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1.
Biochimica Clinica ; 46(3):S115, 2022.
Article in English | EMBASE | ID: covidwho-2168939

ABSTRACT

A 75-year-old man with a history of chronic ischemic heart disease with a previously normal blood count, presented to the emergency room with fever and tachycardia. There was no hepatosplenomegaly or lymphadenopathy. An electrocardiogram showed left bundle branch block. Because of the fever the patient underwent SARS-CoV-2 RNA testing with positive result. The patient's blood count showed a WBC of 10.46 x 109/L, lymphocytes 4.51 x 109/L, hemoglobin 129 g/ L, and platelet count 233 x 109/L. D-Dimer was 659 mug/L (normal range <500) and IL6 was 76.3 pg/ml (normal range <6.4). A computed tomography scan of the chest showed bilateral interstitial infiltrates associated with multiple enlarged mediastinal lymph nodes. Following a rapid and unexpected increase of the WBC to 17.49 x 109/L with lymphocyte count of 8.37 x 109/L, a blood film and immunotyping were performed. The film showed small/medium sized lymphocytes, with a variable N: C ratio and moderately basophilic cytoplasm. Smear cells were present. About 25% of the lymphocytes showed the negative images of one to three rodshaped crystals (average 2 per cell). Some immature monocytes and neutrophils showed mild toxic granulation or abnormal nuclear shapes, consistent with COVID-19. Flow cytometric immunotyping showed an increased number of circulating B cells (93% of lymphocytes, 7.78 x 109/L) with lambda light chain restriction and expressing CD19, CD5, CD23, weak CD20, CD43, and CD200;CD10, CD79b, CD81, FMC7, and CD38 were negative. At this stage the clinical picture could not be distinguished from chronic lymphocytic leukemia (CLL). Two months later the WBC and lymphocyte count returned to normal and immuno typing showed only 0.63 x 109/L CD5-positive clonal B cells. Lymphocytes with cytoplasmic crystals were still present. A diagnosis of monoclonal B-cell lymphocytosis (MBCL) was made. Patients with CLL in whom COVID-19 led to a marked but transient increase in the lymphocyte count have been reported. In our case, COVID-19 in a patient with MBCL led to an increase in the lymphocyte count simulating CLL but follow-up indicated the correct diagnosis. We report here the observation of endocellular crystals, attributable to crystallization of immunoglobulin, in MCBL, a phenomenon previously reported in CLL.

2.
BMC Proceedings. Conference: 6th International Conference on Molecular Diagnostics and Biomarker Discovery, MDBD ; 16(Supplement 7), 2022.
Article in English | EMBASE | ID: covidwho-2196279

ABSTRACT

Background Severe Acute Respiratory Disease 2 (SARS-CoV-2) has been identified as the causal factor to the recent COVID-19 pandemic [1]. One of the capacities required to study this virus is to isolate and propagate them according to the requirement of the studies. To date, the majority of studies utilized monkey-derived Vero cells to rapidly propagate the virus. However, due to its highly susceptibility to SARS-CoV-2 infection and limited capacity to metabolize drugs, this cell line is not suitable for pathophysiology and anti-viral research. Vero cells are not preferred for investigation of pathological mechanism of host cell's response to virus infection because they are not derived from human lung tissue. Furthermore, they are difficult to be used in the study to assess cytopathic effects as they do not express type 1 interferon genes [2]. In this study, we adapted the SARS-CoV-2 isolates into selected human lung-derived cell lines, i.e., MRC-5 and A549, to investigate the capacity of these cell lines as a platform for the characterization of SARS-CoV-2 isolated in Malaysia. MRC-5 has already been identified to be highly susceptible to the infection of various human coronaviruses, including HCoV-OC43, HCoV-229E and Middle East respiratory syndrome coronavirus (MERS-CoV) [3]. Meanwhile, A549 was selected due to its origin of lung derived. Methodology To determine the growth profile of SARS-CoV-2 in human-derived cell lines, a local SARS-CoV-2 clinical isolate obtained from the IMR archive was first adapted into Vero E6, thus resulting in the generation of passage 1 (P1). The P1 isolate was used to study the growth profile of the virus in MRC-5 and A549 cell lines. P1 isolate was subjected to Whole Genome Sequencing (WGS) to identify the genomic sequence of the isolate. The replication kinetics of these isolates in MRC-5 and A459 were evaluated based on the on the formation of cytopathic effect (CPE), Cq value, plaque forming unit (pfu) as well as observation by electron microscope (EM). Whole Genome Sequencing (WGS) was repeated on the propagated SARS-CoV-2 passages to examine the genomic stability. Results and Discussion There was no formation of CPE observed after inoculation of SARSCoV- 2 into MRC-5 and A549 cells after 7 days of culture. In parallel, the qRT-PCR results suggested that the virus did not multiply well in these cells. Plaque assay and EM images further supported these findings. In terms of genome stability, WGS data revealed several genetic polymorphisms in the genome of the virus adapted in MRC- 5 and A549 cells. Conclusion MRC-5 and A549 are not susceptible to SARS-CoV-2 infection.

