Nasal Microbiome in COVID-19: A Potential Role of Corynebacterium in Anosmia.

The evolution and the development of the symptoms of Coronavirus disease 19 (COVID-19) are due to different factors, where the microbiome plays a relevant role. The possible relationships between the gut, lung, nasopharyngeal, and oral microbiome with COVID-19 have been investigated. We analyzed the nasal microbiome of both positive and negative SARS-CoV-2 individuals, showing differences in terms of bacterial composition in this niche of respiratory tract. The microbiota solution A (Arrow Diagnostics) was used to cover the hypervariable V1-V3 regions of the bacterial 16S rRNA gene. MicrobAT Suite and MicrobiomeAnalyst program were used to identify the operational taxonomic units (OTUs) and to perform the statistical analysis, respectively. The main taxa identified in nasal microbiome of COVID-19 patients and in Healthy Control subjects belonged to three distinct phyla: Proteobacteria (HC = 14%, Cov19 = 35.8%), Firmicutes (HC = 28.8%, Cov19 = 30.6%), and Actinobacteria (HC = 56.7%, Cov19 = 14.4%) with a relative abundance > 1% in all groups. A significant reduction of Actinobacteria in Cov19 group compared to controls (P < 0.001, FDR = 0.01) was found. The significant reduction of Actinobacteria was identified in all taxonomic levels down to the genus (P < 0.01) using the ANOVA test. Indeed, a significantly reduced relative abundance of Corynebacterium was found in the patients compared to healthy controls (P = 0.001). Reduced abundance of Corynebacterium has been widely associated with anosmia, a common symptom of COVID-19 as suffered from our patients. Contrastingly, the Corynebacterium genus was highly represented in the nasal mucosa of healthy subjects. Further investigations on larger cohorts are necessary to establish functional relationships between nasal microbiota content and clinical features of COVID-19.

Loss of Detection of sgN Precedes Viral Abridged Replication in COVID-19-Affected Patients-A Target for SARS-CoV-2 Propagation.

The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3-7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2'-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.

Case Report: Discovery a Novel SARS-CoV-2 Variant in a Six-Months Long-Term Swab Positive Female Suffering From Non-Hodgkin Lymphoma.

BACKGROUND: We report the case of a woman with non-Hodgkin lymphoma who remained positive on the molecular assay for SARS-CoV-2 for six months: she has never experienced a severe form of COVID-19 although in absence of seroconversion. METHODS: The whole SARS-CoV-2 genome analysis was performed by the CleanPlex SARS-CoV-2 Research and Surveillance NGS Panel (PARAGON GENOMICS, Hayward, USA). RESULTS: We found twenty-two mutations in SARS-CoV-2 genome and a novel deleterious ORF3a frameshift c.766_769del corresponding to a unique and novel lineage. The region affected by this frameshift variant is reported as being important in determining SARS-CoV-2 immunogenicity. Patient's immunophenotype showed the absence of B lymphocytes and significantly reduced T-cell count. Only after the treatment with hyperimmune plasma she finally became negative on the swab. CONCLUSIONS: Our findings could be helpful in the management of patients with immunodeficiency, particularly when novel variants, potentially altering the virus immune response, are present.

Long-chain polyphosphates impair SARS-CoV-2 infection and replication.

Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO4 3-) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano- LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2-infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.

Nasopharyngeal Microbiome Signature in COVID-19 Positive Patients: Can We Definitively Get a Role to Fusobacterium periodonticum?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the pandemic Coronavirus Disease 2019 (COVID-19). This virus is highly transmissible among individuals through both droplets and aerosol leading to determine severe pneumonia. Among the various factors that can influence both the onset of disease and the severity of its complications, the microbiome composition has also been investigated. Recent evidence showed the possible relationship between gut, lung, nasopharyngeal, or oral microbiome and COVID-19, but very little is known about it. Therefore, we aimed to verify the relationships between nasopharyngeal microbiome and the development of either COVID-19 or the severity of symptoms. To this purpose, we analyzed, by next generation sequencing, the hypervariable V1-V2-V3 regions of the bacterial 16S rRNA in nasopharyngeal swabs from SARS-CoV-2 infected patients (n=18) and control (CO) individuals (n=12) using Microbiota solution A (Arrow Diagnostics). We found a significant lower abundance of Proteobacteria and Fusobacteria in COVID-19 patients in respect to CO (p=0.003 and p<0.0001, respectively) from the phylum up to the genus (p<0.001). The Fusobacterium periodonticum (FP) resulted as the most significantly reduced species in COVID-19 patients respect to CO. FP is reported as being able to perform the surface sialylation. Noteworthy, some sialic acids residues on the cell surface could work as additional S protein of SARS-CoV-2 receptors. Consequently, SARS-CoV-2 could use sialic acids as receptors to bind to the epithelium of the respiratory tract, promoting its clustering and the disease development. We can therefore speculate that the significant reduction of FP in COVID-19 patients could be directly or indirectly linked to the modulation of sialic acid metabolism. Finally, viral or environmental factors capable of interfering with sialic metabolism could determine a fall in the individual protection from SARS-CoV-2. Further studies are necessary to clarify the precise role of FP in COVID-19.

