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Although antibody levels progressively decrease following SARS-CoV-2 infection, the immune memory persists for months. Thus, individuals who naturally contracted SARS-CoV-2 are expected to develop a more rapid and sustained response to COVID-19 vaccines than naïve individuals. In this study, we analyzed the dynamics of the antibody response to the BNT162b2 mRNA COVID-19 vaccine in six healthcare workers who contracted SARS-CoV-2 in March 2020, in comparison to nine control subjects without a previous infection. The vaccine was well tolerated by both groups, with no significant difference in the frequency of vaccine-associated side effects, with the exception of local pain, which was more common in previously infected subjects. Overall, the titers of neutralizing antibodies were markedly higher in response to the vaccine than after natural infection. In all subjects with pre-existing immunity, a rapid increase in anti-spike receptor-binding domain (RBD) IgG antibodies and neutralizing antibody titers was observed one week after the first dose, which seemed to act as a booster. Notably, in previously infected individuals, neutralizing antibody titers 7 days after the first vaccine dose were not significantly different from those observed in naïve subjects 7 days after the second vaccine dose. These results suggest that, in previously infected people, a single dose of the vaccine might be sufficient to induce an effective response.

Droplet digital PCR (ddPCR) is a sensitive and reproducible technology widely used for quantitation of several viruses. The aim of this study was to evaluate the 2019-nCoV CDC ddPCR Triplex Probe Assay (BioRad) performance, comparing the direct quantitation of SARS-CoV-2 on nasopharyngeal swab with the procedure applied to the extracted RNA. Moreover, two widely used swab types were compared (UTM 3 mL and ESwab 1 mL, COPAN). A total of 50 nasopharyngeal swabs (n = 25 UTM 3 mL and n = 25 ESwab 1 mL) from SARS-CoV-2 patients, collected during the pandemic at IRCCS Sacro Cuore Don Calabria Hospital (Veneto Region, North-East Italy), were used for our purpose. After heat inactivation, an aliquot of swab medium was used for the direct quantitation. Then, we compared the direct method with the quantitation performed on the RNA purified from nasopharyngeal swab by automated extraction. We observed that the direct approach achieved generally equal RNA copies compared to the extracted RNA. The results with the direct quantitation were more accurate on ESwab with a sensitivity of 93.33% [95% CI, 68.05 to 99.83] and specificity of 100.00% for both N1 and N2. On the other hand, on UTM we observed a higher rate of discordant results for N1 and N2. The human internal amplification control (RPP30) showed 100% of both sensitivity and specificity independent of swabs and approaches. In conclusion, we described a direct quantitation of SARS-CoV-2 in nasopharyngeal swab. Our approach resulted in an efficient quantitation, without automated RNA extraction and purification. However, special care needs to be taken on the potential bias due to the conservation of samples and to the heating treatment, as we used thawed and heat inactivated material. Further studies on a larger cohort of samples are warranted to evaluate the clinical value of this direct approach.

Strongyloides stercoralis infection is a neglected tropical disease which can lead to severe symptoms and even death in immunosuppressed people. Unfortunately, its diagnosis is hampered by the lack of a gold standard, as the sensitivity of traditional parasitological tests (including microscopic examination of stool samples and coproculture) is low. Hence, alternative diagnostic methods, such as molecular biology techniques (mostly polymerase chain reaction, PCR) have been implemented. However, there are discrepancies in the reported accuracy of PCR. A systematic review with meta-analysis was conducted in order to evaluate the accuracy of PCR for the diagnosis of S. stercoralis infection. The protocol was registered with PROSPERO International Prospective Register of Systematic Reviews (record: CRD42016054298). Fourteen studies, 12 of which evaluating real-time PCR, were included in the analysis. The specificity of the techniques resulted high (ranging from 93 to 95%, according to the reference test(s) used). When all molecular techniques were compared to parasitological methods, the sensitivity of PCR was assessed at 71.8% (95% CI 52.2-85.5), that decreased to 61.8% (95% CI 42.0-78.4) when serology was added among the reference tests. Similarly, sensitivity of real-time PCR resulted 64.4% (95% CI 46.2-77.7) when compared to parasitological methods only, 56.5% (95% CI 39.2-72.4) including serology. PCR might not be suitable for screening purpose, whereas it might have a role as a confirmatory test.