3.
Vascular Medicine ; 27(6):NP10, 2022.
Article in English | EMBASE | ID: covidwho-2194544

ABSTRACT

Background: Atraumatic upper extremity arterial thrombosis is uncommon. We present a case of radial and ulnar artery thrombosis with critical limb ischemia, with symptoms resolving after remediation of a black mold home infestation. This case highlights the importance of identifying potential environmental exposures in uncommon clinical presentations. Case presentation: Chart review of all visits in two hospital systems. Informed consent was obtained from the patient. Result(s): A 51-year-old male noted pain and paresthesia of the left hand. Critical ischemia was found with mid-forearm occlusion of the radial and ulnar arteries;surgical care included no distal target for bypass and digital ray amputation. Workup did not reveal autoimmunity, thrombophilia, or source of embolism. Symptoms were recurrent with ongoing ischemia and tissue loss. Further evaluation identified a home water leak. Professional remediation of black mold coincided with resolution of pain, discoloration, and ulceration without return of symptoms at follow up of 22 months. Conclusion(s): Immune-mediated mechanisms can lead to clinical thrombotic events. Vascular occlusion related to COVID-19 has stimulated interest in thrombotic pathways not routinely emphasized. We postulate neutrophil extracellular traps (NETs), triggered by chronic exposure to mold, contributed to persistent digital ischemia. NETs are fibrous extensions of extracellular strings of DNA, antimicrobial peptides, and chromatin that bind pathogenic microbes and provide scaffolding for thrombi, triggering vascular occlusion. This is a novel case of upper extremity arterial occlusion leading to amputation associated with exposure to black mold. Thrombosis may be related to NETs formation and symptoms did not resolve until the environmental mediator was eradicated. Environmental exposure should be considered in otherwise healthy patients who present with atraumatic critical digital ischemia without thrombophilia, autoimmunity, illicit drug use, or vasculitis.

4.
Science Advances ; 8(51):eadd7197, 2022.
Article in English | MEDLINE | ID: covidwho-2193380

ABSTRACT

The oral protease inhibitor nirmatrelvir is of key importance for prevention of severe coronavirus disease 2019 (COVID-19). To facilitate resistance monitoring, we studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) escape from nirmatrelvir in cell culture. Resistant variants harbored combinations of substitutions in the SARS-CoV-2 main protease (Mpro). Reverse genetics revealed that E166V and L50F + E166V conferred high resistance in infectious culture, replicon, and Mpro systems. While L50F, E166V, and L50F + E166V decreased replication and Mpro activity, L50F and L50F + E166V variants had high fitness in the infectious system. Naturally occurring L50F compensated for fitness cost of E166V and promoted viral escape. Molecular dynamics simulations revealed that E166V and L50F + E166V weakened nirmatrelvir-Mpro binding. Polymerase inhibitor remdesivir and monoclonal antibody bebtelovimab retained activity against nirmatrelvir-resistant variants, and combination with nirmatrelvir enhanced treatment efficacy compared to individual compounds. These findings have implications for monitoring and ensuring treatments with efficacy against SARS-CoV-2 and emerging sarbecoviruses.

5.
Critical Care Medicine ; 51(1 Supplement):176, 2023.
Article in English | EMBASE | ID: covidwho-2190521

ABSTRACT

INTRODUCTION: While myopericarditis due to Coxsackie virus-B has been widely reported, multi-organ involvement is rare. We report a unique case of Coxsackie B myopericarditis, which presented with rash, atypical pneumonia, hepatitis, and sepsis. DESCRIPTION: A previously healthy 32-year-old man presented to the emergency department in January 2022 endorsing shortness of breath, high-grade fever (39.2degreeC), non-pruritic rash on extremities, vomiting, and diarrhea. He had tachypnea (24/min), hypoxia (SpO2 93% on air), and mild lymphadenopathy in the neck. Initial evaluation was pertinent for leukocytosis (17.8 thousand/muL) with neutrophil predominance (89.4%), elevated inflammatory markers (D-dimer [4390 ng/mL], procalcitonin [1.79 ng/ mL], CRP [180.7 mg/L], lactate [3.19 mmol/L]), and transamnitis (AST: 160 U/L, ALT: 116 U/L);SARS-CoV-2 and blood cultures were negative. Chest imaging showed bibasilar consolidation, perihilar ground-glass nodules, and pericardial effusion;ultrasound showed acute hepatitis. He was empirically started on ceftriaxone and azithromycin. However, absence of clinical improvement, persistence of high-grade fever, and leukocytosis with low absolute CD3, CD4, and CD8 counts (286 cells/UL, 199 cells/UL and 71 cells/UL, respectively) suggested atypical infection;vancomycin and doxycycline were added. Further infection and autoimmune workup was negative. He developed atrial fibrillation and an echocardiogram was remarkable for ejection fraction of 50-55%, moderate pericardial effusion circumferential to the heart, and minimal collapse of the right atrium. On subsequent testing, Coxsackie virus B type 3 IgM was positive (1:320, reference 1:10). All antibiotics were discontinued, and the patient was managed with diltiazem, colchicine, ibuprofen, and supportive care;anticoagulation was not initiated. After a remarkable improvement in symptoms and rash, he was discharged home. Follow-up imaging showed resolution of bibasilar consolidations and pericardial effusion. DISCUSSION: The likely mechanism of Coxsackie virus B-induced damage to myocytes (and possibly multiorgan involvement) is immune-mediated and direct viral cytotoxicity. Our patient's atypical pneumonia responded well to colchicine and ibuprofen. A high index of suspicion is warranted.

