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Background: Following the pandemic restrictions, the epidemiology of respiratory syncytial virus (RSV) has changed, leading to intense hospitalization peaks., Objectives: This study, conducted at multiple sites in Italy, aimed to describe the temporal dynamics of two post-COVID-19 RSV epidemics. Additionally, the circulating RSV-A and -B lineages were characterized and compared to those found in 2018 and 2019., Study Design: Respiratory specimens and data were collected from RSV-positive patients, both inpatients, and outpatients, of all ages at three sites in north-central Italy. To analyze these samples, roughly one-sixth were sequenced in the attachment glycoprotein G gene and subjected to phylogenetic and mutational analyses, including pre-pandemic sequences from north-central Italy., Results: The first post-pandemic surge of RSV cases was quite intense, occurring from October 2021 to early January 2022. The subsequent RSV epidemic (from November 2022 to early March 2023) also had a high impact, characterized by a rise in elderly patient cases. Post-pandemic cases of RSV-A were caused by various strains present in Italy prior to COVID-19. In contrast, a distinct RSV-B lineage, which was concurrently spreading in other countries, was identified as the main cause of the surge in 2022-2023 but remained undetected in Italy before the pandemic., Conclusions: This study describes the temporal dynamics of post-pandemic RSV subgroups and uncovers a lineage of RSV-B with high genetic divergence that may have increased the impact of decreased population immunity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Cytomegalovirus (CMV) is a leading cause of congenital infections. Dried blood spots (DBS) collected in the first week of life (Guthrie cards) have been used in the diagnosis of CMV infection outside the three-week window period following birth. The present work summarizes the results of a 15-year observational study in which DBS from 1388 children were used for a late diagnosis of congenital CMV infection., Methods: Three groups of children were studied: (i) symptomatic (with symptoms at birth or late sequelae) (N = 779); (ii) born to mothers with serological profile of primary CMV infection (N = 75); (iii) without any information (N = 534). A highly sensitive method of DNA extraction (heat-induced) from the DBS was used. CMV DNA was detected by a nested PCR., Results: In total CMV DNA was detected in 7.5% (104/1388) of children. Symptomatic children showed a low rate of CMV DNA detection (6.7%) than children born to mothers with serological profile of primary CMV infection (13.3%) (p = 0.034). Sensorial hearing loss and encephalopathy were the two clinical manifestations with the highest CMV detection rate (18.3% and 11.1%, respectively). Children whose mothers had a confirmed primary infection showed a higher rate of CMV detection (35.3%) when compared with children whose mothers had a not confirmed primary infection (6.9%) (p = 0.007)., Conclusion: The present work emphasises the importance of testing DBS in symptomatic children even a long time after symptoms onset and in children born to mothers with serologic diagnosis of maternal primary CMV infection when they miss the diagnosis during the three-week window following birth., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Aims: To assess influenza viruses (IVs) circulation and to evaluate A(H3N2) molecular evolution during the 2021-2022 season in Italy., Materials and Methods: 12,393 respiratory specimens (nasopharyngeal swabs or broncho-alveolar lavages) collected from in/outpatients with influenza illness in the period spanning from January 1, 2022 (week 2022-01) to May 31, 2022 (week 2022-22) were analysed to identify IV genome and were molecularly characterized by 12 laboratories throughout Italy. A(H3N2) evolution was studied by conducting an in-depth phylogenetic analysis of the hemagglutinin (HA) gene sequences. The predicted vaccine efficacy (pVE) of vaccine strain against circulating A(H3N2) viruses was estimated using the sequence-based P epitope model., Results: The overall IV-positive rate was 7.2% (894/12,393), all were type A IVs. Almost all influenza A viruses (846/894; 94.6%) were H3N2 that circulated in Italy with a clear epidemic trend, with 10% positivity rate threshold crossed for six consecutive weeks from week 2022-11 to week 2022-16. According to the phylogenetic analysis of a subset of A(H3N2) strains (n=161), the study HA sequences were distributed into five different genetic clusters, all