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Abstract Introduction Mild traumatic brain injury (mTBI) represents a major public health concern and affects millions of people worldwide every year. Diagnosis mainly relies on clinical criteria and computed tomography (CT) scans. GFAP (glial fibrillary acidic protein) and UCH-L1 (ubiquitin carboxyl-terminal hydrolase-L1) have been recently studied as potential biomarkers of mTBI. This study retrospectively evaluated the possible use of these combined biomarkers as negative predictors for excluding brain injuries in patients with suspected mTBI in the emergency department. Methods Adult patients (n = 130) enrolled at Tor Vergata University Hospital (Rome, Italy), consecutively registered at the triage of the emergency department between October 2022 and January 2023, with non-penetrating TBI and Glasgow Coma Scale (GCS) score of 13–15, were considered. All eligible patients underwent intracranial CT scans and blood tests, within 12 h after trauma, for GFAP and UCH-L1 serum concentrations. Results Intracranial CT detected injuries in only seven patients (5%); GFAP and UCH-L1 tested positive in 96 patients and negative in 34 patients (74% vs. 26%). Combined biomarkers had a sensitivity equal to 1.00 (95% CI 0.64-1.00) and a negative predictive value (NPV) of 1.00 (0.99-1.00) in mTBI diagnosis with a negative CT. Conclusions Combined laboratory tests for GFAP and UCH-L1 biomarkers might play a potential clinical role in avoiding unnecessary head CT scans after mTBI in emergency departments.

The emergence of COVID-19 has evolved into a global pandemic, causing an unprecedented public health crisis marked by unprecedented levels of morbidity never seen in the recent past. Considerable research efforts have been made in the scientific community to establish an optimal method to identify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and to understand the induced immune response. This review examined the development of serological tests during the COVID-19 pandemic, considering the factors affecting sensitivity and specificity, which are key to promote an efficient vaccination strategy for public health. The market has witnessed the introduction of various serological tests for the detection of SARS-CoV-2, such as the chemiluminescence immunoassay (CLIA), which emerged as a powerful and rapid tool to monitor the antibody response before and after vaccination or infection. Therefore, developing serological tests by studying antibody trends and persistence is essential for creating long-term strategies. Our analysis underscores the multifaceted applications of serological tests in pandemic management with a focus on the critical insights they provide into antibody dynamics that help in managing the ongoing pandemic and shaping future public health initiatives, providing a basis for optimizing the future response to viral threats.

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 (

Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, has had a significant impact on global health, with severe cases often characterized by a worsening cytokine storm. Since it has been described that the NF-κB signaling pathway, regulated by microRNAs, could play a pivotal role in the inflammatory response, in this study, the role of miR-9 in modulating NF-κB signaling and inflammatory cytokine expression in COVID-19 patients was investigated. This observational retrospective single-center study included 41 COVID-19 patients and 20 healthy controls. Serum samples were analyzed for miR-9, NF-κB, and IκBα expression levels using RT-PCR. The expression levels and production of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α were measured using RT-PCR and ELISA. Statistical analyses, including correlation and regression, were conducted to explore relationships between these variables. COVID-19 patients, particularly non-survivors, exhibited significantly higher miR-9 and NF-κB levels compared to controls. A strong positive correlation was found between miR-9 and NF-κB expression (r = 0.813, p < 0.001). NF-κB levels were significantly correlated with IL-6 (r = 0.971, p < 0.001), IL-1β (r = 0.968, p < 0.001), and TNF-α (r = 0.968, p < 0.001). Our findings indicate that miR-9 regulates NF-κB signaling and inflammation in COVID-19. Elevated miR-9 levels in non-survivors suggest its potential as a severity biomarker. While COVID-19 cases have decreased, targeting miR-9 and NF-κB could improve outcomes for other inflammatory conditions, including autoimmune diseases, highlighting the need for continued research in this area.

The studies on the development of fusion–fission hybrid reactors (FFHR) have gained consensus in recent years as an intermediate step before fusion energy. This work proposes a possible approach to FFHRs based on the coupling of a Reversed Field Pinch fusion machine and a Molten Salt Subcritical fission test bed. The proposed test bed is characterized by the coexistence of a fast-neutron fission core and a dedicated thermal-neutron zone, allowing the performing of tritium breeding and actinides transmutation studies. The neutronic design solutions and the results obtained by the irradiation of FLiBe salt (inside the thermal-neutron zone) and of an actinide target (inside the core) are shown. The outcomes of the analysis reveal the potential of FFHR systems as breeding/burner systems. In particular, the results regarding tritium breeding are very encouraging as the system is demonstrated to be able to reach a very high Tritium Breeding Ratio.

