Introduction:Elevated inflammation and immune dysregulation are the main consequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The dysregulated inflammatory state persists after coronavirus disease 2019 (COVID-19), establishing the post-acute sequelae of SARS-CoV-2 infection in individuals with long COVID (LC). The role of CD169+ monocytes in the early diagnosis of SARS-CoV-2 infection and their association with severe outcomes were demonstrated in COVID-19 patients (COV). We aimed to delineate specific myeloid activation that characterizes the acute and post-acute phases of SARS-CoV-2 infection, evaluating the correlation between cellular and extracellular vesicles (EVs). Methods:Blood samples from COV, LC, and healthy donors (HD) were collected at Tor Vergata University Hospital in Rome. Plasmatic EVs were isolated by differential centrifugation and evaluated by flow cytometry and atomic force microscopy (AFM). Leukocyte subpopulations and different sizes of circulating EVs (100-200, 240-500, >500 nm) were characterized for HLA-DR and CD169 expression in COV, LC, and HD through flow cytometry. Serum inflammatory markers were assessed by the ELLA immunoassay system. The analyzed markers were associated with clinical and biochemical parameters in COV and LC. Results:The analysis of HLA-DR+, CD169+, and HLA-DR+CD169+ leukocytes confirmed our previous results in which the activated monocytes CD169+HLA-DR+ were found significantly high in COV, persisting in LC, and correlated differently with coagulation markers and inflammatory cytokines. Similar to cellular levels, the percentage and number of HLA-DR+CD169+ EVs were significantly elevated in COV and persisted in LC compared to HD. Different HLA-DR and CD169 expressions were found according to EV size in COV, LC, and HD, and correlations with biochemical parameters and circulating inflammatory markers were found. A positive correlation of HLA-DR and CD169 expression among monocytes and circulating EVs was found, supporting a possible connection between the two compartments and circulating inflammatory mediators. Moreover, the characterization by flow cytometry of EV cell derivation and cytokine cargo revealed EVs as sensitive indicators of both acute and persistent immune perturbations, bridging viral antigen persistence with inflammatory signaling in long COVID. Conclusion:Myeloid activation markers and inflammatory cytokines are dynamically expressed between blood cells and circulating extracellular vesicles, underlining multilevel cell-to-cell communications, opening new possibilities to monitor COVID-19 and long COVID-associated sequelae.

Lower respiratory tract (LRT) infections represent a major cause of mortality, particularly among critically ill patients. Molecular diagnostic tests have improved the detection of respiratory pathogens; however, most commercial assays are validated exclusively in upper RT (URT) specimens, limiting their applicability in LRT samples, which better reflect disease severity. This study evaluated the diagnostic performance of two commercially available assays, the BioFire Respiratory Panel 2.1 Plus and the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay, on bronchoalveolar lavage (BAL) specimens, using the Allplex Respiratory Panel 1/2/3 and the Allplex SARS-CoV-2 assay as reference methods validated for both matrices. Overall, 132 BAL samples were analyzed. BioFire identified more positives than Allplex, particularly for human rhinovirus/enterovirus (HRV/EV), human parainfluenza virus (HPIV), and non-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The overall agreement was fair (κ = 0.237), and pathogen-specific concordance was almost perfect for SARS-CoV-2 (κ = 0.841), influenza A/B (κ = 0.808), and HPIV (κ = 0.884). The Panther assay showed substantial agreement with Allplex (κ = 0.719) and near-perfect concordance for SARS-CoV-2 and influenza viruses, while BioFire and Panther exhibited almost perfect interassay agreement (κ = 0.903). These findings demonstrate that assays validated for URT specimens can perform reliably on BAL samples, underlining the diagnostic potential of LRT matrices and the need for expanded validation of molecular respiratory panels across specimen types.