SARS-CoV-2 induces a spectrum of clinical conditions ranging from asymptomatic infection to life threatening severe disease. Host microRNAs have been involved in the cytokine storm driven by SARS-CoV-2 infection and proposed as candidate biomarkers for COVID-19. To discover signatures of circulating miRNAs associated with COVID-19, disease severity and mortality, small RNA-sequencing was performed on serum samples collected from 89 COVID-19 patients (34 severe, 29 moderate, 26 mild) at hospital admission and from 45 healthy controls (HC). To search for possible sources of miRNAs, investigation of differentially expressed (DE) miRNAs in relevant human cell types . COVID-19 patients showed upregulation of miRNAs associated with lung disease, vascular damage and inflammation and downregulation of miRNAs that inhibit pro-inflammatory cytokines and chemokines, angiogenesis, and stress response. Compared with mild/moderate disease, patients with severe COVID-19 had a miRNA signature indicating a profound impairment of innate and adaptive immune responses, inflammation, lung fibrosis and heart failure. A subset of the DE miRNAs predicted mortality. In particular, a combination of high serum miR-22-3p and miR-21-5p, which target antiviral response genes, and low miR-224-5p and miR-155-5p, targeting pro-inflammatory factors, discriminated severe from mild/moderate COVID-19 (AUROC 0.88, 95% CI 0.80-0.95, p<0.0001), while high leukocyte count and low levels of miR-1-3p, miR-23b-3p, miR-141-3p, miR-155-5p and miR-4433b-5p predicted mortality with high sensitivity and specificity (AUROC 0.95, 95% CI 0.89-1.00, p<0.0001). experiments showed that some of the DE miRNAs were modulated directly by SARS-CoV-2 infection in permissive lung epithelial cells. We discovered circulating miRNAs associated with COVID-19 severity and mortality. The identified DE miRNAs provided clues on COVID-19 pathogenesis, highlighting signatures of impaired interferon and antiviral responses, inflammation, organ damage and cardiovascular failure as associated with severe disease and death.

Background: Public health measures for COVID-19 mitigation influenced the circulation of Respiratory Syncytial Virus (RSV) during the 2020–2021 winter season. In the following autumn, an unprecedented resurgence of RSV occurred. Our study monitored RSV pediatric infections one and two years after the relaxation of containment measures for the COVID-19 pandemic. Methods: We analyzed diagnostic molecular data for SARS-CoV-2, flu, and RSV infections and clinical data from children with respiratory symptoms referring to our hospital during the 2021–2022 and 2022–2023 seasons. Results: In the 2021–2022 season, the number of RSV-affected children was very high, especially for babies <1 year. The outbreak appeared in a shorter interval of time, with a high clinical severity. In the 2022–23 season, a reduced number of infected pediatric patients were detected, with a similar hospitalization rate (46% vs. 40%), and RSV accounted for 12% of the infections. Coinfections were observed in age <2 years. In RSV patients, symptoms were similar across the two seasons. Conclusions: The clinical presentation of RSV in the two post-COVID seasons suggests that the pathophysiology of the virus did not change across these two years. Further studies are needed to continuously monitor RSV to support an effective prevention strategy.

The host immune response has a prominent role in the progression and outcome of SARS-CoV-2 infection. Lymphopenia has been described as an important feature of SARS-CoV-2 infection and has been associated with severe disease manifestation. Lymphocyte dysregulation and hyper-inflammation have been shown to be associated with a more severe clinical course; however, a T cell subpopulation whose dysfunction correlate with disease progression has yet to be identify. We performed an immuno-phenotypic analysis of T cell sub-populations in peripheral blood from patients affected by different severity of COVID-19 (n=60) and undergoing a different clinical evolution. Clinical severity was established based on a modified WHO score considering both ventilation support and respiratory capacity (PaO2/FiO2 ratio). The ability of circulating cells at baseline to predict the probability of clinical aggravation was explored through multivariate regression analyses. The immuno-phenotypic analysis performed by multi-colour flow cytometry confirmed that patients suffering from severe COVID-19 harboured significantly reduced circulating T cell subsets, especially for CD4 T, Th1, and regulatory T cells. Peripheral T cells also correlated with parameters associated with disease severity, i.e., PaO2/FiO2 ratio and inflammation markers. CD4 T cell subsets showed an important significant association with clinical evolution, with patients presenting markedly decreased regulatory T cells at baseline having a significantly higher risk of aggravation. Importantly, the combination of gender and regulatory T cells allowed distinguishing between improved and worsened patients with an area under the ROC curve (AUC) of 82%. The present study demonstrates the association between CD4 T cell dysregulation and COVID-19 severity and progression. Our results support the importance of analysing baseline regulatory T cell levels, since they were revealed able to predict the clinical worsening during hospitalization. Regulatory T cells assessment soon after hospital admission could thus allow a better clinical stratification and patient management.

