ABSTRACT
Background: Institutions across the country are experiencing delays in receipt of essential infant formula and feeding supplies due to a supply chain crisis. The supply chain crisis commenced during the COVID-19 pandemic and has continued into present day, late 2022. The supply chain crisis led to an unstable supply of ready to feed (RTF) infant formulas for a children's hospital within a medical center containing a neonatal ICU, pediatric ICU, pediatric floor and newborn nursery. RTF formulas are the recommended inpatient infant feeding due to their sterility. Method(s): To address these RTF formula supply challenges, interprofessional leadership from Clinical Nutrition, Nursing and Supply Chain developed a local infant formula committee. The committee convened based on the needs of the institution, ranging from daily to weekly beginning October 2021 to present day. A shared, live spreadsheet allowed for real time inventory of RTF formula on the Nursing units and amount of product pending receipt in supply chain. Upon identification of low RTF supply, increased usage or RTF outage, the committee implemented a three-tiered action plan for each unit. For the first tier, the formula roomdiluted a higher calorie RTF liquid with water to the desired calorie density (example RTF 24 to RTF 20 calorie/oz). The medical team had an infant formula substitution list to guide feeding alternatives for specialty preparations. In the second tier, the formula room prepared stock formula for each unit daily, with a 24-hour expiration time, to accommodate potential for rapid census changes outside of the formula room operation. As a third layer of safety, powder emergency stock was pre-measured and sent with instructions for Nursing to reconstitute with sterile water, in a dedicated space, if all stock RTF formula was used. The powder emergency stock expired in 30 days, which allowed for a longer shelf life than the stock RTF formula. Result(s): It is practical for institutions without a formula room to implement similar processes using dedicated infant formula preparation space and storage. It also worth mentioning during this time there was a national shortage of powdered infant formulas due to a recall issued in May of 2022 by a major formula manufacturer. The national shortage included elemental powdered formulas for which there is no RTF alternative. Management of elemental formula outages were managed on a case-by-case basis by the Clinical Nutrition department. The Committee also convened to discuss allocations and identify substitutions for other neonatal and pediatric specialty items including sterile water, feeding preparation bottles, ENFit syringes and syringe caps, breastmilk collection containers and infant feeding nipples. Conclusion(s): Using this three-tiered process, the medical center provided sterile RTF formula to infants when available, remained consistent with best practices, predicted inventory needs consistent with usage and prevented waste of powdered infant formula in a time of scarcity. Technology and the anticipatory interprofessional leadership using a three-tiered action plan equipped the medical center for this most extraordinary infant formula crisis nationally.
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Background: The unprecedented scale of the COVID-19 pandemic and rapid evolution of SARS-CoV-2 variants underscores the need for broadly active inhibitors with a high barrier to resistance. The coronavirus main protease (Mpro) is an essential viral enzyme required for viral polyprotein processing and is highly conserved across human coronaviruses. Pomotrelvir (PBI-0451) is a novel Mpro inhibitor currently completing phase 2 clinical trial. Here we describe the mechanism of action, broad activity against SARS-CoV-2 clinical isolates, combination studies with other SARS-CoV-2 inhibitors and favorable resistance profile of pomotrelvir. Method(s): The kinetic parameters of pomotrelvir Mpro inhibition and its interaction with nirmaltrevir were determined in a kinetic protease assay. The IC50s of pomotrelvir on mutant Mpro proteins were measured in an endpoint Mpro assay. Combination studies of pomotrelvir with remdesivir and molnupiravir were carried out in A549-hACE2 cells infected with SARS-CoV-2 NLuc virus. Activity against SARS-CoV-2 clinical variants was assessed by infection of A549-ACE2-TMPRSS2 cells followed by immunostaining of the viral nucleocapsid protein. Result(s): Pomotrelvir is a potent competitive inhibitor of SARS-CoV-2 Mpro (Ki =2.7 nM). Binding of pomotrelvir and the Mpro inhibitor nirmatrelvir to the active site is mutually exclusive. In the SARS-CoV-2 NLuc assay, pomotrelvir is additive when combined with remdesivir or molnupiravir, two nucleoside analogs targeting viral RNA synthesis. When the effect of Mpro substitutions previously selected in a resistance study of pomotrelvir were analyzed in an enzyme assay, only Mpro-N133H showed a significant increase in IC50 (45-fold). The catalytic efficiency of Mpro-N133H is reduced by 10-fold and the recombinant virus SARSCoV-2 (WA1) -N133H is not viable, suggesting that N133H has lower replicative fitness. Lastly, pomotrelvir exhibits broad activity against all SARS-CoV-2 clinical isolates tested to date, including five omicron variants. Conclusion(s): PBI-0451 is a potent competitive inhibitor of SARS-CoV-2 Mpro and is broadly active against SARS-CoV-2 clinical isolates including omicron variants. Results from inhibitor interaction studies support the potential combination of pomotrelvir with remdesivir and molnupiravir but not nirmatrelvir. Enzymatic characterization of in vitro selected pomotrelvir resistant variants indicates they either confer no resistance or have reduced fitness.
