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With numbers of COVID-19 cases having substantially increased at the end of 2022 in China, some countries have started or expanded testing and genomic surveillance of travellers. We report screening results in Italy in late December 2022 of 556 flight passengers in provenance from two Chinese provinces. Among these passengers, 126 (22.7%) tested SARS-CoV-2 positive. Whole genome sequencing of 61 passengers’ positive samples revealed Omicron variants, notably sub-lineages BA.5.2.48, BF.7.14 and BQ.1.1, in line with data released from China.

The SARS-CoV-2 variant of concern (VOC) “Delta” is currently defined by PANGOLIN as a cluster of 33 different AY sublineages. Delta (in particular B.1.617.2) is largely and rapidly replacing the Alpha VOC as the dominant clade in most countries. To date, variations in the Spike protein of the Delta VOC have largely been limited. We report here the results of a genomic surveillance programme from Northern Italy. We identified several Delta sublineages harbouring mutations previously reported in GISAID at extremely low frequencies and in different combinations. Two patients (one of them vaccinated) tested positive for a Delta sublineage harbouring S71F, T250I, T572I and K854N. More patients tested positive for G769 V plus C1248F, A352S, and R158G and C1248F, respectively. Genomic surveillance of Delta variants should be encouraged to anticipate immune escape and deploy countermeasures.

The Delta variant of concern of severe acute respiratory syndrome coronavirus 2 is dominant worldwide. We report a case cluster caused by Delta sublineage B.1.617.2 harboring the mutation E484K in Italy during July 11-July 29, 2021. This mutation appears to affect immune response and vaccine efficacy; monitoring its appearance is urgent.

Aims/hypothesis Enterovirus (EV) infection of pancreatic islet cells is one possible factor contributing to type 1 diabetes development. We have reported the presence of EV genome by PCR and of EV proteins by immunohistochemistry in pancreatic sections. Here we explore multiple human virus species in the Diabetes Virus Detection (DiViD) study cases using innovative methods, including virus passage in cell cultures. Methods Six recent-onset type 1 diabetes patients (age 24–35) were included in the DiViD study. Minimal pancreatic tail resection was performed under sterile conditions. Eleven live cases (age 43–83) of pancreatic carcinoma without diabetes served as control cases. In the present study, we used EV detection methods that combine virus growth in cell culture, gene amplification and detection of virus-coded proteins by immunofluorescence. Pancreas homogenates in cell culture medium were incubated with EV-susceptible cell lines for 3 days. Two to three blind passages were performed. DNA and RNA were extracted from both pancreas tissue and cell cultures. Real-time PCR was used for detecting 20 different viral agents other than EVs (six herpesviruses, human polyomavirus [BK virus and JC virus], parvovirus B19, hepatitis B virus, hepatitis C virus, hepatitis A virus, mumps, rubella, influenza A/B, parainfluenza 1–4, respiratory syncytial virus, astrovirus, norovirus, rotavirus). EV genomes were detected by endpoint PCR using five primer pairs targeting the partially conserved 5′ untranslated region genome region of the A, B, C and D species. Amplicons were sequenced. The expression of EV capsid proteins was evaluated in cultured cells using a panel of EV antibodies. Results Samples from six of six individuals with type 1 diabetes (cases) and two of 11 individuals without diabetes (control cases) contained EV genomes (p Conclusions/interpretation Sensitive assays confirm that the pancreases of all DiViD cases contain EVs but no other viruses. Analogous EV strains have been found in pancreases of two of 11 individuals without diabetes. The detected EV strains can be passaged in series from one cell culture to another in the form of poorly replicating live viruses encoding antigenic proteins recognised by multiple EV-specific antibodies. Thus, the early phase of type 1 diabetes is associated with a low-grade infection by EVs, but not by other viral agents. Graphical abstract

We report here an imported case of SARS-CoV-2 variant of concern B.1.1.351 (also known as 20H/501Y.V2 or “South African variant” or VOC 202012/02) in a 66-years old symptomatic male who returned from Malawi to Italy.

SARS-CoV-2 variants are usually a consequence of random mutations in humans or other hosts, but accelerated evolution can also occur under selective pressure from therapeutic interventions with neutralizing antibodies1. Bamlanivimab has been recently withdrawn from the vendor as a monotherapy because of failure against E484K-positive SARS-CoV-2 variants, but emergency use authorization remains in place for the bamlanivimab/etesevimab cocktail2, for which no completely resistant variant has been reported to date. We report here the first in vivo case of a Spike escape mutation conferring combined resistance to both bamlanivimab and etesevimab.

