Sequence analysis of respiratory syncytial virus cases reveals a novel subgroup -B strain circulating in north-central Italy after pandemic restrictions.

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.

Molecular characterization of emerging Echovirus 11 (E11) shed light on the recombinant origin of a variant associated with severe hepatitis in neonates.

Echovirus 11 (E11) has gained attention owing to its association with severe neonatal infections. Due to the limited data available, the World Health Organization (WHO) considers public health risk to the general population to be low. The present study investigated the genetic variation and molecular evolution of E11 genomes collected from May to December 2023. Whole genome sequencing (WGS) was performed for 16 E11 strains. Phylogenetic analysis on WG showed how all Italian strains belonged to genogroup D5, similarly to other E11 strains recently reported in France and Germany all together aggregated into separate clusters. A cluster-specific recombination pattern was also identified using phylogenetic analysis of different genome regions. Echovirus 6 was identified as the major recombinant virus in 3Cpro and 3Dpol regions. The molecular clock analysis revealed that the recombination event probably occurred in June 2018 (95% HPD interval: Jan 2016-Jan 2020). Shannon entropy analyses, within P1 region, showed how 11 amino acids exhibited relatively high entropy. Five of them were exposed on the canyon region which is responsible for receptor binding with the neonatal Fc receptor. The present study showed the recombinant origin of a new lineage of E11 associated with severe neonatal infections.

Impact of Whole Genome Sequencing to investigate transmission of Serratia marcescens in Neonatal Intensive Care Unit.

Newborns admitted to neonatal intensive care units (NICU) are at increased risk of health care-associated infections. Serratia marcescens represent the third most common pathogen in NICU outbreaks. Here we present an outbreak investigation performed using Whole Genome Sequencing (WGS) analyses and the control measures implemented to limit the spread of S. marcescens in the NICU of an Italian hospital. In February 2023 S. marcescens was isolated from six newborns, when in 2022 this pathogen was isolated only from two samples in the same ward. Measures for infection prevention were adopted. Routinary surveillance screening, performed with rectal swabs collected at admission and weekly thereafter, was implemented to search for S. marcescens presence. Environmental samples were collected. All the isolates, obtained from the conjunctival swab of six newborns, from rectal swab of two newborns who did not develop infections, as well as from the aerators of two faucets, were sequenced. WGS analyses showed no correlation between the isolates from newborns and environmental isolates. The implementation of the measures for infection prevention and control had enabled us to successfully control the outbreak within a short period. WGS analyses proved to be crucial in outbreak investigation to limit the spreading of the pathogens.

Long-term dynamics of natural killer cells in response to SARS-CoV-2 vaccination: Persistently enhanced activity postvaccination.

Natural Killer (NK) cells play a significant role in the early defense against virus infections and cancer. Recent studies have demonstrated the involvement of NK cells in both the induction and effector phases of vaccine-induced immunity in various contexts. However, their role in shaping immune responses following SARS-CoV-2 vaccination remains poorly understood. To address this matter, we conducted a comprehensive analysis of NK cell phenotype and function in SARS-CoV-2 unexposed individuals who received the BNT162b2 vaccine. We employed a longitudinal study design and utilized a panel of 53 15-mer overlapping peptides covering the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein to assess NK cell function at 0 and 20 days following the first vaccine, and 30 and 240 days following booster. Additionally, we evaluated the levels of total IgG anti-Spike antibodies and their potential neutralizing ability. Our findings revealed an increased NK cell activity upon re-exposure to RBD when combined with IL12 and IL18 several months after booster. Concurrently, we observed that the frequencies of NKG2A + NK cells declined over the course of the follow-up period, while NKG2C increased only in CMV positive subjects. The finding that NK cell functions are inducible 9 months after vaccination upon re-exposure to RBD and cytokines, sheds light on the role of NK cells in contributing to SARS-CoV-2 vaccine-induced immune protection and pave the way to further studies in the field.

Dynamics of viral DNA shedding and culture viral DNA positivity in different clinical samples collected during the 2022 mpox outbreak in Lombardy, Italy.

