Journal: Microbiology Spectrum

Article Title: Diagnosis and Treatment of Bacterial Pneumonia in Critically Ill Patients with COVID-19 Using a Multiplex PCR Assay: A Large Italian Hospital’s Five-Month Experience

doi: 10.1128/Spectrum.00695-21

Figure Lengend Snippet: Comparison between the FilmArray pneumonia plus panel and standard-of-care reference testing results for LRT samples from COVID-19 patients with bacterial pneumonia ^a

Article Snippet: In September 2020, when the second wave of the SARS-CoV-2 pandemic was raging in Italy, we implemented the multiplexed, semiquantitative FilmArray pneumonia plus (FA-PP) panel (BioFire, Salt Lake City, UT, USA) assay ( ) for routine clinical use at our institution (a large Italian university hospital).

Techniques: Comparison

Journal: Microbiology Spectrum

Article Title: Diagnosis and Treatment of Bacterial Pneumonia in Critically Ill Patients with COVID-19 Using a Multiplex PCR Assay: A Large Italian Hospital’s Five-Month Experience

doi: 10.1128/Spectrum.00695-21

Figure Lengend Snippet: Quantitative result agreement between the FilmArray pneumonia plus (FA-PP) panel and standard-of-care (SoC) culture testing methods for 202 bacterial organisms detected in bronchoalveolar lavage (BAL) fluid or endotracheal aspirate (ETA) samples from ICU patients. All but 22 (which did not grow in culture) organisms were detected at or above the 1 × 10 4 CFU/ml (BAL fluid sample) or 1 × 10 5 CFU/ml (ETA sample) thresholds for clinically relevant quantification by both methods. The diagonal dashed line connects dots that represent bacterial organisms with fully concordant results (71/202; 35.1%), whereas dots above or below the line represent bacterial organisms with SoC culture loads that exceeded (18/202, 8.9%) or did not exceed (113/202, 56.0%), respectively, those of the FA-PP panel by ≥1 log 10 . Shades of gray are used to depict the different numbers of samples corresponding to each dot.

Article Snippet: In September 2020, when the second wave of the SARS-CoV-2 pandemic was raging in Italy, we implemented the multiplexed, semiquantitative FilmArray pneumonia plus (FA-PP) panel (BioFire, Salt Lake City, UT, USA) assay ( ) for routine clinical use at our institution (a large Italian university hospital).

Techniques:

Journal: Microbiology Spectrum

Article Title: Diagnosis and Treatment of Bacterial Pneumonia in Critically Ill Patients with COVID-19 Using a Multiplex PCR Assay: A Large Italian Hospital’s Five-Month Experience

doi: 10.1128/Spectrum.00695-21

Figure Lengend Snippet: Overview of antimicrobial therapy interventions performed according to FilmArray pneumonia plus (FA-PP) panel results. Interventions were stratified into three relevant groups based on the full concordance of positive (A) or negative (B) FA-PP testing results or the partial or full discordance (C) of FA-PP testing results, respectively, with the results by standard-of-care (SoC) culture for the lower respiratory tract (LRT) samples tested. For each LRT sample, the type(s) of interventions and the antibiotics involved (one or more per intervention) are reported, together with whether the interventions resulted in appropriate antimicrobial escalations/initiations (93 in panel A and 22 in panel C) or deescalations (10 in panel A and 6 in panel C), appropriate antimicrobial discontinuations (5 in panel B), and inappropriate antimicrobial escalations/initiations (4 in panel C). Cases with no antimicrobial therapy interventions (5 in panel A, 85 in panel B, and 4 in panel C) are reported as well.

Article Snippet: In September 2020, when the second wave of the SARS-CoV-2 pandemic was raging in Italy, we implemented the multiplexed, semiquantitative FilmArray pneumonia plus (FA-PP) panel (BioFire, Salt Lake City, UT, USA) assay ( ) for routine clinical use at our institution (a large Italian university hospital).

Techniques:

Journal: Microbiology Spectrum

Article Title: Diagnosis and Treatment of Bacterial Pneumonia in Critically Ill Patients with COVID-19 Using a Multiplex PCR Assay: A Large Italian Hospital’s Five-Month Experience

doi: 10.1128/Spectrum.00695-21

Figure Lengend Snippet: Characteristics of bacterial pneumonia episodes detected or not detected by the FilmArray pneumonia plus panel in 97 mechanically ventilated patients with COVID-19 ^a

Article Snippet: In September 2020, when the second wave of the SARS-CoV-2 pandemic was raging in Italy, we implemented the multiplexed, semiquantitative FilmArray pneumonia plus (FA-PP) panel (BioFire, Salt Lake City, UT, USA) assay ( ) for routine clinical use at our institution (a large Italian university hospital).