6.
Open Forum Infectious Diseases ; 9(Supplement 2):S447, 2022.
Article in English | EMBASE | ID: covidwho-2189712

ABSTRACT

Background. Quantifying neutralising capacity of circulating SARS-COV-2 antibodies is critical in evaluating protective humoral immune responses generated postinfection/post-vaccination. Here we describe a novel medium-throughput flow cytometry based micro-neutralisation assay to evaluate Neutralising Antibody (NAb) responses against live SARS-CoV-2 Wild Type (D641G) and Variants of Concern (VoC) in convalescent/vaccinated populations. Methods. Micro-Neutralisation assay (Micro-NT) was performed in 96-well plates using clinical isolate 2019-nCoV/Italy-INMI1, D641G (SARS-CoV-2/human/ IRL/AIIDV1446/2020) and/or VOCs Beta (SARS-CoV-2/human/IRL/AIIDV1752/ 2021) and Omicron (SARS-Cov-2/human/IRL/AIIDV2326/2021). Plasma samples (All Ireland Infectious Diseases (AIID) Cohort) were serially diluted (8 points, halflog) from 1/20 and pre-incubated with SARS-CoV-2 (1h, 37degreeC). Virus-plasma mixture were added onto VERO E6/VERO-E6 TMPRSS2 cells for 18h. Percentage infected cells was analysed by automated flow cytometry following trypsinisation,fixation and SARS-CoV-2 Nucleoprotein intracellular staining. Half-maximal Neutralisation Titres (NT50) was determined using four-parameter logistic regression. Our assay was compared to Plaque Reduction Neutralisation Test (PRNT) and validated against WHO anti-SARS-CoV-2 Immunoglobulin Standards. Results. Using WHO Standards with low, medium or high anti-SARS-CoV-2 IgG, both Micro-NT and PRNT achieved comparable NT50 values (Table 1). Micro-NT was found to be highly reproducible (inter-assay CV of 11.39%). Screening 190 convalescent samples and 11 COVID-19 naive controls (AIID cohort) we achieved an assay sensitivity of 90% and specificity of 81%. We demonstrated that Micro-NT has broad dynamic range differentiating NT50s < 1/20 to > 1/5000 (Figure 1). We could also characterise immune-escape VoC, observing up to 10-fold reduction in NT50 against Beta (Figure 2). Table 1: NT50s of Low, Medium and High Titre Anti-SARS-CoV-2 IgG Standards measured against Live SARS-CoV-2 using PRNT and Micro-NT Neutralising Capacity of low, medium and high-titre anti-SARS-CoV-2 IgG (WHO, International Standards) against live SARS-CoV-2 (2019-nCoV/Italy-INMI1) measured using PRNT and Micro-NT Assays on Vero E6 cells, as well as the potency of NAbs in each sample in International Units (IU/ml) as determined by the WHO. Figure 1: Dynamic Range of Micro-NT Micro-NT has a broad Dynamic Range, distinguishing low (A), medium (B) and high (C) neutralising plasma samples against live SARS-CoV-2 (2019-nCoV/Italy-INMI1) from a cohort of COVID-19 convalescent individuals (AIID cohort), as well as negative samples from COVID-19 naive samples (D). Graphs show 3 representative samples of each NT50 range. (E) shows the population distribution of 190 Convalescent plasma samples as measured by Micro-NT on Vero E6 cells. Figure 2: Reduced Neutralisation Capacities measured against SARS-CoV-2 VoC using Micro-NT Low (A), Medium (B) and High (C) anti-SARS-CoV-2 IgG (WHO Standards) show different neutralising capacities against WT (D614G) SARS-CoV-2 and variants Beta and Omicron, measured using Micro-NT on Vero-E6-TMPRSS2 cells. Conclusion. Our flow-cytometry-based Micro-NT is a robust and reliable assay to quantify NAb titres, an important evaluation endpoint in clinical trials. It has higher throughput (96 well format versus 12 well) and reduced infection time (18h vs 48-96h) compared to the gold standard PRNT.

7.
Open Forum Infectious Diseases ; 9(Supplement 2):S205, 2022.
Article in English | EMBASE | ID: covidwho-2189627

ABSTRACT

Background. Rapid COVID-19 tests can offer significant advantages and reduce health disparities. The LumiraDx SARS-CoV-2 platform can perform microfluidic fluorescence assays for the rapid detection of SARS-CoV-2 antigen (Ag) and antibodies (Ab). We evaluated both tests in a longitudinal cohort to evaluate performance during acute SARS-CoV-2 infection and recovery. Methods. We collected nasal samples from 71 unique participants at four clinic visits spanning 0-21 days since symptom onset (DSSO);blood samples were collected from the same participants over six visits spanning 0-87 DSSO. For Ag testing, 232 anterior nasal swabs were assayed by: 1) the LumiraDx Ag test, 2) a laboratory-based electrochemiluminescence immunoassay for N Ag, 3) RT-PCR (Hologic Panther Fusion), and 4) culture (growth in VeroE6AT cells). For Ab testing, 308 serum samples were assayed by: 1) the LumiraDx Ab test and 2) Roche Elecsys Anti-S SARS-CoV-2 total Ab test. Measures of concordance [positive predictive agreement (PPA), negative predictive agreement (NPA), and Cohen's Kappa (K)] were estimated for qualitative results of the LumiraDx tests versus corresponding lab reference tests. Confidence intervals were estimated via bootstrapping. Results. LumiraDx Ag results had strong agreement with lab N-Ag results (K > 0.80) across all samples. Between 0-5 days, agreement was perfect, except for one sample resulting positive by LumiraDx Ag and negative by lab Ag. Agreement with PCR results was moderate overall (K=0.60), though substantial (K > 0.6) for both 0-5 DSSO (PPA=0.96/NPA=0.80) and 6-10 DSSO (PPA=0.96/NPA=0.59). Agreement with culture results was moderate overall (K=0.46): substantial (K=0.6) between 0-5 DSSO (PPA=0.96/NPA=0.60) and fair (K=0.29) between 6-10 DSSO (PPA=1.0/NPA=0.32). LumiraDx Ab results showed almost perfect agreement with lab Ab results across all samples (K=0.88), with substantial agreement (K > 0.7) for samples collected 0-10 DSSO (PPA=0.93/NPA=0.89) and 11-28 DSSO (PPA=0.99/NPA=0.69). Longitudinal agreement of LumiraDx antigen test result and culture positivity, by PCR Ct value. Nasal samples grouped by participant (lines) and agreement of results between LumiraDx antigen test result and culture positivity (proxy for infectiousness). Conclusion. LumiraDx rapid tests perform well compared to more costly and time-consuming lab methods of Ag and Ab detection. The rapid Ag test may be helpful in identifying patients infectious between 0-5 DSSO, given the substantial concordance of the rapid Ag test and culture positivity.

