Oropouche fever cases diagnosed in Italy in two epidemiologically non-related travellers from Cuba, late May to early June 2024.

Oropouche fever is caused by Oropouche virus (OROV), transmitted primarily through the bite of infected midges, particularly of the genus Culicoides. The virus is mainly circulating in Central and South America where several countries reported an ongoing outbreak. We report here two imported cases of OROV infection identified in Italy, late May-early June 2024. These cases indicate that in the shadow of a massive dengue outbreak in the Americas, the Oropouche outbreak might be more widespread than previously estimated.

Wide Real-Life Data Support Reduced Sensitivity of Antigen Tests for Omicron SARS-CoV-2 Infections.

With the continuous spread of new SARS-CoV-2 variants of concern (VOCs), the monitoring of diagnostic test performances is mandatory. We evaluated the changes in antigen diagnostic tests' (ADTs) accuracy along the Delta to Omicron VOCs transition, exploring the N protein mutations possibly affecting ADT sensitivity and assessing the best sampling site for the diagnosis of Omicron infections. In total, 5175 subjects were enrolled from 1 October 2021 to 15 July 2022. The inclusion criteria were SARS-CoV-2 ADT combined with a same-day RT-PCR swab test. For the sampling site analysis, 61 patients were prospectively recruited during the Omicron period for nasal and oral swab analyses by RT-PCR. Next-Generation Sequencing data were obtained to evaluate the different sublineages. Using RT-PCR as a reference, 387 subjects resulted in becoming infected and the overall sensitivity of the ADT decreased from 63% in the Delta period to 33% in the Omicron period. This decrease was highly statistically significant (p < 0.001), and no decrease in viral load was detected at the RNA level. The nasal site presented a significantly higher viral load than the oral site during the Omicron wave. The reduced detection rate of Omicron infections by ADT should be considered in the global testing strategy to preserve accurate diagnoses across the changing SARS-CoV-2 variants.

Exploring Viral Genome Profile in Mpox Patients during the 2022 Outbreak, in a North-Eastern Centre of Italy.

In 2022, an unprecedented outbreak of mpox raged in several nations. Sequences from the 2022 outbreak reveal a higher nucleotide substitution if compared with the estimated rate for orthopoxviruses. Recently, intra-lesion SNVs (single nucleotide variants) have been described, and these have been suggested as possible sources of genetic variation. Until now, it has not been clear if the presence of several SNVs could represents the result of local mutagenesis or a possible co-infection. We investigated the significance of SNVs through whole-genome sequencing analysis of four unrelated mpox cases. In addition to the known mutations harboured by the circulating strains of virus (MPXV), 7 novel mutations were identified, including SNVs located in genes that are involved in immune evasion mechanisms and/or viral fitness, six of these appeared to be APOBEC3-driven. Interestingly, three patients exhibited the coexistence of mutated and wild-type alleles for five non-synonymous variants. In addition, two patients, apparently unrelated, showed an analogous pattern for two novel mutations, albeit with divergent frequencies. The coexistence of mixed viral populations, harbouring non-synonymous mutations in patients, supports the hypothesis of possible co-infection. Additional investigations of larger clinical cohorts are essential to validating intra-patient viral genome heterogeneity and determining the possibility of co-presence events of slightly divergent MPXV strains.

Changes in the Adaptive Cellular Repertoire after Infection with Different SARS-CoV-2 VOCs in a Cohort of Vaccinated Healthcare Workers.

