Evaluation of BioFire® FilmArray® Pneumonia Panel in Bronchoalveolar Lavage Samples From Immunocompromised Patients With Suspected Pneumonia.

Objectives Immunocompromised patients, specifically those with solid organ transplants or cancer on chemotherapy, are at particularly high risk of severe pneumonia and opportunistic infections. In select patients, bronchoalveolar lavage (BAL) is performed to provide high-quality samples for analysis. We compare BioFire® FilmArray® Pneumonia Panel (BioFire Diagnostics, Salt Lake City, Utah, United States), a multiplex polymerase chain reaction (PCR) assay, with standard of care diagnostics in BAL samples from immunocompromised patients to identify opportunities for this test to affect clinical decision making. Methods Patients hospitalized with pneumonia based on clinical and radiographic findings who underwent evaluation with bronchoscopy between May 2019 to January 2020 were reviewed. Among those patients undergoing bronchoscopy, those who were immunocompromised were selected for inclusion in the study. BAL specimens submitted to the microbiology laboratory were chosen based on as part of the internal validation of the panel in comparison with sputum culture at our hospitals. We compared the outcomes of the multiplex PCR assay with traditional culture methods and evaluated the role of PCR assay in de-escalating antimicrobial therapy. Results Twenty-four patients were identified for testing with the multiplex PCR assay. Of the 24 patients, 16 were immunocompromised, all with solid or hematological malignancy or a history of organ transplant. Seventeen individual BAL samples from the 16 patients were reviewed. BAL culture results and the multiplex PCR assay were in agreement in 13 samples (76.5%). In four cases, the multiplex PCR assay identified a possible causative pathogen not detected by standard workup. The median time to de-escalation of antimicrobials was three days (interquartile range (IQR) 2-4) from the day of collection of the BAL samples. Conclusions Studies have established the additive role of multiplex PCR testing in addition to traditional diagnostic tools like sputum culture in diagnosing the etiology of pneumonia. Limited data exist specifically looking at immunocompromised patients, in whom a timely and accurate diagnosis is particularly important. There is a potential benefit for performing multiplex PCR assays as an additive diagnostic tool in BAL samples for these patients.

Identifying key genes related to inflammasome in severe COVID-19 patients based on a joint model with random forest and artificial neural network.

Background: The coronavirus disease 2019 (COVID-19) has been spreading astonishingly and caused catastrophic losses worldwide. The high mortality of severe COVID-19 patients is an serious problem that needs to be solved urgently. However, the biomarkers and fundamental pathological mechanisms of severe COVID-19 are poorly understood. The aims of this study was to explore key genes related to inflammasome in severe COVID-19 and their potential molecular mechanisms using random forest and artificial neural network modeling. Methods: Differentially expressed genes (DEGs) in severe COVID-19 were screened from GSE151764 and GSE183533 via comprehensive transcriptome Meta-analysis. Protein-protein interaction (PPI) networks and functional analyses were conducted to identify molecular mechanisms related to DEGs or DEGs associated with inflammasome (IADEGs), respectively. Five the most important IADEGs in severe COVID-19 were explored using random forest. Then, we put these five IADEGs into an artificial neural network to construct a novel diagnostic model for severe COVID-19 and verified its diagnostic efficacy in GSE205099. Results: Using combining P value < 0.05, we obtained 192 DEGs, 40 of which are IADEGs. The GO enrichment analysis results indicated that 192 DEGs were mainly involved in T cell activation, MHC protein complex and immune receptor activity. The KEGG enrichment analysis results indicated that 192 GEGs were mainly involved in Th17 cell differentiation, IL-17 signaling pathway, mTOR signaling pathway and NOD-like receptor signaling pathway. In addition, the top GO terms of 40 IADEGs were involved in T cell activation, immune response-activating signal transduction, external side of plasma membrane and phosphatase binding. The KEGG enrichment analysis results indicated that IADEGs were mainly involved in FoxO signaling pathway, Toll-like receptor, JAK-STAT signaling pathway and Apoptosis. Then, five important IADEGs (AXL, MKI67, CDKN3, BCL2 and PTGS2) for severe COVID-19 were screened by random forest analysis. By building an artificial neural network model, we found that the AUC values of 5 important IADEGs were 0.972 and 0.844 in the train group (GSE151764 and GSE183533) and test group (GSE205099), respectively. Conclusion: The five genes related to inflammasome, including AXL, MKI67, CDKN3, BCL2 and PTGS2, are important for severe COVID-19 patients, and these molecules are related to the activation of NLRP3 inflammasome. Furthermore, AXL, MKI67, CDKN3, BCL2 and PTGS2 as a marker combination could be used as potential markers to identify severe COVID-19 patients.