8.
Open Forum Infectious Diseases ; 9(Supplement 2):S124, 2022.
Article in English | EMBASE | ID: covidwho-2189546

ABSTRACT

Background. Molnupiravir is an orally available prodrug of the antiviral nucleoside analog N-Hydroxycytidine (NHC). In preclinical studies NHC has shown broad-spectrum antiviral activity against multiple RNA viruses including SARS-CoV-2. Incorporation of NHC by viral polymerases impairs replication by introducing errors into the viral genome. NHC has been shown to have a high barrier to the development of resistance in vitro with RSV, Influenza and Venezualen Equine Encephalitis viruses. In these studies, we have explored the potential for SARS-CoV-2 to develop resistance to NHC in cell culture. Methods. Vero E6 cells were infected with SARS-CoV-2 (WA-1) in triplicate in the presence of NHC or a C3L-protease inhibitor (MRK-A). Culture supernatants from wells with the highest drug concentration exhibiting a cytopathic effect (CPE) score of>=2+ were repassaged and at each passage, IC50 values were estimated based on CPE scoring. At each passage, full genome next generation sequencing (NGS) was performed on the viral RNA Results. No change in susceptibility to NHC (EC50 fold change <= 1.1) was noted in 2 of 3 cultures and a 2-fold change was observed in one culture after 30 passages. In contrast, a 3- to 4-fold decreases in susceptibility to the 3CL protease inhibitor were seen by passage by 12, with increasing resistance of 4.6- to 15.7-fold observed by passage 30. NHC passaged viruses exhibited 53 to 99 amino acid changes, including substitutions and deletions (both in-frame and frameshift), across 25 different viral proteins as compared with 10 to 13 changes in 13 proteins in the MRK-A cultures. With NHC, 3 to 4 changes were observed in the viral polymerase;however, these were randomly distributed, and none were observed more than once. In contrast, the 3CL protease passaged virus had a nsp5 T21I substitution detected in all 3 cultures. Conclusion. No evidence of SARS-CoV-2 phenotypic or genotypic resistance was observed following 30 passages with NHC. A random pattern of amino acid changes were observed across multiple proteins consistent with the mechanism of action of NHC. In the same study, resistance was readily selected to a control 3CL protease inhibitor. Together these data support previous reports demonstrating the high barrier to resistance of NHC.

9.
Medical Mycology ; 60(Supplement 1):234-235, 2022.
Article in English | EMBASE | ID: covidwho-2189372

ABSTRACT

Objectives: Mucormycosis is an aggressive, life-threatening infection caused by fungi in the order Mucorales. There was an explosion of new cases of rhino-sino-orbital mucormycosis following the COVID pandemic in India, and the need for easy and rapid diagnostics was felt. The current diagnosis of mucormycosis relies on mycological cultures, radiology, and histopathology. These methods lack sensitivity and are most definitive later in the course of infection, resulting in the failure of timely intervention. A real-time multiplex PCR platform is commercially available for the detection of Rhizopus spp., Mucor spp.Rhizomucor spp., Lichtheimia spp., and Cunninghamella spp. (PN-700, MucorGenius , PathoNostics , Maastricht, The Netherlands) This real-time PCR has been validated to identify these fungal pathogens from bronchoalveolar lavage, tissue, and serum samples. This study aimed to validate this PCR-based system to detect Mucorales from nasal swab samples and evaluate its utility in the detection of Mucorales from nasal cavities of high-risk patients developing signs and symptoms of mucormycosis. Method(s): A single-center cross-sectional observational study was conducted on 50 hospitalized adult patients with signs and symptoms of mucormycosis. Nasal swabs were taken for PCR analysis once there was a clinical suspicion and were com-pared with the results of the gold standard.The gold standard for the diagnosis of mucormycosis was the conventional method (KOHmountedmicroscopy/HPE).Demographicdetails andrisk factorsfor thesepatients wererecorded, andthe RTPCR-based test was run on the nasal swab samples of all these 50 patients. The workflow is depicted graphically in Fig. 1 (Created with BioRender.com). Result(s): The study population mean (SD) age was 50 (16) years and consisted of 32 (64%) males. A total of 39 (78%) patients were known cases of diabetes mellitus, 48 (96%) patients had amphotericin B intake, and 20 (40%) had posaconazole intake. In all, 21 (42%) patients had a past history of COVID-19 infection;14 patients had received steroids and 10 patients received oxygen support. PCR for Mucorales was positive in 15 (30%) patients while the KOH mount was positive in 4 (8%) patients. Conclusion(s): These results are not encouraging for the use of nasal swabs as the sample for diagnosis of mucormyco-sis. Though the PCR performed better on the swab samples than KOH preparation and culture techniques, it highlights the importance of using standard sampling methods.

