Since SARS-CoV-2-based disease (COVID-19) spreads as a pandemic, the necessity of a highly sensitive molecular diagnosis that can drastically reduce false negatives reverse transcription PCR (rtPCR) results, raises as a major clinical need. Here we evaluated the performance of a ddPCR-based assay to quantify SARS-CoV-2 titer in 55 suspected COVID-19 cases with negative rtPCR results thanks to in-house ddPCR assay (targeting RdRp and host RNaseP). Samples were collected at ASST-GOM Niguarda between February and May 2020 at hospital admission. Clinical and imaging data were obtained for clinical staging and definition of disease severity. Patients were mainly female (45.5%) with a median age of 73 (57-84) years. ddPCR-based assay detected SARS-CoV-2 genome in nasopharyngeal samples of 19 (34.5%) patients (median viral-load: 128 copies/mL, IQR: 72-345). In 15 of them (78.9%), chest CT showed a classical COVID-19 bilateral interstitial pneumonia; 14 patients (73.7%) showed severe COVID-19 manifestations. ddPCR did not identify any trace of SARS-CoV-2 genome in the respiratory samples of the remaining 36 patients. The serological assay performed in a subgroup of 34 patients at the later stage of illness (from 3 days to 90 days after) confirmed the presence of SARS-CoV-2 antibodies in all patients tested positive for SARS-CoV-2 in ddPCR (100%). Contrariwise, negative tests were observed in 95.0% ddPCR negative patients (P<0.001). Thanks to a ddPCR-based assay, we achieved a rapid and accurate SARS-CoV-2 diagnosis in rtPCR-negative respiratory

Oxidative stress is involved in the pathogenesis of numerous diseases. Nevertheless, no optimal natural antioxidant has been found for therapeutics, therefore polyphenol antioxidants have been looked for in myrtle leaves, a plant that in folk medicine has been used as anti-inflammatory drug. Antioxidant-rich fractions were prepared from myrtle (Myrtus communis L.) leaves liquid-liquid extraction (LLE) with different solvents. All myrtle extracts were very rich in polyphenols. In particular, hydroalcoholic extracts contain galloyl-glucosides, ellagitannins, galloyl-quinic acids and flavonol glycosides; ethylacetate extract and aqueous residues after LLE are enriched in flavonol glycosides and hydrolysable tannins (galloyl-glucosides, ellagitannins, galloyl-quinic acids), respectively. Qualitative and quantitative analysis for the single unidentified compound was also performed. Human LDL exposed to copper ions was used to evaluate the antioxidant activity of the myrtle extracts. Addition of these extracts did not affect the basal oxidation of LDL but dose-dependently decreased the oxidation induced by copper ions. Moreover, the myrtle extracts reduce the formation of conjugated dienes. The antioxidant effect of three myrtle extracts decreased in the following order: hydroalcoholic extracts, ethylacetate and aqueous residues after LLE. The extracts had the following IC50: 0.36, 2.27 and 2.88 microM, when the sum of total phenolic compounds was considered after the correction of molecular weight based on pure compounds. Statistical analysis showed a significant difference among hydroalcoholic extracts vs. the ethylacetate and aqueous residues after LLE. These results suggest that the myrtle extracts have a potent antioxidant activity mainly due to the presence of galloyl derivatives.

In patients failing cART with LLV, HIV-1 genotyping provides reliable and reproducible results that are informative about emerging drug resistance.

In vitro studies show that some individual minor polar phenolic compounds (MPC) present in virgin olive oil prevent oxidation of human low-density lipoproteins (LDL), but few data are available on the antioxidant effect of whole oil extract. Thus, whole virgin olive extracts were studied to determine whether they maintain the antioxidant activity and whether this last is linked to MPC composition of a single virgin oil. Using HPLC-DAD the MPC content in Taggiasca and Seggianese virgin olive oils was measured. Taggiasca oil was less rich in total MPC (208.5 mg/L) than Seggianese oil (441.9 mg/L). In addition, the major compounds of Taggiasca oil were lignan derivatives, whereas the major compounds in Seggianese oils were secoiridoid derivatives. Moreover, Taggiasca oil was practically free of 5-hydroxytyrosol and 5-hydroxytyrosol derivatives, deacetoxy-oleuropein aglycone and oleuropein aglycone. The antioxidant activity of the oils on human LDL was evaluated by measuring malondialdehyde and conjugate diene generation induced by copper ions. In both tests, the oil extracts dose-dependently reduced malondialdehyde and conjugate diene generation. Moreover, antioxidant potency correlated with total MPC; thus, Seggianese extract was more active. The two oils differed quantitatively and qualitatively, and these differences influenced their biological activities; thus clinical trials focused on studying the effects of olive oils should specify the oils used.

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Our findings prove the reliability of HIV-1 integrase genotyping and reinforce the concept that this assay may be useful in the management of failures even at LLV.

Sequenicing cOronavirus and Variability Analysis (SCoVA) Study Group. The authors also thank all of the staff of the Microbiology and Virology Laboratory of ASST Grande Ospedale Metropolitano Niguarda for their outstanding technical support in processing swab samples and performing laboratory analyses and data management.Disclaimer. The funding source had no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication.Financial support.