Identification and high-throughput quantification of baicalein and its metabolites in plasma and urine

Ethnopharmacological relevance: Scutellaria baicalensis Georgi. contains varieties of function compounds, and it has been used as traditional drug for centuries. Baicalein is the highest amount of flavonoid found in Scutellaria baicalensis Georgi., which exerts various pharmacological activities and might be a promising drug to treat COVID-19. Aim of the study: The present work aims to investigate the metabolism of baicalein in humans after oral administration, and study the pharmacokinetics of BA and its seven metabolites in plasma and urine. Materials and methods: The metabolism profiling and the identification of baicalein metabolites were performed on HPLC-Q-TOF. Then a column-switching method named MPX™-2 system was applied for the high-throughput quantificationof BA and seven metabolites. Results: Seven metabolites were identified using HPLC-Q-TOF, including sulfate, glucuronide, glucoside, and methyl-conjugated metabolites. Pharmacokinetic study found that BA was extensively metabolized in vivo, and only 5.65% of the drug remained intact in the circulatory system after single dosing. Baicalein-7-O-sulfate and baicalein-6-O-glucuronide-7-O-glucuronide were the most abundant metabolites. About 7.2% of the drug was excreted through urine and mostly was metabolites. Conclusion: Seven conjugated metabolites were identified in our assay. A high-throughput HPLC-MS/MS method using column switch was established for quantifying BA and its metabolites. The method has good sensitivity and reproducibility, and successfully applied for the clinical pharmacokinetic study of baicalein and identified metabolites. We expect that our results will provide a metabolic and pharmacokinetic foundation for the potential application of baicalein in medicine. © 2022

Immunotoxicity In Vitro Assays for Environmental Pollutants under Paradigm Shift in Toxicity Tests.

With the outbreak of COVID-19, increasingly more attention has been paid to the effects of environmental factors on the immune system of organisms, because environmental pollutants may act in synergy with viruses by affecting the immunity of organisms. The immune system is a developing defense system formed by all metazoans in the course of struggling with various internal and external factors, whose damage may lead to increased susceptibility to pathogens and diseases. Due to a greater vulnerability of the immune system, immunotoxicity has the potential to be the early event of other toxic effects, and should be incorporated into environmental risk assessment. However, compared with other toxicity endpoints, e.g., genotoxicity, endocrine toxicity, or developmental toxicity, there are many challenges for the immunotoxicity test of environmental pollutants; this is due to the lack of detailed mechanisms of action and reliable assay methods. In addition, with the strong appeal for animal-free experiments, there has been a significant shift in the toxicity test paradigm, from traditional animal experiments to high-throughput in vitro assays that rely on cell lines. Therefore, there is an urgent need to build high-though put immunotoxicity test methods to screen massive environmental pollutants. This paper reviews the common methods of immunotoxicity assays, including assays for direct immunotoxicity and skin sensitization. Direct immunotoxicity mainly refers to immunosuppression, for which the assays mostly use mixed immune cells or isolated single cells from animals with obvious problems, such as high cost, complex experimental operation, strong variability and so on. Meanwhile, there have been no stable and standard cell lines targeting immune functions developed for high-throughput tests. Compared with direct immunotoxicity, skin sensitizer screening has developed relatively mature in vitro assay methods based on an adverse outcome pathway (AOP), which points out the way forward for the paradigm shift in toxicity tests. According to the experience of skin sensitizer screening, this paper proposes that we also should seek appropriate nodes and establish more complete AOPs for immunosuppression and other immune-mediated diseases. Then, effective in vitro immunotoxicity assay methods can be developed targeting key events, simultaneously coordinating the studies of the chemical immunotoxicity mechanism, and further promoting the paradigm shift in the immunotoxicity test.

A high-throughput fully automatic biosensing platform for efficient COVID-19 detection.