Identification of natural compounds as SARS-CoV-2 inhibitors via molecular docking and molecular dynamic simulation.

Background: Base mutations increase the contagiousness and transmissibility of the Delta and Lambda strains and lead to the severity of the COVID-19 pandemic. Molecular docking and molecular dynamics (MD) simulations are frequently used for drug discovery and relocation. Small molecular compounds from Chinese herbs have an inhibitory effect on the virus. Therefore, this study used computational simulations to investigate the effects of small molecular compounds on the spike (S) protein and the binding between them and angiotensin-converting enzyme 2 (ACE2) receptors. Methods: In this study, molecular docking, MD simulation, and protein-protein analysis were used to explore the medicinal target inhibition of Chinese herbal medicinal plant chemicals on SARS-CoV-2. 12,978 phytochemicals were screened against S proteins of SARS-CoV-2 Lambda and Delta mutants. Results: Molecular docking showed that 65.61% and 65.28% of the compounds had the relatively stable binding ability to the S protein of Lambda and Delta mutants (docking score ≤ -6). The top five compounds with binding energy with Lambda and Delta mutants were clematichinenoside AR2 (-9.7), atratoglaucoside,b (-9.5), physalin b (-9.5), atratoglaucoside, a (-9.4), Ochnaflavone (-9.3) and neo-przewaquinone a (-10), Wikstrosin (-9.7), xilingsaponin A (-9.6), ardisianoside G (-9.6), and 23-epi-26-deoxyactein (-9.6), respectively. Four compounds (Casuarictin, Heterophylliin D, Protohypericin, and Glansrin B) could interact with S protein mutation sites of Lambda and Delta mutants, respectively, and MD simulation results showed that four plant chemicals and spike protein have good energy stable complex formation ability. In addition, protein-protein docking was carried out to evaluate the changes in ACE2 binding ability caused by the formation of four plant chemicals and S protein complexes. The analysis showed that the binding of four plant chemicals to the S protein could reduce the stability of the binding to ACE2, thereby reducing the replication ability of the virus. Conclusion: To sum up, the study concluded that four phytochemicals (Casuarictin, Heterophylliin D, Protohypericin, and Glansrin B) had significant effects on the binding sites of the SARS-CoV-2 S protein. This study needs further in vitro and in vivo experimental validation of these major phytochemicals to assess their potential anti-SARS-CoV-2. Graphical abstract.

Protective effect of plasma neutralization from prior SARS-CoV-2 Omicron infection against BA.5 subvariant symptomatic reinfection (preprint)

