Droplet digital PCR assay as an innovative and promising highly sensitive assay to unveil residual and cryptic HBV replication in peripheral compartment.

Droplet digital PCR is an innovative and promising approach for highly sensitive quantification of nucleic acids that is being increasingly used in the field of clinical virology, including the setting of hepatitis B virus (HBV). Here, we comprehensively report a robust and reproducible ddPCR assay for the highly sensitive quantification of serum HBV-DNA. The assay showed a limit of detection of 4 copies/ml (<1IU/ml) by Probit analysis, showed a good linearity (R2 = 0.94) and a high intra- and inter-run reproducibility with differences between the values obtained in the same run or in two independent runs never exceeding 0.14logcopies/mL and 0.21logcopies/mL, respectively. By analysing serum samples from chronically HBV infected patients (mostly under antiviral treatment), ddPCR successfully quantified serum HBV-DNA in 89.8% of patients with detectable serum HBV-DNA < 20 IU/mL [equivalent to <112copies/ml] by classical Real-Time PCR assay, with a median (IQR) of 8(5-14)IU/mL [45(28-78)copies/ml], and in 66.7% of patients with undetectable serum HBV-DNA, with a median (IQR) of 5(4-9)IU/mL [28(20-50)copies/ml]. Similarly, by analysing serum samples from patients with a serological profile compatible with occult HBV infection (anti-HBc+/HBsAg-), ddPCR successfully quantified serum HBV-DNA in 40% of patients with a median (IQR) value of 1(1-2)IU/mL [5(5-11)copies/ml], in line with the extremely limited viral replication typically observed in occult HBV infection. Overall, the availability of assays for the highly sensitive quantification of serum HBV-DNA can provide an added value in optimizing the diagnosis of occult hepatitis B infection, improving the therapeutic management of chronically HBV infected patients, also in the light of innovative drugs (upcoming in clinical practise) aimed at achieving HBV functional cure.

Prevalence of hepatitis D virus infection in Central Italy has remained stable across the last 2 decades with dominance of subgenotypes 1 and characterized by elevated viral replication.

OBJECTIVES: Here we investigate Hepatitis D virus (HDV)-prevalence in Italy and its fluctuations over time and we provide an extensive characterization of HDV-infected patients. METHODS: The rate of HDV seroprevalence and HDV chronicity was assessed in 1579 hepatitis B surface antigen (HBsAg)+ patients collected from 2005 to 2022 in Central Italy. RESULTS: In total, 45.3% of HBsAg+ patients received HDV screening with an increasing temporal trend: 15.6% (2005-2010), 45.0% (2011-2014), 49.4% (2015-2018), 71.8% (2019-2022). By multivariable model, factors correlated with the lack of HDV screening were alanine-aminotransferase (ALT) less than two times of upper limit of normality (<2ULN) and previous time windows (P <0.002). Furthermore, 13.4% of HDV-screened patients resulted anti-HDV+ with a stable temporal trend. Among them, 80.8% had detectable HDV-ribonucleic acid (RNA) (median [IQR]:4.6 [3.6-5.6] log copies/ml) with altered ALT in 89.3% (median [IQR]:92 [62-177] U/L). Anti-HDV+ patients from Eastern/South-eastern Europe were younger than Italians (44 [37-54] vs 53 [47-62] years, P <0.0001), less frequently nucleos(t)ide analogs (NUC)-treated (58.5% vs 80%, P = 0.026) with higher HDV-RNA (4.8 [3.6-5.8] vs 3.9 [1.4-4.9] log copies/ml, P = 0.016) and HBsAg (9461 [4159-24,532] vs 4447 [737-13,336] IU/ml, P = 0.032). Phylogenetic analysis revealed the circulation of HDV subgenotype 1e (47.4%) and -1c (52.6%). Notably, subgenotype 1e correlated with higher ALT than 1c (168 [89-190] vs 58 [54-88] U/l, P = 0.015) despite comparable HDV-RNA. CONCLUSIONS: HDV-screening awareness is increasing over time even if some gaps persist to achieve HDV screening in all HBsAg+ patients. HDV prevalence in tertiary care centers tend to scarcely decline in native/non-native patients. Detection of subgenotypes, triggering variable inflammatory stimuli, supports the need to expand HDV molecular characterization.