We propose a label-free biosensor based on a porous silicon resonant microcavity and localized surface plasmon resonance. The biosensor detects SARS-CoV-2 antigen based on engineered trimeric angiotensin converting enzyme-2 binding protein, which is conserved across different variants. Robotic arms run the detection process including sample loading, incubation, sensor surface rinsing, and optical measurements using a portable spectrometer. Both the biosensor and the optical measurement system are readily scalable to accommodate testing a wide range of sample numbers. The limit of detection is 100 TCID50/ml. The detection time is 5 min, and the throughput of one single robotic site is up to 384 specimens in 30 min. The measurement interface requires little training, has standard operation, and therefore is suitable for widespread use in rapid and onsite COVID-19 screening or surveillance.

The application of a novel 5-in-1 multiplex RT-PCR assay for rapid detection of SARS-CoV-2 and differentiation between variants of concern.

OBJECTIVES: To rapid detect for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and immediately distinguish whether positive samples represent variants of concern (VOCs), we established a novel 5-in-1 VOC assay. METHODS: This assay could distinguish among five VOCs: Alpha, Beta, Gamma, Delta, and Omicron, in a single reaction tube. The five variants exhibit different single nucleotide polymorphisms (SNPs) in their viral genome, which can be exploited to distinguish them. We selected target SNPs in the spike gene, including N501Y, P681R, K417N, and deletion H69/V70 for the assay. RESULTS: The limit of detection of each gene locus was 80 copies per PCR reaction. We observed a high consistency among the results when comparing the performance of our 5-in-1 VOC assay, whole gene sequencing, and the Roche VirSNiP SARS-CoV-2 test in retrospectively analyzing 150 clinical SARS-CoV-2 variant positive samples. The 5-in-1 VOC assay offers an alternative and rapid high-throughput test for most diagnostic laboratories in a flexible sample-to-result platform. CONCLUSION: The assay can also be applied in a commercial platform with completion of the SARS-CoV-2 confirmation test and identification of its variant within 2.5 hours.

Array-Based Multiplex and High-Throughput Serology Assays.

The detection of antibody responses using serological tests provides means to diagnose infections, follow disease transmission, and monitor vaccination responses. The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, highlighted the need for rapid development of robust and reliable serological tests to follow disease spreading. Moreover, the rise of SARS-CoV-2 variants emphasized the need to monitor their transmission and prevalence in the population. For this reason, multiplex and flexible serological assays are needed to allow for rapid inclusion of antigens representing new variants as soon as they appear. In this chapter, we describe the generation and application of a multiplex serological test, based on bead array technology, to detect anti-SARS-CoV-2 antibodies in a high-throughput manner, using only a few microliters of sample. This method is currently expanding to include a multi-disease antigen panel that will allow parallel detection of antibodies towards several infectious agents.

Visual detection of SARS-CoV-2 with a CRISPR/Cas12b-based platform

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic, highlighting the unprecedented demand for rapid and portable diagnostic methods. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins-based platforms have been used for the detection of pathogens. However, in further applications and research, due to multiple steps needed, many methods showed an increased risk of cross-reactivity. The thermostable Cas12b enables the combination of isothermal amplification and CRISPR-mediated detection, which could decrease the risk of cross-contamination. In this study, we developed a portable and specific diagnostic method that combined the gold nanoparticle (AuNP) with thermal stable CRISPR/Cas12b-enhanced reverse transcription loop-mediated isothermal amplification (RT-LAMP), which is called SCAN, to distinguish the N gene of SARS-CoV-2 from flu gene. We validated our method using RNA from cells transfected by plasmids. We could easily distinguish the positive results by the naked eye based on the strong molar absorption coefficient of AuNP. Moreover, SCAN has the potential for high-throughput tests owing to its convenient operation. In sum, SCAN has broken the site and equipment restrictions of traditional detection methods and could be applied outside of hospitals and clinical laboratories, greatly expanding the test of COVID-19. [Display omitted] • The CRISPR/Cas12 b was employed to realize one-tube detection. • The SCAN assay is isothermal that requires minimal equipment. • The SCAN assay has a high-throughput potential for large-scale population screening. [ FROM AUTHOR]

Present status of microfluidic PCR chip in nucleic acid detection and future perspective