BACKGROUND: Currently approved vaccines are highly effective in protecting against hospitalization and severe COVID-19 infections. How pre-existing immunity responds to new variants with mutated antigens is crucial information for elucidating the functional interplay between antibodies and B and T cell responses during infection with new SARS-CoV-2 variants. METHODS: In this study, we monitored the dynamics and persistence of the immune response versus different SARS-CoV-2 variants of concern that emerged during the pandemic period (2021-2022) in a cohort of vaccinated healthcare workers, who experienced breakthrough infection in the Pre-Delta, Delta, and Omicron waves. We evaluated both the humoral and cell-mediated responses after infection. We also evaluated the anti-SARS-CoV-2 antibodies levels produced by infection in comparison with those produced after vaccination. RESULTS: Our results highlighted that the immune response against the Delta VOC mainly involved an adaptive humoral and switched memory B cells component, even 3 months after the last vaccine dose, conversely showing a high percentage of depleted adaptive T cells. Omicron infections triggered a consistent production of non-vaccine-associated anti-N antibodies, probably to balance the spike epitope immune escape mechanisms. CONCLUSION: Our results suggest a direct dependence between the VOC and different humoral and B and T cell balances in the post-infection period, despite the administration of a different number of vaccine doses and the elapsed time since the last vaccination.

Urine: A Pitfall for Molecular Detection of Toscana Virus? An Analytical Proof-of-Concept Study.

Toscana virus (TOSV), a sandfly-borne virus, is an important etiological agent in human acute meningitis and meningoencephalitis in the Mediterranean area during the summer. However, the actual number of TOSV infections is underestimated. Laboratory confirmation is necessary because TOSV infection has overlapping clinical features with other neuro-invasive viral infections. Nowadays, the reference test for direct diagnosis in the acute phase of TOSV infection is the PCR based method for detecting TOSV in cerebrospinal fluid and/or plasma, serum, or blood. Although poorly employed, urine is another helpful biological matrix for TOSV detection. Urine is a matrix rich in PCR inhibitors that affect PCR efficiency; consequently, false negatives could be generated. To investigate the potential effect of urine PCR inhibitors on TOSV detection, we compared undiluted and diluted urine using 10-fold series of spiked TOSV. The results showed a significant improvement in TOSV detection performance in diluted urine (1 TCID50 vs. 1 × 104 TCID50 limit of detection and 101.35% vs. 129.62% efficiency, respectively, in diluted and undiluted urine). In conclusion, our data provide preliminary important insights into the use of diluted urine to limit the impact of the inhibitory effects of urine on the detection of TOSV in RT-PCR-based approaches.

Comparative analysis of bioinformatics tools to characterize SARS-CoV-2 subgenomic RNAs.

During the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), positive-sense genomic RNA and subgenomic RNAs (sgRNAs) are synthesized by a discontinuous process of transcription characterized by a template switch, regulated by transcription-regulating sequences (TRS). Although poorly known about makeup and dynamics of sgRNAs population and function of its constituents, next-generation sequencing approaches with the help of bioinformatics tools have made a significant contribution to expand the knowledge of sgRNAs in SARS-CoV-2. For this scope to date, Periscope, LeTRS, sgDI-tector, and CORONATATOR have been developed. However, limited number of studies are available to compare the performance of such tools. To this purpose, we compared Periscope, LeTRS, and sgDI-tector in the identification of canonical (c-) and noncanonical (nc-) sgRNA species in the data obtained with the Illumina ARTIC sequencing protocol applied to SARS-CoV-2-infected Caco-2 cells, sampled at different time points. The three software showed a high concordance rate in the identification and in the quantification of c-sgRNA, whereas more differences were observed in nc-sgRNA. Overall, LeTRS and sgDI-tector result to be adequate alternatives to Periscope to analyze Fastq data from sequencing platforms other than Nanopore.

sgRNAs: A SARS-CoV-2 emerging issue.

Like for other coronaviruses, SARS-CoV-2 gene expression strategy is based on the synthesis of a nested set of subgenomic mRNA species (sgRNAs). These sgRNA are synthesized using a "discontinuous transcription" mechanism that relies on template switching at Transcription Regulatory Sequences (TRS). Both canonical (c-sgRNA) and non-canonical (nc-sgRNA, less numerous) subgenomic RNA species can be produced. Currently, sgRNAs are investigated on the basis of sequence data obtained through next generation sequencing (NGS), and bioinformatic tools are crucial for their identification, characterization and quantification. To date, few software have been developed to this aim, whose reliability and applicability to all the available NGS platforms need to be established, to build confidence on the information resulting from such tools. In fact, these information may be crucial for the in depth elucidation of viral expression strategy, particularly in respect of the significance of nc-sgRNAs, and for the possible use of sgRNAs as potential markers of virus replicative activity in infected patients.