ABSTRACT
Pulmonary aspergillosis (PA) is a category of respiratory illnesses that significantly impacts the lives of immunocompromised individuals. However, new classifications of secondary infections like influenza associated aspergillosis (IAA) and COVID-19 associated pulmonary aspergillosis (CAPA) only exacerbate matters by expanding the demographic beyond the immunocompromised. Meanwhile anti-fungal resistant strains of Aspergillus are causing current treatments to act less effectively. Symptoms can range from mild (difficulty breathing, and expectoration of blood) to severe (multi organ failure, and neurological disease). Millions are affected yearly, and mortality rates range from 20-90% making it imperative to develop novel medicines to curtail this evolving group of diseases. Chalcones and imidazoles are current antifungal pharmacophores used to treat PA. Chalcones are a group of plant-derived flavonoids that have a variety of pharmacological effects, such as, antibacterial, anticancer, antimicrobial, and anti-inflammatory activities. Imidazoles are another class of drug that possess antibacterial, antiprotozoal, and anthelmintic activities. The increase in antifungal resistant Aspergillus and Candida species make it imperative for us to synthesize novel pharmacophores for therapeutic use. Our objective was to synthesize a chalcone and imidazole into a single pharmacophore and to evaluate its effectiveness against three different fungi from the Aspergillus or Candida species. The chalcones were synthesized via the Claisen-Schmidt aldol condensation of 4-(1H-Imizadol-1-yl) benzaldehyde with various substituted acetophenones using aqueous sodium hydroxide in methanol. The anti-fungal activity of the synthesized chalcones were evaluated via a welldiffusion assay against Aspergillus fumigatus, Aspergillus niger, and Candida albicans. The data obtained suggests that chalcone derivatives with electron-withdrawing substituents are moderately effective against Aspergillus and has the potential for further optimization as a treatment for pulmonary aspergillosis. This project was supported by grants from the National Institutes of Health (NIH), National Institute of General Medicine Sciences (NIGMS), IDeA Networks of Biomedical Research Excellence (INBRE), Award number: P20GM103466. The content is solely the responsibility of the authors and do not necessarily represent the official views of the NIH.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
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Background: Remdesivir (RDV) is a broad-spectrum nucleotide analog antiviral approved for the treatment of COVID-19 in patients who are hospitalized or non-hospitalized and at risk of progressing to severe disease. Here we present SARS-CoV-2 resistance analyses from the Phase 3 PINETREE trial. Method(s): PINETREE was a double-blind, placebo-controlled trial of nonhospitalized participants (N=562) with COVID-19 and >=1 risk factor for disease progression, randomized to receive RDV or placebo once-daily for 3 days. The whole genome of SARS-CoV-2 was sequenced from nasopharyngeal swabs collected at days 1 (baseline), 2, 3, 7, and 14 using next-generation sequencing. Emergent amino acid substitutions in SARS-CoV-2 from participants treated with RDV were tested in a replicon system to determine if they alter sensitivity to RDV. Result(s): Resistance analysis criteria included all participants in the RDV group and 50% in the placebo group with viral load above the lower limit of detection for the viral load assay. Of 281 participants who met these criteria, baseline and postbaseline sequencing data were available for 115/130 (88.5%) participants in the RDV group and 129/151 (85.4%) participants in the placebo group (Table 1). Among these, emergent substitutions in Nsp12 were observed in 8/115 (7.0%) in the RDV group and 7/129 (5.4%) in the placebo group. A total of 7 emergent amino acid substitutions in Nsp12 were observed in the RDV group, but not in the placebo group. Among these, only one substitution from one participant (A376V;first detected at day 14), showed reduced in vitro susceptibility to RDV, with a half-maximal effective concentration (EC50) fold-change of 12.6 compared with a wildtype reference. The participant achieved clinical recovery by day 14. None of the other substitutions impacted RDV susceptibility (EC50 fold-change <=1.4). Emergent substitutions in Nsp8, Nsp10, Nsp13, or Nsp14 were detected in 10/115 (8.7%) of participants in the RDV group and 10/129 (7.8%) in the placebo group, with substitutions in the RDV group showing similar susceptibility to RDV as the wildtype reference (EC50 fold-change <=2.3). Conclusion(s): Overall, emergent substitutions in the SARS-CoV-2 replication complex including Nsp12 were observed with similar frequency in the RDV and placebo groups, with only one participant developing a substitution associated with reduced in vitro RDV susceptibility, indicating a high barrier to the development of RDV resistance in COVID-19 patients.
ABSTRACT
Background: Recent SARS-CoV-2 variants of concern (VOCs) have shown a progressive loss of sensitivity to monoclonal antibody therapeutics. Remdesivir (RDV) is a nucleotide analog prodrug that targets the viral RNA-dependent RNA polymerase (RdRp) Nsp12 and is approved to treat COVID-19 in hospitalized and non-hospitalized patients. Nsp12 is highly conserved across VOCs to date and RDV antiviral activity against previous VOCs (Alpha to Omicron BA.1) has been maintained. Here, we conduct a structural analysis of Nsp12 substitutions observed in recent Omicron subvariants (BA.2, BA.2.12.1, BA.4, BA.5 and BA.2.75) and assess RDV antiviral activity against clinical isolates and sitedirected mutants (SDMs) in a replicon system. Method(s): The prevalence of Nsp12 substitutions in Omicron subvariants was evaluated by analysis of sequences from the Global Initiative on Sharing Avian Influenza Data (GISAID) EpiCoV database. Structural analysis of identified substitutions was conducted on a prior cryo-electron microscopy-based model of the replication-transcription complex. Antiviral activity against subvariant clinical isolates was assessed by nucleoprotein ELISA in A549-hACE2-TMPRSS2 cells and by SDMs in the replicon system. Result(s): Genomic analysis of >1.4 million Omicron subvariant sequences revealed unique substitutions in Nsp12 compared to the ancestral WA1 strain. Besides P323L, present in all subvariants, G671S was observed in 95.9% of BA.2.75 sequences, F694Y was observed in <=1.9% of BA.4, BA.5 and BA.2.75 sequences, and Y521C was observed in 1.7% of BA.5 sequences. As anticipated, structural analysis of these substitutions showed no direct interaction with the incoming RDV nucleotide triphosphate or the viral RNA. Phenotyping of clinical isolates of Omicron subvariants BA.2, BA.2.12.1, BA.4, BA.5, and BA.2.75 consistently resulted in mean RDV EC50 values of 24.5 nM (BA.2) to 106.0 nM (BA.5). This represented 0.15-to 0.66-fold changes compared to WA1, indicating no loss of in vitro RDV antiviral activity against these VOCs. P323L, G671S, and F694Y were shown previously to have no impact on RDV antiviral activity. Similarly, the individual substitution Y521C showed no change in RDV susceptibility in the SARS-CoV-2 replicon system. Conclusion(s): RDV retained potent in vitro antiviral activity against all tested Omicron VOCs with potencies comparable to the WA1 isolate. These data support the continued use of RDV in patients infected with Omicron subvariants.