Polymerase chain reaction (PCR) amplifies specific fragment of DNA molecules and has been extensively applied in fields of pathogens and gene mutation detection, food safety and clinical diagnosis which on the other hand, holds the drawbacks of large size instrument, high heat dissipation etc. It has been demonstrated that microfluidics technique coupling with PCR reaction exhibits characteristics of integration, automatization, miniaturization, and portability. Meanwhile, various designed fabrication of microchip could contribute to diverse applications. In this review, we summarized major works about a variety of microfluidic chips equipped with several kinds of PCR techniques (PCR, RT-PCR, mPCR, dPCR) and detection methods like fluorescence, electrochemistry, and electrophoresis detection. The development and application of PCR-based microfluidic chip in pathogen and gene mutation detection, diseases prevention and diagnosis, DNA hybridization and low-volume sample treatment were also discussed. Copyright © 2022 Elsevier B.V.

Prolonged fever and inflammation in a young child in the midst of the pandemic - it's not always MIS-C

Case Report: Prolonged fever in children is a symptom that is seen in many different diseases, infections, malignancies, and autoimmune conditions. This can, at times, make the correct diagnosis challenging. A previously healthy 10-year-old male was transferred to our institution with one week history of fever, fatigue, abdominal pain, and vomiting. Laboratory studies demonstrated pancytopenia, transaminitis, electrolyte abnormalities, elevated pro-inflammatory markers & D-Dimer, and hypoalbuminemia. COVID-19 IgG was reactive. Due to the severity in presentation the patient was transferred to the ICU with a presumptive diagnosis of MIS-C. Hewas started on IVIG as well as a five-day course of high-dose methylprednisolone per protocol. Aspirin was added, but later discontinued, due to worsening thrombocytopenia. CT imaging with contrast showed small bilateral pleural effusions & periportal edema, mild splenomegaly, and echocardiogram showed diffuse dilation of the left main and left anterior descending arteries. Given the laboratory findings the differential diagnosis was expanded, Ehrlichia caffeensis serology was sent and empiric Doxycycline started. EBV Nuclear Antigen IgG antibody and EBV Viral Capsid Antigen IgM Antibody resulted as positive suggesting recent or reactivated infection. Respiratory viral PCR with COVID-19, Cytomegalovirus and Parvovirus PCR were negative. Despite initial treatment, the patient continued to have persistent fever, severe pancytopenia, and high ferritin up to 24 426 ng/mL, raising suspicion for Haemophagocytic Lymphohistiocytosis (HLH). Soluble interleukin-2 level was elevated & his presentation was then considered to be more consistent with HLH given that he met 6/8 criteria. Screening for primary HLH including CD107a, perforin and granzyme B, SAP, and XIAP resulted in the latter three being normal but CD107a was abnormal. Next generation sequencing for primary criteria was negative. E. Chaffeensis resulted positive: IgM 1:80, IgG 1:256. MIS-C and HLH have overlapping features but differ in some clinical manifestations. Timely recognition and management is paramount as the management differs. This case illustrates the importance of performing a broad search for potential causes, allowing for appropriate and timely treatment. COVID-19 serology alone should not be the basis for diagnosis of MIS-C in a patient with fever and inflammation. This is important as SARS-CoV2 becomes endemic. Infections such as EBV and Ehrlichiosis should be on the differential particularly in endemic areas and during seasons of higher prevalence for the latter, as these have been well documented to cause HLH. Copyright © 2023 Southern Society for Clinical Investigation.

COVID-19 pandemic and neonatal health: What we know so far?

COVID-19 first time appeared in December 2019 in Wuhan, China. The number of cases increased rapidly in china and outside and the World Health Organization declared a pandemic on 11th March 2020. The pregnant and postpartum women, child, and neonatal populations are vulnerable to this disease due to immunological and physiological changes. This paper analyzed the published evidence for assessing the effect of COVID-19 on neonatal health and health care. Online published literature was searched from PubMed, Google Scholar, and other official webpages using keywords: "coronavirus/COVID-19/new coronavirus 2019"/SARS-CoV-2 and neonatal health/care/outcomes" and reviewed to prepare this article. COVID-19 is the potential to transmit either mother to fetus or mother/caregiver to neonates. However, neonates born from infected mothers did not show significant clinical features. Pharyngeal-swab, amniotic-fluid, cord-blood, and breast-milk test results were not found positive. Health facility-based vaginal/caesarian delivery was considered a low risk of transmission. However, recommended to separate neonates with infected mothers/caregivers and test immediately after birth to avoid the possible transmission. Mothers/caregivers should take routine preventive measures such as washing hands frequently and avoiding contact with infected people. If neonates suffered from the server acute respiratory distress requires intensive care urgently. Despite the possibility of the intrauterine transmission of COVID-19 direct evidence is still lacking so it needs more studies for further confirmation. The International Pediatric Association suggested preventive programs, curative care, vaccination, and telemedicine care as the minimum services and called on its members to address these cares during the pandemic. Copyright © 2020, Kathmandu University. All rights reserved.

