Genomic surveillance of SARS-CoV-2 positive passengers on flights from China to Italy, December 2022.

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.

Detection of Torquetenovirus and Redondovirus DNA in Saliva Samples from SARS-CoV-2-Positive and -Negative Subjects.

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.

Expansion of L452R-Positive SARS-CoV-2 Omicron Variant, Northern Lombardy, 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 <0.5% worldwide. Many laboratories are using previously developed L452R-specific PCRs to discriminate Omicron from Delta mutations, but these tests may be unreliable.

Performance Assessment of the LIAISON® SARS-CoV-2 Antigen Assay On Nasopharyngeal Swabs

The SARS-CoV-2 pandemic is ongoing worldwide, causing prolonged pressure on molecular diagnostics. Viral antigen (Ag) assays have several advantages, ranging from lower cost to shorter turnaround time to detection. Given the rare occurrence of low-load viremia, antigen assays for SARSCoV-2 have focused on nasopharyngeal swab and saliva as biological matrices, but their effectiveness must be validated. We assayed here the performances of the novel quantitative Liaison® SARSCoV-2 Ag assay on 119 nasopharyngeal swabs and obtained results were compared with Hologic Panther and Abbott m2000 RT-qPCR. The Ag assay demonstrated a good correlation with viral load, shorter turnaround time, and favorable economics. The best performance was obtained in the acute phase of disease.

Immune-Mediated Mechanisms in Patients Testing Positive for SARS-CoV-2: Protocol for a Multianalysis Study.

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.

A CLUSTER OF SARS-COV-2 DELTA VARIANT OF CONCERN ADDITIONALLY HARBORING F490S, NORTHERN LOMBARDY, ITALY.

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.

Spike protein evolution in the SARS-CoV-2 Delta variant of concern: a case series from Northern Lombardy.

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.

Emergence of SARS-COV-2 Spike Protein Escape Mutation Q493R after Treatment for COVID-19.

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.

Breakthrough Infections of E484K-Harboring SARS-CoV-2 Delta Variant, Lombardy, Italy.

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.

Pilot Study: Long-Term Shedding of SARS-CoV-2 in Urine: A Threat for Dispersal in Wastewater.

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.

Symptomatic SARS-CoV-2 infections after full schedule BNT162b2 vaccination in seropositive healthcare workers: a case series from a single institution.

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.

Previous Humoral Immunity to the Endemic Seasonal Alphacoronaviruses NL63 and 229E Is Associated with Worse Clinical Outcome in COVID-19 and Suggests Original Antigenic Sin.

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.

Imported SARS-CoV-2 Variant P.1 in Traveler Returning from Brazil to Italy.

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.

Introduction of SARS-CoV-2 variant of concern 20h/501Y.V2 (B.1.351) from Malawi to Italy.

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 on Ocular Surfaces in a Cohort of Patients With COVID-19 From the Lombardy Region, Italy.

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.

Saliva is a reliable tool to detect SARS-CoV-2.

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.