From December 2022 to January 2023, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections caused by BA.5 and BF.7 subvariants of B.1.1.529 (Omicron) swept across mainland China. It is crucial to estimate the protective effect of the neutralizing antibodies generated by such mass infections against the next potential SARS-CoV-2 reinfection wave, especially if driven by CH.1.1 or XBB.1.5. Previously, we recruited and continuously followed a cohort of individuals that experienced Omicron BA.1, BA.2, and BA.5 breakthrough infections, as well as a control cohort with no history of SARS-CoV-2 infection. In the previously uninfected cohort, the total symptomatic infection rate surveyed during the outbreak was 91.6%, while the symptomatic reinfection rate was 32.9%, 10.5%, and 2.8% among individuals with prior Omicron BA.1, BA.2 and BA.5 infection, respectively, with median intervals between infections of 335, 225 and 94 days. Pseudovirus neutralization assays were performed in plasma samples collected from previously Omicron BA.1-infected individuals approximately 3 months before the outbreak. Results indicate a robust correlation between the plasma neutralizing antibody titers and the protective effect against symptomatic reinfection. The geometric mean of the 50% neutralizing titers (NT50) against D614G, BA.5, and BF.7 were 2.0, 2.5, and 2.3-fold higher in individuals without symptomatic reinfection than in those with symptomatic reinfection (p < 0.01). Low plasma neutralizing antibody titer (below the geometric mean of NT50) was associated with an enhanced cumulative risk of symptomatic reinfection, with a hazard ratio (HR) of 23.55 (95% CI: 9.23-60.06) against BF.7 subvariant. Importantly, neutralizing antibodies titers post one month after BF.7/BA.5 breakthrough infections against CH.1.1 and XBB.1.5 are similar to that against BF.7 from individuals with prior BA.1 infection while not experiencing a symptomatic BF.7/BA.5 reinfection (plasma collected 3 months before the outbreak), suggesting that the humoral immunity generated by the current BF.7/BA.5 breakthrough infection may provide protection against CH.1.1 and XBB.1.5 symptomatic reinfection wave for 4 months. Of note, the higher hACE2 binding of XBB.1.5 may reduce the protection period since the potential increase of infectivity.

ZDHHC11 Suppresses Zika Virus Infections by Palmitoylating the Envelope Protein.

Zika virus (ZIKV) is an RNA-enveloped virus that belongs to the Flavivirus genus, and ZIKV infections potentially induce severe neurodegenerative diseases and impair male fertility. Palmitoylation is an important post-translational modification of proteins that is mediated by a series of DHHC-palmitoyl transferases, which are implicated in various biological processes and viral infections. However, it remains to be investigated whether palmitoylation regulates ZIKV infections. In this study, we initially observed that the inhibition of palmitoylation by 2-bromopalmitate (2-BP) enhanced ZIKV infections, and determined that the envelope protein of ZIKV is palmitoylated at Cys308. ZDHHC11 was identified as the predominant enzyme that interacts with the ZIKV envelope protein and catalyzes its palmitoylation. Notably, ZDHHC11 suppressed ZIKV infections in an enzymatic activity-dependent manner and ZDHHC11 knockdown promoted ZIKV infection. In conclusion, we proposed that the envelope protein of ZIKV undergoes a novel post-translational modification and identified a distinct mechanism in which ZDHHC11 suppresses ZIKV infections via palmitoylation of the ZIKV envelope protein.

Phillyrin for COVID-19 and Influenza Co-infection: A Potential Therapeutic Strategy Targeting Host Based on Bioinformatics Analysis.

Background: The risk of co-epidemic between COVID-19 and influenza is very high, so it is urgent to find a treatment strategy for the co-infection. Previous studies have shown that phillyrin can not only inhibit the replication of the two viruses, but also has a good anti-inflammatory effect, which is expected to become a candidate compound against COVID-19 and influenza. Objective: To explore the possibility of phillyrin as a candidate compound for the treatment of COVID-19 and influenza co-infection and to speculate its potential regulatory mechanism. Methods: We used a series of bioinformatics network pharmacology methods to understand and characterize the pharmacological targets, biological functions, and therapeutic mechanisms of phillyrin in COVID-19 and influenza co-infection and discover its therapeutic potential. Results: We revealed potential targets, biological processes, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and upstream pathway activity of phillyrin against COVID-19 and influenza co-infection. We constructed protein-protein interaction (PPI) network and identified 50 hub genes, such as MMP9, IL-2, VEGFA, AKT, and HIF-1A. Furthermore, our findings indicated that the treatment of phillyrin for COVID-19 and influenza co-infection was associated with immune balance and regulation of hypoxia-cytokine storm, including HIF-1 signaling pathway, PI3K-Akt signaling pathway, Ras signaling pathway, and T cell receptor signaling pathway. Conclusion: For the first time, we uncovered the potential targets and biological pathways of phillyrin for COVID-19 and influenza co-infection. These findings should solve the urgent problem of co-infection of COVID-19 and influenza that the world will face in the future, but clinical drug trials are needed for verification in the future.