BACKGROUND: Mpox virus (MPXV) has recently spread outside of sub-Saharan Africa. This large multicentre study was conducted in Lombardy, the most densely populated Italian region accounting for more than 40% of Italian cases. The present study aims to: i) evaluate the presence and the shedding duration of MPXV DNA in different body compartments correlating the MPXV viability with the time to onset of symptoms; ii) provide evidence of MPXV persistence in different body compartment as a source of infection and iii) characterize the MPXV evolution by whole genome sequencing (WGS) during the outbreak occurred in Italy. MATERIAL AND METHODS: The study included 353 patients with a laboratory-confirmed diagnosis of MPXV infection screened in several clinical specimens in the period May 24th - September 1st, 2022. Viral isolation was attempted from different biological matrices and complete genome sequencing was performed for 61 MPXV strains. RESULTS: MPXV DNA detection was more frequent in the skin (94.4%) with the longest median time of viral clearance (16 days). The actively-replicating virus in cell culture was obtained for 123/377 (32.6%) samples with a significant higher viral quantity on isolation positive samples (20 vs 31, p < 0.001). The phylogenetic analysis highlighted the high genetic identity of the MPXV strains collected, both globally and within the Lombardy region. CONCLUSION: Skin lesion is gold standard material and the high viral load and the actively-replicating virus observed in genital sites confirms that sexual contact plays a key role in the viral transmission.

Enterobacter asburiae ST229: an emerging carbapenemases producer.

Enterobacter asburiae, member of the Enterobacter cloacae complex (ECC) group, shows an increasing clinical relevance being responsible for infections like pneumonia, urinary tract infections and septicemia. The aim of the present study was the investigation of the genomic features of two XDR E. asburiae ST229 clinical strains co-carrying blaNDM-1 and blaVIM-1 determinants, collected in October 2021 and in June 2022, respectively. Two E. asburiae strains were collected from rectal swabs of as many patients admitted to the cardiopulmonary intensive care unit of Fondazione I.R.C.C.S. "Policlinico San Matteo" in Pavia, Italy. Based on the antibiotic susceptibility profile results, both isolates showed an XDR phenotype, retaining susceptibility only to fluoroquinolones. Both isolates shared identical resistome, virulome, plasmid content, and belonged to ST229, a rarely reported sequence type. They co-harbored blaNDM-1 and blaVIM-1 genes, that resulted located on transferable plasmids by conjugation and transformation. Moreover, both strains differed in 24 SNPs and showed genetic relatedness with E. asburiae ST709 and ST27. We described the first case of ST229 E. asburiae co-harboring blaNDM-1 and blaVIM-1 in Italy. This study points out the emergence of carbapenemases in low-risk pathogens, representing a novel challenge for public health, that should include such types of strains in dedicated surveillance programs. Antimicrobial susceptibility testing was carried out using Thermo Scientific™ Sensititre™ Gram Negative MIC Plates DKMGN. Both strains underwent whole-genome sequencing (WGS) using Illumina Miseq platform. Resistome, plasmidome, virulome, MLST, plasmid MLST and a SNPs-based phylogenetic tree were in silico determined.

Epidemiological and clinical insights into the enterovirus D68 upsurge in Europe 2021/22 and the emergence of novel B3-derived lineages, ENPEN multicentre study.

Enterovirus D68 (EV-D68) infections are associated with severe respiratory disease and acute flaccid myelitis (AFM). The European Non-Polio Enterovirus Network (ENPEN) aimed to investigate the epidemiological and genetic characteristics of EV-D68 and its clinical impact during the fall-winter season of 2021/22. From 19 European countries, 58 institutes reported 10,481 (6.8%) EV-positive samples of which 1,004 (9.6%) were identified as EV-D68 (852 respiratory samples). Clinical data was reported for 969 cases. 78.9% of infections were reported in children (0-5 years); 37.9% of cases were hospitalised. Acute respiratory distress was commonly noted (93.1%) followed by fever (49.4%). Neurological problems were observed in 6.4% of cases with six reported with AFM. Phylodynamic/Nextstrain and phylogenetic analyses based on 694 sequences showed the emergence of two novel B3-derived lineages, with no regional clustering. In conclusion, we describe a large-scale EV-D68 European upsurge with severe clinical impact and the emergence of B3-derived lineages.

Increased circulation of echovirus 11 in the general population and hospital patients as elicited by the non-polio enterovirus laboratory-based sentinel surveillance in northern Italy, 2023.