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Introduction: Itolizumab, a CD6 inhibitor has been found to be effective in COVID-19 in some studies [1] but there is no randomised controlled trial at present to prove its effectiveness. Method(s): The study population was adults (> 18 years) with severe COVID-19 pneumonia admitted in the ICU who received either tocilizumab or itolizumab in their course of stay in ICU. The primary outcome of the study was a clinical improvement (CI). The secondary outcomes were time for clinical improvement, improvement in PO2/ FiO2 ratio, best PO2/ FiO2 ratio, need for mechanical ventilation (MV) after administration of study drugs, time to discharge and survival days. Result(s): 126 patients were included in the study;92 received tocilizumab, and 34 received itolizumab. CI was seen in 46.7% and 61.7% of the patients in the tocilizumab and itolizumab groups, respectively and was statistically non-significant. The time to CI was also non-significant between the tocilizumab and itolizumab groups (median 12 vs 11 days). The number of days required to achieve the improvement of 100 in the PO2/ FiO2 ratio was significantly less with itolizumab as compared to tocilizumab. (6 vs 8 days, p value = 0.028). The best PO2/ FiO2 ratio achieved was also significantly better with itolizumab as compared to tocilizumab (315 vs 250, p value = 0.043). The incidence of serious adverse events due to the study drugs was significantly higher with itolizumab as compared to tocilizumab (14.7 vs 3.26%). The estimated median time for CI was 12 days and 11 days in the tocilizumab and itolizumab groups, respectively and was non-significant (log-rank p value = 0.262) (Fig. 1). Conclusion(s): The clinical improvement and survival rates with itolizumab are similar to tocilizumab. Better oxygenation can be achieved with itolizumab and can be a substitute for tocilizumab in managing severe COVID-19 infection.
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Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the current global pandemic of the COVID-19, which has persisted partly through the emergence of new variants. A non-infectious, convenient, and reproducible in vitro system is needed to assess drug susceptibility of new variants of concern and potential drug resistance mutations. Method(s): The SARS-CoV-2 replicon protocol was adapted and optimized based on {Zhang 2021}. The replicon RNA was produced by in vitro transcription of full-length replicon DNA assembled by ligation of plasmid fragments encoding for the SARS-CoV-2 non-structural proteins (Nsps), nucleoprotein and gaussia luciferase reporter protein. Wild-type and mutant replicon RNAs were transfected into Huh7-1CN cells by electroporation and treated with remdesivir (RDV). To determine EC50 values, luciferase activity was determined at 48 hours post transfection. A recombinant SARS-CoV-2 virus rescue system {Xie 2020} was used to generate matching Nsp mutants for comparison with the replicon system. Result(s): The selected substitutions reflective of Omicron BA.5 sub-lineage BF.7 variant: the triple mutants (Nsp12 (P323L) +Nsp13 (R392C) + Nsp14 (I42V), and a single Nsp12 L247F mutant as well as several specific Nsp12 mutations identified by in vitro resistance selection with RDV or RDV parent nucleoside analog GS-441524 were cloned into the replicon and tested for susceptibility to RDV. RDV inhibited the SARS-CoV-2 wild-type replicon with a mean EC50 value of 14.7 +/- 3.5 nM (N=9). The Nsp12 P323L substitution, a common polymorphism in all major variants of concern including Omicron, was fully susceptible to RDV with a 0.6-fold change in EC50 from the wild-type. The Omicron BF.7 triple mutants and L247F were also fully susceptible to RDV with 0.5- and 0.4-fold changes, respectively. Nsp12 substitutions F480L, V557L, V792I, S759A+V792I, and C799F resulting from in vitro resistance selections with RDV showed minimal to moderate levels of reduced susceptibility to RDV (1.8 to 18.3-fold change) (Table 1). The RDV EC50 fold changes correlated between the noninfectious replicon and recombinant infection virus system (Table 1). Conclusion(s): The replicon system is a convenient and reproducible model to test the susceptibility of SARS-CoV-2 mutant variants to RDV and potentially other antivirals. The common Nsp12 polymorphisms in all variants including the highly transmissible Omicron variant were fully susceptible to RDV.