10.
Medical Mycology ; 60(Supplement 1):83-84, 2022.
Article in English | EMBASE | ID: covidwho-2189360

ABSTRACT

Background: Mucormycosis is a deadly fungal infection that emerges in patients affected with COVID-19. All fungal illnesses are caused by dysregulated adaptive immunity, but Myeloid-derived suppressor cells (MDSC) have added a new di-mension to the chronic inflammatory response. Objective(s): We attempted to enumerate the MDSC immune response in rhino-orbital mucormycosis patients before and after treatment and compared the data with healthy control. Method(s): A total of 3 ml of blood samples were taken in an EDTA vial from 20 patients with mucormycosis and 20 age-matched healthy control. A second blood sample was collected to examine the immune system post three months of treatment. Mycological identification was performed on nasal crust retrieved aftersurgery using KOH/culture.The expression of the MDSC marker was analyzed by immunostaining with the antibodies against CD14, HLA-DR, CD11b, CD33, CD66 (Biolegend). Flu-orescence profiles were recorded by Flow Cytometer (BD FACSAria TM III) and analyzed by Flow Jo s oftware (BD Biosciences). The percentage of positive cells is used to express the results.The GraphPad Prism (version 8, GraphPad s oftware, LaJolla, CA, USA) was used to analyze the data. All of the results were considered significant when P <.05. Result(s): All of the patients tested positive for Rhizopus arrhizus, which was confirmed by the culture. The percentages of Monocytic-MDSC (mMDSC: CD14 + HLA-DR-/low) cells were significantly high in patients compared to healthy control. In post-3-month treatment, the percentages of mMDSC were found significantly low and comparable with healthy control. Granulocytic MDSC (gMDSC: HLA-DR-/low CD33 + CD11b + CD66 +) cell population was higher in patients compared with healthy control and patients with post-3-month treatment. Conclusion(s): MDSC regulates T cells and other immune cells with a different mode of action. The findings in this study imminently indicatethe mechanism of immunedysregulation involvingMDSCpathways inmucormycosis andprovide evidence that restoration of immune balance causes reduction of MDSCcells may be considered a therapeutic option for long-term benefit.

11.
Antimicrobial Stewardship and Healthcare Epidemiology ; 2(S1):s9, 2022.
Article in English | ProQuest Central | ID: covidwho-2184927

ABSTRACT

Background: Understanding SARS-CoV-2 persistence on surfaces can help inform transmission risk from surfaces in healthcare and community settings. A sensitive viral infectivity assay is crucial for the detection of infective virus in environmental investigations. The conventional cell culture-based infectivity assay is limited by the time dependence, subjectivity, and insensitivity of cytopathic effect (CPE) scoring. We validated an integrated cell-culture and reverse-transcription quantitative RT-PCR method (cc-RT-qPCR) to improve SARS-CoV-2 detection and reduce detection time. We compared cc-RT-qPCR with CPE-scored cell culture to evaluate assay sensitivity of recovered virus from stainless-steel coupons simulating nonporous healthcare surfaces. Method: Human β-coronavirus OC43, a model strain for SARS-CoV-2, was propagated on HRT-18G cells in growth medium at 33°C in a 5% CO2 incubator. The OC43 infectivity was determined by cell culture with a 10-fold dilution series of viral samples in 96-well plates, and incubation for 7 days at 33°C to confirm CPE. Plates were CPE-scored and TCID50 was calculated using the Reed-Muench method. For the cc-RT-qPCR assay, CPE-negative wells were interrogated for viral intracellular replication using RT-PCR;infectivity was based on a titer increase of ≥ 2 logs 7 days after inoculation using RT-qPCR. CPE-positive or replicative virus-harboring cells were enumerated to determine TCID50. The sensitivity of both CPE-scored cell culture and cc-RT-qPCR assays were evaluated by inoculating 105 TCID50/mL OC43 in infection media and artificial saliva matrices onto coupons and dried in an environmental chamber at 26°C and 57% relative humidity for 6 hours. Viral eluates from coupons served as test samples. Results: Low-titer infectious OC43 (0.75 log10) was detected by both methods 7 days after incubation;however, infectivity confirmation required 4 and 6 days after incubation, respectively, for cc-RT-qPCR and CPE-scored cell culture methods. When cells were inoculated with OC43 at titer range 1.75–4.75 log10, CPE presented at 4–5 days after incubation, while viral replication was already detected at 3 days after incubation via RT-PCR. Upon virus titration, cc-RT-qPCR demonstrated greater sensitivity, detecting up to 1 log10 higher of infectious OC43 than cell culture alone at 0 and 6 hours (P ≤ .05) dried in infection medium and 0 hours (P ≤ .05) in saliva. Conclusions: Our data demonstrated greater sensitivity and shorter times to detect viral replication by cc-RT-qPCR, minimizing potential for false-negative results with cell culture alone. This sensitive assay may provide investigators with quicker results for informing infection control practices to reduce risk of transmission from deposited bodily fluids on surfaces, eg, coughing and sneezing.Funding: NoneDisclosures: None

12.
Biochimica et Biophysica Acta - Bioenergetics ; Conference: EBEC2022, 2022.
Article in English | EMBASE | ID: covidwho-2176722