Unexpected rise in the circulation of complex HBV variants enriched of HBsAg vaccine-escape mutations in HBV genotype-D: potential impact on HBsAg detection/quantification and vaccination strategies.

Specific HBsAg mutations are known to hamper HBsAg recognition by neutralizing antibodies thus challenging HBV-vaccination efficacy. Nevertheless, information on their impact and spreading over time is limited. Here, we characterize the circulation of vaccine-escape mutations from 2005 to 2019 and their correlation with virological parameters in a large cohort of patients infected with HBV genotype-D (N = 947), dominant in Europe. Overall, 17.7% of patients harbours ≥1 vaccine-escape mutation with the highest prevalence in subgenotype-D3. Notably, complex profiles (characterized by ≥2 vaccine-escape mutations) are revealed in 3.1% of patients with a prevalence rising from 0.4% in 2005-2009 to 3.0% in 2010-2014 and 5.1% in 2015-2019 (P = 0.007) (OR[95%CI]:11.04[1.42-85.58], P = 0.02, by multivariable-analysis). The presence of complex profiles correlates with lower HBsAg-levels (median[IQR]:40[0-2905]IU/mL for complex profiles vs 2078[115-6037]IU/ml and 1881[410-7622]IU/mL for single or no vaccine-escape mutation [P < 0.02]). Even more, the presence of complex profiles correlates with HBsAg-negativity despite HBV-DNA positivity (HBsAg-negativity in 34.8% with ≥2 vaccine-escape mutations vs 6.7% and 2.3% with a single or no vaccine-escape mutation, P < 0.007). These in-vivo findings are in keeping with our in-vitro results showing the ability of these mutations in hampering HBsAg secretion or HBsAg recognition by diagnostic antibodies. In conclusion, vaccine-escape mutations, single or in complex profiles, circulate in a not negligible fraction of HBV genotype-D infected patients with an increasing temporal trend, suggesting a progressive enrichment in the circulation of variants able to evade humoral responses. This should be considered for a proper clinical interpretation of HBsAg-results and for the development of novel vaccine formulations for prophylactic and therapeutic purposes.

PaO2/FiO2 ratio forecasts COVID-19 patients' outcome regardless of age: a cross-sectional, monocentric study.

We studied the predictive value of the PaO2/FiO2 ratio for classifying COVID-19-positive patients who will develop severe clinical outcomes. One hundred fifty patients were recruited and categorized into two distinct populations ("A" and "B"), according to the indications given by the World Health Organization. Patients belonging the population "A" presented with mild disease not requiring oxygen support, whereas population "B" presented with a severe disease needing oxygen support. The AUC curve of PaO2/FiO2 in the discovery cohort was 0.838 (95% CI 0.771-0.908). The optimal cut-off value for distinguishing population "A" from the "B" one, calculated by Youden's index, with sensitivity of 71.79% and specificity 85.25%, LR+4.866, LR-0.339, was < 274 mmHg. The AUC in the validation cohort of 170 patients overlapped the previous one, i.e., 0.826 (95% CI 0.760-0.891). PaO2/FiO2 ratio < 274 mmHg was a good predictive index test to forecast the development of a severe respiratory failure in SARS-CoV-2-infected patients. Moreover, our work highlights that PaO2/FiO2 ratio, compared to inflammatory scores (hs-CRP, NLR, PLR and LDH) indicated to be useful in clinical managements, results to be the most reliable parameter to identify patients who require closer respiratory monitoring and more aggressive supportive therapies. Clinical trial registration: Prognostic Score in COVID-19, prot. NCT04780373 https://clinicaltrials.gov/ct2/show/NCT04780373 (retrospectively registered).

Three Cases of COVID-19 Pneumonia in Female Patients in Italy Who Had Pulmonary Fibrosis on Follow-Up Lung Computed Tomography Imaging.