The use of vaccines has allowed the containment of coronavirus disease 2019 (COVID-19) at a global level. The present work aims to add data on vaccination by evaluating the level of neutralizing antibodies in individuals who have received a three-vaccination series. For this purpose, we ran a surveillance program directed at measuring the level of IgG Abs against the Receptor Binding Domain (RBD) and surrogate virus neutralizing Ab (sVNT) anti-SARS-CoV-2 in the serum of individuals undergoing vaccination. This study was performed on employees from the University of Rome Tor Vergata and healthcare workers from the University Hospital who received the Vaxzevria vaccine (n = 56) and Comirnaty vaccine (n = 113), respectively. After the second dose, an increase in both RBD and sVNT Ab values was registered. In individuals who received the Comirnaty vaccine, the antibody titer was about one order of magnitude higher after 6 months from the first dose. All participants in this study received the Comirnaty vaccine as the third dose, which boosted the antibody response. Five months after the third dose, nearly one year from the first injection, the antibody level was >1000 BAU/mL (binding antibody units/mL). According to the values reported in the literature conferring protection against SARS-CoV-2 infection, our data indicate that individuals undergoing three vaccine doses present a low risk of infection.

Background: It has been reported that mid-regional proadrenomedullin (MR-proADM) could be considered a useful tool to stratify the mortality risk in COVID-19 patients upon admission to the emergency department (ED). During the COVID-19 outbreak, computed tomography (CT) scans were widely used for their excellent sensitivity in diagnosing pneumonia associated with SARS-CoV-2 infection. However, the possible role of CT score in the risk stratification of COVID-19 patients upon admission to the ED is still unclear. Aim: The main objective of this study was to assess if the association of the CT findings alone or together with MR-proADM results could ameliorate the prediction of in-hospital mortality of COVID-19 patients at the triage. Moreover, the hypothesis that CT score and MR-proADM levels together could play a key role in predicting the correct clinical setting for these patients was also evaluated. Methods: Epidemiological, demographic, clinical, laboratory, and outcome data were assessed and analyzed from 265 consecutive patients admitted to the triage of the ED with a SARS-CoV-2 infection. Results and conclusions: The accuracy results by AUROC analysis and statistical analysis demonstrated that CT score is particularly effective, when utilized together with the MR-proADM level, in the risk stratification of COVID-19 patients admitted to the ED, thus helping the decision-making process of emergency physicians and optimizing the hospital resources.

Merkel cell polyomavirus (MCPyV) is the etiological agent of the majority of Merkel cell carcinoma (MCC): a rare skin tumor. To improve our understanding of the role of MCPyV in MCCs, the detection and analysis of MCPyV DNA and transcripts were performed on primary tumors and regional lymph nodes from two MCC patients: one metastatic and one non-metastatic. MCPyV-DNA was searched by a quantitative polymerase chain reaction (qPCR), followed by the amplification of a Large T Antigen (LTAg), Viral Protein 1 (VP1) and Non-Coding Control Region (NCCR). LTAg and VP1 transcripts were investigated by reverse-transcription PCR (RT-PCR). Viral integration was also studied, and full-length LTAg sequencing was performed. qPCR revealed that the primary tumor of both patients and the lymph node of one patient was positive for the small t-antigen, with an average value of 7.0 × 102 copies/µg. The same samples harbored LTAg, NCCR and VP1 DNA. Sequencing results showed truncated LTAg with the conserved retinoblastoma (Rb) protein binding motif and VP1 and NCCR sequences identical to the MCC350 strain. RT-PCR detected LTAg but not VP1 transcripts. The MCPyV genome was integrated into the primary tumor of both patients. The results confirmed the connection between MCPyV and MCC, assuming integration, LTAg truncation and Rb sequestration as key players in MCPyV-mediated oncogenesis.