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Background: SARS-CoV-2 evolution has contributed to successive waves of infections and severely compromised the efficacy of available SARS-CoV-2 monoclonal antibodies. Decaying vaccine-induced immunity, vaccine hesitancy, and limited vaccine protection in older and immunocompromised populations further compromises vaccine efficacy at the population level. Early antiviral treatments, including intravenous remdesivir (RDV), reduce hospitalization and severe disease due to COVID-19. An orally bioavailable RDV analog could facilitate earlier widespread administration to non-hospitalized COVID-19 patients. Method(s): We synthesized monoalkyl glyceryl ether phosphodiesters of GS-441524 (RVn), lysophospholipid analogs which allow for oral bioavailability and stability in plasma. We evaluated the in vivo efficacy of our lead compound, 1-O-octadecyl-2-O-benzyl-sn-glyceryl-3-phospho-RVn (V2043), in an oral treatment model of murine SARS-CoV-2 infection. We then synthesized numerous phospholipid analogs of RVn and determined which modifications enhanced in vitro antiviral activity and selectivity. The most effective compounds against SARS-CoV-2 were then evaluated for antiviral activity against other RNA viruses. Result(s): Oral treatment of SARS-CoV-2 infected BALB/c mice with V2043 (60 mg/kg once daily for 5 days, starting 12 hrs after infection) reduced lung viral load by more than 100-fold versus vehicle at day 2 and to below the LOD at day 5. V2043 inhibited previous and contemporary SARS-CoV-2 Variants of concern to a similar degree, as measured by the half maximal effective concentration (EC50) in a human lung epithelial cell line (Calu-3). Evaluation of multiple RVn analogs with hydrophobic esters at the sn-2 of glycerol revealed that in vitro antiviral activity was improved by the introduction of a 3-fluoro-4-methoxysubstituted benzyl or a 3-or 4-cyano-substituted benzyl. These compounds showed a 2-to 6-fold improvement in antiviral activity compared to analogs having an unsubstituted benzyl, such as V2043, and were more active than RDV. These compounds also showed enhanced antiviral activity against multiple contemporary and emerging RNA viruses. Conclusion(s): Collectively, our data support the development of RVn phospholipid prodrugs as oral antiviral agents for prevention and treatment of SARS-CoV-2 infections and as preparation for future outbreaks of pandemic RNA viruses.
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Objective(s): The objectives were to provide an overview of the current practices of Near East (NE) healthcare practitioners (HCPs) by probing their prescribing decisions, to report the COVID-19 impacts on neurologists' prescribing habits, and to explore the future relevance of current medication used in MS management among other newcomers Material(s) and Method(s): A cross-sectional study was carried out using an online survey from April 27, 2022, to July 5, 2022. The questionnaire was designed with the input of five neurologists representing five NE countries (Iran, Iraq, Lebanon, Jordan & Palestine). They identified several factors that play a crucial role in the optimal care of MS patients. The link was shared among neurologists using snowball sampling Result(s): The survey included 98 neurologists from the included NE countries, the majority of whom had more than 15 years of experience in the field, and 39% were seeing more than 40 MS patients a month. Effectiveness and safety balance was the most important factor considered when selecting the MS treatment. In the treatment of mild to moderate RRMS in men, Interferon beta 1a SC, Fingolimod, and Glatiramer acetate were the most commonly recommended treatments. Dimethyl fumarate substituted fingolimod in female patients. According to 80.7% of participants, interferon beta 1a SC was the safest treatment for mild to moderate RRMS. Interferon beta 1a SC was preferred over other treatments for patients with mild to moderate MS and planning for pregnancy (56.6%) or breastfeeding (60.2%). Fingolimod was not a choice for these patients. Neurologists seemed to discuss the top three treatments of Natalizumab, Ocrelizumab, and Cladribine with patients with highly active MS. Conclusion(s): Most neurologists in the NE region followed MENACTRIMS recommendations for prescribing treatment. The treatment choice also depended on the availability of DMTs in the region. Regarding the use of upcoming DMTs such as Ofatumumab, Siponimod, Ozanimod, and BTK inhibitors, there is a clear need for real-world data, long-term extension studies, and comparative studies to support their efficacy and safety profiles in treating patients with MSCopyright © 2022
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Background: In the year earlier part of 2020, many scientists urged to discover novel drugs against for the treatments of COVID-19. Coronavirus Disease 2019 (COVID-19), a life-threatening viral disease, was discovered first in China and quickly spread throughout the world. Objective(s): In the present article, some novel chalcone substituted 9-anilinoacridines (1a-z) were developed by in silico studies for their COVID19 inhibitory activity. Molecular docking studies of the ligands 1a-z were performed against COVID19 (PDB id-5R82) targeting the coronavirus using Schrodinger suite 2019-4. Method(s): The molecular docking studies were performed by the Glide module and the binding energy of ligands was calculated using the PRIME MM-GB/SA module of Schrodinger suite 2019-4. Result(s): From the results, many compounds are significantly active against COVID19 with a Glide score of more than-5.6 when compared to the currently used drug for the treatment of COVID19, Hy-droxychloroquine (-5.47). The docking results of the compounds exhibited similar mode of interactions with COVID19 and the residues, THR25, THR26, LEU27, SER46, MET49, HIE41, GLN189, ARG188, ASP187, VAL186, HIE164, ASN142, and GLY143 play a crucial role in binding with ligands. MM-GBSA binding calculations of the most potent inhibitors are more stably favourable. Conclusion(s): From the results of in-silico studies, it provides strong evidence for the consideration of valuable ligands in chalcone substituted 9-anilinoacridines as potential COVID19 inhibitors and the compounds, 1x,a,r,s with significant Glide scores may produce significant COVID19 activity for further development, which may prove their therapeutic potential.Copyright © 2020 Bentham Science Publishers.