ABSTRACT

Molecular hydrogen H2 has been reported to be an antioxidative, anti-inflammatory, and antiapoptotic agent with therapeutic potential for various diseases such as cardiac arrest, asthma, chronic obstructive pulmonary disease (COPD), and, most recently, COVID-19 [1]. In previous studies, H2 is typically administered repeatedly or over longer periods of time (hours to days) via inhalation of H2 gas, drinking H2-rich water, or injection of H2 saline, wherefore the observed effects, e.g. on mitochondrial metabolism [2], might be either directly or indirectly related to H2. To investigate a direct short-term effect of H2 on mitochondrial function, we measured mitochondrial respiration and H2O2 production in permeabilized HEK 293T cells upon sequential changes of H2 concentration cH2 in the experimental medium. O2 and H2O2 flux were measured simultaneously in the O2k with the Fluo-Module (Oroboros Instruments). Increase of cH2 was accomplished by injecting H2 into the gas phase of the open O2k-chamber. This causes not only an increase of cH2 but also a decrease of oxygen concentration cO2. As mitochondrial ROS production is a continuous function of cO2, we used the conventionally applied N2 gas as a control to distinguish between cO2- and cH2-dependent effects. Measurements were started near air saturation (~160 muM of oxygen). The plasma membrane was permeabilized with digitonin and the NADH-linked substrates pyruvate & malate were titrated to measure O2 and H2O2 flux in the LEAK state (without ADP). Upon transition of cO2 from ~160 to ~25 muM, a decrease in O2 and H2O2 flux was observed. This was comparable between regimes with increased cH2 or cN2. Further transitions by re-oxygenation and injection of H2 or N2 yielded the same results. Similarly, cO2-dependent changes in mitochondrial respiration and H2O2 production in the OXPHOS state (kinetically saturating [ADP]) were independent of the increase in cH2 or cN2. These results indicate that short-term exposure to increased cH2 does not affect mitochondrial respiration or H2O2 production. [1] Y. Tian, Y. Zhang, Y. Wang, Y. Chen, Hydrogen, a Novel Therapeutic Molecule, Regulates Oxidative Stress, Inflammation, and Apoptosis, Frontiers in Physiology, 12 (2021) 1-14 [2] A. Gvozdjakova, J. Kucharska, B. Kura, O. Vancova O, A new insight into the molecular hydrogen effect on coenzyme Q and mitochondrial function of rats, J Physiol Pharmacol., 1 (2020) 29-34 Copyright © 2022

13.
Journal for ImmunoTherapy of Cancer ; 10(Supplement 2):A1169, 2022.
Article in English | EMBASE | ID: covidwho-2161956

ABSTRACT

Background Messenger ribonucleic acid (mRNA) is a powerful tool for transferring genetic information. Its advantages include potent but transient gene expression without risk of genomic insertion, tailorable immunogenicity to match therapeutic application, and the potential for efficient, scalable manufacturing.1 The recent success of mRNA-based SARSCoV- 2 vaccines has inspired interest in mRNA as a cancer therapy to deliver immunostimulatory molecules and tumor antigens. However, clinical translation is limited by mRNA instability at physiological conditions and inefficient in vivo delivery.2 A reliable, non-toxic, and stabilizing in vivo delivery system for immunotherapeutic mRNA would help to advance mRNA as a viable cancer therapy. Here, we utilized calcium phosphate mineral-coated microparticles (MCMs) as a delivery system for mRNA-lipid complexes (lipoplexes) to transfect melanoma cells. Methods MCMs were prepared as previously described3 by suspending beta-tricalcium phosphate particles in modified simulated body fluid under rotation for 7 days at 37degreeC, refreshing the media daily. MCMs were then washed in deionized water and freeze dried. Custom-synthesized reporter or therapeutic mRNA constructs were complexed with a lipidic transfecting agent through mixing, then resulting lipoplexes were incubated briefly with MCMs to facilitate electrostatic binding to the porous CaP coating (figure 1a). Loaded MCMs or soluble lipoplexes were added to B16F10 murine melanoma cell culture, and transfection was measured through various assays, including fluorescence microscopy, bioluminescence, and enzymelinked immunosorbent assays. Results Scanning electron microscopy was used to verify platelike, porous coating morphology following MCM fabrication (figure 1b). MCMs enhanced transfection of B16F10 melanoma cells compared to soluble mRNA lipoplex delivery. This was demonstrated with reporter constructs encoding enhanced green fluorescent protein (eGFP, figure 1c) and Gaussia luciferase (G-Luc), as well as with a therapeutic construct encoding interleukin 15 (IL-15), a T cell growth factor. Timelapse imaging also revealed more rapid transfection with MCMs. A close proximity of cells to MCMs was observed as necessary for transfection. Conclusions We demonstrated that MCMs efficiently and locally deliver mRNA lipoplexes to melanoma cells and cause elevated levels of protein expression compared to soluble lipoplex delivery. This enhanced delivery profile makes MCMs a potential drug delivery platform for future in vivo tumor studies and clinical translation. (Figure Presented).

14.
Bmj ; 379, 2022.
Article in English | ProQuest Central | ID: covidwho-2152959

ABSTRACT

Among 3000 people with hypertension inadequately controlled by medication, who were treated with renal denervation, there were sustained reductions in systolic blood pressure and fewer major cardiovascular events over 36 months of follow-up (J Am Coll Cardiol doi:10.1016/j.jacc.2022.08.802). PACE labels Ten worksite cafeterias in England were randomised in the order in which they introduced labels containing information about physical activity calorie equivalents (PACE labels) on selected food and drinks. Experiments in transgenic mice and in cell culture now link the APOE gene with faulty lipid processing in oligodendrocytes.