OBJECTIVES: Following the alert of echovirus 11 (E-11) infection in neonates in EU/EEA Member States, we conducted an investigation of E-11 circulation by gathering data from community and hospital surveillance of enterovirus (EV) in northern Italy from 01 August 2021 to 30 June 2023. METHODS: Virological results of EVs were obtained from the regional sentinel surveillance database for influenza-like illness (ILI) in outpatients, and from the laboratory database of ten hospitals for inpatients with either respiratory or neurological symptoms. Molecular characterization of EVs was performed by sequence analysis of the VP1 gene. RESULTS: In our ILI series, the rate of EV-positive specimens showed an upward trend from the end of May 2023, culminating at the end of June, coinciding with an increase in EV-positive hospital cases. The E-11 identified belonged to the D5 genogroup and the majority (83%) were closely associated with the novel E-11 variant, first identified in severe neonatal infections in France since 2022. E-11 was identified sporadically in community cases until February 2023, when it was also found in hospitalized cases with a range of clinical manifestations. All E-11 cases were children, with 14 out of 24 cases identified through hospital surveillance. Of these cases, 60% were neonates, and 71% had severe clinical manifestations. CONCLUSION: Baseline epidemiological data collected since 2021 through EV laboratory-based surveillance have rapidly tracked the E-11 variant since November 2022, alongside its transmission during the late spring of 2023.

Fourth dose of microneedle array patch of SARS-CoV-2 S1 protein subunit vaccine elicits robust long-lasting humoral responses in mice.

The COVID-19 pandemic has underscored the pressing need for safe and effective booster vaccines, particularly in considering the emergence of new SARS-CoV-2 variants and addressing vaccine distribution inequalities. Dissolving microneedle array patches (MAP) offer a promising delivery method, enhancing immunogenicity and improving accessibility through the skin's immune potential. In this study, we evaluated a microneedle array patch-based S1 subunit protein COVID-19 vaccine candidate, which comprised a bivalent formulation targeting the Wuhan and Beta variant alongside a monovalent Delta variant spike proteins in a murine model. Notably, the second boost of homologous bivalent MAP-S1(WU + Beta) induced a 15.7-fold increase in IgG endpoint titer, while the third boost of heterologous MAP-S1RS09Delta yielded a more modest 1.6-fold increase. Importantly, this study demonstrated that the administration of four doses of the MAP vaccine induced robust and long-lasting immune responses, persisting for at least 80 weeks. These immune responses encompassed various IgG isotypes and remained statistically significant for one year. Furthermore, neutralizing antibodies against multiple SARS-CoV-2 variants were generated, with comparable responses observed against the Omicron variant. Overall, these findings emphasize the potential of MAP-based vaccines as a promising strategy to combat the evolving landscape of COVID-19 and to deliver a safe and effective booster vaccine worldwide.

Clinical isolates of ST131 blaOXA-244-positive Escherichia coli, Italy, December 2022 to July 2023.

The dissemination of carbapenemase-producing Escherichia coli, although still at low level, should be continuously monitored. OXA-244 is emerging in Europe, mainly in E. coli. In Italy, this carbapenemase was reported from an environmental river sample in 2019. We report clinical isolates of OXA-244-producing ST131 E. coli in four patients admitted to an acute care hospital in Pavia, Italy. The association of this difficult-to-detect determinant with a globally circulating high-risk clone, ST131 E. coli, is of clinical relevance.

Second Boost of Omicron SARS-CoV-2 S1 Subunit Vaccine Induced Broad Humoral Immune Responses in Elderly Mice.

Currently approved COVID-19 vaccines prevent symptomatic infection, hospitalization, and death from the disease. However, repeated homologous boosters, while considered a solution for severe forms of the disease caused by new SARS-CoV-2 variants in elderly individuals and immunocompromised patients, cannot provide complete protection against breakthrough infections. This highlights the need for alternative platforms for booster vaccines. In our previous study, we assessed the boost effect of the SARS-CoV-2 Beta S1 recombinant protein subunit vaccine (rS1Beta) in aged mice primed with an adenovirus-based vaccine expressing SARS-CoV-2-S1 (Ad5.S1) via subcutaneous injection or intranasal delivery, which induced robust humoral immune responses (1). In this follow-up study, we demonstrated that a second booster dose of a non-adjuvanted recombinant Omicron (BA.1) S1 subunit vaccine with Toll-like receptor 4 (TLR4) agonist RS09 (rS1RS09OM) was effective in stimulating strong S1-specific immune responses and inducing significantly high neutralizing antibodies against the Wuhan, Delta, and Omicron variants in 100-week-old mice. Importantly, the second booster dose elicits cross-reactive antibody responses, resulting in ACE2 binding inhibition against the spike protein of SARS-CoV-2 variants, including Omicron (BA.1) and its subvariants. Interestingly, the levels of IgG and neutralizing antibodies correlated with the level of ACE2 inhibition in the booster serum samples, although Omicron S1-specific IgG level showed a weaker correlation compared to Wuhan S1-specific IgG level. Furthermore, we compared the immunogenic properties of the rS1 subunit vaccine in young, middle-aged, and elderly mice, resulting in reduced immunogenicity with age, especially an impaired Th1-biased immune response in aged mice. Our findings demonstrate that the new variant of concern (VOC) rS1 subunit vaccine as a second booster has the potential to offer cross-neutralization against a broad range of variants and to improve vaccine effectiveness against newly emerging breakthrough SARS-CoV-2 variants in elderly individuals who were previously primed with the authorized vaccines.