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BACKGROUND: Nirsevimab is an extended half-life monoclonal antibody to the respiratory syncytial virus (RSV) fusion protein that has been developed to protect infants for an entire RSV season. Previous studies have shown that the nirsevimab binding site is highly conserved. However, investigations of the geotemporal evolution of potential escape variants in recent (ie, 2015-2021) RSV seasons have been minimal. Here, we examine prospective RSV surveillance data to assess the geotemporal prevalence of RSV A and B, and functionally characterise the effect of the nirsevimab binding-site substitutions identified between 2015 and 2021. METHOD(S): We assessed the geotemporal prevalence of RSV A and B and nirsevimab binding-site conservation between 2015 and 2021 from three prospective RSV molecular surveillance studies (the US-based OUTSMART-RSV, the global INFORM-RSV, and a pilot study in South Africa). Nirsevimab binding-site substitutions were assessed in an RSV microneutralisation susceptibility assay. We contextualised our findings by assessing fusion-protein sequence diversity from 1956 to 2021 relative to other respiratory-virus envelope glycoproteins using RSV fusion protein sequences published in NCBI GenBank. FINDINGS: We identified 5675 RSV A and RSV B fusion protein sequences (2875 RSV A and 2800 RSV B) from the three surveillance studies (2015-2021). Nearly all (25 [100%] of 25 positions of RSV A fusion proteins and 22 [88%] of 25 positions of RSV B fusion proteins) amino acids within the nirsevimab binding site remained highly conserved between 2015 and 2021. A highly prevalent (ie, >40.0% of all sequences) nirsevimab binding-site Ile206Met:Gln209Arg RSV B polymorphism arose between 2016 and 2021. Nirsevimab neutralised a diverse set of recombinant RSV viruses, including new variants containing binding-site substitutions. RSV B variants with reduced susceptibility to nirsevimab neutralisation were detected at low frequencies (ie, prevalence <1.0%) between 2015 and 2021. We used 3626 RSV fusion-protein sequences published in NCBI GenBank between 1956 and 2021 (2024 RSV and 1602 RSV B) to show that the RSV fusion protein had lower genetic diversity than influenza haemagglutinin and SARS-CoV-2 spike proteins. INTERPRETATION: The nirsevimab binding site was highly conserved between 1956 and 2021. Nirsevimab escape variants were rare and have not increased over time. FUNDING: AstraZeneca and Sanofi.Copyright © 2023 Elsevier Ltd. All rights reserved.
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The development of COVID-globulin, a COVID-19-specific human immunoglobulin preparation, involved choosing a method to quantify antibodies to SARS-CoV-2. Antibody titre determination by virus neutralisation (VN) is labour-intensive and unsuitable for large-scale application. To enable routine testing, it was necessary to develop a less demanding method;the enzyme-linked immunosorbent assay (ELISA) was the most appropriate of solutions. The lack of international and industry reference standards (RS) prompted the preparation and certification of an RS for COVID-globulin potency control. The aim of the study was to examine the possibility of substituting ELISA for VN and to develop an RS for SARS-CoV-2 antibody quantification in immunoglobulin preparations. Material(s) and Method(s): the authors used commercial ELISA kits by several manufacturers, COVID-globulin by Microgen (48 batches), and human plasma samples from multiple sources (1499 samples). The tests were performed by VN, ELISA, and chemiluminescent microparticle immunoassay. Result(s): the authors validated an ELISA method for SARSCoV-2 antibody quantification with the selected reagent kits by the National Medical Research Center for Hematology (NMRC for Hematology) and Euroimmun AG. The authors demonstrated the possibility of using ELISA instead of VN (with a correlation coefficient of more than 0.9). They developed and characterised an in-house RS for SARS-CoV-2 antibody content in human immunoglobulin preparations. The RS was certified in newly introduced anti-COVID units (ACU) and in international binding antibody units (BAU) using the World Health Organisation (WHO) international reference panel (NIBSC code: 20/268). The RS's potency was measured in terms of its neutralising activity in ACU (320 ACU/mL) and BAU (2234.8 BAU/mL). The authors established the relationship between ACU and BAU units. For the selected ELISA reagent kits, the conversion factors were 6.4 (NMRC for Hematology) and 7.0 (Euroimmun AG). Conclusion(s): the ELISA method for SARS-CoV-2 antibody quantification and the RS for SARS-CoV-2 antibody content can be applied to determine the potency of human anti-COVID-19 immunoglobulins.Copyright © 2023 Safety and Risk of Pharmacotherapy. All rights reserved.