To date only one case of SARS-COV-2 B.1.1.7 reinfection has been reported. We report here two more such reinfection cases in Lombardy residents.

Antibody-dependent enhancement (ADE) of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) infection has been hypothesized. However, to date, there has been no in vitro or in vivo evidence supporting this. Cross-reactivity exists between SARS CoV-2 and other Coronaviridae for both cellular and humoral immunity. We show here that IgG against nucleocapsid protein of alphacoronavirus NL63 and 229E correlate with the World Health Organization’s (WHO) clinical severity score ≥ 5 (incidence rate ratios was 1.87 and 1.80, respectively, and 1.94 for the combination). These laboratory findings suggest possible ADE of SARS CoV-2 infection by previous alphacoronavirus immunity.

Background: Hashimoto’s thyroiditis and Graves’ disease are autoimmune thyroid disorders (AITD) of unknown origin. Enterovirus (EV) infection of thyroid cells has been implicated as a possible initiator of cell damage and of organ-specific autoimmunity. We asked whether persistent infection of human epithelial cells with EV strains obtained from thyroid tissue of AITD patients could be associated with transcriptional changes capable of fostering immunopathology. Methods: EV isolates obtained from thyroid tissue of AITD cases were used to infect the AV3 epithelial cell line. AV3 cells incubated with a virus-free medium from thyroid tissue of subjects without evidence of thyroid autoimmunity were used as uninfected controls. Transcripts of immune-related genes were compared in infected vs. uninfected cells. Results: The EV genome and antigens were detected only in the cells exposed to AITD-derived virus isolates, not in control cells. Persistent EV infection, while suppressing transcription of several type I IFN and cytokine determinants, was associated with enhanced transcription of NFKB1/RELA, IFNAR1, JAK1/STAT1, i.e., the determinants that play key immunologic roles. Infection also led to upregulation of the CCL2 chemokine and the IL-18 pro-inflammatory interleukin. Conclusion: As in the case of EV strains obtained from autoimmune diabetes, results show that the EV strains that are present in the thyroid of AITD cases do repress IFN and cytokine pathways. JAK1/STAT1 upregulation supports activation of TLR pathways and aberrant T cell signaling. In the early phases of AITD, our results highlight the potential benefit of interventions aimed at blocking the viral infection and easing the inflammatory response.

Importance Since February 2020, coronavirus disease 2019 (COVID-19) has spread rapidly all over the world, with an epidemiological cluster in Lombardy, Italy. The viral communicability may be mediated by various body fluids, but insufficient information is available on the presence of the virus in human tears. Objectives To investigate the rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in tears collected from patients with COVID-19 by means of real-time reverse transcriptase–polymerase chain reaction (rRT-PCR) assay and to assess the association of virus presence with concomitant clinical conditions. Design, Setting, and Participants Cross-sectional study conducted between April 9 and May 5, 2020. The setting was intensive care units at Azienda Socio-Sanitaria Territoriale (ASST) Sette-Laghi Hospital, University of Insubria, in Varese, Lombardy, Italy. A conjunctival swab was performed in 91 patients hospitalized for COVID-19, which was clinically diagnosed by rRT-PCR assay on nasopharyngeal swabs and by radiological imaging. Conjunctival swabs from 17 additional healthy volunteer participants with no symptoms of COVID-19 were examined to evaluate the availability and applicability of the conjunctival swab test. Exposure SARS-CoV-2 detection by means of rRT-PCR assay performed on the collected samples obtained by conjunctival swabs. Main Outcomes and Measures Conjunctival swab and nasopharyngeal swab results are reported, as well as demographic and clinical data. Results A total of 108 participants (mean [SD] age, 58.7 [14.2] years; 55 female and 53 male) were tested for SARS-CoV-2 using rRT-PCR assay, including 91 patients hospitalized with COVID-19 and 17 were healthy volunteers. SARS-CoV-2 was found on the ocular surface in 52 of 91 patients with COVID-19 (57.1%; 95% CI, 46.3%-67.5%), with a wide variability in the mean viral load from both eyes. Among a subset of 41 patients, concordance of 63.0% (95% CI, 41.0%-81.0%) was found between positive conjunctival and nasopharyngeal swab test results when performed within 2 days of each other. In 17 of these patients, nasopharyngeal swab results were negative for SARS-CoV-2. In 10 of these 17 patients, conjunctival swab results were positive for the virus. Conclusions and Relevance In this study, SARS-CoV-2 RNA was found on the ocular surface in a large part of this cohort of patients with COVID-19, although the infectivity of this material could not be determined. Because patients may have positive test results with a conjunctival swab and negative results with a nasopharyngeal swab, use of the slightly invasive conjunctival swab may be considered as a supplementary diagnostic test.