of them belonging to the clade 3C.2a, sub-clade 3C.2a1 and the genetic subgroup 3C.2a1b.2a.2. The selective pressure analysis of A(H3N2) sequences showed evidence of diversifying selection particularly in the amino acid position 156. The comparison between the predicted amino acid sequence of the 2021-2022 vaccine strain (A/Cambodia/e0826360/2020) and the study strains revealed 65 mutations in 59 HA amino acid positions, including the substitution H156S and Y159N in antigenic site B, within major antigenic sites adjacent to the receptor-binding site, suggesting the presence of drifted strains. According to the sequence-based P epitope model, antigenic site B was the dominant antigenic site and the p(VE) against circulating A(H3N2) viruses was estimated to be -28.9%., Discussion and Conclusion: After a long period of very low IV activity since public health control measures have been introduced to face COVID-19 pandemic, along came A(H3N2) with a new phylogenetic makeup. Although the delayed 2021-2022 influenza season in Italy was characterized by a significant reduction of the width of the epidemic curve and in the intensity of the influenza activity compared to historical data, a marked genetic diversity of the HA of circulating A(H3N2) strains was observed. The identification of the H156S and Y159N substitutions within the main antigenic sites of most HA sequences also suggested the circulation of drifted variants with respect to the 2021-2022 vaccine strain. Molecular surveillance plays a critical role in the influenza surveillance architecture and it has to be strengthened also at local level to timely assess vaccine effectiveness and detect novel strains with potential impact on public health., Competing Interests: Conflict of interest Nothing to declare., (Copyright © 2022. Published by Elsevier B.V.)

Background: Polyomavirus (PyV) infection is common, ranging from 60% to 100% depending on the virus. The urinary excretion rates of JC virus (JCV) and BK virus (BKV) have been extensively studied, but less is known about the more recently discovered PyVs., Objectives: To investigate the urinary excretion of Merkel cell PyV (MCPyV), which is associated with Merkel cell carcinoma (MCC), in kidney transplant recipients and healthy subjects, as well as those of lymphotropic polyomavirus (LPV), which was isolated from the B-cells of African green monkeys but has also been found in human blood, JCV and BKV., Study Design: Urine samples were collected from 62 adult (ATP) and 46 pediatric (PTP) kidney transplant recipients and from 67 adult (AHC) and 40 pediatric (PHC) healthy controls. DNA was isolated and analyzed using real-time PCR (Q-PCR) to determine the viral loads of MCPyV, LPV, JCV and BKV., Results: MCPyV DNA was more frequently detected (p<0.05) in the PTP (36.9%) and PHC (30.0%) groups compared to JCV (8.7% in PTP, 12.5% in PHC), BKV (6.5% in PTP, 2.5% in PHC), and LPV (2.2% in PTP, 5% in PHC) and in the AHC (47.8%) group compared to BKV (13.4%) and LPV (0%)., Conclusions: Based on the results, it could be concluded that: (a) Despite the rarity of MCC, MCPyV is a common infection; (b) MCPyV demonstrates an excretion pattern similar to those of JCV and BKV, persisting in the kidney and infecting skin cells upon reactivation, with subsequent integration and transformation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Congenital cytomegalovirus infection causes 20-30% of congenital sensorineural hearing loss (SNHL) cases. Early identification of CMV attributable cases and their successful treatment are often hampered by the late appearance of the damage in a high proportion of children both symptomatic and asymptomatic at birth., Objective: To discuss the feasibility of a screening program aimed at finding congenitally infected babies followed by their audiological monitoring., Study Design: Opinion-review article., Results and Conclusions: Frequency and severity of hearing loss due to congenital CMV suggest it maybe worthwhile setting up neonatal screening campaigns. Structures where SNHL can be diagnosed and treated exist already in many countries as part of universal neonatal audiological screening schemes. A test based on viral DNA extraction from neonatal dried blood spots on Guthrie cards and its amplification by means of a nested PCR (DBS test) seems to offer the best characteristics for use in screening. Clearly it will be necessary to calculate whether the costs of screening, diagnosis and follow-up, and the financial and emotional burden on the families of infected children, are up to the potential gain.