Extracellular vesicles (EVs) from human body fluids are valuable tools for biomarker discovery and for exploring the mechanisms underlying various pathologies, including infectious diseases. The translation of EV research into clinical practice is however hindered by the variability in EV pre-clinical investigations. Therefore, standardisation of analytical procedures and reporting policies is essential. Human serum is a key biological matrix for biomarker discovery and is commonly stored within biobanks. Here, we investigated different strategies for EV enrichment from small volumes of biobanked serum and evaluated their impact on EVs’ downstream analyses. EVs were obtained from 250 μL of biobanked serum using ultracentrifugation (UC), size-exclusion chromatography-based methods (ExoSpin-ES, qEV1-35 nm, and qEV1-70 nm), or ExoRNeasy (ER). The resulting EVs were subsequently characterised for morphology, concentration, surface phenotype, and multi-omics profiles. All methods successfully enriched small EVs expressing tetraspanins on their surface, although at different concentrations. The most efficient method for proteomics analyses was qEV1-70 nm, followed by ES, which was more susceptible to contamination by serum proteins. EV-miRNA cargo was effectively profiled in UC-, ES-, and ER-EVs, with the latter providing the broadest miRNA coverage. Our results support the feasibility of using biobanked serum for EV-based research and further highlight the importance of selecting appropriate EV enrichment methods, since they influence both miRNA and protein cargo characterisation.

On 27 May 2024, the Cuban Ministry of Health reported the first outbreak of Oropouche fever on the island. The etiologic agent, Oropouche virus (OROV), is a poorly understood arbovirus that has been known since the 1960s and represents a public health burden in Latin America. We report the whole-genome characterization of the first European OROV isolate from a returning traveler from Cuba with Oropouche fever-like symptoms. The isolate was obtained from the patient’s serum; whole-genome sequencing was performed by next-generation sequencing, followed by phylogenetic analysis and genetic variability studies. The analysis showed that the most closely related sequence was from the French Guiana 2020 outbreak. Interestingly, our isolate is a reassortant virus, included in a highly supported monophyletic clade containing recent OROV cases (Brazil 2015–Colombia 2021), separated from the other four previously known genotypes. More deeply, it was found to be included in a distinct branch containing the sequences of the Brazil 2022–2024 outbreak. The reassortment event involved the S and L segments, which have high similarity with sequences belonging to a new cluster (here defined as OROV_SCDC_2024), while the M segment shows high similarity with older sequences. These results likely describe the viral strain responsible for the current outbreak in Cuba, which may also reflect the ongoing outbreak in Latin America. Further studies are needed to understand how OROV evolves towards traits that facilitate its spread and adaptation outside its original basin, and to track its spread and evolution in the European continent.

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.

Data from recent studies support the hypothesis that infections by human gastrointestinal (GI) helminths impact, directly and/or indirectly, on the composition of the host gut microbial flora. However, to the best of our knowledge, these studies have been conducted in helminth-endemic areas with multi-helminth infections and/or in volunteers with underlying gut disorders. Therefore, in this study, we explore the impact of natural mono-infections by the human parasite Strongyloides stercoralis on the faecal microbiota and metabolic profiles of a cohort of human volunteers from a non-endemic area of northern Italy ( S +), pre- and post-anthelmintic treatment, and compare the findings with data obtained from a cohort of uninfected controls from the same geographical area ( S− ). Analyses of bacterial 16S rRNA high-throughput sequencing data revealed increased microbial alpha diversity and decreased beta diversity in the faecal microbial profiles of S + subjects compared to S− . Furthermore, significant differences in the abundance of several bacterial taxa were observed between samples from S + and S− subjects, and between S + samples collected pre- and post-anthelmintic treatment. Faecal metabolite analysis detected marked increases in the abundance of selected amino acids in S + subjects, and of short chain fatty acids in S− subjects. Overall, our work adds valuable knowledge to current understanding of parasite-microbiota associations and will assist future mechanistic studies aimed to unravel the causality of these relationships.