We report 11 cases of SARS-CoV-2 infection in healthcare workers (HCW) naïve for COVID-19 and seropositive after the second dose of the BNT162b2 mRNA vaccine. Based on voluntary-based surveillance, they tested positive for different strains of SARS-CoV-2, as Spike gene sequencing showed. Five of them reported mild symptoms. Given the risk for SARS-CoV-2 introduction from asymptomatic vaccinees, this case series suggests the need to continue nasopharyngeal screening programmes.

Only 4 months after the beginning of SARS-CoV-2 epidemic, the world is facing a global pandemic due to a complex and insidious virus that today constantly poses new challenges. In this study, we highlight a persistent shedding of SARS-CoV-2 RNA into the urine, even in patients with a negative nasopharyngeal swab and in patients considered recovered. What does it mean? Besides the fact that the kidney is a probable site of viral replication, the prolonged viral excretion is a matter of great concern for our drainage system contamination.

Intestinal ischemia/reperfusion (I/R) injury has severe consequences on myenteric neurons, which can be irreversibly compromised resulting in slowing of transit and hindered food digestion. Myenteric neurons synthesize hyaluronan (HA) to form a well-structured perineuronal net, which undergoes derangement when myenteric ganglia homeostasis is perturbed, i.e. during inflammation. In this study we evaluated HA involvement in rat small intestine myenteric plexus after in vivo I/R injury induced by clamping a branch of the superior mesenteric artery for 60 min, followed by 24 h of reperfusion. In some experiments, 4-methylumbelliferone (4-MU, 25 mg/kg), a HA synthesis inhibitor, was intraperitoneally administered to normal (CTR), sham-operated (SH) and I/R animals for 24 h. In longitudinal muscle myenteric plexus (LMMP) whole-mount preparations, HA binding protein staining as well as HA levels were significantly higher in the I/R group, and were reduced after 4-MU treatment. HA synthase 1 and 2 (HAS1 and HAS2) labelled myenteric neurons and mRNA levels in LMMPs increased in the I/R group with respect to CTR, and were reduced by 4-MU. The efficiency of the gastrointestinal transit was significantly reduced in I/R and 4-MU-treated I/R groups with respect to CTR and SH groups. In the 4-MU-treated I/R group gastric emptying was reduced with respect to the CTR, SH and I/R groups. Carbachol (CCh) and electrical field (EFS, 0.1–40 Hz) stimulated contractions and EFS-induced (10 Hz) NANC relaxations were reduced in the I/R group with respect to both CTR and SH groups. After I/R, 4-MU treatment increased EFS contractions towards control values, but did not affect CCh-induced contractions. NANC on-relaxations after I/R were not influenced by 4-MU treatment. Main alterations in the neurochemical coding of both excitatory (tachykinergic) and inhibitory pathways (iNOS, VIPergic) were also observed after I/R, and were influenced by 4-MU administration. Overall, our data suggest that, after an intestinal I/R damage, changes of HA homeostasis in specific myenteric neuron populations may influence the efficiency of the gastrointestinal transit. We cannot exclude that modulation of HA synthesis in these conditions may ameliorate derangement of the enteric motor function preventing, at least in part, the development of dysmotility.