Background: Limited information is available on the extent of HDV genetic diversification in ribozyme (critical for HDV replication) and HDAg domains (crucial for viral morphogenesis and containing cytotoxic T lymphocytes epitopes [CTLE]) and their correlation with virological and biochemical parameters.Methods: 103 individuals with chronic HDV infection were included. Full-length HDV genome sequences were obtained by Illumina (median [IQR] reads/seq: 62045[30460-91899]). Sub-genotypes 1 were defined by phylogenetic analysis. Amino acid (aa) residues were defined conserved if not mutated in 99% of sequences. HDAg domains and CTLE (N=18) were defined according to Pascarella 2010 and Kohsar 2021.Results:Individuals were mostly males with a median age of 54 (44-60) years, mainly from Eastern Europe (EE, 51%) and Italy (IT, 44.8%). Serum HDV-RNA and ALT were 5.6 (4.9-6.2)logIU/ml and 94 (65-152)U/L. Sub-genotypes 1c and 1e were the most prevalent (47.1% and 45.2%): 1c predominated in individuals from EE(77.6% vs 22.4%, P<0.001) while 1e in IT(77.5% vs 22.5%, P<0.001).HDV ribozyme was characterised by a high degree of genetic conservation. An opposite scenario was observed for HDAg in which the number of conserved aa in HDAg dropped to 36.7% (79/214), with 18.2% in coiled coil sequence (CCS), 27.3% RNA binding domain (RDB)2, 30.8% in RBD1, 33.3% in nuclear localization sequence (NLS), 40% in virus assembly signal (VAS) and 63.3% in RBD3. The degree of genetic conservation of CTLEs ranges from 11.1% in CTLEs 46-54 and 43-51 to 60.0% in 140-149.Notably, CTLE 170-179 from individuals with HDV-RNA >5logIU/ml (70% of conserved aa with vs 20% without HDV-RNA <5 logIU/ml, P=0.025) showed lower genetic conservation, suggesting that an enrichment of mutations in this CTLE can enhance viral replication.Finally, despite a comparable degree of genetic conservation between sub-genotypes 1e and 1c, they were characterized by divergent genetic pathways. In particular, sub-genotype 1c was significantly associated with the selection of 7 specific mutations (I16T/V, N22S, D47E, R112K, T180A, A202S, prevalence ranging from 26.5% to 44.9% vs 0% in 1e, P<0.001). Conversely, sub-genotype 1e was significantly associated with the selection of 6 specific mutations (D29E, D46E, K113R, R131K, M171L, I188V prevalence ranging from 23.9% to 43.5% vs 0% in 1c, P<0.009). Notably, in sub-genotype 1c, the co-presence of I16V/T+D47E+A202S correlated with ALT>3ULN (100% vs 27.5%, P=0.001)Conclusion: Sub-genotypes 1 are characterized by a conspicuous degree of genetic diversification in HDAg that has contributed to the selection of divergent genetic signatures. The enrichment of mutations in specific CTL epitopes could potentially hamper HDV recognition by immune response and in turn enhancing viral replication.Overall, the role of the high degree of genetic variability in affecting the proper HDV detection by the currently available diagnostic assays deserves further investigation.

Human T-Lymphotropic virus type 1 (HTLV-1) lifelong infects at least 5-10 million people worldwide, a minority of whom develop severe lethal diseases including adult T-cell Leukemia/lymphoma and HTLV-1-associated myelopathy or tropical spastic paraparesis. Currently, no vaccines or curative therapies to fight HTLV-1 infection or diseases are available. Recently we found that a tributyltin molecule, Bu3SnOCOCF3 (TBT), which is more potent than cisplatin in inducing cytotoxic effects towards a panel of cell lines including high-tumorigenic cells, also exerted potent cytotoxic effects even towards HTLV-1-infected cell lines, mimicking different states of virus-driven transformation. The type of cell death involved was elusive. In the present study, the effects of TBT on virological and cell death parameters were investigated in HTLV-1-infected immortalized lymphocytes generated by in vitro infection and rendered with or without progressive independence from interleukin-2 as a growth factor. Molecular studies demonstrated that TBT affected HTLV-1 viral gene expression, especially HBZ. TBT confirmed its high cytotoxic potential on the HTLV-1-infected cell lines assayed, especially towards the IL-2-independent HTLV-1-infected cells. Investigation of mechanisms involved in cell death induced by TBT in HTLV-1-infected cells confirmed that caspase 3 and 8 activation, as well as apoptotic response, were relevant. In addition, pyroptosis as well as other unspecifed forms of lytic death presumably contribute to cell death induced by TBT in HTLV-1-infected cells, while a concomitant activation of an autophagic response by this compound seems to mitigate it. Overall, these experimental results outline a particular profile of TBT-induced cell death in HTLV-1-infected cells that is useful for future studies aimed at verifying the real potential of tin-based compounds to contrast diseases caused by HTLV-1.