15.
Revista Cubana de Medicina Tropical ; 74(2) (no pagination), 2022.
Article in Spanish | EMBASE | ID: covidwho-2147758

ABSTRACT

Introduction: Collection media of clinical samples with the capacity to denature viruses reduce the risk of contagion during transportation and processing. Objective(s): To use the nucleic acids transport media (NATM) in nasopharyngeal swab samples collected for the diagnosis of SARS-CoV-2. Method(s): An experimental study was conducted to demonstrate the medium capacity to inactivate viral infectivity. Zika virus (ZIKV), of biosafety level 2, was used as an enveloped virus model. The clinical performance of the NATM for the diagnosis of SARS-CoV-2 was evaluated. A ZIKV strain propagated in the Vero cell line was used and, prior to cells infection, ZIKV was in contact at different intervals (2;15, and 30 min) with pure NATM;subsequently, serial dilutions (10-1-10-4) were performed. Viral inactivation was evaluated by RT-PCR in the supernatant and the collected cells when the propagation period was completed. CITOSWAB VTM was used as reference to estimate the clinical performance of the NATM in 30 nasopharyngeal swabs collected for the diagnosis of SARS-CoV-2 infection. Result(s): ZIKV remained infectious at inoculum dilutions of >= 10-2, regardless of contact time. Clinical specificity and sensitivity of the NATM for the diagnosis of SARS-CoV-2 were 100%, respectively. Conclusion(s): Results suggest that ZIKV positive clinical samples at dilutions <= 10-1 of the NATM can be safely handled, which could potentially be applied to the molecular diagnosis of SARS-CoV-2. Copyright © 2022, Editorial Ciencias Medicas. All rights reserved.

16.
Medical Journal of Malaysia ; 77(Supplement 4):46, 2022.
Article in English | EMBASE | ID: covidwho-2147523

ABSTRACT

Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) a virus from the Coronaviridae family that causes the Coronavirus disease (COVID-19), has emerged and spread since December 2019. Since then, many in vitro and in vivo models for COVID-19 research has been developed. Objective(s): This study aimed to determine infectivity rate of various SARS-CoV-2 strains in the Vero E6 cell line. Material(s) and Method(s): Four SARS-CoV-2 strains (Wuhan, Alpha, Beta, and Delta) were isolated from clinical samples. Virus titre concentration of all strains were measured using Tissue Culture Infectious Dose (TCID50) assay and Plaque assay. At similar virus titre concentration, all strains were incubated in the Vero E6 cells at 37degreeC for 72 hours. At the end of incubation period, all virus cultures were terminated and analysed using TCID50 assay. Result(s) and Conclusion(s): It was found that the Wuhan strain has the highest infectivity rate (3601 PFU/mL/72hours) towards the Vero E6 cells, followed by Alpha (2946 PFU/mL/72hours), Beta (1780 PFU/mL/72hours) and Delta (571 PFU/mL/72hours). Vero E6 cell is commonly used for virus isolation and propagation, however this cell does not mimic the primary entry sites in the human respiratory track. The successful isolation and culture of SARS-CoV-2 in the Vero E6 cell is multifactorial, with high viral titre in source clinical samples and low passage number of cell culture as key factors. Vero E6 cell is susceptible towards all SARS-CoV-2 strains and can be used as in vitro COVID-19 culture model. Further studies can be conducted to determine the influence of different cell lines on the COVID-19 infectivity.

17.
30th International Conference on Software, Telecommunications and Computer Networks, SoftCOM 2022 ; 2022.
Article in English | Scopus | ID: covidwho-2146141

ABSTRACT

VR apps in the categories of gaming, leisure, social, fitness, work and education are becoming increasingly popular. The aim of this paper is to determine the potential for increasing efficiency and conserving resources in laboratory processes. For this purpose, literature research was conducted on the one hand, and on the other hand, a virtual simulation environment in the field of cell culture was created within a Laminar Flow Hood (Flow) using the Unity development environment and the Oculus Quest 2 VR goggles. Outstanding features of the app are that, in addition to the possibility of being used via cable using a PC (PCVR), it can also be operated wireless as an Android standalone package, and that it can be used with controllers as well as with hand tracking, i.e., freehand. The Laminar Flow application was extensively tested on a group of 10 employees and students of the Department of Biomedical Engineering. The data obtained from the app, as well as questionnaires completed by the test persons, show that a usability-friendly application can be used to train workflows efficiently and, in a resource saving manner. Furthermore, the more often the workflow has been worked through by a person, a greater improvement in the workflow was achieved. In addition, a strong acceptance and high commitment of the users in the technical environment can be seen. Regarding comparable studies and although the test group is subject to some limitations the enormous potential that IVR offers to support location-based training sessions in companies and especially in laboratories and university facilities can be seen. In view of the decreasing prices, the technical advancement of VR devices and the paradigm shift of work and education towards increased home office driven by COVID-19, intensified future research in the field of IVR can be justified in combination with these positive results. © 2022 University of Split, FESB.