Safety and efficacy of COVID-19 vaccines in patients on dialysis: a multicentre cohort study in Italy.

BACKGROUND: The aim of this study was to evaluate the efficacy and safety of COVID-19 vaccines in patients undergoing haemodialysis in Italy compared to the general population. METHODS: In this cohort study, 118 dialysis centres from 18 Italian Regions participated. Individuals older than 16 years on dialysis treatment for at least 3 months, who provided informed consent were included. We collected demographic and clinical information, as well as data on vaccination status, hospitalisations, access to intensive care units and adverse events. We calculated the incidence, hospitalisation, mortality, and fatality rates in the vaccinated dialysis cohort, adjusted for several covariates. The incidence rates of infection in the dialysis cohort and the general population were compared through Standardised Incidence Rate Ratio. RESULTS: The study included 6555 patients vaccinated against SARS-CoV-2 infection according to the schedule recommended in Italy. Between March 2021 and May 2022, there were 1096 cases of SARS-CoV-2 infection, with an incidence rate after completion of the three-dose vaccination cycle of 37.7 cases per 100 person-years. Compared to the general population, we observed a 14% reduction in the risk of infection for patients who received three vaccine doses (Standardised Incidence Rate Ratio: 0.86; 95% Confidence Interval: 0.81-0.91), whereas no statistically significant differences were found for COVID-19-related hospitalisations, intensive care unit admissions or death. No safety signals emerged from the reported adverse events. CONCLUSIONS: The vaccination program against SARS-CoV-2 in the haemodialysis population showed an effectiveness and safety profile comparable to that seen in the general population.

Functional CVIDs phenotype clusters identified by the integration of immune parameters after BNT162b2 boosters.

Introduction: Assessing the response to vaccinations is one of the diagnostic criteria for Common Variable Immune Deficiencies (CVIDs). Vaccination against SARS-CoV-2 offered the unique opportunity to analyze the immune response to a novel antigen. We identify four CVIDs phenotype clusters by the integration of immune parameters after BTN162b2 boosters. Methods: We performed a longitudinal study on 47 CVIDs patients who received the 3rd and 4th vaccine dose of the BNT162b2 vaccine measuring the generation of immunological memory. We analyzed specific and neutralizing antibodies, spike-specific memory B cells, and functional T cells. Results: We found that, depending on the readout of vaccine efficacy, the frequency of responders changes. Although 63.8% of the patients have specific antibodies in the serum, only 30% have high-affinity specific memory B cells and generate recall responses. Discussion: Thanks to the integration of our data, we identified four functional groups of CVIDs patients with different B cell phenotypes, T cell functions, and clinical diseases. The presence of antibodies alone is not sufficient to demonstrate the establishment of immune memory and the measurement of the in-vivo response to vaccination distinguishes patients with different immunological defects and clinical diseases.

Evaluation of a ddPCR Commercial Assay for the Absolute Quantification of the Monkeypox Virus West Africa in Clinical Samples.