Background & Aim: The entry of Hepatitis D Virus (HDV) depends on HBV surface proteins (HBsAg) composed of three isoforms: large-, middle, and small HBsAg. Here, we investigate the levels of total HBsAg and HBsAg isoforms and their correlations with HDV-RNA, HBcrAg, and transaminases in the setting of untreated chronic hepatitis D (CHD). Methods: This study includes 316 HBeAg-negative patients: 192 CHD and 124 with chronic hepatitis B (CHB) as a control group. HBsAg isoforms were quantified by ad hoc-designed ELISAs. Results: The composition of HBsAg isoforms varied between the two groups of patients, with remarkably higher small HBsAg, middle-HBsAg, and large HBsAg in CHD than in CHB. This data was confirmed by multivariable analysis (p < 0.0001). Among CHD, HBsAg isoforms positively correlated with HDV-RNA (p < 0.0001) and HBcrAg (p < 0.0001) but not with HBV-DNA. The results were confirmed by stratifying patients according to HDV-RNA (< or >1000 IU/mL) and HBcrAg (< or >3 logU/mL). Furthermore, CHD patients with ALT > upper limit of normal presented significantly higher S-HBsAg and M-HBsAg levels. Conclusions: CHD is characterized by a more elevated HBsAg isoform production, paralleling HDV-RNA and HBcrAg release. This may suggest a preferential recruitment of HBsAg isoforms in HDV virions at the expense of HBV virions. The association of HBsAg isoforms with higher ALT also suggests their potential contribution in supporting HDV-induced pro-inflammatory stimuli.

OBJECTIVES:To describe the clinical, radiological, and microbiological characteristics of community-acquired pneumonia (CAP) managed outside the ICU and evaluate the impact of rapid microbiological diagnostics on hospital outcomes in a multicenter Italian cohort. METHODS:In this prospective observational study conducted across seven Italian centers (October 2024-October 2025), 176 adults hospitalized with CAP outside the ICU and a PaO₂/FiO₂ ratio <300 were enrolled. Demographic, clinical, laboratory, radiological, microbiological, therapeutic, and outcome data were collected. Syndromic respiratory panels were performed in a subset of patients. Secondary outcomes included factors associated with in-hospital mortality and time to live discharge within 28 days. RESULTS:Patients were predominantly elderly (mean age 69.7 ± 14.4 years) with high comorbidity burden (Charlson Comorbidity Index 4.9 ± 2.1). Dyspnea, fever, and cough were the most frequent symptoms. Microbiological testing was performed in 48% of cases; viral pathogens were detected more often than bacteria (42.3% vs 27.0%), with viral-bacterial co-detection in 14.1%. In-hospital mortality was 2%, and 3% required ICU transfer. Rapid microbiological testing was independently associated with earlier discharge (HR: 1.67, 95% CI: 1.17-2.38; P = 0.005), while chronic corticosteroid therapy predicted longer hospitalization. CONCLUSIONS:CAP outside the ICU mainly affects elderly multimorbid patients. Rapid molecular diagnostics may facilitate earlier discharge by supporting clinical decision-making.

Among the metal-derived complexes, recently, tin derivatives have been investigated as promising anti-cancer drug candidates. Our previous study showed that the tin-based compound Bu3SnOCOCF3 (TBT) exerts cytotoxic activity on solid tumor cell lines. In the present study, the effects of TBT were evaluated in vitro on HTLV-1-infected human lymphocytic cell lines at different stages of viral transformation, consisting of IL-2-dependent (PB2/IL-2) and IL-2-independent (PB2/NO-IL-2) cells, generated in our laboratory by HTLV-1 in vitro infection of lymphocytes from the same donor, and the C91/PL cell line established by co-cultivation with T cells from a patient with HTLV-1-positive leukemia. TBT induced a reliable and reproducible dose-dependent inhibition of metabolic activity and viability in the HTLV-1-infected cells. The effect was cell-type-dependent, with C91/PL cells being quite resistant. An investigation into the cytotoxic effects induced by TBT in HTLV-1-infected cells and data on caspase inhibitors/caspase activation indicated that apoptotic cell death was involved, but also that the possible involvement of other forms of cell death could not be excluded. Taken together, the results show for the first time that the tin-based compound, although not devoid of a certain cytotoxicity toward uninfected cells, can induce typical and potent effects on HTLV-1-infected cells.