The burden, microbial etiology and clinical impact of hospital-acquired respiratory infections (HARIs) were determined at an Italian teaching hospital over a 12-month period. For this purpose, overall ordinary hospitalizations ≥ 2 days of subjects over 18 years old with discharge from 1 January 2018 to 31 December 2018 were examined by cross-referencing demographic and clinical data from hospital discharge forms with microbiological data from the computer system of the Microbiology Unit. We identified 329 individuals with HARIs (96 females and 233 males; median age 70 years, range 18–93), who represented ¼ of the total hospital-acquired infections (HAIs) in the period. The inpatient setting was medical and surgical in similar proportions (169 vs. 160, respectively) and the mean hospital stay was 38.9 ± 33.6 days. One hundred and forty patients (42.6% of the total sample) were suffering from one or more chronic diseases. A total of 581 microorganisms (82 antibiotic-resistant and 499 non-resistant) were detected in HARI patients. The most common isolated species were Staphylococcus aureus (16.7%), Klebsiella pneumoniae (13.3%), Pseudomonas spp. (12.6%) and Acinetobacter baumannii (10.5%), followed by Enterobacter spp. (5.3%), Escherichia coli (5.2%) and Enterococcus spp. (4.8%). One hundred and sixty-seven individuals (49.0% of the total) had polymicrobial infections. One hundred thirty-one patients (39.8% of the total) underwent endotracheal intubation and mechanical ventilation and 62.6% of them died, compared to 17.7% of the non-intubated patients. Multivariable analysis confirmed a positive correlation between death and increased age (p = 0.05), surgical MDC (p = 0.007), number of microorganisms over the sample mean (p = 0.001), the presence of chronic diseases (p = 0.046), and intubation and mechanical ventilation (p < 0.0001). A positive correlation between intubation and antibiotic-resistant organisms (p = 0.003) was also found. HARIs are still a major public health problem and require constant surveillance due to their severe clinical outcome.

Since it was clearly established that HIV/AIDS predisposes to the infection, persistence or reactivation of latent viruses, the prevalence of human polyomaviruses (HPyVs) among HIV-1-infected patients and a possible correlation between HPyVs and HIV sero-status were investigated. PCR was performed to detect and quantify JCPyV, BKPyV, MCPyV, HPyV6, HPyV7 and QPyV DNA in the urine and plasma samples of 103 HIV-1-infected patients. Subsequently, NCCR, VP1 and MCPyV LT sequences were examined. In addition, for MCPyV, the expression of transcripts for the LT gene was investigated. JCPyV, BKPyV and MCPyV’s presence was reported, whereas HPyV6, HPyV7 and QPyV were not detected in any sample. Co-infection patterns of JCPyV, BKPyV and MCPyV were found. Archetype-like NCCRs were observed with some point mutations in plasma samples positive for JCPyV and BKPyV. The VP1 region was found to be highly conserved among these subjects. LT did not show mutations causing stop codons, and LT transcripts were expressed in MCPyV positive samples. A significant correlation between HPyVs’ detection and a low level of CD4+ was reported. In conclusion, HPyV6, HPyV7 and QPyV seem to not have a clinical relevance in HIV-1 patients, whereas further studies are warranted to define the clinical importance of JCPyV, BKPyV and MCPyV DNA detection in these subjects.

Coronavirus disease 2019 (COVID-19) presents a clinical spectrum that ranges from a mild condition to critical illness. Patients with critical illness present respiratory failure, septic shock and/or multi-organ failure induced by the so called “cytokine storm”. Inflammatory cytokines affect iron metabolism, mainly inducing the synthesis of hepcidin, a hormone peptide not routinely measured. High levels of hepcidin have been associated with the severity of COVID-19. The aim of this study was to analyze, retrospectively, the levels of hepcidin in a group of COVID-19 patients admitted to the intensive care unit (ICU) of the Policlinico Tor Vergata of Rome, Italy. Thirty-eight patients from November 2020 to May 2021 were enrolled in the study. Based on the clinical outcome, the patients were assigned to two groups: survivors and non-survivors. Moreover, a series of routine laboratory parameters were monitored during the stay of the patients in the ICU and their levels correlated to the outcome. Statistical differences in the level of hepcidin, D-dimer, IL-6, LDH, NLR, neutrophils level, CRP, TNF-α and transferrin were observed between the groups. In particular, hepcidin values showed significantly different median concentrations (88 ng/mL vs. 146 ng/mL) between survivors and non-survivors. In addition, ROC curves analysis revealed sensitivity and specificity values of 74% and 76%, respectively, at a cut-off of 127 (ng/mL), indicating hepcidin as a good biomarker in predicting the severity and mortality of COVID-19 in ICU patients.