SARS-CoV-2 Subgenomic N (sgN) Transcripts in Oro-Nasopharyngeal Swabs Correlate with the Highest Viral Load, as Evaluated by Five Different Molecular Methods.

The COVID-19 pandemic has forced diagnostic laboratories to focus on the early diagnostics of SARS-CoV-2. The positivity of a molecular test cannot respond to the question regarding the viral capability to replicate, spread, and give different clinical effects. Despite the fact that some targets are covered by commercially-available assays, the identification of new biomarkers is desired in order to improve the quality of the information given by these assays. Therefore, since the subgenomic transcripts (sgN and sgE) are considered markers of viral activity, we evaluated these subgenomic transcripts in relation to the genomic amplification obtained using five different commercial CE-IVD tools. Methods: Five CE-IVD kits were compared in terms of their capability to detect both synthetic SARS-CoV-2 viral constructs (spiked in TMB or PBS medium) and targets (N, E, RdRp and Orf1ab genes) in twenty COVID-19-positive patients' swabs. The sgN and sgE were assayed by real-time RT-qPCR and digital PCR. Results: None of the diagnostic kits missed the viral target genes when they were applied to targets spiked in TMB or PBS (at dilutions ranging from 100 pg to 0.1 pg). Nevertheless, once they were applied to RNA extracted from the patients' swabs, the superimposability ranged from 50% to 100%, regardless of the extraction procedure. The sgN RNA transcript was detected only in samples with a higher viral load (Ct ≤ 22.5), while sgE was within all of the Ct ranges. Conclusions: The five kits show variable performances depending on the assay layout. It is worthy of note that the detection of the sgN transcript is associated with a higher viral load, thus representing a new marker of early and more severe infection.

BRCA testing delay during the COVID-19 pandemic: How to act?

Recently, our lab, part of a referral center in Italy, reported its experience regarding the execution of germline BRCA1/2 (gBRCA) testing during the first months of the coronavirus disease-2019 (COVID-19) pandemic, which highlights a substantial reduction (about 60%) compared with the first 2 months of the current year. This evidence appeared to be a lockdown effect due to extraordinary restriction measures to slow down the spread of SARS-CoV-2. In this study, we aimed to evaluate the overall effects of the ongoing pandemic on gBRCA testing in our institution and to understand how COVID-19 has influenced testing after the complete lockdown (March 8-May 5, 2020). Additionally, we compared this year's trend with trends of the last 3 years to better monitor gBRCA testing progress. This detailed analysis highlights two important findings: (1) gBRCA testing did not increase significantly after the lockdown period (May-October 2020) compared with the lockdown period (March-April 2020), emphasizing that even after the lockdown period testing remained low. (2) Comparing the total tests per year (January-October 2017, 2018, 2019, with 2020), the impact of COVID-19 on gBRCA testing is apparent, with similarities of trends registered in 2017. These evidences reveal a gBRCA testing delay for cancer patients and healthy patients at this moment, and the new era of gBRCA testing in the management of ovarian, breast, pancreas and prostate cancer patients has been seriously questioned due to the COVID-19 pandemic. As consequence, we underline that measures to guarantee oncogenetic testing (e.g., gBRCA testing) along with new diagnostic/clinic strategies are mandatory. For these reasons, several proposals are presented in this study.

PARP-inhibitors in a non-oncological indication as COVID-19: Are we aware about its potential role as anti-thrombotic drugs? The discussion is open.

In the last three months, the whole scientific community has shifted its focus to the fight against the COVI-2 infection (COVID-19) trying to use different medications to save the patients' life. In some studies, the results were completely inconclusive, as in the case of chloroquine. However, the recent discovery on benefits deriving from use of such anticoagulants for Covid-19 patients, has increased the success of patients' treatment. Among lots of old and new drugs, PARP-inhibitors were not considered as possible option in the treatment of Covi-2 infection, being the latter able to induce the inflammatory and thrombotic cascades. Since PARP-inhibitors are able to reduce and block mechanisms leading to thrombosis and inflammation, they could be used as antithrombotic medications. Therefore, the present brief report is aimed to open the discussion on the potentials of PARP-inhibitors in non-oncological settings, like Covid-19.