HERE A CLOT, THERE A CLOT, EVERYWHERE A CLOT: A CASE OF VENOUS AND ARTERIAL THROMBI IN A PATIENT WITH COVID-19

TOPIC: Chest Infections TYPE: Medical Student/Resident Case Reports INTRODUCTION: Venous and arterial thrombi are both common complications seen in patients with COVID-19. This phenomenon may be due to the hyper-inflammatory state seen in COVID-19 infection. While venous thrombosis has been reported in 21% of patients with COVID-19, arterial thrombosis is more infrequent, reported in 2% of patients [1]. We present a patient with COVID-19 infection found to have pulmonary embolism (PE), left ventricular (LV) thrombus, and renal infarcts. CASE PRESENTATION: A 54-year-old male with a history of hypertension, diabetes mellitus, coronary artery disease, and heart failure with EF of 40% presented with worsening shortness of breath 10 days after having tested positive for SARS-COVID-19. The patient was placed on 100% FiO2 via high flow nasal cannula for acute hypoxic respiratory failure and started on dexamethasone, remdesivir, and enoxaparin for VTE prophylaxis.On day 6 of admission, the patient had acute onset pleuritic chest pain. ECG was unchanged from baseline and troponin level remained undetectable, however the d-dimer level increased from 338 ng/mL on admission to >10,000 ng/mL. CT chest with IV contrast revealed right upper lobe pulmonary emboli (Figure 1) and suspicion of left ventricular thrombus. The patient was started on therapeutic heparin infusion.TTE showed an akinetic left ventricular apex with echo density in apex suggestive of apical thrombus (Figure 2) and a 30% EF not seen previously. The patient was initiated on a heparin-warfarin bridge. On day 12 of admission, the patient experienced acute severe right lower quadrant pain. CT with IV contrast revealed multifocal large right renal infarcts with patent renal vessels (Figure 3). Because his renal function remained stable, no further intervention was taken. The patient's respiratory status began to improve and the patient was discharged home on warfarin. DISCUSSION: LV thrombus is a serious complication most often seen after acute myocardial infarction, but it can be found in 6% of patients with low ejection fraction and ischemic heart disease [2]. There have been reports of it in patients with COVID-19, particularly those with pre-existing cardiomyopathy such as our patient. LV thrombus would be unlikely to cause the PE seen in this patient, indicating the patient had unrelated venous and arterial thrombi due to COVID-19. However, the renal infarcts found after the patient had acute abdominal pain suggest that they are embolic from the LV thrombus. Early diagnosis and treatment of LV thrombus with warfarin as gold standard is important due to the high risk of stroke or systemic embolism [3]. CONCLUSIONS: There is an increased risk of both venous and arterial thrombi in COVID-19 despite prophylaxis. Patients with existing ischemic cardiomyopathy may be at risk for LV thrombus. Prompt recognition and management is critical to preventing additional venous and arterial embolic events and improving mortality. REFERENCE #1: Malas MB, Naazie IN, Elsayed N, Mathlouthi A, Marmor R, Clary B. Thromboembolism risk of COVID-19 is high and associated with a higher risk of mortality: A systematic review and meta-analysis. EClinicalMedicine. 2020 Dec;29:100639. doi: 10.1016/j.eclinm.2020.100639. Epub 2020 Nov 20. REFERENCE #2: Aljaber NN, Mattash ZA, Alshoabi SA, Alhazmi FH. The prevalence of left ventricular thrombus among patients with low ejection fraction by trans-thoracic echocardiography. Pak J Med Sci. 2020 May-Jun;36(4):673-677. doi: 10.12669/pjms.36.4.1972. REFERENCE #3: Robinson AA, Trankle CR, Eubanks G, Schumann C, Thompson P, Wallace RL, Gottiparthi S, Ruth B, Kramer CM, Salerno M, Bilchick KC, Deen C, Kontos MC, Dent J. Off-label Use of Direct Oral Anticoagulants Compared With Warfarin for Left Ventricular Thrombi. JAMA Cardiol. 2020 Jun 1;5(6):685-692. doi: 10.1001/jamacardio.2020.0652. DISCLOSURES: No relevant relationships by Tony Li-Geng, source=Web Response no disclosure on file for Carrie Mahowald;

SARS-CoV-2 N protein promotes NLRP3 inflammasome activation to induce hyperinflammation.