Lactoferrin (Lf) is a cationic glycoprotein synthetized by exocrine glands and is present in all human secretions. It is also secreted by neutrophils in infection and inflammation sites. This glycoprotein possesses antimicrobial activity due to its capability to chelate two ferric ions per molecule, as well as to interact with bacterial and viral anionic surface components. The cationic features of Lf bind to cells, protecting the host from bacterial and viral injuries. Its anti-inflammatory activity is mediated by the ability to enter inside the nucleus of host cells, thus inhibiting the synthesis of proinflammatory cytokine genes. In particular, Lf down-regulates the synthesis of IL-6, which is involved in iron homeostasis disorders and leads to intracellular iron overload, favoring viral replication and infection. The well-known antiviral activity of Lf has been demonstrated against DNA, RNA, and enveloped and naked viruses and, therefore, Lf could be efficient in counteracting also SARS-CoV-2 infection. For this purpose, we performed in vitro assays, proving that Lf exerts an antiviral activity against SARS-COV-2 through direct attachment to both SARS-CoV-2 and cell surface components. This activity varied according to concentration (100/500 μg/ml), multiplicity of infection (0.1/0.01), and cell type (Vero E6/Caco-2 cells). Interestingly, the in silico results strongly supported the hypothesis of a direct recognition between Lf and the spike S glycoprotein, which can thus hinder viral entry into the cells. These in vitro observations led us to speculate a potential supplementary role of Lf in the management of COVID-19 patients.

In the past two pandemic years, Emergency Departments (ED) have been overrun with COVID-19-suspicious patients. Some data on the role played by laboratory biomarkers in the early risk stratification of COVID-19 patients have been recently published. The aim of this study is to assess the potential role of the new biomarker mid-regional proadrenomedullin (MR-proADM) in stratifying the in-hospital mortality risk of COVID-19 patients at the triage. A further goal of the present study is to evaluate whether MR-proADM together with other biochemical markers could play a key role in assessing the correct care level of these patients. Data from 321 consecutive patients admitted to the triage of the ED with a COVID-19 infection were analyzed. Epidemiological; demographic; clinical; laboratory; and outcome data were assessed. All the biomarkers analyzed showed an important role in predicting mortality. In particular, an increase of MR-proADM level at ED admission was independently associated with a threefold higher risk of IMV. MR-proADM showed greater ROC curves and AUC when compared to other laboratory biomarkers for the primary endpoint such as in-hospital mortality, except for CRP. This study shows that MR-proADM seems to be particularly effective for early predicting mortality and the need of ventilation in COVID-19 patients admitted to the ED.

Background: In the last two years, the SARS-CoV-2 pandemic has determined radical changes in human behaviors and lifestyles, with a drastic reduction in socialization due to physical distancing and self-isolation. These changes have also been reflected in the epidemiological patterns of common respiratory viruses. For this reason, early discrimination of respiratory viruses is important as new variants emerge. Methods: Nasopharyngeal swabs of 2554 patients, with clinically suspected Acute Respiratory Infections (ARIs) from October 2019 to November 2021, were collected to detect 1 or more of the 23 common respiratory pathogens, especially viruses, via BioFilmArray RP2.1plus, including SARS-CoV-2. Demographical characteristics and epidemiological analyses were performed as well as a laboratory features profile of positive patients. Results: An observational study on 2300 patients (254 patients were excluded because of missing data) including 1560 men and 760 women, median age of 64.5 years, was carried out. Considering the respiratory virus research request, most of the patients were admitted to the Emergency Medicine Department (41.2%, of patients), whereas 29.5% were admitted to the Infectious Diseases Department. The most frequently detected pathogens included SARS-CoV-2 (31.06%, 707/2300, from March 2020 to November 2021), InfA-B (1.86%, 43/2300), HCoV (2.17% 50/2300), and HSRV (1.65%, 38/2300). Interestingly, coinfection rates decreased dramatically in the SARS-CoV-2 pandemic period. The significative decrease in positive rate of SARS-CoV-2 was associated with the massive vaccination. Conclusion: This study represents a dynamic picture of the epidemiological curve of common respiratory viruses during the two years of pandemic, with a disregarded trend for additional viruses. Our results showed that SARS-CoV-2 had a preferential tropism for the respiratory tract without co-existing with other viruses. The possible causes were attributable either to the use of masks, social isolation, or to specific respiratory receptors mostly available for this virus, external and internal lifestyle factors, vaccination campaigns, and emergence of new SARS-CoV-2 variants.