Liposome-based high-throughput and point-of-care assays toward the quick, simple, and sensitive detection of neutralizing antibodies against SARS-CoV-2 in patient sera.

The emergence of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) in 2019 caused an increased interest in neutralizing antibody tests to determine the immune status of the population. Standard live-virus-based neutralization assays such as plaque-reduction assays or pseudovirus neutralization tests cannot be adapted to the point-of-care (POC). Accordingly, tests quantifying competitive binding inhibition of the angiotensin-converting enzyme 2 (ACE2) receptor to the receptor-binding domain (RBD) of SARS-CoV-2 by neutralizing antibodies have been developed. Here, we present a new platform using sulforhodamine B encapsulating liposomes decorated with RBD as foundation for the development of both a fluorescent, highly feasible high-throughput (HTS) and a POC-ready neutralizing antibody assay. RBD-conjugated liposomes are incubated with serum and subsequently immobilized in an ACE2-coated plate or mixed with biotinylated ACE2 and used in test strip with streptavidin test line, respectively. Polyclonal neutralizing human antibodies were shown to cause complete binding inhibition, while S309 and CR3022 human monoclonal antibodies only caused partial inhibition, proving the functionality of the assay. Both formats, the HTS and POC assay, were then tested using 20 sera containing varying titers of neutralizing antibodies, and a control panel of sera including prepandemic sera and reconvalescent sera from respiratory infections other than SARS-CoV-2. Both assays correlated well with a standard pseudovirus neutralization test (r = 0.847 for HTS and r = 0.614 for POC format). Furthermore, excellent correlation (r = 0.868) between HTS and POC formats was observed. The flexibility afforded by liposomes as signaling agents using different dyes and sizes can hence be utilized in the future for a broad range of multianalyte neutralizing antibody diagnostics.

Machine-learning assisted scheduling optimization and its application in quantum chemical calculations.

Easy and effective usage of computational resources is crucial for scientific calculations, both from the perspectives of timeliness and economic efficiency. This work proposes a bi-level optimization framework to optimize the computational sequences. Machine-learning (ML) assisted static load-balancing, and different dynamic load-balancing algorithms can be integrated. Consequently, the computational and scheduling engine of the ParaEngine is developed to invoke optimized quantum chemical (QC) calculations. Illustrated benchmark calculations include high-throughput drug suit, solvent model, P38 protein, and SARS-CoV-2 systems. The results show that the usage rate of given computational resources for high throughput and large-scale fragmentation QC calculations can primarily profit, and faster accomplishing computational tasks can be expected when employing high-performance computing (HPC) clusters.

Visual Detection of COVID-19 from Materials Aspect.

Abstract: In the recent COVID-19 pandemic, World Health Organization emphasized that early detection is an effective strategy to reduce the spread of SARS-CoV-2 viruses. Several diagnostic methods, such as reverse transcription-polymerase chain reaction (RT-PCR) and lateral flow immunoassay (LFIA), have been applied based on the mechanism of specific recognition and binding of the probes to viruses or viral antigens. Although the remarkable progress, these methods still suffer from inadequate cellular materials or errors in the detection and sampling procedure of nasopharyngeal/oropharyngeal swab collection. Therefore, developing accurate, ultrafast, and visualized detection calls for more advanced materials and technology urgently to fight against the epidemic. In this review, we first summarize the current methodologies for SARS-CoV-2 diagnosis. Then, recent representative examples are introduced based on various output signals (e.g., colorimetric, fluorometric, electronic, acoustic). Finally, we discuss the limitations of the methods and provide our perspectives on priorities for future test development.