BACKGROUND Since December 2019, an outbreak caused by a novel coronavirus infection (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) occurred in Wuhan, China, and it rapidly spread all over the world. The clinical spectrum of coronavirus disease 2019 (COVID-19) is wide, with acute respiratory distress syndrome (ARDS) occurring in 15% of patients affected, requiring high oxygen support. Currently, there is no clearly effective antiviral therapy. Steroids and immunomodulators are under investigation for potential activity. Little is known about middle and long-term sequelae on respiratory function. According to some authors, COVID-19 could cause pulmonary fibrosis. We report 3 cases of pulmonary fibrosis detected on follow-up computed tomography (CT) imaging in 3 female patients who recovered from COVID-19 pneumonia in Italy (L'Aquila, Abruzzo). CASE REPORT All patients were female and had no significant previous respiratory disease or history of smoke exposure, and none had received high-flow oxygen support during treatment of the disease. In all cases, late onset of mild dyspnea, slow and incomplete respiratory recovery, and early evidence of fibrous signs on chest CT scan were characteristic of the clinical course. CONCLUSIONS This report focuses on a possible scenario of long-term lung damage in COVID-19 pneumonia survivors. Limitations are lack of long-term follow-up and functional data in the very early phase. It is advantageous that all COVID-19 pneumonia patients undergo serial chest CT and spirometry long-term follow-up for at least 1 year to assess residual damage. This is particularly relevant in those with slow respiratory recovery and long hospitalization.

A Hyper-Glycosylation of HBV Surface Antigen Correlates with HBsAg-Negativity at Immunosuppression-Driven HBV Reactivation in Vivo and Hinders HBsAg Recognition in Vitro.

Immune-suppression driven Hepatitis B Virus (HBV)-reactivation poses serious concerns since it occurs in several clinical settings and can result in severe forms of hepatitis. Previous studies showed that HBV strains, circulating in patients with HBV-reactivation, are characterized by an enrichment of immune-escape mutations in HBV surface antigen (HBsAg). Here, we focused on specific immune-escape mutations associated with the acquisition of N-linked glycosylation sites in HBsAg (NLGSs). In particular, we investigated profiles of NLGSs in 47 patients with immunosuppression-driven HBV-reactivation and we evaluated their impact on HBsAg-antigenicity and HBV-replication in vitro. At HBV-reactivation, despite a median serum HBV-DNA of 6.7 [5.3-8.0] logIU/mL, 23.4% of patients remained HBsAg-negative. HBsAg-negativity at HBV-reactivation correlated with the presence of >1 additional NLGSs (p < 0.001). These NLGSs are located in the major hydrophilic region of HBsAg (known to be the target of antibodies) and resulted from the single mutation T115N, T117N, T123N, N114ins, and from the triple mutant S113N+T131N+M133T. In vitro, NLGSs strongly alter HBsAg antigenic properties and recognition by antibodies used in assays for HBsAg-quantification without affecting HBsAg-secretion and other parameters of HBV-replication. In conclusion, additional NLGSs correlate with HBsAg-negativity despite HBV-reactivation, and hamper HBsAg-antigenicity in vitro, supporting the role of NGSs in immune-escape and the importance of HBV-DNA for a proper diagnosis of HBV-reactivation.

Key mutations in the C-terminus of the HBV surface glycoprotein correlate with lower HBsAg levels in vivo, hinder HBsAg secretion in vitro and reduce HBsAg structural stability in the setting of HBeAg-negative chronic HBV genotype-D infection.

Increasing evidences suggest that HBsAg-production varies across HBV-genotypes. HBsAg C-terminus plays a crucial role for HBsAg-secretion. Here, we evaluate HBsAg-levels in different HBV-genotypes in HBeAg-negative chronic infection, the correlation of specific mutations in HBsAg C-terminus with HBsAg-levels in-vivo, their impact on HBsAg-secretion in-vitro and on structural stability in-silico.HBsAg-levels were investigated in 323 drug-naïve HBeAg-negative patients chronically infected with HBV genotype-D(N = 228), -A(N = 65) and -E(N = 30). Genotype-D was characterized by HBsAg-levels lower than genotype-A and -E (3.3[2.7-3.8]IU/ml; 3.8[3.5-4.2]IU/ml and 3.9[3.7-4.2]IU/ml, P < 0.001). Results confirmed by multivariable analysis correcting for patients'demographics, HBV-DNA, ALT and infection-status.In genotype-D, specific C-terminus mutations (V190A-S204N-Y206C-Y206F-S210N) significantly correlate with HBsAg<1000IU/ml(P-value from <0.001 to 0.04). These mutations lie in divergent pathways involving other HBsAg C-terminus mutations: V190A + F220L (Phi = 0.41, P = 0.003), S204N + L205P (Phi = 0.36, P = 0.005), Y206F + S210R (Phi = 0.47, P < 0.001) and S210N + F220L (Phi = 0.40, P = 0.006). Notably, patients with these mutational pairs present HBsAg-levels 1log lower than patients without them(P-value from 0.003 to 0.02). In-vitro, the above-mentioned mutational pairs determined a significant decrease in HBsAg secretion-efficiency compared to wt(P-value from <0.001 to 0.02). Structurally, these mutational pairs reduced HBsAg C-terminus stability and determined a rearrangement of this domain.In conclusion, HBsAg-levels in genotype-D are significantly lower than in genotype-A and -E in HBeAg-negative patients. In genotype-D, specific mutational clusters in HBsAg C-terminus correlate with lower HBsAg-levels in-vivo, hamper HBsAg-release in-vitro and affect its structural stability, supporting their detrimental role on HBsAg-secretion. In this light, genotypic-testing can be a valuable tool to optimize the clinical interpretation of HBsAg in genotype-D and to provide information on HBV-pathogenicity and disease-progression.