18.
Tissue Engineering - Part A ; 28(Supplement 3):335, 2022.
Article in English | EMBASE | ID: covidwho-2134755

ABSTRACT

Nanomedicine has already revolutionized medicine through the development of materials that can prolong circulation in the body, avoid immune system clearance, penetrate cells and bacteria, invade tumors, promote tissue growth, inhibit infection, and so much more. New fields have emerged such as 4D printing which can enhance the performance of nanomaterials by 3D printing them into desirable shapes, implant them, and control their shape through external stimuli (such as near infrared excitation, temperature control, and others). This presentation will provide an overview of 25 years of commercializing University based research into real products helping human health. It will cover the promises and pitfalls of commercializing University based research and even discuss if this is the right model to advance science and research into the medical industry. It will also highlight new areas emerging for commcerialization such as the use of 4D printing in medicine for straightening the spine for scoliosis patients, closing of sphincters that weaken as one ages (for example, to decrease acid reflux from the stomach to the esophagus), promote intervertebral tissue growth by increasing pressure on such tissue during regeneration, deliver stem cells on the same materials in which they are cultured to enhance stem cell viability, and more. It will cover additional new areas like picomedicine, implantable sensors and more. It will cover in vitro and in vivo assessment of such materials and discuss what is needed to experience full application of nanomedicine throughout all of medicine.

19.
Research and Practice in Thrombosis and Haemostasis Conference ; 6(Supplement 1), 2022.
Article in English | EMBASE | ID: covidwho-2128221

ABSTRACT

Background: Neutrophil extracellular traps (NETs) release is the one of the main mechanisms behind hypercoagulability and disease severity in severe acute respiratory syndromes. The identification of drugs capable of inhibiting this pathological mechanism is mandatory. Aim(s): Neutrophil extracellular traps (NETs) release is the one of the main mechanisms behind hypercoagulability and disease severity in severe acute respiratory syndromes. The identification of drugs capable of inhibiting this pathological mechanism is mandatory. Method(s): Healthy neutrophils (20 x 103/well) were stimulated with phorbol myristate acetate (PMA) or sera from severe COVID-19 patients (n = 16) in the presence or absence of dipyridamole (10 muM), aspirin (1 mM) and heparin (50 mug/mL). Neutrophils nuclei were stained with nuclear red and incubated with a medium containing the non-permeable cell membrane marker Sytox Green. Cell images were obtained using IncuCyte ZOOM and the number of cells that suffered netosis was monitored over time. NETs release was determined after 1 h of incubation and the percentage of NETs was calculated dividing the number of green cells by the total number of cells per well. Result(s): COVID-19 induced NETs was lower in neutrophils pretreated with heparin (median 2.6%, IQR 2.6-2.9) than in non-treated neutrophils (median 3.6%, IQR 3.2-4.0, p < 0.0001). Pretreatment with dipyridamole and aspirin did not change the effect of COVID-19 sera in inducing NETs. A similar pattern of inhibition was observed with PMA stimulation, in which heparin decreased NETs by 3 times (NETs after PMA 43.2% and NETs after PMA and heparin 14.8%) while dipyridamole and aspirin did not significantly affect the release of PMA-induced NETs (Figure 1). Figure 2 illustrates the identification of NETs. Conclusion(s): Heparin was capable of inhibiting in vitro NETs release induced by COVID-19, while dipyridamole and aspirin had no significant effect on this process. Such findings are in line with evidence that heparin use can improve COVID-19 prognosis. (Figure Presented).

20.
Research and Practice in Thrombosis and Haemostasis Conference ; 6(Supplement 1), 2022.
Article in English | EMBASE | ID: covidwho-2128158

ABSTRACT

Background: Angiostatin is a break-down product of plasmin(ogen). Physiologically, angiostatin is generated by platelets in an urokinase (uPA)-dependent manner. During normoxia angiostatin has anti-angiogenic/ anti-inflammatory effects and protects against lung injury, however during hypoxia/acidosis it is pro-apoptotic. In SARS-CoV infected mice, the uPA-plasminogen pathway was shown to be the most transcriptionally enriched regulating sublethal vs. lethal infection. Similarly, uPA has been shown to be transcriptionally upregulated in SARS-CoV- 2;however, the role of angiostatin has not been investigated. Aim(s): To assess role of angiostatin in COVID-19. Method(s): Plasma samples from COVID-negative controls and from hospitalized COVID-19 patients (n = 30) were collected (day 1, 7, 14, 28, 70) via the COVID-19 Surveillance Collaboration study. WHO clinical progression scale was used to assess COVID-19 severity. Angiostatin and plasminogen were quantified by immunoblot. VeroE6 cells were infected with SARS-CoV- 2 and treated with angiostatin (140 mug/ml) for 24h at pH 7.5 or 6.9. Cell death was quantified by both TUNEL and the percentage of detached cells. Immunofluorescent staining against the spike protein was used to confirm cellular infection. Result(s): Plasma angiostatin level was elevated in COVID-19 patients compared to COVID-negative controls at baseline. Both angiostatin and plasminogen increased with time of hospitalization in patients with severe COVID-19, but not with mild-to- moderate disease (p = 0.05;Fig.1). In preliminary cell culture experiments, at pH = 7.5 angiostatin decreased the percentage of detached (26 +/- 8% vs 67 +/- 5%;p = 0.0004) and TUNEL-positive VeroE6 (6 +/- 6 vs 11 +/- 7%;p = 0.07) following infection. Conversely, at pH = 6.9 angiostatin increased the percentage of detached cells following infection. Interestingly, angiostatin lowered the percentage of spike protein-positive Vero E6 at both pH (Fig.2). Conclusion(s): Angiostatin concentrations increase with disease progression in severe COVID-19. This likely reflects angiostatin's complex role in COVID-19 pathophysiology. Angiostatin promotes cell death in acidotic microenvironments (associated with severe Covid-19). Conversely, at physiological pH, angiostatin may have protective effects possibly by reducing viral entry and/or replication. (Figure Presented).

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