To date, 14 human polyomaviruses (HPyVs) have been identified using high-throughput technologies. Among them, MCPyV, HPyV6, HPyV7 and TSPyV present a skin tropism, but a causal role in skin diseases has been established only for MCPyV as a causative agent of Merkel cell carcinoma (MCC) and TSPyV as an etiological agent of Trichodysplasia Spinulosa (TS). In the search for a possible role for cutaneous HPyVs in the development of skin malignant lesions, we investigated the prevalence of MCPyV, HPyV6, HPyV7 and TSPyV in actinic keratosis (AK), a premalignant skin lesion that has the potential to progress towards a squamous cell carcinoma (SCC). One skin lesion and one non-lesion skin from nine affected individuals were analyzed by qualitative PCR. MCPyV was detected in 9 out of 9 lesion biopsies and 6 out of 8 non-lesion biopsies. HPyV6 was detected only in healthy skin, while HPyV7 and TSPyV were not detected in any skin sample. These findings argue against a possible role of cutaneous HPyVs in AK. However, considering the small sample size analyzed, a definitive conclusion cannot be drawn. Longitudinal studies on large cohorts are warranted.

Background: Progressive multifocal leukoencephalopathy (PML) caused by the JC virus is the main limitation to the use of disease modifying therapies for treatment of multiple sclerosis (MS). Methods: To assess the PML risk in course of ocrelizumab, urine and blood samples were collected from 42 MS patients at baseline (T0), at 6 (T2) and 12 months (T4) from the beginning of therapy. After JCPyV-DNA extraction, a quantitative-PCR (Q-PCR) was performed. Moreover, assessment of JCV-serostatus was obtained and arrangements’ analysis of non-coding control region (NCCR) and of viral capsid protein 1 (VP1) was carried out. Results: Q-PCR revealed JCPyV-DNA in urine at all selected time points, while JCPyV-DNA was detected in plasma at T4. From T0 to T4, JC viral load in urine was detected, increased in two logarithms and, significantly higher, compared to viremia. NCCR from urine was archetypal. Plasmatic NCCR displayed deletion, duplication, and point mutations. VP1 showed the S269F substitution involving the receptor-binding region. Anti-JCV index and IgM titer were found to statistically decrease during ocrelizumab treatment. Conclusions: Ocrelizumab in JCPyV-DNA positive patients is safe and did not determine PML cases. Combined monitoring of ocrelizumab’s effects on JCPyV pathogenicity and on host immunity might offer a complete insight towards predicting PML risk.

The aim of this study was to investigate the persistence of SARS-CoV-2 in post-mortem swabs of subjects who died from SARS-CoV-2 infection. The presence of the virus was evaluated post-mortem from airways of 27 SARS-CoV-2 positive patients at three different time points (T1 2 h; T2 12 h; T3 24 h) by real-time PCR. Detection of antibodies to SARS-CoV-2 was performed by Maglumi 2019-nCoV IgM/IgG chemiluminescence assay. SARS-CoV-2 viral RNA was still detectable in 70.3% of cases within 2 h after death and in 66,6% of cases up to 24 h after death. Our data showed an increase of the viral load in 78,6% of positive individuals 24 h post-mortem (T3) in comparison to that evaluated 2 h after death (T1). Noteworthy, we detected a positive T3 post-mortem swab (24 h after death) from 4 subjects who were negative at T1 (2 h after death). The results of our study may have an important value in the management of deceased subjects not only with a suspected or confirmed diagnosis of SARS-CoV-2, but also for unspecified causes and in the absence of clinical documentation or medical assistance.

Accurate measurement of the hepatitis B virus (HBV) DNA is important for the management of patients with chronic HBV infection. Here, the performance of the Xpert® HBV Viral Load test (Xpert HBV Viral Load) versus the Roche COBAS® Ampliprep/COBAS® TaqMan® system (CAP/CTM HBV) HBV test v2.0 was evaluated. From September 2017 to December 2017, a total of 876 prospectively collected or archived serum or EDTA plasma specimens from subjects chronically infected with HBV were tested using the Xpert HBV Viral Load and the CAP/CTM HBV v2.0 assays. Of the 876 specimens tested, 560 were within the quantitative range of both assays. The agreement between the two methods was 90.0%. No difference in plasma or serum samples was observed. Deming regression analysis showed a good correlation of the Xpert HBV Viral Load assay with the CAP/CTM HBV v2.0 assay. The Bland–Altman analysis showed a good agreement between the results of the Xpert HBV Viral Load assay and the CAP/CTM HBV assay, with a mean difference (±1.96 standard deviation) of 0.0091 ± 0.3852 Log IU/mL. Comparing the two assays, only nineteen specimens (2.1%) had a difference greater than 1.96 times the standard deviation. The Xpert® HBV Viral Load test is suitable for monitoring patients with HBV infection and is useful in diagnostic settings.

