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Quantification of Torquetenovirus (TTV) viremia is becoming important for evaluating the status of the immune system in solid organ transplant recipients, monitoring the appearance of post-transplant complications, and controlling the efficacy of maintenance immunosuppressive therapy. Thus, diagnostic approaches able to scale up TTV quantification are needed. Here, we report on the development and validation of a real-time PCR assay for TTV quantification on the Hologic Panther Fusion® System by utilizing its open-access channel. The manual real-time PCR previously developed in our laboratories was optimized to detect TTV DNA on the Hologic Panther Fusion® System. The assay was validated using clinical samples. The automated TTV assay has a limit of detection of 1.6 log copies per ml of serum. Using 112 samples previously tested via manual real-time PCR, the concordance in TTV detection was 93% between the assays. When the TTV levels were compared, the overall agreement between the methods, as assessed using Passing–Bablok linear regression and Bland–Altman analyses, was excellent. In summary, we validated a highly sensitive and accurate method for the diagnostic use of TTV quantification on a fully automated Hologic Panther Fusion® System. This will greatly improve the turnaround time for TTV testing and better support the laboratory diagnosis of this new viral biomarker.

IntroductionMethimazole (MMI) represents the conventional therapeutic agent for Graves’ disease (GD) hyperthyroidism, but MMI efficacy is limited since it marginally affects the underlying autoimmune process. In a previous study, we randomly assigned 42 newly diagnosed GD patients with insufficient vitamin D (VitD) and selenium (Se) levels to treatment with MMI alone (standard) or combined with selenomethionine and cholecalciferol (intervention) and observed a prompter resolution of hyperthyroidism in the intervention group.MethodsIn the present study, we aimed to explore changes in peripheral T regulatory (Treg) and circulating natural killer (NK) cell frequency, circulating NK cell subset distribution and function, during treatment.ResultsAt baseline, circulating total CD3-CD56+NK cells and CD56bright NK cells were significantly higher in GD patients than in healthy controls (HC) (15.7 ± 9.6% vs 9.9 ± 5.6%, p=0.001; 12.2 ± 10.3% vs 7.3 ± 4.1%, p=0.02, respectively); no differences emerged in Treg cell frequency. Frequencies of total NK cells and CD56bright NK cells expressing the activation marker CD69 were significantly higher in GD patients than in HC, while total NK cells and CD56dim NK cells expressing CD161 (inhibitory receptor) were significantly lower. When co-cultured with the K562 target cell, NK cells from GD patients had a significantly lower degranulation ability compared to HC (p

We report in vivo selection of a severe acute respiratory syndrome coronavirus 2 spike mutation (Q493R) conferring simultaneous resistance to bamlanivimab and etesivimab. This mutation was isolated from a patient who had coronavirus disease and was treated with these drugs.

IntroductionEvidence points to viral infections as possible triggers of autoimmune thyroid disease (AITD), but little is known about the prevalence of common viruses in the thyroid gland. Using a novel approach based on virus enrichment in multiple cell lines followed by detection of the viral genome and visualization of viral proteins, we investigated the presence of multiple human viruses in thyroid tissue from AITD patients and controls.MethodsThyroid tissue was collected by core needle biopsy or during thyroid surgery from 35 patients with AITD (20 Graves’ disease and 15 Hashimoto’s thyroiditis). Eighteen thyroid tissue specimens from patients undergoing neck surgery for reasons other than thyroid autoimmunity served as controls. Specimens were tested for the presence of ten different viruses. Enteroviruses and human herpesvirus 6 were enriched in cell culture before detection by PCR and immunofluorescence, while the remaining viruses were detected by PCR of biopsied tissue.ResultsForty of 53 cases (75%) carried an infectious virus. Notably, 43% of all cases had a single virus, whereas 32% were coinfected by two or more virus types. An enterovirus was found in 27/53 cases (51%), human herpesvirus 6 in 16/53 cases (30%) and parvovirus B19 in 12/53 cases (22%). Epstein-Barr virus and cytomegalovirus were found in a few cases only. Of five gastroenteric virus groups examined, only one was detected in a single specimen. Virus distribution was not statistically different between AITD cases and controls.ConclusionCommon human viruses are highly prevalent in the thyroid gland. This is the first study in which multiple viral agents have been explored in thyroid. It remains to be established whether the detected viruses represent causal agents, possible cofactors or simple bystanders.

