoV-2 infection via real-time reverse transcription–polymerase chain reaction (RT-PCR) performed on nasopharyngeal swabs and tests for serum anti-SARS-CoV-2 immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies. Results None of the patients enrolled tested positive for SARS-CoV-2 infection by RT-PCR at the first or the second clinical evaluation. One patient who had previously received a liver transplant and who had a history of symptomatic SARS-CoV-2 infection that occurred 4 months before hospital admission tested positive for anti-SARS-CoV-2 IgG but not IgM antibodies at each of the two hospital admissions. Conclusions The results of our study suggest that patients with liver disease are at no increased risk of nosocomial SARS-CoV-2 infection. These data support the policy of maintaining clinical hospital checks that will be necessary until or possibly even after the completion of the current SARS-CoV-2 vaccination campaign.

BACKGROUND & AIMS:A strategy to improve the low rate of anti-SARS-CoV-2 mRNA vaccine-induced immunogenicity in liver transplant recipients (LTs) is urgently needed.METHODS:We analysed the rate of positive (≥0.8 U/ml) anti-SARS-CoV-2 receptor domain-binding protein (RBD) antibody response 2 months after a third dose of the BNT16b2 vaccine in 107 LTs who completed the second vaccine dose 7 months earlier.RESULTS:A positive anti-SARS-CoV-2-s-RBD antibody response after the third vaccine dose was detected in 98 (91.6%) LTs compared to 82 (76.6%) after the second vaccine dose (p = .003). The median of anti-SARS-CoV-2 RBD antibody titres increased from 22.9 U/ml 6 months after the second to 3500 U/ml 2 months after the third vaccine dose (p < .001). Fourteen (14.3%) responder patients presented antibody titres <100 U/ml, 57 (58.2%) between 100 and 9999 U/ml and 27 (27.6%) ≥10 000 U/ml. Seropositivity after the second dose was maintained after the third dose. Independent predictors of antibody response failure after the third vaccine dose were taking a higher daily dose of mycophenolate mofetil (MMF, p < .001) and had a lower (<60 ml/min/1.73 m2 ) estimated glomerular filtration rate (p = .007). Nine (9.1%) LTs experienced symptomatic SARS-CoV-2 infection after the third vaccine dose. Median antibody titres were not statistically different between infected and not infected LTs (1325 vs 3515 U/ml, p = .678).CONCLUSIONS:The third dose of the BNT16b2 vaccine increased the number of LTs who developed a positive anti-SARS-CoV-2 s-RBD antibody response. A proportion of patients remained unresponsive, mainly for modifiable factors, such as the use of MMF or multiple immunosuppressants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. The presence of viral RNA in samples by nucleic acid (NA) molecular analysis is the only method available to diagnose COVID-19 disease and to assess patients' viral load. Since the demand for laboratory reagents has increased, there has been a worldwide shortage of RNA extraction kits. We, therefore, developed a fast and cost-effective viral genome isolation method that, combined with quantitative RT-PCR assay, detects SARS-CoV-2 RNA in patient samples. The method relies on the addition of Proteinase K followed by a controlled heat-shock incubation and, then, E gene evaluation by RT-qPCR. It was validated for sensitivity, specificity, linearity, reproducibility, and precision. It detects as low as 10 viral copies/sample, is rapid, and has been characterized in 60 COVID-19-infected patients. Compared to automated extraction methods, our pretreatment guarantees the same positivity rate with the advantage of shortening the time of the analysis and reducing its cost. This is a rapid workflow meant to aid the healthcare system in the rapid identification of infected patients, such as during a pathogen-related outbreak. For its intrinsic characteristics, this workflow is suitable for large-scale screenings.