Sepsis remains a leading cause of death in the intensive care units and in all age groups worldwide. Early recognition and diagnosis are key to achieving improved outcomes. Therefore, novel biomarkers that might better inform clinicians treating such patients are surely needed. The main attributes of successful biomarkers would be high sensitivity, specificity, possibility of bedside monitoring and financial accessibility. A panel of sepsis biomarkers along with currently used laboratory tests will facilitate earlier diagnosis, timely treatment and improved outcome may be more effective than single biomarkers. In this review, we summarize the most recent advances on sepsis biomarkers evaluated in clinical and experimental studies.

Context: Human immunodeficiency virus type 2 (HIV-2) infections are mainly restricted to West Africa; however, in the recent years, the prevalence of HIV-2 is a growing concern in some European countries and the Southwestern region of India. Despite the presence of different HIV-2 groups, only A and B Groups have established human-to-human transmission chains. Aims: This work aimed to evaluate the phylogeographic inference of HIV-2 Group B worldwide to estimate their data of origin and the population dynamics. Materials and Methods: The evolutionary rates, the demographic history for HIV-2 Group B dataset, and the phylogeographic analysis were estimated using a Bayesian approach. The viral gene flow analysis was used to count viral gene out/in flow among different locations. Results: The root of the Bayesian maximum clade credibility tree of HIV-2 Group B dated back to 1957. The demographic history of HIV-2 Group B showed that the epidemic remained constant up to 1970 when started an exponential growth. From 1985 to early 2000s, the epidemic reached a plateau, and then it was characterized by two bottlenecks and a new plateau at the end of 2000s. Phylogeographic reconstruction showed that the most probable location for the root of the tree was Ghana. Regarding the viral gene flow of HIV-2 Group B, the only observed viral gene flow was from Africa to France, Belgium, and Luxembourg. Conclusions: The study gives insights into the origin, history, and phylogeography of HIV-2 Group B epidemic. The growing number of infections of HIV-2 worldwide indicates the need for strengthening surveillance.

Merkel cell polyomavirus (MCPyV) viral protein 1 (VP1) is the capsid protein that mediates virus attachment to host cell receptors and is the major immune target. Given the limited data on MCPyV VP1 mutations, the VP1 genetic variability was examined in 100 plasma and 100 urine samples from 100 HIV+ individuals. Sequencing of VP1 DNA in 17 urine and 17 plasma specimens, simultaneously MCPyV DNA positive, revealed that 27 samples displayed sequences identical to VP1 of MCC350 strain. VP1 from two urine specimens had either Thr47Ser or Ile115Phe substitution, whereas VP1 of one plasma contained Asp69Val and Ser251Phe substitutions plus deletion (∆) of Tyr79. VP1 DNA in the remaining samples had mutations encoding truncated protein. Three-dimensional prediction models revealed that Asp69Val, Ser251Phe, and Ile115Phe caused neutral effects while Thr47Ser and Tyr79∆ produced a deleterious effect reducing VP1 stability. A549 cells infected with urine or plasma samples containing full-length VP1 variants with substitutions, sustained viral DNA replication and VP1 expression. Moreover, medium harvested from these cells was able to infect new A549 cells. In cells infected by samples with truncated VP1, MCPyV replication was hampered. In conclusion, MCPyV strains with unique mutations in the VP1 gene are circulating in HIV+ patients. These strains display altered replication efficiency compared to the MCC350 prototype strain in A549 cells.