HDV Can Constrain HBV Genetic Evolution in HBsAg: Implications for the Identification of Innovative Pharmacological Targets.

Chronic HBV + HDV infection is associated with greater risk of liver fibrosis, earlier hepatic decompensation, and liver cirrhosis hepatocellular carcinoma compared to HBV mono-infection. However, to-date no direct anti-HDV drugs are available in clinical practice. Here, we identified conserved and variable regions in HBsAg and HDAg domains in HBV + HDV infection, a critical finding for the design of innovative therapeutic agents. The extent of amino-acid variability was measured by Shannon-Entropy (Sn) in HBsAg genotype-d sequences from 31 HBV + HDV infected and 62 HBV mono-infected patients (comparable for demographics and virological-parameters), and in 47 HDAg genotype-1 sequences. Positions with Sn = 0 were defined as conserved. The percentage of conserved HBsAg-positions was significantly higher in HBV + HDV infection than HBV mono-infection (p = 0.001). Results were confirmed after stratification for HBeAg-status and patients' age. A Sn = 0 at specific positions in the C-terminus HBsAg were correlated with higher HDV-RNA, suggesting that conservation of these positions can preserve HDV-fitness. Conversely, HDAg was characterized by a lower percentage of conserved-residues than HBsAg (p < 0.001), indicating higher functional plasticity. Furthermore, specific HDAg-mutations were significantly correlated with higher HDV-RNA, suggesting a role in conferring HDV replicative-advantage. Among HDAg-domains, only the virus-assembly signal exhibited a high genetic conservation (75% of conserved-residues). In conclusion, HDV can constrain HBsAg genetic evolution to preserve its fitness. The identification of conserved regions in HDAg poses the basis for designing innovative targets against HDV-infection.

Prevalence of Single and Multiple Natural NS3, NS5A and NS5B Resistance-Associated Substitutions in Hepatitis C Virus Genotypes 1-4 in Italy.

Natural resistance-associated substitutions (RASs) are reported with highly variable prevalence across different HCV genotypes (GTs). Frequency of natural RASs in a large Italian real-life cohort of patients infected with the 4 main HCV-GTs was investigated. NS3, NS5A and NS5B sequences were analysed in 1445 HCV-infected DAA-naïve patients. Sanger-sequencing was performed by home-made protocols on 464 GT1a, 585 GT1b, 92 GT2c, 199 GT3a, 16 GT4a and 99 GT4d samples. Overall, 20.7% (301/1455) of patients showed natural RASs, and the prevalence of multiclass-resistance was 7.3% (29/372 patients analysed). NS3-RASs were particularly common in GT1a and GT1b (45.2-10.8%, respectively), mainly due to 80K presence in GT1a (17%). Almost all GTs showed high prevalence of NS5A-RASs (range: 10.2-45.4%), and especially of 93H (5.1%). NS5A-RASs with fold-change >100x were detected in 6.8% GT1a (30H/R-31M-93C/H), 10.3% GT1b (31V-93H), 28.4% GT2c (28C-31M-93H), 8.5% GT3a (30K-93H), 45.5% GT4a (28M-30R-93H) and 3.8% GT4d (28V-30S-93H). Sofosbuvir RAS 282T was never detected, while the 159F and 316N RASs were found in GT1b (13.4-19.1%, respectively). Natural RASs are common in Italian patients infected with HCV-GTs 1-4. High prevalence of clinically-relevant RASs (such as Y93H) supports the appropriateness of HCV resistance-test to properly guide DAA-based therapy.

