Detection of a characteristic melting profile of a SARS-CoV-2 Kappa variant in Italy using the SARS-CoV-2 Variants ELITe MGB® Kit.

BACKGROUND: Although more than a year has passed since the start of the pandemic, SARS-CoV-2 infection still represents a major challenge for public health all over the world due to viral genome capability of gaining rapid mutations. Whole-genome sequencing (WGS) is the gold standard for variant identification, but it is time consuming and relatively expensive. For this reason, assays targeting multiple regions of the SARS-CoV-2 genome may be useful for a rapid traceability of either known or new variants, anyway, not all the manufacturers are able to sustain the rapid development of variants. OBJECTIVE: We tested forty nasopharyngeal swabs, resulted positive for the presence of SARS-CoV-2 RNA at low cycle threshold (CT < 25), with SARS-CoV-2 Variants ELITe MGB® Kit, which was designed to identify Nigerian variant, possible UK variant and South African or Brazilian variant. RESULTS: During the analysis, we noted an atypical melting curve, different from the other variants recognizable by the kit. The subsequent WGS reported this variant as Kappa, so we assess the possibility of "suspecting" the presence of a Kappa variant using SARS-CoV-2 Variants ELITe MGB® Kit. CONCLUSIONS: Rapid variant screening followed by WGS offers the opportunity to study mutation dynamics and quickly identify possible variants of interest (VOI) and/or variants of concern (VOC), which is crucial in virus spreading control. Furthermore, an accurate analysis of the melting peak could be useful to suspect the presence of new variants.

The antibiotic polymyxin B modulates P2X7 receptor function.

The natural peptide polymyxin B (PMB) is a well-known and potent antibiotic that binds and neutralizes bacterial endotoxin (LPS), thus preventing its noxious effects among LPS-mediated endotoxin shock in animal models. We have investigated the effect of PMB on responses mediated by the P2X(7)R in HEK293 and K562 cells transfected with P2X(7) cDNA and in mouse and human macrophages. In addition, in view of the potential exploitation of P2X(7)-directed agonists in antitumor therapy, we also investigated the effect of PMB in B lymphocytes from patients affected by chronic lymphocytic leukemia. PMB, at an optimal concentration dependent on the given cell type, greatly potentiated the effect of nucleotide-mediated P2X(7) stimulation. In particular, ATP-mediated Ca(2+) influx, plasma membrane permeabilization, and cytotoxicity were enhanced to an extent that, in the presence of PMB, cells were killed by otherwise ineffective nucleotide concentrations. The synergistic effect due to the combined application of ATP and PMB was prevented by incubation with the irreversible P2X blocker oxidized ATP (oATP), but not with the reversible antagonist 1-(N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl)-4-phenilpiperazine (KN-62). Cells lacking P2X(7) were fully insensitive to the combined stimulation with PMB and ATP. Furthermore, PMB at the concentrations used had no untoward effects on cell viability. These results point to PMB as a useful tool for the modulation of P2X(7)R function and suggest that care should be used in the evaluation of ATP-stimulated immune cell responses in the presence of PMB as they may not solely be affected by removal of contaminating LPS.