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.

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.

Prompt and stable control of hyperthyroidism is fundamental to avoid the detrimental effects of thyroid hormone excess, and antithyroid drugs, mainly methimazole (MMI), represent the first-line treatment for Graves’ disease (GD) hyperthyroidism. Decreased serum concentrations of selenium (Se) and calcifediol (25(OH)D, VitD) have been reported in newly diagnosed GD patients in observational studies. Low Se levels might exacerbate oxidative stress by compromising the antioxidant machinery’s response to reactive oxygen species, and low VitD levels might hamper the anti-inflammatory immune response. We performed a randomized controlled clinical trial (EudraCT 2017-00505011) to investigate whether Se and cholecalciferol (VitD) addition to MMI is associated with a prompter control of hyperthyroidism. Forty-two consecutive patients with newly-onset GD and marginal/insufficient Se and VitD levels were randomly assigned to treatment with either MMI monotherapy or MMI combined with Se and VitD. Se treatment was withdrawn after 180 days, while the other treatments were continued. Combination therapy resulted in a significantly greater reduction in serum FT4 concentration at 45 days (-37.9 pg/ml, CI 95%, -43.7 to -32.2 pg/ml) and 180 days (-36.5 pg/ml, CI 95%, -42 to -30.9 pg/ml) compared to MMI monotherapy (respectively: -25.7 pg/ml, CI 95%, -31.6 to -19.7 pg/ml and -22.9 pg/ml, CI 95%, -28 to -17.3 pg/ml, p 0.002). Data at 270 days confirmed this trend (-37.8 pg/ml, CI 95%, -43.6 to -32.1 pg/ml vs -24.4 pg/ml, CI 95%, -30.3 to -18.4 pg/ml). The quality of life (QoL) score was investigated by the validated “Thyroid-related Patient-Reported Outcome” questionnaire (ThyPRO). ThyPRO composite score showed a greater improvement in the intervention group at 45 days (-14.6, CI 95%, -18.8 to -10.4), 180 (-9, CI 95%, -13.9 to -4.2) and 270 days (-14.3, CI 95%, -19.5 to -9.1) compared to MMI group (respectively, -5.2, CI 95%, -9.5 to -1; -5.4, CI 95%, -10.6 to -0.2 and -3.5, CI 95%, -9 to -2.1, p 0-6 months and 6-9 months

We report an imported case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant P.1 detected in an asymptomatic traveler who arrived in Italy on an indirect flight from Brazil. This case shows the risk for introduction of SARS-CoV-2 variants from indirect flights and the need for continued SARS-CoV-2 surveillance.

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.

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.

We report 25 cases of infection with SARS-CoV-2 Omicron variant containing spike protein L452R mutation in northern Lombardy, Italy. Prevalence of this variant was >30% in this region, compared with