Highlights • Saliva is a reliable tool to detect SARS-Cov-2 by RT-rPCR analysis. • Saliva may provide information about the clinical evolution of the disease. • Saliva could represent a valid instrument in COVID-19 diagnosis. • Patients should be checked for salivary viral load at hospital discharge., Summary Objectives This study analyzed salivary samples of COVID-19 patients and compared the results with their clinical and laboratory data. Methods Salivary samples of 25 COVID-19 patients were analyzed by rRT-PCR. The following data were collected: age, sex, comorbidities, drugs. Lactate dehydrogenase (LDH) and ultrasensitive reactive C protein (usRCP) values were registered on the same day when a salivary swab was collected. Prevalence of positivity in saliva and association between clinical data and the cycle threshold as a semiquantitative indicator of viral load were considered. Results Twenty-five subjects were recruited into this study, 17 males and 8 females. The mean age was 61.5 +/− 11.2 years. Cardiovascular and/or dysmetabolic disorders were observed in 65.22% of cases. All the samples tested positive for the presence of SARS-CoV-2, while there was an inverse association between LDH and Ct values. Two patients showed positive salivary results on the same days when their pharyngeal or respiratory swabs showed conversion. Conclusions Saliva is a reliable tool to detect SARS-CoV-2. The role of saliva in COVID-19 diagnosis could not be limited to a qualitative detection of the virus, but it may also provide information about the clinical evolution of the disease.

Background The novel coronavirus has a high mortality rate (over 1% for patients older than 50 years). This can only be partially ascribed to other comorbidities. A possible explanation is a factor that assures a prompt response to SARS-CoV-2 in younger people, independent from the novelty of the virus itself. A factor is believed to stimulate the immune system and provide immunity against more antigens. The only external stimulation received by healthy people is vaccination (eg, the diphtheria, tetanus, and pertussis [DTP] vaccine). One hypothesis is that vaccination helps develop specific immunity but generates sprouting immunity against antigens in transit. The underlying immunological phenomena are the “bystander effect” and “trained immunity.” The developed immunity gives protection for years until it naturally fades out. After the fifth decade of life, the immune system is almost incompetent when a viral infection occurs, and thus, at this stage, the novel coronavirus can enter the body and cause acute respiratory distress syndrome. Objective The initial aim is to demonstrate that blood monocytes and natural killer cells show overpowering hyperactivity, while CD4+ and CD8+ T cells experience impediments to their defensive functions in patients with severe SARS-CoV-2 infection. The secondary objectives are to correlate clinical data and vaccination history with laboratory immune patterns in order to identify protective factors. Subsequently, we are also interested in characterizing the phenotypes and state of the degree of activation of peripheral blood mononuclear cells, including monocytes, natural killer cells, and CD4+ and CD8+ T cells, in healthy subjects vaccinated with the Pfizer vaccine. Methods Data will be collected using the following 3 approaches: (1) an experimental analysis to study the innate immune response and to identify genetic profiles; (2) an epidemiological analysis to identify the patients’ vaccination history; and (3) a clinical analysis to detect the immunological profile. Results The protocol was approved by the Ethics Committee on April 16, 2020, and the study started on April 27, 2020. As of February 2021, enrollment has been completed. Immunological analysis is ongoing, and we expect to complete this analysis by December 2022. Conclusions We will recognize different populations of patients, each one with a specific immunological pattern in terms of cytokines, soluble factor serum levels, and immune cell activity. Anamnestic data, such as preceding vaccinations and comorbidities, biochemical findings like lymphocyte immunophenotyping, and pre-existing persistent cytomegalovirus infection, allow depicting the risk profile of severe COVID-19. Proof of the roles of these immunological phenomena in the development of COVID-19 can be the basis for the implementation of therapeutic immunomodulatory treatments. Trial Registration ClinicalTrials.gov NCT04375176; https://clinicaltrials.gov/ct2/show/NCT04375176 International Registered Report Identifier (IRRID) DERR1-10.2196/29892

Background Typically, hand-foot-and-mouth disease (HFMD) is a mild childhood illness associated with coxsackievirus (CV)-A16, CV-A6, enterovirus (EV)-A71. Objectives To identify the viral agents associated with severe cases of atypical HFMD in Italy. Study design Epidemiologically unrelated cases of severe atypical HFMD admitted to the Emergency Room (ER) of IRCCS San Martino IST (Genoa, Italy) in 2014–2016 were investigated. Serologic screening for viral positivity was performed against exanthem-inducing agents. Ten cases with serology indicative of recent EV infection were selected. Molecular assays were used to detect viral genomes in blood [EVs, Parvovirus B19 (PVB19), herpesviruses (CMV; EBV, HHV-6, -7, -8)]. Results CV-A6 was detected in 10 cases of severe atypical HFMD. Two cases were also infected with PVB19. Herpesviruses were not detected. Phylogenetic analysis mapped the CV-A6 strains into a single cluster related to two recent isolates from a German and an Asian child. Fever, systemic symptoms, severe vasculitis-like rash, and enanthem were predominant at presentation. Spontaneous recovery occurred in 1–3 weeks. Conclusions CV-A6 is emerging as a frequent cause of severe atypical HFMD in Italian adults. This viral agent is disseminating worldwide. Dermatologists must identify the manifold alterations caused by EVs and understand the diagnostic power of current virology methods.