BACKGROUND: Monkeypox virus (MPXV) is a double-stranded DNA virus belonging to the orthopoxvirus genus in the family Poxviridae. Distinct clades are identified: the clade I belonging to the Central African (or Congo Basin) clade and the subclades IIa and IIb belonging to the West African clade. Here, a commercial droplet digital PCR (ddPCR) assay was evaluated for the quantification of the MPXV West Africa clade in clinical samples. METHODS: The ddPCR reaction was assessed as a duplex assay using RPP30 as an internal amplification control. A total of 60 clinical specimens were tested, 40 positives (skin lesions, n=10; rectal swabs, n = 10; pharyngeal swabs, n = 10; and whole blood, n = 10), and 20 negatives (n = 5 for each biological matrix) were found at the routine molecular diagnostics (orthopoxvirus qPCR followed by confirmation with Sanger sequencing). To evaluate the analytical sensitivity, the ddPCR reaction was first analyzed on serial dilutions of synthetic DNA spiked in water and in negative biological matrices, achieving a limit of detection of 3.5 copy/µL. RESULTS: Regarding the clinical samples, compared to routine molecular diagnostics, the ddPCR duplex assay showed 100% of specificity for all biological matrices and 100% sensitivity (10/10) for lesions, 100% (10/10) for rectal swabs, 90% (9/10) for pharyngeal swabs, and 60% (6/10) for whole blood. CONCLUSION: Overall, our data showed that the commercial ddPCR assay allowed the DNA detection of MPXV in 87.5% (35/40) of our cohort, highlighting useful technical indications for the different specimens with a potential greatest performance for skin lesions and rectal swabs.

Emerging viral infections in immunocompromised patients: A great challenge to better define the role of immune response.

The immune response to invading pathogens is characterized by the rapid establishment of a complex network of cellular interactions and soluble signals. The correct balancing of activating and regulating pathways and tissue-homing signals determines its effectiveness and persistence over time. Emerging viral pathogens have always represented a great challenge to the immune system and an often uncontrolled/imbalanced immune response has been described (e.g. cytokine storm, immune paralysis), contributing to the severity of the disease. Several immune biomarkers and cell subsets have been identified as major players in the cascade of events leading to severe diseases, highlighting the rationale for host-directed intervention strategy. There are millions of immunocompromised pediatric and adult patients worldwide (e.g. transplant recipients, hematologic patients, subjects with primary immune-deficiencies), experiencing an impaired immune reactivity, due to diseases and/or to the medical treatments. The reduced immune reactivity could have two paradoxical non-exclusive effects: a weak protective immunity on one hand, and a reduced contribution to immune-mediated pathogenetic processes on the other hand. In these sensitive contexts, the impact of emerging infections represents a still open issue to be explored with several challenges for immunologists, virologists, physicians and epidemiologists. In this review, we will address emerging infections in immunocompromised hosts, to summarize the available data concerning the immune response profile, its influence on the clinical presentation, the possible contribution of persistent viral shedding in generating new viral variants with improved immune escape features, and the key role of vaccination.

Subjects who developed SARS-CoV-2 specific IgM after vaccination show a longer humoral immunity and a lower frequency of infection.

BACKGROUND: We have previously shown that eliciting SARS-CoV-2-specific IgM after vaccination is associated with higher levels of SARS-CoV-2 neutralizing IgG. This study aims to assess whether IgM development is also associated with longer-lasting immunity. METHODS: We analysed anti-SARS-CoV-2 spike protein IgG and IgM (IgG-S, IgM-S), and anti-nucleocapsid IgG (IgG-N) in 1872 vaccinees at different time points: before the first dose (D1; w0), before the second dose (D2; w3) at three (w6) and 23 weeks (w29) after D2; moreover, 109 subjects were further tested at the booster dose (D3, w44), at 3 weeks (w47) and 6 months (w70) after D3. Two-level linear regression models were used to evaluate the differences in IgG-S levels. FINDINGS: In subjects who had no evidence of a previous infection at D1 (non-infected, NI), IgM-S development after D1 and D2 was associated with higher IgG-S levels at short (w6, p < 0.0001) and long (w29, p < 0.001) follow-up. Similar IgG-S levels were observed after D3. The majority (28/33, 85%) of the NI subjects who had developed IgM-S in response to vaccination did not experience infection. INTERPRETATION: The development of anti-SARS-CoV-2 IgM-S following D1 and D2 is associated with higher IgG-S levels. Most individuals who developed IgM-S never became infected, suggesting that IgM elicitation may be associated with a lower risk of infection. FUNDING: "Fondi Ricerca Corrente" and "Progetto Ricerca Finalizzata" COVID-2020 (Italian Ministry of Health); FUR 2020 Department of Excellence 2018-2022 (MIUR, Italy); the Brain Research Foundation Verona.