Conversion of monoclonal IgG to dimeric and secretory IgA restores neutralizing ability and prevents infection of Omicron lineages.

The emergence of Omicron lineages and descendent subvariants continues to present a severe threat to the effectiveness of vaccines and therapeutic antibodies. We have previously suggested that an insufficient mucosal immunoglobulin A (IgA) response induced by the mRNA vaccines is associated with a surge in breakthrough infections. Here, we further show that the intramuscular mRNA and/or inactivated vaccines cannot sufficiently boost the mucosal secretory IgA response in uninfected individuals, particularly against the Omicron variant. We thus engineered and characterized recombinant monomeric, dimeric, and secretory IgA1 antibodies derived from four neutralizing IgG monoclonal antibodies (mAbs 01A05, rmAb23, DXP-604, and XG014) targeting the receptor-binding domain of the spike protein. Compared to their parental IgG antibodies, dimeric and secretory IgA1 antibodies showed a higher neutralizing activity against different variants of concern (VOCs), in part due to an increased avidity. Importantly, the dimeric or secretory IgA1 form of the DXP-604 antibody significantly outperformed its parental IgG antibody, and neutralized the Omicron lineages BA.1, BA.2, and BA.4/5 with a 25- to 75-fold increase in potency. In human angiotensin converting enzyme 2 (ACE2) transgenic mice, a single intranasal dose of the dimeric IgA DXP-604 conferred prophylactic and therapeutic protection against Omicron BA.5. Thus, dimeric or secretory IgA delivered by nasal administration may potentially be exploited for the treatment and prevention of Omicron infection, thereby providing an alternative tool for combating immune evasion by the current circulating subvariants and, potentially, future VOCs.

Relationship of human cytomegalovirus-infected endothelial cells and circulating leukocytes in the pathogenesis of disseminated human cytomegalovirus infection: A narrative review.

Among the leucocyte subpopulations circulating in peripheral blood of immune-compromised patients with disseminated Human cytomegalovirus (HCMV) infection, polymorphonuclear leuckocytes (PMNL) and M/M may carry infectious virus. While only in PMNL early HCMV replicative events do occur, monocytes are susceptible to complete virus replication when they enter human organs, where as macrophages become a site of active complete virus replication. In vivo leucocytes and endothelial cells interact continuously, as suggested by several in vitro experimental findings showing the bidirectional HCMV transmission from leucocytes to and from endothelial cells with the critical aid of adhesion molecules. Recently, the neutralising antibody response in sera from subjects with primary HCMV infection was reported to be much higher and earlier than in human embryonic lung fibroblasts (HELF) cells when measured in endothelial cells and epithelial cells, where virus entry is mediated mostly by the pentamer complex gH/gL/pUL128/pUL130/pUL131, whereas it was much lower and delayed when determined in HELF, where virus entry is mediated mostly by the trimer complex gH/gL/gO. Thus, these results suggested that products of UL128L were the molecules primary responsible for the differential neutralising antibody response. This conclusion was confirmed by a series of polyclonal and monoclonal antibodies directed to the components of pUL128L. Very recently, based on two sets of experiments including inhibition and immunoblotting assays, the pentamer complex/trimer complex ratio has been finally identified as the main factor of the neutralising antibody response. This ratio may change with the virus suspension producer and target cell system as well as number of cell culture passages.

Phenotypic and Genotypic Assays to Evaluate Coagulase-Negative Staphylococci Biofilm Production in Bloodstream Infections.

