Human Immunodeficiency Virus (HIV) interacts with a wide array of host factors at each stage of its lifecycle to facilitate replication and circumvent the immune response. Identification and characterization of these host factors is critical for elucidating the mechanism of viral replication and for developing next-generation HIV-1 therapeutic and curative strategies. Recent advances in CRISPR-Cas9-based genome engineering approaches have provided researchers with an assortment of new, valuable tools for host factor discovery and interrogation. Genome-wide screening in a variety of in vitro cell models has helped define the critical host factors that play a role in various cellular and biological contexts. Targeted manipulation of specific host factors by CRISPR-Cas9-mediated gene knock-out, overexpression, and/or directed repair have furthermore allowed for target validation in primary cell models and mechanistic inquiry through hypothesis-based testing. In this review, we summarize several CRISPR-based screening strategies for the identification of HIV-1 host factors and highlight how CRISPR-Cas9 approaches have been used to elucidate the molecular mechanisms of viral replication and host response. Finally, we examine promising new technologies in the CRISPR field and how these may be applied to address critical questions in HIV-1 biology going forward. All viruses, from the simplest to the most complex, rely on host cell machinery for replication and immune evasion. Human Immunodeficiency Virus (HIV) is a lentivirus that replicates by reverse transcribing its roughly 10 kb RNA genome into DNA, which is then integrated into the human genome. This integrated provirus is transcribed, and the resultant RNA transcripts are translated and processed to produce the 15 viral proteins required for successful production of new, infectious virions. Given this limited repertoire of viral proteins, every step of the HIV lifecycle—including viral entry, reverse transcription, cytoplasmic transport, uncoating, nuclear entry, proviral integration, transcription, translation, assembly and egress—is mediated by, and dependent on, host proteins, nucleic acids, and metabolites collectively known as host factors ,3.Host factors play critical roles in viral replication and pathogenesis, and so have emerged as promising targets for the development of next-generation therapeutics ,4. For eGiven their vital functions in HIV biology and their potential use as next-generation therapeutic or curative targets, the identification and functional characterization of HIV host factors remains a high priority goal in HIV research. Both of these goals have been significantly advanced by the maturation of CRISPR-Cas9 gene editing technologies for the efficient, targeted manipulation of human gene expression . The devHIV replication is impacted by a broad array of host factors that vary by cell type, cellular environment, and viral genotype . These fThe principle behind a genetic screen for viral host factors is as follows: the genetic perturbation of a functionally relevant host gene will result in a significant change in a quantifiable viral phenotype. These perturbations can be driven by systematic mutagenesis, RNA interference, or through targeted gene editing approaches such as CRISPR-Cas9 ,14. The In a pooled screen, all genetic perturbations are carried out in a single population of cells . This cePooled screens can be exceptionally powerful tools for initial discovery of genes of interest . Pre-exiAs opposed to pooled screens, arrayed screens are formatted such that every gene target or perturbation occurs in an independent well . Since tDespite these many challenges, several targeted and genome-wide screens, both arrayed and pooled, have been conducted for the identification of HIV host factors . These hEarly efforts to identify HIV-1 host factors via genetic screening employed high-throughput RNA interference (RNAi) technologies to conduct loss-of-function screens. RNAi suppresses the expression of genes via introduction of short (20–25 nucleotides) RNA species that are complementary to an mRNA target. Binding results in inhibition of mRNA translation and/or degradation of the mRNA target, silencing gene expression . These aThe first three genome-wide screens for HIV host factors by Brass et al., König et al., and Zhou et al. used an arrayed siRNA screening approach to systematically knock-down genes in TZM-bl, HEK293T, and HeLa cells, respectively ,20,21. TWhile these landmark studies revealed a wealth of information regarding host proteins critical to HIV-1 pathogenesis, a meta-analysis of these screens revealed that there was little concordance between the candidates discovered for each screen . CompariFollowing up on the poor concordance observed in these initial results from the HIV-1 host factor RNAi screens, Zhu et al. suggested a number of methods to increase the robustness of such screens, including the use of multiple orthologous RNAi oligonucleotides and standardized downstream bioinformatics analysis to mitigate issues with high false positives, false negatives, and off-target effects . Since tThe advent of CRISPR-Cas9 genome editing has led to substantial advances in genetic screening methods. CRISPR-Cas9 gene editing technology is adapted from a naturally occurring process in the adaptive immune system of bacteria and archaea ,40,41,42CRISPR-Cas9 technology has since been adapted to elicit other genetic perturbations besides gene knock-out. For instance, CRISPR-Cas9 has been adapted to repress gene expression via CRISPR interference (CRISPRi) ,47. CRISThe CRISPR-Cas9 system can also be used to induce gene upregulation through CRISPR activation (CRISPRa). CRISPRa leverages a catalytically dead Cas9 fused to a transcriptional activation domain (such as the VP64 transactivator domain) to induce overexpression of target genes . CRISPRaCRISPR-Cas9 approaches have a few advantages over RNAi-mediated methods for high-throughput screening. In general, there are fewer off-target effects compared to RNAi, though the use of multiple independent guide RNA per gene target is still required for experimental rigor ,41. CRISCD4, the coreceptor CCR5 and three novel factors TPST2, SLC35B2, and ALCAM [One of the first genome-wide CRISPR screens for HIV host factors leveraged a pooled, lentiviral library of sgRNA containing nearly 10 sgRNA per gene targeting over 18,000 gene targets . This scnd ALCAM . Both TPin trans with budding HIV virions from infected cells. Thus, HIV viruses produced after infection of a given knock-out cell will carry the sgRNA from that cell as