Background: Spontaneous bacterial peritonitis (SBP) remains a major complication of cirrhosis. However, the incidence and the real impact of SBP in determining patient survival rates remain unclear. This study aims to evaluate the incidence and risk factors for SBP development and the role of SBP in predicting transplant-free survival. Methods: Two hundred two consecutive patients underwent 492 paracenteses with biochemical and microbiological analysis of the ascitic fluid. When multiple paracenteses had been performed on a given patient, the first SBP-positive paracentesis or the first paracentesis conducted when none was diagnostic for SBP was included in the study. Results: SBP was detected in 28 of 202 (13.9%) patients; in 26 of 28 patients, the neutrophil count in the ascitic fluid was $250 cells/ml, and in 15 of 28 patients, the cultures were positive. Variables independently associated with SBP were as follows: a higher model of end-stage liver disease (MELD) score, the serum glucose value, elevated CRP serum levels, and higher potassium serum levels. Overall, the median (range) transplant-free survival was 289 (54-1253) days. One hundred (49.5%) patients died, whereas 35 patients (17.3%) underwent liver transplantation. Independent predictors of death or liver transplantation were a higher MELD score and the development of SBP, especially if it was antibiotic-resistant or recurrent SBP. Conclusion: The occurrence of SBP is associated with more severe liver dysfunction in conjunction with the presence of inflammation. Unlike the occurrence of SBP per se, failure of first-line antibiotic treatment and SBP recurrence appear to strongly influence the mortality rate.

Mass screening aimed at detecting, in asymptomatic subjects, the presence of SARS-CoV-2 is considered a strategic measure for the control of the present pandemic. It allows virus carriers to be identified and quarantined, thus preventing local spread and protecting vulnerable individuals. Although the screening strategy should be determined by the epidemiological situation, the size of the population that can be screened is indeed limited by the availability of resources. Here we present the implementation of an 8-sample pool strategy that relies on protocols, reagents and equipment currently used in clinical diagnostics. The method permitted to identify, with 100% sensitivity, specificity and accuracy, samples with low viral load, being the limit of detection of 11 viral copies extracted from the equivalent of 133μl of nasopharyngeal sample-pool. When the protocol has been applied, as a proof of principle, in a real population of 3592 consecutive nasopharyngeal swabs collected by healthcare providers in asymptomatic subjects, 20 positive pools were detected and in 100% of cases the positive specimens identified. Considering these performances, the 8-sample pool will allow, in populations with an expected positive rate of less than 1%, reducing costs by at least 80%, being a suitable method for a sustainable mass screening strategy in a population of asymptomatic subjects. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement None ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Consent CEUR-2020-Os-033 All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data referred to in the manuscript are available

Objectives Transjugular intrahepatic portosystemic shunt (TIPS) is adopted to treat refractory complications of portal hypertension, such as variceal bleeding and ascites. This study aimed to assess predictors of hepatic encephalopathy (HE) development and cumulative transplant-free survival after TIPS placement in patients with cirrhosis complicated by refractory ascites and major gastroesophageal bleeding. Materials and methods Sixty-three cirrhotic patients who underwent TIPS positioning as a secondary prophylaxis of major upper gastroesophageal bleeding (N=30) or to control refractory ascites (N=33) were enrolled. Results After a median follow-up of 26 months following TIPS insertion, only 1/30 (3.3%) patients developed reoccurrence of bleeding. Complete control of refractory ascites was recorded in 19/23 (82.6%) patients. Within the first month after TIPS placement, 34/63 (53.9%) patients developed clinically significant HE, which was associated with the baseline presence of type 2 hepatorenal syndrome (P=0.022). At the end of 90 months of follow-up, 35 (55.6%) patients were alive, 12 (19.0%) patients underwent liver transplantation, and 16 (25.4%) patients died. Independent predictors of transplant-free survival were a model for end-stage liver disease score up to 15 (P<0.001), the absence of a history of spontaneous bacterial peritonitis (P=0.010) pre-TIPS, and no HE within 1 month post-TIPS (P=0.040). Conclusion TIPS insertion can be considered a safe and effective treatment in patients with cirrhosis and severe complications of portal hypertension that are not manageable with standard treatments. Interestingly, if confirmed in future studies, the history of spontaneous bacterial peritonitis pre-TIPS could be added to the model for end-stage liver disease score as a strong baseline predictor of post-TIPS mortality.