In the context of long-term therapy in virologically suppressed people living with HIV-1 (PLWH), the identification of new biomarkers associated with immuno-virological discordance, and the risk of disease progression is needed. Herein we investigated HERVs expression in association with immuno-virological discordance parameters for the identification of novel markers for the clinical monitoring of virologically suppressed PLWH. It is known the human endogenous retroviruses (HERVs), relics of ancestral exogenous retroviral infections comprising 8% of human genome, could be reactivated by exogenous viruses including HIV-1. The study included 31 virologically suppressed PLWH and 10 healthy donors; blood HIV-DNA levels and residual plasma viremia were quantified by droplet digital-PCR, the expression of HERVs by RT-Real time PCR, and immunophenotyping by flow cytometry. The results revealed a dynamic association of HERVs with several virological and immunological parameters such as the HIV-1 reservoir, CD4 cell count, CD4 nadir and with CD8+ and CD19 lymphocyte activation. In an era of searching innovative biomarkers for people living with HIV-1, the interconnection of HERVs with the HIV-1 reservoir and lymphocyte activation opens to further investigation on HERVs role in persistent immune activation in virologically suppressed PLWH, proposing them as potential new markers for clinical monitoring.

The human T-cell leukemia virus type 1 (HTLV-1) was first described in 1980. It is spread in highly endemic regions in the world, such as the Southwestern part of Japan, sub-Saharan Africa and South America, Caribbean, Middle East, and Australo-Melanesia regions. HTLV-1 causes adult T cell leukemia and is associated with many inflammatory conditions, most notably HTLV-1-associated myelopathy/tropic spastic paraparesis. HTLV-2, first isolated in 1982, was recognized as a common infection in intravenous drug users, but a clear association with disease remains elusive. The first estimate of HTLV-1-positive individuals worldwide, in 1993, was around 10-20 millions. Due to the lack of global population-based prevalence studies, this is considered an underestimate at the moment. Furthermore, HTLV-1 prevalence in Europe is impacted by changing migration flows. Particularly, no data on HTLV-1 prevalence in the general population in Italy are available. Here, we report a systematic literature review of studies conducted in Italy on HTLV-1/2 from 1980 to 2023. Based on the criteria we adopted a total of 426 publications were found (64 reviews, 99 epidemiological, and 263 translational studies). The contents of some representative publications are summarized and discussed. Moreover, an approximate estimation of about 26,000 HTLV-1 positive foreigners living in Italy was obtained from updated data of foreigners from each single country officially registered as resident in Italy and from data on HTLV-1 prevalence among the general population in the corresponding countries.

Introduction Human Immunodeficiency virus (HIV) is effectively suppressed in the blood by the Antiretroviral Therapy in people living with HIV, but in rare cases can be present in some tissues and body fluids. In recent years, integrated systems were validated for detecting HIV-1 in plasma or serum. but not in cerebrospinal fluid (CSF). We evaluated the performance of ELITE InGenius® in comparison with the cobas® in this area. Methods To test the diagnostic accuracy of the HIV-1 ELITe MGB® kit on CSF samples, we tested CSF samples previously characterised with the cobras® HIV1 test. Archived CSF samples were also spiked with serial dilutions of the 4th WHO International Standard for HIV-1 NAT and assays and tested to assess the repeatability and reproducibility of the ELITechGroup assay. Results The HIV-1 ELITe MGB® Kit confirmed all the HIV-1 negative CSF samples from patients HIV positive in plasma and from non-HIV1 patients. All the CSF samples that were HIV-1 positive by the cobas®, were confirmed positive by the ELITe InGenius®. Concordance across the methods was also observed when processing the CSF dilutions spiked at medium-low titre, mimicking HIV-1 low-load infections. Conclusions The two systems were equivalent in the detection and quantification of HIV-1 RNA in CSF samples.