Exploration of Computational Aids for Effective Drug Designing and Management of Viral Diseases: A Comprehensive Review.

BACKGROUND: Microbial diseases, specifically originating from viruses are the major cause of human mortality all over the world. The current COVID-19 pandemic is a case in point, where the dynamics of the viral-human interactions are still not completely understood, making its treatment a case of trial and error. Scientists are struggling to devise a strategy to contain the pandemic for over a year and this brings to light the lack of understanding of how the virus grows and multiplies in the human body. METHODS: This paper presents the perspective of the authors on the applicability of computational tools for deep learning and understanding of host-microbe interaction, disease progression and management, drug resistance and immune modulation through in-silico methodologies which can aid in effective and selective drug development. The paper has summarized advances in the last five years. The studies published and indexed in leading databases were included in the review. RESULTS: Computational systems biology works on an interface of biology and mathematics and intends to unravel the complex mechanisms plying between the biological systems and the inter and intra species dynamics using computational tools, and high-throughput technologies developed on algorithms, networks and complex connections to simulate cellular biological processes. CONCLUSION: Computational strategies and modelling integrate and prioritize microbial-host interactions and may predict the conditions in which the fine-tuning attenuates. These microbial-host interactions and working mechanisms are important from the aspect of effective drug designing and fine-tuning the therapeutic interventions.

High-throughput screening of SARS-CoV-2 main and papain-like protease inhibitors.

The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (Mpro) and papain like protease (PLpro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851Mpro inhibitors and 205 PLpro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both Mpro and PLpro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of Mpro inhibitors and over 20% of PLpro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 Mpro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.

Reads2Vec: Efficient Embedding of Raw High-Throughput Sequencing Reads Data.

The massive amount of genomic data appearing for SARS-CoV-2 since the beginning of the COVID-19 pandemic has challenged traditional methods for studying its dynamics. As a result, new methods such as Pangolin, which can scale to the millions of samples of SARS-CoV-2 currently available, have appeared. Such a tool is tailored to take as input assembled, aligned, and curated full-length sequences, such as those found in the GISAID database. As high-throughput sequencing technologies continue to advance, such assembly, alignment, and curation may become a bottleneck, creating a need for methods that can process raw sequencing reads directly. In this article, we propose Reads2Vec, an alignment-free embedding approach that can generate a fixed-length feature vector representation directly from the raw sequencing reads without requiring assembly. Furthermore, since such an embedding is a numerical representation, it may be applied to highly optimized classification and clustering algorithms. Experiments on simulated data show that our proposed embedding obtains better classification results and better clustering properties contrary to existing alignment-free baselines. In a study on real data, we show that alignment-free embeddings have better clustering properties than the Pangolin tool and that the spike region of the SARS-CoV-2 genome heavily informs the alignment-free clusterings, which is consistent with current biological knowledge of SARS-CoV-2.

Metagenomic Next-Generation Sequencing of Nasopharyngeal Swabs in Acute Febrile Illness in Cambodia