Excessive inflammatory responses induced upon SARS-CoV-2 infection are associated with severe symptoms of COVID-19. Inflammasomes activated in response to SARS-CoV-2 infection are also associated with COVID-19 severity. Here, we show a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation to induce hyperinflammation. N protein facilitates maturation of proinflammatory cytokines and induces proinflammatory responses in cultured cells and mice. Mechanistically, N protein interacts directly with NLRP3 protein, promotes the binding of NLRP3 with ASC, and facilitates NLRP3 inflammasome assembly. More importantly, N protein aggravates lung injury, accelerates death in sepsis and acute inflammation mouse models, and promotes IL-1ß and IL-6 activation in mice. Notably, N-induced lung injury and cytokine production are blocked by MCC950 (a specific inhibitor of NLRP3) and Ac-YVAD-cmk (an inhibitor of caspase-1). Therefore, this study reveals a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation and induces excessive inflammatory responses.

Protection of K18-hACE2 mice and ferrets against SARS-CoV-2 challenge by a single-dose mucosal immunization with a parainfluenza virus 5-based COVID-19 vaccine.

Transmission-blocking vaccines are urgently needed to reduce transmission of SARS-CoV 2, the cause of the COVID-19 pandemic. The upper respiratory tract is an initial site of SARS-CoV-2 infection and, for many individuals, remains the primary site of virus replication. An ideal COVID-19 vaccine should reduce upper respiratory tract virus replication and block transmission as well as protect against severe disease. Here, we optimized a vaccine candidate, parainfluenza virus 5 (PIV5) expressing the SARS-CoV-2 S protein (CVXGA1), and then demonstrated that a single-dose intranasal immunization with CVXGA1 protects against lethal infection of K18-hACE2 mice, a severe disease model. CVXGA1 immunization also prevented virus infection of ferrets and blocked contact transmission. This mucosal vaccine strategy inhibited SARS-CoV-2 replication in the upper respiratory tract, thus preventing disease progression to the lower respiratory tract. A PIV5-based mucosal vaccine provides a strategy to induce protective innate and cellular immune responses and reduce SARS-CoV-2 infection and transmission in populations.

Research progress and challenges to coronavirus vaccine development.

Coronaviruses (CoVs) are nonsegmented, single-stranded, positive-sense RNA viruses highly pathogenic to humans. Some CoVs are known to cause respiratory and intestinal diseases, posing a threat to the global public health. Against this backdrop, it is of critical importance to develop safe and effective vaccines against these CoVs. This review discusses human vaccine candidates in any stage of development and explores the viral characteristics, molecular epidemiology, and immunology associated with CoV vaccine development. At present, there are many obstacles and challenges to vaccine research and development, including the lack of knowledge about virus transmission, pathogenesis, and immune response, absence of the most appropriate animal models.

Coronavirus infections and immune responses.

Coronaviruses (CoVs) are by far the largest group of known positive-sense RNA viruses having an extensive range of natural hosts. In the past few decades, newly evolved Coronaviruses have posed a global threat to public health. The immune response is essential to control and eliminate CoV infections, however, maladjusted immune responses may result in immunopathology and impaired pulmonary gas exchange. Gaining a deeper understanding of the interaction between Coronaviruses and the innate immune systems of the hosts may shed light on the development and persistence of inflammation in the lungs and hopefully can reduce the risk of lung inflammation caused by CoVs. In this review, we provide an update on CoV infections and relevant diseases, particularly the host defense against CoV-induced inflammation of lung tissue, as well as the role of the innate immune system in the pathogenesis and clinical treatment.