Background: Detection of viral DNA in dried blood spots using the Guthrie card (DBS test) is a reliable and practical method of diagnosing congenital cytomegalovirus (CMV) infection. The test lends itself to epidemiological studies to establish the prevalence of the infection, but also to neonatal screening for secondary prevention of sequelae. These applications would be facilitated if it were possible to use smaller samples and do the test on pools of individual cases., Objective: To ascertain whether doing the test on smaller, pooled samples still accurately identifies neonates with congenital CMV infection., Study Design: We tested DBS from: (A) 39 laboratory reference cases; (B) 156 neonates suspected of having congenital CMV infection; (C) 119 children examined for the retrospective diagnosis of congenital CMV; (D) mock specimens prepared with known amounts of viral DNA., Results: The test using only one third of the usual amount of dried blood was 100% sensitive and specific compared to the standard DBS test (A) and to viral isolation (A and B). Pools of three single cases gave the same results as viral isolation (B) and the small-sample test (B and C). All the versions of the test gave a detection limit of 400 copies/ml., Conclusions: The modified procedure can accurately diagnose congenital CMV infection. It achieves savings in both the patient material and the costs of testing.

Background: The role of the virulence of the infecting cytomegalovirus (CMV) strain in the transmission of the virus from mother to fetus and the outcome of the fetal infection has not received much attention yet. Molecular analysis of the gene coding for the surface glycoprotein B (gB) has been used to investigate the relationship between genotype and virulence in groups of immunosuppressed patients., Objectives: (1) to assess the prevalence of different gB genotypes in babies with congenital CMV infection; (2) to investigate the possible relationship between genotype and severity of congenital CMV disease; (3) to evaluate the possibility of using dried blood on Guthrie cards (DBS) for genotyping., Study Design: CMV DNA was extracted from DBS and from urine/saliva samples collected in the first two weeks of life of 98 congenitally infected babies, half of which were symptomatic at birth. Genotyping was performed through RFLP analysis of the region corresponding to the cleavage site of the gB protein., Results: The most prevalent genotype was gB1 (42%) followed by gB3 (26%), gB2 (19%) and gB4 (13%). Rates of disease and CNS damages were higher among children infected by gB1 (35%, 17%) and gB3 (31%, 28%) than in those infected by gB2 and gB4 (20%, 17% and 13%, 15%, respectively). These differences however did not reach the statistical significance. The parallel typing of DBS and urine/saliva strains gave a full concordance of results., Conclusions: All four major CMV gB genotypes (gB1-4) can cause a congenital infection but none seems to be associated to the development and the severity of disease. The possibility of using the neonatal DBS for genotyping opens a way to the examination of large numbers of cases of congenital CMV infection.

Background: A simple and reliable diagnosis of congenital cytomegalovirus infection is necessary both for clinical and epidemiological purposes. This could be accomplished through the demonstration of cytomegalovirus (CMV) DNA in blood spots (DBS) on Guthrie cards., Objectives: (1) To assess the sensitivity and specificity of the method (DBS test) in diagnosing congenital CMV infection compared with viral isolation and (2) to evaluate the applications of the test to the late diagnosis of congenital CMV., Study Design: The method was tested on the cards of (1) 509 babies examined through viral isolation within their third week of life (72 positive cases) and (2) 191 children studied after 3 weeks of life (25 days to 5 years). Blood was eluted from Guthrie cards and heat extracted. The products of a nested polymerase chain reaction (PCR) amplifying one region in the CMV glycoprotein B (gB) gene were detected by agarose gel electrophoresis., Results: DBS test was positive in all 72 congenitally infected babies and in four of the 437 negative at cytomegalovirus isolation (sensitivity 100%, specificity 99%). Infection in 16 of the 92 infants with a late viral isolation was demonstrated to be congenital by the test, which also detected congenital infection in 18 of 83 children in whom viral culture was not performed (13 with and five without symptoms). Fifty-six additional control cases tested negative., Conclusions: DBS test is a reliable assay for diagnosing congenital cytomegalovirus infection and could be used as an alternative to viral culture. It is able to reveal whether ascertained CMV infection is congenital or postnatal at an age when viral isolation is not able to do so. It can assess the role of risky procedures such as transfusion and it can ascertain the etiology of morbid conditions diagnosed late or of controversial origin.