Coagulase-negative staphylococci (CoNS) are commensal on human body surfaces and, for years, they were not considered a cause of bloodstream infection and were often regarded as contamination. However, the involvement of CoNS in nosocomial infection is increasingly being recognized. The insertion of cannulas and intravascular catheters represents the primary source of CoNS entry into the bloodstream, causing bacteremia and sepsis. They owe their pathogenic role to their ability to produce biofilms on surfaces, such as medical devices. In this study, we evaluate the adhesive capacity of CoNS isolated from blood cultures by comparing a spectrophotometric phenotypic assay with genotypic analysis based on the evidence of the ica operon. We retrospectively reviewed the database of CoNS isolated from blood cultures from January to December 2021 that were considered responsible for 361 bloodstream infections. Eighty-nine CoNS were selected among these. Our data show that Staphylococcus epidermidis was the predominant species isolated, expressing greater adhesive capacities, especially those with the complete operon. Knowledge of the adhesive capabilities of a microorganism responsible for sepsis can be useful in implementing appropriate corrective and preventive measures, since conventional antibiotic therapy cannot effectively eradicate biofilms.

Mpox Virus in the Pharynx of Men Having Sex with Men: A Case Series.

The recent Mpox virus (MPV) outbreak in Europe and North America, primarily among men who have sex with men (MSM), raised concerns about various transmission sources. We examined patients with Mpox from an urban STI center in Lombardy, Italy, between May and August 2022. Demographic, transmission, and clinical data were collected using a standardized form. Initial and subsequent tests were conducted using the RealStar Orthopoxvirus PCR Kit 1.0 (Altona Diagnostics, Hamburg, Germany) for skin lesions and oropharyngeal swabs. A total of 15 patients were recruited, all MSM, with 40% being HIV-positive. Almost all reported recent unprotected sexual activity. Oropharyngeal symptoms were observed in a minority, and oral cavity lesions were present in 20% of cases. MPV DNA was detected in skin lesions of 93% of patients and in oropharyngeal swabs of 87%. Skin samples exhibited a higher viral load than pharyngeal samples, with the latter persisting longer. Prospective follow-up of 11 individuals revealed an average pharyngeal persistence of 5.3 days beyond skin lesion clearance, reaching up to 80 days in an immunosuppressed case. Our findings indicate that MPV replication can persist in the pharynx asymptomatically and for an extended period.

Concomitant Resistance to Cefiderocol and Ceftazidime/Avibactam in Two Carbapenemase-Producing Klebsiella pneumoniae Isolates from Two Lung Transplant Patients.

In this study, we present two cases of Klebsiella pneumoniae, one KPC-33- and one NDM-1-producing, showing resistance to cefiderocol and ceftazidime/avibactam, collected in the intensive care unit of a hospital in Northern Italy from two patients who had recently undergone lung transplantation. Whole-genome sequencing was performed to investigate the molecular features of these strains.

The immunogenicity and the safety of the adjuvanted glycoprotein E (gE)-based recombinant vaccine against herpes zoster (RZV) in cancer patients during immunotherapy.

Herpes zoster (HZ) is caused by the reactivation of latent varicella zoster virus (VZV). Severe immunocompromising conditions, such as solid tumors, have been largely associated with an increased risk for HZ due to waning VZV-specific cellular immunity. With the approval of the adjuvanted glycoprotein E (gE)-based recombinant vaccine (RZV; Shingrix™, GSK) also in immunocompromised subjects, HZ is considered a vaccine-preventable disease changing perspectives in immunocompromised subjects. To date, no clinical trial has evaluated the immunogenicity in the patients with cancer undergoing immunotherapy. In this study, we describe the humoral and cell-mediated immune responses in 38 cancer patients treated with immune checkpoint inhibitors (ICIs) and receiving RZV. We used samples collected at baseline (T0), 3 weeks (T2), and 6 months (T3) after the complete RV vaccination schedule. Our data showed that a significant proportion (40,5%) of RZV recipients mounted a stronger humoral and cell-mediated immune response at 3 weeks (T2) after complete RZV vaccination schedule. Interestingly, both humoral and cell-mediated immune responses were mostly stable over 6 months (T3). Interestingly, the overall IFNγ-producing lymphocytes was mainly associated with CD4 T cell response (p = .0012). In conclusion, data from our pilot study suggest a strong and long-lasting immunogenicity of RZV in ICI-treated patients. Prospective analyses at 1 year after vaccination will be performed in order to evaluate the long-term persistence of humoral and cell-mediated response against RZV.