Comparative analysis of the neutralizing activity against SARS-CoV-2 Wuhan-Hu-1 strain and variants of concern: Performance evaluation of a pseudovirus-based neutralization assay.

Objectives: Emergence of new variants of SARS-CoV-2 might affect vaccine efficacy. Therefore, assessing the capacity of sera to neutralize variants of concern (VOCs) in BSL-2 conditions will help evaluating the immune status of population following vaccination or infection. Methods: Pseudotyped viruses bearing SARS-CoV-2 spike protein from Wuhan-Hu-1/D614G strains (wild type, WT), B.1.617.2 (Delta), or B.1.1.529 (Omicron) VOCs were generated to assess the neutralizing antibodies (nAbs) activity by a pseudovirus-based neutralization assay (PVNA). PVNA performance was assessed in comparison to the micro-neutralization test (MNT) based on live viruses. Sera collected from COVID-19 convalescents and vaccinees receiving mRNA (BNT16b2 or mRNA-1273) or viral vector (AZD1222 or Ad26.COV2.S) vaccines were used to measure nAbs elicited by two-dose BNT16b2, mRNA-1273, AZD1222 or one-dose Ad26.CO2.S, at different times from completed vaccination, ~ 1.5 month and ~ 4-6 months. Sera from pre-pandemic and unvaccinated individuals were analyzed as controls. Neutralizing activity following booster vaccinations against VOCs was also determined. Results: PVNA titers correlated with the gold standard MNT assay, validating the reliability of PVNA. Sera analyzed late from the second dose showed a reduced neutralization activity compared to sera collected earlier. Ad26.CO2.S vaccination led to very low or absent nAbs. Neutralization of Delta and Omicron BA.1 VOCs showed significant reduction of nAbs respect to WT strain. Importantly, booster doses enhanced Omicron BA.1 nAbs, with persistent levels at 3 months from boosting. Conclusions: PVNA is a reliable tool for assessing anti-SARS-CoV-2 nAbs helping the establishment of a correlate of protection and the management of vaccination strategies.

External quality assessment of SARS-CoV-2 serology in European expert laboratories, April 2021.

BackgroundCountries worldwide are focusing to mitigate the ongoing SARS-CoV-2 pandemic by employing public health measures. Laboratories have a key role in the control of SARS-CoV-2 transmission. Serology for SARS-CoV-2 is of critical importance to support diagnosis, define the epidemiological framework and evaluate immune responses to natural infection and vaccine administration.AimThe aim of this study was the assessment of the actual capability among laboratories involved in sero-epidemiological studies on COVID-19 in EU/EEA and EU enlargement countries to detect SARS-CoV-2 antibodies through an external quality assessment (EQA) based on proficiency testing.MethodsThe EQA panels were composed of eight different, pooled human serum samples (all collected in 2020 before the vaccine roll-out), addressing sensitivity and specificity of detection. The panels and two EU human SARS-CoV-2 serological standards were sent to 56 laboratories in 30 countries.ResultsThe overall performance of laboratories within this EQA indicated a robust ability to establish past SARS-CoV-2 infections via detection of anti-SARS-CoV-2 antibodies, with 53 of 55 laboratories using at least one test that characterised all EQA samples correctly. IgM-specific test methods provided most incorrect sample characterisations (24/208), while test methods detecting total immunoglobulin (0/119) and neutralising antibodies (2/230) performed the best. The semiquantitative assays used by the EQA participants also showed a robust performance in relation to the standards.ConclusionOur EQA showed a high capability across European reference laboratories for reliable diagnostics for SARS-CoV-2 antibody responses. Serological tests that provide robust and reliable detection of anti-SARS-CoV-2 antibodies are available.

Monkeypox 2022 outbreak in non-endemic countries: Open questions relevant for public health, nonpharmacological intervention and literature review.