Background: Since limited data are available about the prevalence of Merkel cell polyomavirus (MCPyV) and the genetic variability of its noncoding control region (NCCR) in the context of immunosuppression, this study aimed to investigate the distribution of MCPyV in anatomical sites other than the skin and the behavior of NCCR among an HIV-1-positive population. Methods: Urine, plasma, and rectal swabs specimens from a cohort of 66 HIV-1-positive patients were collected and subjected to quantitative real-time polymerase chain reaction (qPCR) for MCPyV DNA detection. MCPyV-positive samples were amplified by nested PCR targeting the NCCR, and NCCRs alignment was carried out to evaluate the occurrence of mutations and to identify putative binding sites for cellular factors. Results: MCPyV DNA was detected in 10/66 urine, in 7/66 plasma, and in 23/66 rectal samples, with a median value of 5 × 102 copies/mL, 1.5 × 102 copies/mL, and 2.3 × 103 copies/mL, respectively. NCCR sequence analysis revealed a high degree of homology with the MCC350 reference strain in urine, whereas transitions, transversions, and single or double deletions were observed in plasma and rectal swabs. In these latter samples, representative GTT and GTTGA insertions were also observed. Search for putative binding sites of cellular transcription factors showed that in several strains, deletions, insertions, or single base substitutions altered the NCCR canonical configuration. Conclusions: Sequencing analysis revealed the presence of numerous mutations in the NCCR, including insertions and deletions. Whether these mutations may have an impact on the pathogenic features of the virus remains to be determined. qPCR measured on average a low viral load in the specimens analyzed, with the exception of those with the GTTGA insertion.

Respiratory tract infections account for high morbidity and mortality around the world. Fragile patients are at high risk of developing complications such as pneumonia and may die from it. Limited information is available on the extent of the circulation of respiratory viruses in the hospital setting. Most knowledge relates to influenza viruses (FLU) but several other viruses produce flu-like illness. The study was conducted at the University Hospital Policlinico Tor Vergata, Rome, Italy. Clinical and laboratory data from hospitalized patients with respiratory tract infections during the period October 2016–March 2019 were analysed. The retrospective analysis included 17 viral agents detected by FilmArray test and clinical data from medical records and hospital discharge sheets. Models were adjusted for relevant confounders such as clinical severity and risk of death, socio-demographic characteristics and surgical procedures. From a total of 539 specimens analysed, 180 (33.39%) were positive for one or more respiratory viruses. Among them, 83 (46.1 %) were positive for influenza viruses (FLU), 36 (20%) rhino/enteroviruses (RHV/EV), 17 (9.44%) human coronaviruses (HCOV-229E, -HKU1, -NL63, and -OC43), 17 (9.44%) respiratory syncytial virus, 15 (8.33%) human metapneumovirus (HMPV), 8 (4.44%) parainfluenza viruses (PIV) and 4 (2.22%) adenoviruses (ADV). The distribution of viral agents varied across age groups and month of detection. The positive specimens were from 168 patients [102 M, 66 F; median age (range): 64 years (19−93)]. Overall, 40% of them had a high-grade clinical severity and a 27% risk of death; 27 patients died and 22 of them (81.5%) had received a clinical diagnosis of pneumonia. Respiratory viral infections may have a severe course and a poor prognosis in hospitalized patients, due to underlying comorbidities. Monitoring the circulation of respiratory viruses in hospital settings is important to improve diagnosis, prevention and treatment.

To investigate an association between KI and WU polyomavirus (KIPyV and WUPyV) infections and CD4+ cell counts, we tested HIV-1–positive patients and blood donors. No association was found between cell counts and virus infections in HIV-1–positive patients. Frequency of KIPyV infection was similar for both groups. WUPyV was more frequent in HIV-1–positive patients.

BACKGROUND: Because of the extreme genetic variability of hepatitis C virus (HCV), we analyzed whether specific HCV-genotypes are differently prone to develop resistance to linear and macrocyclic protease-inhibitors (PIs). METHODS: The study includes 1568 NS3-protease sequences, isolated from PI-naive patients infected with HCV-genotypes 1a (N = 621), 1b (N = 474), 2 (N = 72), 3 (N = 268), 4 (N = 54) 5 (N = 6), and 6 (N = 73). Genetic-barrier was calculated as the sum of nucleotide-transitions (score = 1) and/or nucleotide-transversions (score = 2.5) required for drug-resistance-mutations emergence. Forty-three mutations associated with PIs-resistance were analyzed (36A/M/L/G-41R-43S/V-54A/S/V-55A-Q80K/R/L/H/G-109K-138T-155K/Q/T/I/M/S/G/L-156T/V/G/S-158I-168A/H/T/V/E/I/G/N/Y-170A/T-175L). Structural analyses on NS3-protease and on putative RNA-models have been also performed. RESULTS: Overall, NS3-protease was moderately conserved, with 85/181 (47.0%) amino-acids showing 94% HCV-2-4; 175L present in 100% HCV-1a-3-5 and >97% HCV-2-4). Furthermore, HCV-3 specifically showed non-conservative polymorphisms (R123T-D168Q) at two drug-interacting positions. Regardless of HCV-genotype, 13 PIs resistance-mutations were associated with low genetic-barrier, requiring only 1 nucleotide-substitution (41R-43S/V-54A-55A-80R-156V/T: score = 1; 54S-138T-156S/G-168E/H: score = 2.5). By contrast, by using HCV-1b as reference genotype, nucleotide-heterogeneity led to a lower genetic-barrier for the development of some drug-resistance-mutations in HCV-1a (36M-155G/I/K/M/S/T-170T), HCV-2 (36M-80K-155G/I/K/S/T-170T), HCV-3 (155G/I/K/M/S/T-170T), HCV-4-6 (155I/S/L), and HCV-5 (80G-155G/I/K/M/S/T). CONCLUSIONS: The high degree of HCV genetic variability makes HCV-genotypes, and even subtypes, differently prone to the development of PIs resistance-mutations. Overall, this can account for different responsiveness of HCV-genotypes to PIs, with important clinical implications in tailoring individualized and appropriate regimens.