Techniques:

Journal: bioRxiv

Article Title: Ubiquitin Ligase ITCH Regulates Life Cycle of SARS-CoV-2 Virus

doi: 10.1101/2024.12.04.624804

Figure Lengend Snippet: (A) Culture medium from vT2-WT and vT2-ITCH-KO cells expressing HA-tagged ubiquitin (Ub) and Flag-2×Strep tagged E was harvested for Strep AP. A decrease of the extracellular E protein induced by ITCH ablation was visualized by immunoblot (left) and dot blot analysis (right). (B) Lysates from HEK293 cells expressing Flag-tagged E with ITCH or ITCH-CS were subjected to Flag IP, followed by immunoblotting for autophagosome cargo receptors. E specifically precipitated p62 and ITCH promoted their interaction. (C-E) HEK293 cells were transfected with Flag-tagged E with ITCH or ITCH-CS. 24 h later, cells were analyzed by immunofluorescence with Flag, ITCH and p62 or LC3B or LAMP1 antibodies. ITCH enhanced the colocalization between E and p62 (C) or LC3B (D), while no change in the colocalization between E and LAMP1 (E) was noted. Scale bar, 10 μm. (F) Culture media and denatured lysates from control (CTRL) and p62 knock down (#1, #2) HEK293 cells expressing HA-tagged ubiquitin (Ub), Flag-tagged E were subjected to Flag IP (with incorporation of a washing step with urea before elution for culture media samples), followed by immunoblotting or dot blot analysis. p62 depletion resulted in the accumulation of intracellular E (both unmodified and ubiquitinated), while decreasing the level of extracellular E (n=3). (G, H) vT2-WT and vT2-ITCH-KO cells infected with SARS-CoV-2 at 1 MOI for 10 h were subjected to immunofluorescence analysis with E and p62 or LC3B antibodies. ITCH-ablation decreased the colocalization between E and p62 (G) or LC3B (H). Scale bar, 10 μm. (I) A model of the function of ITCH in promoting autophagosome-mediated SARS-CoV-2 virion egress. ITCH-dependent ubiquitin modification enhances E binding with S and M binding with non-ubiquitinated E, resulting in the increase in virion formation and p62-dependent autophagosome targeting for release.

Article Snippet: The following primary antibodies were used: Flag (Sigma, F1804); Flag (Sigma, F3165); Flag (Cell Signaling Technology, 14793S); GAPDH (Invitrogen, MA5-27912); β-tubulin (Cell Signaling Technology, 2128S);s (BioLegend, 688102); Strep (Invitrogen, MA5-17283); CBD (New England BioLabs, E8034S); ubiquitin (Cell Signaling Technology, 58395S); K63-linkage-specific antibody (Enzo Life Sciences, BML-PW0600-0100); K48-linkage-specific antibody (Cell Signaling Technology, 8081S); Spike (Proteintech, 28867-1-AP); M (Proteintech, 28882-1-AP); E (Proteintech, 28904-1-AP); ITCH (Santa Cruz, sc-28367); ITCH (Novus Biologicals, NB100-68142); p62 (Cell Signaling Technology, 88588S and 7695S); GM130 (Proteintech, 11308-1-AP); LAMP1 (Cell Signaling Technology, 9091S); OPTN (Cayman Chemical, 100002); LC3 (Proteintech,14600-1-AP); LC3B (Cell Signaling Technology, 3868S); SARS-CoV-2 Membrane protein (Cell Signaling Technology, 15333S); SARS-CoV-2 Envelope protein (Cell Signaling Technology, 74698S); furin (Proteintech, 18413-1-AP); Cathepsin L (Proteintech, 10938-1-AP); NDP52 (Proteintech, 12229-1-AP); NBR1 (Proteintech, 16004-1-AP); FAM134B (Proteintech, 21537-1-AP); RTN3 (Proteintech, 12055-2-AP) and NIX (Proteintech, 12986-1-AP).

Techniques: Expressing, Western Blot, Dot Blot, Transfection, Immunofluorescence, Control, Knockdown, Infection, Modification, Binding Assay