Starting from mid-May 2022, cases of human monkeypox started to rise in several non-endemic countries. By mid-July, more than 17000 confirmed/suspect cases have been reported by at least 82 countries worldwide, with a regular incremental trend. In order to contain the disease diffusion, risk evaluation is crucial to undertake informed decisions and effective communication campaigns. However, since orthopoxvirus infections so far have attracted low attention, due to the eradication of smallpox 40 years ago, and to the confinement of human monkeypox almost exclusively to endemic areas, several unresolved issues concerning natural history, ecology and pathogenesis remain. To this respect, we identified some open questions and reviewed the relevant literature on monkeypoxvirus and/or related orthopoxviruses. The results will be discussed in the perspective of their relevance to public health decisions, particularly those related to non-pharmacological interventions.

Safety and immune response kinetics of GRAd-COV2 vaccine: phase 1 clinical trial results.

Despite the successful deployment of efficacious vaccines and therapeutics, the development of novel vaccines for SARS-CoV-2 remains a major goal to increase vaccine doses availability and accessibility for lower income setting. We report here on the kinetics of Spike-specific humoral and T-cell response in young and old volunteers over 6 months follow-up after a single intramuscular administration of GRAd-COV2, a gorilla adenoviral vector-based vaccine candidate currently in phase-2 of clinical development. At all three tested vaccine dosages, Spike binding and neutralizing antibodies were induced and substantially maintained up to 3 months, to then contract at 6 months. Potent T-cell responses were readily induced and sustained throughout the study period, with only minor decline. No major differences in immune response to GRAd-COV2 vaccination were observed in the two age cohorts. In light of its favorable safety and immunogenicity, GRAd-COV2 is a valuable candidate for further clinical development and potential addition to the COVID-19 vaccine toolbox to help fighting SARS-CoV-2 pandemic.

Coordinated innate and T-cell immune responses in mild COVID-19 patients from household contacts of COVID-19 cases during the first pandemic wave.

Objective: To better define the immunopathogenesis of COVID-19, the present study aims to characterize the early immune responses to SARS-CoV-2 infection in household contacts of COVID-19 cases. In particular, innate, T- and B-cell specific responses were evaluated over time. Methods: Household contacts of COVID-19 cases screened for SARS-CoV-2 infection by nasopharyngeal swab for surveillance purposes were enrolled (T0, n=42). Of these, 28 subjects returned for a follow-up test (T1). The innate response was assessed by detecting a panel of soluble factors by multiplex-technology in plasma samples. Cell-mediated response was evaluated by measuring interferon (IFN)-γ levels by ELISA in plasma harvested from whole-blood stimulated with SARS-CoV-2 peptide pools, including spike (S), nucleocapsid (N) and membrane (M) proteins. The serological response was assessed by quantifying anti-Receptor-Binding-Domain (RBD), anti-Nucleocapsid (N), whole virus indirect immunofluorescence, and neutralizing antibodies. Results: At T0, higher levels of plasmatic IFN-α, IL-1ra, MCP-1 and IP-10, and lower levels of IL-1ß, IL-9, MIP-1ß and RANTES were observed in subjects with positive swab compared to individuals with a negative one (p<0.05). Plasmatic IFN-α was the only cytokine detectable in subjects with positive SARS-CoV-2 swabs with high accuracy for swab score positivity (0.93, p<0.0001). Among subjects with positive swabs, significant negative correlations were found among the RT-PCR cycle threshold values reported for genes S and N and IFN-α or IP-10 levels. At T0, the IFN-γ T-cell specific response was detected in 50% (5/10) of subjects with positive swab, while anti-RBD/anti-N antibodies showed a positivity rate of 10% (1/10). At T1, the IFN-γ T-cell specific response was detected in most of the confirmed-infection subjects (77.8%, 7/9), whereas the serological response was still observed in a minority of them (44.4%, 4/9). Overall, the swab test showed a moderate concordance with the T-cell response (78.6%, k=0.467), and a scarce concordance with the serological one (72.9%, k=0.194). Conclusions: Plasmatic IFN-α and the IFN-γ T-cell specific response appear early even in the absence of seroconversion, and show a greater positivity rate than the serological response in household contacts with positive swab.