The use of CD169 as a marker of viral infection has been widely discussed in the context of COVID-19, and in particular, its crucial role in the early detection of SARS-CoV-2 infection and its association with the severity and clinical outcome of COVID-19 were demonstrated. COVID-19 patients show relevant systemic alteration and immunological dysfunction that persists in individuals with post-acute sequelae of SARS-CoV-2 infection (PASC). It is critical to implement the characterization of the disease, focusing also on the possible impact of the different COVID-19 waves and the consequent effects found after infection. On this basis, we evaluated by flow cytometry the expression of CD169 and HLA-DR on monocytes from COVID-19 patients and PASC individuals to better elucidate their involvement in immunological dysfunction, also evaluating the possible impact of different pandemic waves. The results confirm CD169 RMFI is a good marker of viral infection. Moreover, COVID-19 patients and PASC individuals showed high percentage of CD169+ monocytes, but low percentage of HLA-DR+ monocytes and the alteration of systemic inflammatory indices. We have also observed alterations of CD169 and HLA-DR expression and indices of inflammation upon different COVID-19 waves. The persistence of specific myeloid subpopulations suggests a role of CD169+ monocytes and HLA-DR in COVID-19 disease and chronic post-infection inflammation, opening new opportunities to evaluate the impact of specific pandemic waves on the immune response impairment and systemic alterations with the perspective to provide new tools to monitoring new variants and diseases associated to emerging respiratory viruses.

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.

Infectious diseases such as HIV and HBV are a global concern for their impact in terms of public health and costs for national health services. A central role in contrasting the spread of the infections is represented by timely diagnosis. The speed of detection depends on several factors including the type of test used. Antibody response to hepatitis B surface antigens (anti-HBs) is an important serological marker used for HBV-infection detection. The aim of this study was to compare the performance of the Abbott system and of the new analyser Mindray 1200i in the detection of HBV- and HIV-infections. Clinical serum samples were collected from patients randomly selected from PTV University Hospital of University of Rome "Tor Vergata" and tested for HBV and HIV antibodies. Samples were evaluated by Mindray Cl 1200i CLIA screening tests for HBV and HIV and the results were compared with the Abbott Architect analytical system, the routine instrument of the hospital clinical biochemistry laboratory. Precision study, linearity, and carryover were performed on the results obtained. The agreement between the results of the Abbott and Mindray CLIA ranged from 99% to 100% and the discrepancy rate from 0% to 1%. The measurements demonstrated that the Mindray CL-1200i platform offers high-level performance with accurate and consistent test results and could represent a valuable tool if implemented in routine analysis.

The complex alterations of the immune system and the immune-mediated multiorgan injury plays a key role in host response to SARS-CoV-2 infection and in the pathogenesis of COVID-19, being also associated with adverse outcomes. Thymosin alpha 1 (Tα1) is one of the molecules used in the treatment of COVID-19, as it is known to restore the homeostasis of the immune system during infections and cancer. The use of Tα1 in COVID-19 patients had been widely used in China and in COVID-19 patients, it has been shown to decrease hospitalization rate, especially in those with greater disease severity, and reduce mortality by restoring lymphocytopenia and more specifically, depleted T cells. Persistent dysregulation with depletion of naive B and T cell subpopulations and expansion of memory T cells suggest a chronic stimulation of the immune response in individuals with post-acute sequelae of SARS-CoV-2 infection (PASC). Our data obtained from an ex vivo study, showed that in PASC individuals with a chronically altered immune response, Tα1 improve the restoration of an appropriate response, most evident in those with more severe illness and who need respiratory support during acute phase, and in those with specific systemic and psychiatric symptoms of PASC, confirming Tα1 treatment being more effective in compromised patients. The results obtained, along with promising reports on recent trials on Tα1 administration in patients with COVID-19, offer new insights into intervention also for those patients with long-lasting inflammation with post-infectious symptoms, some of which have a delayed onset.