Background. There is growing recognition of metagenomic next-generation sequencing (mNGS) as a valuable diagnostic tool capable of providing unbiased pathogen detection, but data on performance in low-resource settings remains scant. Here, we use mNGS of nasopharyngeal (NP) swabs taken from subjects in Cambodia to identify potential pathogens causing acute febrile illness. Methods. Febrile subjects aged 2 months to 65 years were enrolled in a crosssectional study conducted across 4 tertiary hospitals in Cambodia. NP swabs were collected at hospital presentation. Depending on reported symptom constellations, sera was also taken in a subset of subjects for comparison of mNGS results. RNA was isolated from biosamples, converted to cDNA libraries, and sequenced on a NextSeq2000 (Illumina). Raw sequence reads were stripped for host reads and aligned to NCBI nucleotide and protein databases using a cloud-based bioinformatics platform (CZID). Results. NP swabs were collected from 97 subjects between April 2020 and June 2021. Subjects were predominantly male (53.6%) and young (median age 3 years [IQR 1-25]). Pathogens were identified in 42 (43.2%) NP swabs;of these, 26 (61.9%) were respiratory viruses including 9 rhinovirus, 7 coronavirus (1 SARS-CoV-2), and 5 respirovirus cases. Co-infection was identified in 3 subjects with coronavirus and respirovirus (N=2) and coronavirus and rhinovirus (N=1). Of subjects with paired sera and NP samples (N=61), 18 (29.5%) had positive NP swabs but negative sera, 7 (11.5%) had negative NP swabs but positive sera, 12 (19.7%) had positive NP swabs and sera, and 24 (39.3%) had negative NP swabs and sera. Pathogen hits correlated in NP swabs and sera in 10 of 12 subjects, including six subjects with chikungunya. Conclusion. mNGS can be successfully implemented in low-resource settings to identify emerging pathogens and common respiratory pathogens, including coinfecting pathogens, from NP swabs of febrile patients. mNGS may also be able to detect chikungunya from NP swab alone, raising the possibility of non-invasive diagnostics for infections associated with high viremic states.

Treatment-Emergent Viral Variants in the Phase 3 TACKLE Trial Investigating Efficacy and Safety of AZD7442 (Tixagevimab/Cilgavimab) for the Treatment of Mild to Moderate COVID-19 in Adults

Background. AZD7442 (tixagevimab/cilgavimab) is a combination of neutralizing monoclonal antibodies (mAbs) that bind to distinct epitopes on the SARS-CoV-2 spike protein, with neutralization activity against variants including Omicron. In the Phase 3 TACKLE study, AZD7442 significantly reduced severe disease progression or death and was well-tolerated through Day 29. Viral evolution during treatment has the potential for resistance selection, such as variants exhibiting reduced mAb binding. We report genotypic analysis and phenotypic characterization of variants identified over 15 days after AZD7442 treatment in TACKLE. Methods. In TACKLE (NCT04723394), non-hospitalized adults with mild to moderate COVID19 were randomized and dosed <=7 days from symptom onset with a single 600-mg AZD7442 dose (2 consecutive intramuscular injections, 300 mg of each antibody;n=452) or placebo (n=451). Next-generation sequencing of the spike gene was performed on SARS-CoV-2 reverse-transcription polymerase chain reaction-positive nasal swabs (at baseline and Days 3, 6, and 15). SARS-CoV-2 lineages were assigned using spike nucleotide sequences. Amino acid substitutions, insertions, and deletions were analyzed at allele fractions (AF, % of sequence reads represented by mutation) >=25% and 3-25%. Results. Baseline spike sequences were available from 744 participants (82.4%) (AZD7442, n=380;placebo, n=364);87% of sequences corresponded to variants of concern/interest;these were balanced between AZD7442 and placebo groups (Table 1). Treatment-emergent (post-dosing) viral variants were rare, with 11 (4.5%) AZD7442 and 3 (1.3%) placebo participants showing the emergence of >=1 mutation at tixagevimab/cilgavimab binding sites, with an AF >=25% (Table 2). At AF 3- 25%, treatment-emergent viral variants in the AZD7442 binding site were observed in 16 (6.6%) AZD7442 and 15 (6.5%) placebo participants. Conclusion. Following AZD7442 treatment, low levels of SARS-CoV-2 variants bearing mutations at tixagevimab/cilgavimab binding sites were identified. These data indicate that combination of two antibodies creates a high genetic barrier for resistance, supporting the use of mAb combinations that bind to distinct epitopes for the treatment of COVID-19.