At the time of the clinical onset of type 1 diabetes (T1D), we investigated 82 pediatric cases in parallel with 117 non-diabetic controls matched by age, geographic area, and time of collection. The occurrence of an enteroviral infection was evaluated in peripheral blood using a sensitive method capable of detecting virtually all human enterovirus (EV) types. While non-diabetic controls were consistently EV-negative, 65% of T1D cases carried EVs in blood. The vitamin D status was assessed by measuring the concentration of 25-hydroxyvitamin D [25(OH)D] in serum. Levels of 25(OH)D were interpreted as deficiency (≤50 nmol/L), insufficiency (52.5-72.5 nmol/L), and sufficiency (75-250 nmol/L). In T1D cases, the median serum concentration of 25(OH)D was 54.4 ± 27.3 nmol/L vs 74.1 ± 28.5 nmol/L in controls (P = .0001). Diabetic children/adolescents showed deficient levels of vitamin D 25(OH)D (ie, 72.5 nmol/L) in 48.8% cases vs 17.9% in non-diabetic controls (P = .0001). Unexpectedly, the median vitamin D concentration was significantly reduced in virus-positive vs virus-negative diabetics (48.2 ± 22.5 vs 61.8 ± 31.2 nmol/L; P = .015), with deficient levels in 58.5% vs 31.0%, respectively. Thus, at the time of clinical onset, EV-positive cases had reduced vitamin D levels compared with EV-negative cases. This could indicate either that the virus-negative children/adolescents had been hit by a non-infectious T1D-triggering event, or that children/adolescents with proper levels of vitamin D had been able to rapidly clear the virus. Thus, it would be important to assess whether adequate vitamin D supplementation before or during the prediabetic phase of T1D may counteract the diabetogenic potential of infectious pathogens.

Acute promyelocytic leukemia (APL) is a rare subtype of acute myeloid leukemia (AML) and constitutes 5–8% of all AML cases. About 98% of APL patients exhibits the specific chromosomal translocation...

Several forms of sensorineural hearing loss (SNHL) have been imputated to connexins mutations and prevalently to connexin 26 (Cx26), codified by the GJB2 gene (gap junction protein, beta 2). Here, we report the first familiar case (heterozygous p. G130V mutation) of non-syndromic (without any dermatological manifestation) dominant profound SNHL. Proband was a 6-years-old male with post-lingual bilateral profound SNHL, clinically identified at the age of 3 with diagnosis of severe SNHL. We confirm that the p. G130V variant of the GJB2 gene is causative of autosomal dominant form of SNHL, although it is not always associated with the presence of skin diseases.

Human enteroviruses (EVs) comprise >100 different types. Research suggests a non-chance association between EV infections and type 1 diabetes. Immunohistochemical studies with the anti-EV antibody 5D-8.1 have shown that the EV capsid antigen is present in pancreatic islet cells of diabetic subjects. When it was noticed that 5D-8.1 may cross-react with human proteins, doubt was casted on the significance of the above histopathologic findings. To address this issue, properties of EV antibodies 5D-8.1 and 9D5 have been investigated using peptide microarrays, peptide substitution scanning, immunofluorescence of EV-infected cells, EV neutralization assays, bioinformatics analysis. Evidence indicates that the two antibodies bind to distinct non-neutralizing linear epitopes in VP1 and are specific for a vast spectrum of EV types (not for other human viruses). However, their epitopes may align with a few human proteins at low expected values. When tested by immunofluorescence, high concentrations of 5D-8.1 yelded faint cytoplasmic staining in uninfected cells. At reduced concentrations, both antibodies produced dotted staining only in the cytoplasm of infected cells and recognized both acute and persistent EV infection. Thus, the two monoclonals represent distinct and independent probes for hunting EVs in tissues of patients with diabetes or other endocrine conditions.