ABSTRACT
The development of COVID-globulin, a COVID-19-specific human immunoglobulin preparation, involved choosing a method to quantify antibodies to SARS-CoV-2. Antibody titre determination by virus neutralisation (VN) is labour-intensive and unsuitable for large-scale application. To enable routine testing, it was necessary to develop a less demanding method;the enzyme-linked immunosorbent assay (ELISA) was the most appropriate of solutions. The lack of international and industry reference standards (RS) prompted the preparation and certification of an RS for COVID-globulin potency control. The aim of the study was to examine the possibility of substituting ELISA for VN and to develop an RS for SARS-CoV-2 antibody quantification in immunoglobulin preparations. Materials and methods: the authors used commercial ELISA kits by several manufacturers, COVID-globulin by Microgen (48 batches), and human plasma samples from multiple sources (1499 samples). The tests were performed by VN, ELISA, and chemiluminescent microparticle immunoassay. Results: the authors validated an ELISA method for SARSCoV-2 antibody quantification with the selected reagent kits by the National Medical Research Center for Hematology (NMRC for Hematology) and Euroimmun AG. The authors demonstrated the possibility of using ELISA instead of VN (with a correlation coefficient of more than 0.9). They developed and characterised an in-house RS for SARS-CoV-2 antibody content in human immunoglobulin preparations. The RS was certified in newly introduced anti-COVID units (ACU) and in international binding antibody units (BAU) using the World Health Organisation (WHO) international reference panel (NIBSC code: 20/268). The RS's potency was measured in terms of its neutralising activity in ACU (320 ACU/mL) and BAU (2234.8 BAU/mL). The authors established the relationship between ACU and BAU units. For the selected ELISA reagent kits, the conversion factors were 6.4 (NMRC for Hematology) and 7.0 (Euroimmun AG). Conclusions: the ELISA method for SARS-CoV-2 antibody quantification and the RS for SARS-CoV-2 antibody content can be applied to determine the potency of human anti-COVID-19 immunoglobulins.
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Case Report: Tsukamurella species are aerobic, partially acid fast saprophytes commonly isolated from soil and water. They are opportunistic pathogens known to infect multiple organs and can contribute to significant pathologies such as bacteremia, peritonitis, and respiratory tract infections. Moreover, Tsukamurella shares certain characteristic properties to Mycobacterium tuberculosis and Actinomyces species, including the acid fast stain, which can contribute to misdiagnosis of patients. A 68 year old female patient presented to the ED for shortness of breath, fatigue, and weight loss for 6 months. The patient's past medical history includes pulmonary fibrosis, type 2 diabetes, coronary artery disease with stent, hyperlipidemia, hypertension, and M. tuberculosis infection when she was 3 years old in Finland. On admission, labs revealed thrombocytosis (reactive 555 000/microL), leukocytosis (14 450/microL), and microcytic anemia (9.4 microg/dl). Moreover, C reactive protein was elevated and procalcitonin was normal (0.06 microg/l);a COVID-19 PCR was negative. An X-ray revealed severe patchy and interstitial infiltrates throughout both lungs with parenchymal scarring and pleural thickening in the periphery of the left mid-lung zone with multifocal pneumonia. Blood and sputum cultures were performed under the impression of pneumonia, and treatment with azithromycin and ceftriaxone was started. A M. tuberculosis infection was suspected due to a positive AFS. Further chest CT suggested multifocal pneumonia within the left lung in addition to apparent cavitary lesions versus bulla, a chronic interstitial lung disease with traction bronchiectasis, calcified right lower lung nodule, and calcified hilar lymph nodes suggesting a history of granulomatosis diseases. A bronchoscopy with Bronchoalveolar lavage was performed. The initial sputum specimen direct smear showed acid-fast stain positive with Actinomyces growth, and Penicillin G was added to the treatment. Samples were sent to the state department lab, and biopsy revealed granulomatous inflammation negative for malignant cells. One month later, the patient's sputum culture showed Tsukamurella for High-performance liquid chromatography (HPLC). Moreover, a rifampicin sensible M. tuberculosis complex by NAA was also positive six weeks later. The patient was started on a complete TB regimen and continued in the outpatient pulmonology clinic with the addition of levofloxacin for three months and rifampicin substituted for rifabutin. As demonstrated in the case above, a Tsukamurella infection can present similarly to a Mycobacterium infection. Patients may be misdiagnosed or potentially be co-infected. Our patient was further tested and appropriately treated for Tsukamurella after further extensive diagnostic screenings. Due to a high rate of missed cases, it is important to keep Tsukamurella infection on the differential diagnosis as the patient presentation may initially appear to be a Mycobacterium or other pulmonary infection. Copyright © 2023 Southern Society for Clinical Investigation.
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New N-containing xanthone analogs of α-mangostin were synthesized via one-pot Smiles rearrangement. Using cesium carbonate in the presence of 2-chloroacetamide and catalytic potassium iodide, α-mangostin (1) was subsequently transformed in three steps to provide ether 2, amide 3, and amine 4 in good yields at an optimum ratio of 1:3:3, respectively. The evaluation of the biological activities of α-mangostin and analogs 2-4 was described. Amine 4 showed promising cytotoxicity against the non-small-cell lung cancer H460 cell line fourfold more potent than that of cisplatin. Both compounds 3 and 4 possessed antitrypanosomal properties against Trypanosoma brucei rhodesiense at a potency threefold stronger than that of α-mangostin. Furthermore, ether 2 gave potent SARS-CoV-2 main protease inhibition by suppressing 3-chymotrypsinlike protease (3CLpro) activity approximately threefold better than that of 1. Fragment molecular orbital method (FMO-RIMP2/PCM) indicated the improved binding interaction of 2 in the 3CLpro active site regarding an additional ether moiety. Thus, the series of N-containing α-mangostin analogs prospectively enhance druglike properties based on isosteric replacement and would be further studied as potential biotically active chemical entries, particularly for anti-lung-cancer, antitrypanosomal, and anti-SARS-CoV-2 main protease applications.