Novel HBsAg mutations correlate with hepatocellular carcinoma, hamper HBsAg secretion and promote cell proliferation in vitro.

BACKGROUND: An impaired HBsAg-secretion can increase HBV oncogenic-properties. Here, we investigate genetic-determinants in HBsAg correlated with HBV-induced hepatocellular carcinoma (HCC), and their impact on HBsAg-secretion and cell-proliferation. METHODS: This study included 128 chronically HBV-infected patients: 23 with HCC (73.9% D; 26.1% A HBV-genotype), and 105 without cirrhosis/HCC (72.4% D, 27.6% A) as reference-group. The impact of mutations on HBsAg-secretion was assessed by measuring the ratio [secreted/intracellular HBsAg] until day 5 post-transfection. The impact of mutations on cell-cycle advancement was assessed by flow-cytometry. RESULTS: Two HBsAg mutations significantly correlated with HCC: P203Q (17.4% [4/23] in HCC vs 1.0% [1/105] in non-HCC, P=0.004); S210R (34.8% [8/23] in HCC vs 3.8% [4/105] in non-HCC, P <0.001); P203Q+S210R (17.4% [4/23] in HCC vs 0% [0/110] in non-HCC, P=0.001). Both mutations reside in trans-membrane C-terminal domain critical for HBsAg-secretion. In in-vitro experiments, P203Q, S210R and P203Q+S210R significantly reduced the ratio [secreted/intracellular HBsAg] compared to wt at each time-point analysed (P <0.05), supporting an impaired HBsAg-secretion. Furthermore, P203Q and P203Q+S210R increased the percentage of cells in S-phase compared to wt, indicating cell-cycle progression (P203Q:26±13%; P203Q+S210R:29±14%; wt:18%±9, P <0.01. Additionally, S210R increased the percentage of cells in G2/M-phase (26±8% for wt versus 33±6% for S210R, P <0.001). CONCLUSIONS: Specific mutations in HBsAg C-terminus significantly correlate with HBV-induced HCC. They hamper HBsAg-secretion and are associated with increased cellular proliferation, supporting their involvement in HCC-development. The identification of viral genetic markers associated with HCC is critical to identify patients at higher HCC-risk that may deserve intensive liver monitoring, and/or early anti-HBV therapy.

Multidisciplinary management of hepatocellular carcinoma in clinical practice.

BACKGROUND: Hepatocellular carcinoma (HCC) patients require different treatment strategies according to disease extension, liver function, and patient's fitness. We evaluated HCC multidisciplinary management in clinical practice. METHODS: Consecutive patients were followed and treated with tailored medical, locoregional, and surgical treatments, according to disease stage and patient's fitness (age, Cumulative Illness Rating Scale (CIRS)). Activity, efficacy, and safety were evaluated. RESULTS: Thirty-eight patients were evaluated: median age, 74; elderly 92%; CIRS secondary 28 (74%); Child-Pugh A 20 (53%), B 11 (29%); and Barcelona Clinic Liver Cancer (BCLC) 0 2 (5%), A 9 (24%), B 10 (26%), C 13 (34%), and D 4 (11%). Overall survival (OS) was 30 months. At 9 months median follow-up, among 25 unresectable HCC, OS was 10 months; BCLC B-D unfit for sorafenib showed OS 3 months. Ten patients (40%) received sorafenib: Child-Pugh A 5 (50%) and B 5 (50%) and disease control rate 89%, progression-free survival 7 months, and OS 9 months. G3-4 toxicities: anorexia, hypertransaminaemia, hyperbilirubinemia, and hypercreatininemia. Limiting toxicity syndromes were 40%, all multiple sites. CONCLUSION: HCC patients require multidisciplinary clinical management to properly select tailored treatments according to disease stage, fitness, and liver function. Patients suitable for sorafenib should be carefully selected, monitored for individual safety, and prevalently characterized by limiting toxicity syndromes multiple sites.