Resistance Analysis in the COMET-TAIL Study: Participants with Mild-to-Moderate COVID-19 Treated with Intramuscular or Intravenous Sotrovimab

Background. Sotrovimab (VIR-7831) is an engineered human monoclonal antibody targeting a conserved region of the SARS-CoV-2 spike protein;it has been shown to have a favorable safety profile and be effective for early treatment of highrisk COVID-19 patients. The COMET-TAIL phase 3 study evaluated sotrovimab administered intravenously (IV) or intramuscularly (IM) for the treatment of participants with mild to moderate COVID-19 who are at high risk of disease progression. Methods. Between June to August 2021, 973 participants were randomized and received sotrovimab by 500 mg IV infusion or by 500 or 250 mg IM injection. Deep sequencing of the spike gene was performed on nasopharyngeal samples. Baseline (BL;Day 1 or Day 3), post-BL (Day 5 or later), treatment-emergent (TE) substitutions at sotrovimab epitope positions, and presence of variants of concern/interest (VOC/ VOI), were evaluated at a >=5% allelic frequency. Phenotypic analyses were conducted using a pseudotyped virus assay. Results. Sequences were available from 764 participants (500 mg IV: 314/393;500 mg IM: 302/387;250 mg IM: 148/193). Consistent with VOC circulation during enrollment, the Delta variant was detected in 88.2% (674/764) of participants. Alpha and Mu variants were also seen at >2% prevalence. Of the 764 participants, 26 met the primary endpoint for clinical progression to hospitalization >24 hours or death due to any cause through day 29 and were infected with Delta (500 mg IV: 4;500 mg IM: 9;250 mg IM: 11), Alpha (500 mg IM: 1), or Mu (500 mg IV: 1) variants. Substitutions at sotrovimab epitope positions were similar across arms and were detected in 82/764 (10.7%) participants at any visit (500 mg IV: 42/314;500 mg IM: 27/302;250 mg IM: 13/148). Of these, 2 participants experienced clinical progression: 1 participant infected with the Mu variant (500 mg IV) carried the characteristic R346K substitution at BL;1 participant infected with the Delta variant (500 mg IM) had P337L and E340K substitutions detected at Day 3 and P337L was enriched at Day 8. The predominant TE epitope substitutions included P337L and E340A/K/V, which confer reduced susceptibility to sotrovimab in vitro. Conclusion. Overall, TE epitope substitutions were not associated with clinical progression.

Resistance Analyses of Patient Viral Samples from the Remdesivir Phase 3 Adaptive COVID-19 Treatment Trial-1 (ACTT-1)

Background. Remdesivir (RDV) is a broad-spectrum nucleotide analog prodrug approved for the treatment of COVID-19 in non-hospitalized and hospitalized adult as well as pediatric patients with clinical benefit demonstrated in multiple Phase 3 trials. Here we present SARS-CoV-2 resistance analyses from the Phase 3 ACTT-1 placebo-controlled clinical trial in hospitalized adults. Methods. Oro- or nasopharyngeal swab samples in ACTT-1 study were collected on Day 1, 3, 5, 8, 11, 15, and 29. All participants with >80th and 50% of participants with < 20th percentile of cumulative viral shedding underwent resistance analysis in both the RDV and placebo arm. The SARS-CoV-2 genome was sequenced using next generation sequencing. Phenotyping was conducted using virus isolation from clinical samples or generation of select site-directed mutants (SDMs) in a SARS-CoV-2 replicon system. Results. The majority of the sequencing data were obtained from participants with 80th percentile of cumulative viral shedding from the RDV and placebo arms as shown in Table 1. Among participants with both baseline and postbaseline sequencing data, emergent substitutions in nsp12 were observed in 12 of 31 participants (38.7%) treated with RDV and 12 of 30 participants (40.0%) in the placebo arm. The nsp12 substitutions that emerged in the RDV arm were only observed in one participant each, and the majority were present as mixtures with wildtype sequence. The following nsp12 mutations emerged in the RDV treatment group and were successfully phenotyped as clinical isolates or SDMs with low to no fold change in RDV susceptibility: A16V (0.8-fold), P323L+V792I (2.2-fold), C799F (2.5-fold), K59N (1.0-fold), and K59N+V792I (3.4-fold). V792I and C799F were identified previously in vitro in resistance selection experiments (Stevens Sci Transl Med 2022). In addition, for D684N and V764L identified in the RDV arm, the recovery of neither clinical isolates nor SDMs for phenotypic analysis were successful. Conclusion. The similar rate of emerging nsp12 substitutions in participants treated with RDV compared to placebo and the minimal to no change in RDV susceptibility among the treatment-emergent nsp12 substitutions support a high barrier to RDV resistance development in COVID-19 patients.