Objectives: Torquetenovirus (TTV) and Redondovirus (ReDoV) are the most prevalent viruses found in the human respiratory virome in viral metagenomics studies. A large-scale epidemiological study was performed to investigate their prevalence and loads in saliva samples according to SARS-CoV-2 status. Methods: Saliva samples from 448 individuals (73% SARS-CoV-2 negative and 27% SARS-CoV-2 positive) aged 23–88 years were tested. SARS-CoV-2 and TTV were determined in saliva by specific qualitative and quantitative real-time PCRs, respectively. A sub-cohort of 377 subjects was additionally tested for the presence and load of ReDoV in saliva, and a different sub-cohort of 120 subjects for which paired saliva and plasma samples were available was tested for TTV and ReDoV viremia at the same timepoints as saliva. Results: TTV in saliva was 72% prevalent in the entire cohort, at a mean DNA load of 4.6 log copies/mL, with no difference regardless of SARS-CoV-2 status. ReDoV was found in saliva from 61% of the entire cohort and was more prevalent in the SARS-CoV-2-negative subgroup (65% vs. 52%, respectively). In saliva, the total mean load of ReDoV was very similar to the one of TTV, with a value of 4.4 log copies/mL. The mean viral loads in subjects infected with a single virus, namely, those infected with TTV or ReDoV alone, was lower than in dually infected samples, and Tukey’s multiple-comparison test showed that ReDoV single-infected samples resulted in the only true outlier (p = 0.004). Differently from TTV, ReDoV was not detected in any blood samples. Conclusions: This study establishes the prevalence and mean value of TTV and ReDoV in saliva samples and demonstrates the existence of differences between these two components of the human virome.

The Delta variant of concern (VOC) of SARS-CoV-2 has become dominant worldwide. In this article, we report a cluster caused by B.1.617.2 harboring the additional mutation of concern (MOC) F490S. We observed that 5 fully vaccinated subjects aged between 47 and 84 years were infected with this variant. The immune escape mutation F490S, first identified in the Lambda VOI, appears to impair vaccine efficacy and is rapidly increasing in prevalence worldwide.

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.

Abstract Enteroviruses (EVs) causing persisting infection are characterized by minimal replication and genetic changes. Typing of these agents may complement disease assessment and shed light on pathogenesis. Here we report an integrated approach for EV detection in human samples that is based on pre-enrichment of virus in cell culture before search for the viral genome and viral antigens. Cases of post-polio syndrome, type 1 diabetes, and chronic cardiomyopathy were investigated. As tissue-based approaches require invasive procedures, information was mainly gleaned from virus in blood. Molecular assays targeting conserved genome regions of all EV types (5′UTR, 2 C, 3Dpol) were employed. As compared to direct assays of plasma or leukocytes, the EV detection rate was significantly enhanced by co-culture of leukocytes with cell lines prior to molecular and immunologic tests. Results of RT-PCR and sequencing were confirmed by staining cell cultures with a panel of EV-specific antibodies. Sequence and phylogenetic analysis showed that EVs of the C species (polioviruses) were associated with the post-polio syndrome, while members of the B species were found in type 1 diabetes and cardiomyopathy. The procedure may be used for investigating the possible association of different EVs with a variety of chronic neurologic, endocrine, and cardiac disorders.

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.

Enterovirus (EV) infection of insulin-producing pancreatic beta cells is associated with type 1 diabetes (T1D), but little is known about the mechanisms that lead the virus to cause a persistent infection and, possibly, to induce beta cell autoimmunity. A cell line susceptible to most enterovirus types was infected with EV isolates from cases of T1D and, for comparison, with a replication-competent strain of coxsackievirus B3. The transcription of immune-related genes and secretion of cytokines was evaluated in infected vs. uninfected cells. Acutely infected cells showed the preserved transcription of type I interferon (IFN) pathways and the enhanced transcription/secretion of IL6, IL8, LIF, MCP1, and TGFB1. On the other hand, infection by defective EV strains obtained from diabetic subjects suppressed IFN pathways and the transcription of most cytokines, while enhancing the expression of IL8, IL18, IL32, and MCP1. IL18 and IL32 are known for their pathogenic role in autoimmune diabetes. Thus, the cytokine profile of AV3 cells infected by diabetes-derived EV strains closely matches that observed in patients at the early stages of T1D. The concordance of our results with clinically verified information reinforces the hypothesis that the immune changes observed in type 1 diabetic patients are due to a hardly noticeable virus infection.