Subject(s)
Antineoplastic Agents , COVID-19 , Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , SARS-CoV-2/metabolism , Antineoplastic Agents/pharmacology , Ethers , Peptide Hydrolases , Protease Inhibitors/chemistry , Molecular Docking Simulation , Antiviral AgentsABSTRACT
Purpose: Kidney transplant recipients (KTR) have inadequate responses to 2-dose COVID vaccination schedules and are at increased risk of severe COVID-19. Formation of T cell memory following vaccination is regulated by mTOR complex 1. mTOR inhibitors have been used in pre-clinical models to boost vaccine-elicited cytotoxic T cell memory responses. In observational studies, KTR receiving mTOR inhibitors had improved serological neutralisation and SARS-CoV-2 reactive T cell responses to 2 doses of COVID-19 vaccine, including cytotoxic T cells and circulating T follicular helper cells. We performed a clinical trial in stable KTR using sirolimus as a substitute for mycophenolate prior to a 3rd dose of COVID-19 vaccine to enhance COVID-19 vaccine responses. Method(s): KTR receiving tacrolimus, mycophenylate and corticosteroid with inadequate response to 2 doses of a COVID vaccine (defined by anti-RBD IgG <100U/ mL) and no history of COVID infection were recruited from 2 Australian transplant centres. Patients were randomised in a 1:1 ratio to continue mycophenolate maintenance or switch to sirolimus (trough level target 6 ng/mL). All patients received a 3rd dose of BNT162b2 COVID-19 vaccine and had immunological responses measured 4-6 weeks later. Result(s): 54 patients were randomised to sirolimus switch (n = 28), or control (n = 26). Patients were 70% male, mean age 57.5 years (SD10.4), with mean graft age 6.2 years (SD 5.4). Mean serum trough concentrations of sirolimus and tacrolimus were 6.4 and 6.1 respectively. There have been no safety or tolerability issues in the sirolimus cohort with stable serum creatinine (mean 117.8 vs 119.3, p=0.6), and mild increase in urinary ACR (mean 5.4 vs 17.4, p=0.1). Final results including immunological testing will be collated March 2022. Conclusion(s): Sirolimus switch is safe and well-tolerated. This trial will determine whether the strategy of mTOR inhibitor therapy peri-vaccination can optimise vaccine immune responses against COVID-19 in KTR.
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Introduction: Comparatively little is known about drug requirements in patients admitted to ICU with COVID-19 pneumonitis. We analysed drug usage for patients admitted during the first wave of the pandemic, comparing these with a retrospective cohort admitted with Influenza pneumonia. Methods: Forty-nine ventilated patients with COVID-19 pneumonitis were identified through ICNARC, ten were excluded as duration of stay < 7 days or not needing ventilation. Further three were excluded due to missing data and one due to ECMO escalation. Results: The median age was 61 years;length of stay 22 days and 68% survived ICU. Table 1 describes the use of Infusions and enteral medications. Discussion: Propofol was used in most (43% patient-hours in ICU/median duration = 234 hours). All patients received opiate infusions (mainly morphine or alfentanil in similar proportions) and 91% received muscle relaxants, for prolonged periods. Over half received Midazolam (median 106 hours) as an adjunct or substitute to Propofol as patients were difficult to sedate, required longer ventilation, paralysis and concerns with Propofol associated hypertriglyceridemia. Over two-third received alpha agonist infusions (median 68.5 hours) as adjunctive sedation or delirium management. Three quarters of patients received a furosemide infusion (median 90 hours), the evidence extrapolated from studies such as FACTT.1 Around three quarters received Human Albumin (median 100 grams over 3 days). Nearly a quarter received nebulized Prostacyclin for refractory hypoxia, often associated with saturation of HME filters and ventilatory difficulties.2 Over half of patients received Carbocisteine (median 13 days). Clonidine and Risperidone to manage delirium were used in a third (median 10.5 and 11 days respectively), as was Acetazolamide to restore pH and aid weaning. Over a third were prescribed enteral opiates and nearly a quarter received benzodiazepines to manage withdrawal symptoms. Just under a half of patients received Melatonin. Antibiotic usage was high with a median of 3 Antibiotics used (median duration 15 days/61% of patient days). Diagnosing superadded infection such as VAP was challenging3 and we did not routinely monitor serum Procalcitonin levels. We also compared prescribing habits with 12 influenza patients (11 survivors) identified using similar inclusion criteria and found patients with COVID-19 were older (61 versus 51 years ) with longer ICU stays (median 22 versus 20 days). They were also more likely to receive enteral Carbocisteine, Clonidine, Acetazolamide, Morphine and Diazepam. Conclusion: We were able to generate valuable data on prescribing in ventilated patients with COVID-19 pneumonitis during the first wave. Through this, we are able to use drug usage as a surrogate for issues such as delirium, drug withdrawal, antibiotic prescribing and nursing workload in general.