The Polyomaviruses KI and WU have been identified in 2007 in nasopharyngeal aspirates of patients with acute respiratory tract infections.They have been further isolated in faecal samples, lymphoid tissue, bronchoalveolar lavages and blood, although their pathological associations, molecular epidemiology and tissue tropism are still not widely known.The aim of this study was to determine the presence of KIV and WUV in tonsillar tissue of immunocompetent patients to assess whether the tonsils may represent a site of infection and/or persistence.The presence of Polyomavirus BKV, JCV and SV40 was also evaluated. The presence of Polyomavirus KI,WU, BK, JC and SV40-DNA was evaluated in a prospective study on tonsillar samples obtained from 29 immunocompetent patients (adults and children undergoing tonsillectomy) by a commercially available Taqman Real-time quantitative PCR (BKV Q-PCR Alert Kit, Nanogen) and a home-made system (for KIV,WUV, SV40, JCV).The amplification conditions were optimized and standardized. KIV-DNA was detected in 2/29 (6.9%) tonsil tissue (obtained from an adult and a child), while WU, BK, JC and SV40 were negative in all cases. Our prevalence results are consistent with those reported in the literature on samples from the respiratory tract and lymphoid tissue such as tonsils. Further studies are needed to understand the significance of the presence of KIV in tonsil tissue and assess the possibility that the virus can establish latent and/or persistent infection at this level.

Viral genotype is an important determinant of the therapeutical outcome of the chronic hepatitis C and is useful in clinical practice to determine the duration of treatment1.While the viral type shows a clear association with therapeutic success, there is currently no evidence to that effect for HCV subtype, whose value is thus confined to epidemiological studies.The Abbott RealTime HCV Genotype II assay, through the use of Minor Groove Binder probes (MGB) is able to distinguish genotypes 1 to 6 (target 5’-UTR region) and subtypes 1a and 1b (NS5B region). In four different Italian centers a comparison between the Abbott RealTime HCV Genotype II assay and the Versant HCV Genotype 2.0 (LIPA) has been performed.A total of 143 non selected samples with the request of HCV genotyping have been analysed. 141/143 samples (98.6%) have provided reportable results with both tests (2 indeterminates with LIPA). Concordance at the type level was 96.5% (136/141). Considering the 136 concordant samples, the distribution was as follows: type 1 = 61 (44.9%), 2 = 36 (26.5%), 3 = 21 (15.4%), 4 = 17 (12.5% ) 5 = 1 (0.7%). Both assays assigned subtype in 56/61 (91.8%) samples of genotype 1 (3 and 2 samples only provided the type for LIPA and Abbott, respectively) and 50/56 (89.3%) had concordant subtype. It is worth to note that 4 of the 5 samples with discordant subtype Abbott 1a/LiPA 1b came from the only center that used for LIPA the 5-UTR amplicon, loosing the benefit of the core region which has been introduced in the version 2 of the test to improve the accuracy in distinguishing between 1a and 1b.There was only one discordant sample at type level (Abbott 4, LIPA 1b) which after sequencing and phylogenetic analysis was resolved as type 4. Four mixed infections were detected, 3 with the Abbott test (two 1a+4 and one 1b+3) and 1 with the LIPA test (1a+3). In all cases the comparison test showed a single genotype 1 infection. The new Abbott RealTime HCV Genotype II assay showed a high correlation with the Versant HCV Genotype 2.0 assay (LIPA).The automation platform m2000 system (Abbott), together with objective interpretation and digital archiving results may be particularly advantageous for the laboratory.