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Background: Nucleoside analog (NAs) drugs are used for the treatment of a variety of diseases, such as cancers and viral infections. After phosphorylation of viral and host kinases, NA drugs compete with the corresponding naturally occurring nucleotide during DNA replication of the infected cell. After incorporation, they can lead to mutations, or chain termination. However, because of their mechanism of action, NA are also potentially mutagenic to the genome of physiologically normal cells. Indeed, we have shown that treatment with the antiviral NA ganciclovir (GCV) after stem cell transplantation induces an increased mutation burden in the hematopoietic stem and progenitor cells (HSPCs) of pediatric leukemia patients. Using mutational signature analysis, we provided evidence that GCV-induced mutagenesis contributes to development of relapses and second malignancies in pediatric patients by inducing driver mutations. Over 30 NA drugs have been approved for clinical use and millions of people receive antiviral treatment worldwide to treat viral infections, including COVID-19. However, the mutagenicity in normal cells and potential carcinogenicity is unclear. Aims: Here, we aimed to systematically assess the mutational consequences of antiviral NAs in human HSPCs and identify underlying mechanisms. Methods: By combining in vitro treatment of umbilical cord blood-derived HSPCs with whole-genome sequencing (WGS) analyses, we provide a compendium of mutational consequences of antiviral NAs in a relevant human tissue (i.e., toxicity to the hematopoietic system is often dose-limiting). We treated HSPCs with IC40-60 concentration of the assessed compound followed by clonal expansions to obtain sufficient DNA for WGS. Using established bioinformatic pipelines, we catalogued the somatic mutations and mutational signatures in these cells. Results: At time of writing, 5 out of 7 tested antiviral NAs induce an enhanced mutation burden in exposed HSPCs. For some of this antiviral NAs we were able to identify unique unreported mutational signatures. Of note, the thymidine analog brivudine showed the highest increase in single base substitutions, which were characterized by a T>C signature, depleted for flanking cytosines. Furthermore, like GCV, we also observed a signature characterized by C>ApA substitution after treatment with the penciclovir, a molecule nearly identical to GCV. Currently we are working on machine learning approach to identify relevant mutation characteristics and modes of action as well as to screen cancer genome databases for mutational signature occurrence. Summary/Conclusion: Many compounds of the NA class currently prescribed for the treatment of viral infections are mutagenic to healthy cells. This calls for more thorough screening of these drugs, incorporation of information on mutagenicity to healthy cells in drug safety guidelines and patient surveillance over time.
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Irritant contact dermatitis (ICD) affects over 20% of health care workers, who manage their condition by substituting soap with an emollient cleanser. It is not clear whether emollient cleansers have the same level of virus eliminating activity as soap. Therefore, we evaluated a range of emollient cleansers for virus eliminating activity against enveloped (coronavirus and herpes simplex virus) and nonenveloped viruses (adenovirus (Ad)). In accordance with European standards a range of cleansers were combined with viruses under different (‘clean’ and ‘dirty’ hand hygiene) conditions. Virus viability and architecture were determined by plaque /TCDI50 assays and transmission electron microscopy. Traditional soaps (natural fatty acid), synthetic soaps and emollient cleansers (e.g., ceramide-containing cleansers) exhibited significant antiviral activity in enveloped viruses. However, the antiviral activity of traditional soaps reduced drastically when combined with hard water. Moreover, nonenveloped viruses were less susceptible to both synthetic soaps and emollients cleansers. Interestingly, traditional soaps inhibited the viability of Ad at high concentrations, but only in soft water. Most emollient cleansers were effective at eliminating enveloped viruses, suggesting that they are an acceptable substitute for soap to control the spread of viruses, like SARS-CoV-2, and protect against ICD. Nonenveloped viruses showed resistance to most of the hand hygiene products tested, except for traditional soap. This suggests that hand washing alone may not be sufficient to control the spread of nonenveloped viruses. Taken together this suggests that different virus strains respond differently to soaps and cleansers, and that this should be considered in the guidance for hand hygiene.
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CASE: An 81-year-old female with multiple co-morbidities including recent covid-19, presented to the emergency room with shortness of breath. On arrival, she was febrile with a temperature of 101F, pulse 100 beats/min, respiratory rate 14, blood pressure 196/163 and saturating at 75% on 10 L non-rebreather mask. Initial blood work showed WBC 10.9, lactic acid 1.7, BUN/creatinine 27/1.7 (consistent with her baseline), ABG showed pH 7.37, PCO2 49, PO2 88, HCO3 27.9. Chest x-ray demonstrated volume loss in the left hemithorax, airspace disease in the left mid lung and lung base. Due to suspicion for superimposed bacterial pneumonia and positive blood cultures for staphylococcus haemolyticus, she was started on vancomycin and azithromycin. Choice of antibiotics was challenging as she was allergic to penicillin and cephalosporins. During hospitalization, her kidney function deteriorated, vancomycin was substituted with tigecycline on day 3. Day 5 of treatment, she developed multiple episodes of vomiting with epigastric pain, lipase was 4523. Acute pancreatitis was diagnosed with tigecycline presumed to be the inciting agent in the absence of other risk factors such as gall stones, chronic alcohol use, elevated triglycerides, previous known episodes of pancreatitis or any other causative medications. Tigecycline was switched back to vancomycin and she received aggressive IV fluid hydration which also improved her kidney function. Within 48 hours, the patient had improved oxygen saturation, resolution of her abdominal pain, and good oral intake marking significant overall clinical progress. She was discharged on home oxygen and few more days of IV vancomycin for bacteremia. IMPACT/DISCUSSION: Tigecycline is a broad-spectrum glycylcycline antimicrobial agent belonging to the tetracycline class of antibiotics. Tetracyclines have been associated with acute pancreatitis in literature, and concerns about tigecycline-induced acute pancreatitis have been raised over the past decade in post marketing surveys, we described one such case above. Using the Naranjo Adverse Drug reaction probability scale, a score of 6 was achieved, indicating that the patient's pancreatitis was probably related to tigecycline. CONCLUSION: We recommend physicians monitor patients for signs and symptoms of pancreatitis including abdominal pain after initiating treatment with tigecycline. There should be a low threshold for ordering lipase levels and abdominal CT imaging where indicated. If the patient has symptoms concerning for acute pancreatitis, consider stopping tigecycline and switching to a different class of antibiotics immediately.