a marker. As a result, if a dependency factor is perturbed, fewer viruses with that sgRNA will be produced and if a restriction factor is perturbed, more viruses with that sgRNA will be produced [HIV library) and assessed interferon-mediated inhibition of HIV-1 in the monocytic THP-1 cell line [To overcome the inherent issue with MOI in screens that rely on HIV infection, Ohainle et al. developed a new CRISPR-Cas9 approach for pooled host factor screening that uses the virus as a readout instead of the cells . Brieflyproduced ,51. In aell line . This scell line . The HIVNotably, no genome-wide, arrayed screens for HIV host factors using CRISPR-Cas9 have yet been reported.A number of CRISPR-Cas9 screens have also been employed to identify host factors involved in the establishment, maintenance, and reversal of HIV latency. This focus has been guided in part by curative strategies aimed at either reversing latency to facilitate killing of infected cells (“shock and kill”) or for inducing cells to enter a deep state of latency in which the virus remains completely dormant (“lock and block”) ,53. RathTo date, two pooled CRISPRko screens have been used to identify host factors that promote viral latency. These screens by Huang et al. and Krasnopolsky et al. utilized cell lines  that harbor transcriptionally inhibited proviral transcripts containing a reporter gene. For these screens, the readout of interest is reactivation of the proviral sequence as determined by reporter activity. These screens uncovered a number of lead candidate host factors which promote viral latency, including MINA53 and ZNF304 ,24. In aALCAM) did not validate in primary CD4+ T cells as it was not expressed in that cell type. To overcome this limitation, recent advances in genome editing technology and primary cell culture techniques have focused on expanding the use of CRISPR-Cas9 gene editing tools into diverse primary cell types.All of the genetic screens for HIV-1 host factors described above relied on the use of immortalized cell line models as these cells are easy to propagate in bulk and easy to manipulate. However, cell lines do not always recapitulate disease and infection-associated phenotypes in a way that translates to studies in humans . ImmortaGEMIN2, KPNA1, KPNA5, and XRCC6 [The use of CRISPR-Cas9 to conduct HIV-1 host factor screens in primary cells was first reported by Hultquist et al. With this screening strategy, in vitro synthesized crRNPs are delivered to activated primary CD4+ T cells isolated from the blood of multiple independent donors by electroporation . Though nd XRCC6 . This wand XRCC6 . Using and XRCC6 .While CRISPR-Cas9 gene editing has revolutionized our ability to manipulate the human genome, there are still a number of limitations inherent to the technology that must be considered during screen design and interpretation. For instance, some genetic loci are not readily targetable by CRISPR-Cas9, potentially due to inaccessible chromatin structure or genome architecture at the target site . Thus, iAdditional challenges for CRISPR-Cas9 HIV host factor screens arise in considerations of scale and scope. Most pooled CRISPR-Cas9 screens utilizing lentiviral delivery of CRISPR libraries require initial transduction at a low multiplicity of infection (MOI) in order to ensure delivery of a single guide RNA to each cell, reducing the false-positive discovery rate . HoweverGiven that arrayed screens require a separate population of cells for each genetic perturbation, these screens can likewise require a vast amount of resources when applied at a genome-wide level . Even moCRISPR-Cas9 genome editing has yet to be utilized to its full potential in the context of HIV-1 host factor screens. To date, only a single pooled CRISPR-Cas9 screen for host factors that act during active HIV-1 replication has been reported . FurtherGiven the critical importance of cell type in screen design, leveraging CRISPR-Cas9 in primary cell models holds the potential to unlock many novel insights into HIV-1 host factor biology. While advances in CRISPR-Cas9 editing of primary cells have opened the door for CRISPR screens in primary cells models of HIV-1 infection, there have been only limited attempts to leverage these technologies for the purposes of screening. New technologies for pooled CRISPRko, CRISPRi, and CRISPRa screening in primary CD4+ T cells should enable more of these studies in the future. Given that HIV-1 is known to infect myeloid cells and that protocols for editing these cells via CRISPR-Cas9 have now been established , conductBeyond its use in genetic screening, CRISPR-Cas9 has also enabled scientists to efficiently validate and mechanistically interrogate complex cellular processes at the host–pathogen interface. In this section, we aim to highlight several studies that have utilized CRISPR-Cas9 systems to better understand the vast network of host–pathogen interactions that drive HIV-1 infection and pathogenesis. Additionally, we discuss how these efforts are being leveraged towards the development of next-generation HIV therapies.Many applications of CRISPR-Cas9 host factor editing with the goal of long-term control of HIV-1 infection have focused on targeting the co-receptors used by HIV-1 during viral entry. The HIV-1 replication cycle begins with attachment of the virus to the host cell surface, followed by fusion of the viral and cell membranes. Attachment and fusion are mediated by the viral glycoprotein Envelope (Env) that is embedded in the virion outer membrane. Upon encountering a target cell, Env binds to its primary receptor, CD4, which induces a conformational change in Env that allows for co-receptor binding . DependiCCR5, have been instrumental in conceptualizing HIV-1 cure strategies [CCR5 harboring a 32 base pair deletion (CCR5∆32) that results in a truncated co-receptor was found to be protective against HIV-1 infection in repeatedly exposed, but uninfected, individuals. In the only two reported cases of HIV-1 cure to date, heterologous transplantation of hematopoietic stem cells carrying this CCR5Δ32 allele into patients resulted in sustained viral suppression after cessation of antiretroviral therapy [CCR5Δ32 mutation may be found [In vivo perturbation of the main HIV receptor, CD4, is not feasible due to the biological significance of CD4 in regulating immune function. However, naturally occurring polymorphisms in the co-receptor, rategies ,69,70. A therapy ,72. Whilbe found   for potential downstream use in autologous stem cell transplantation. In one of the first studies to demonstrate this possibility, Ye et al. utilized a combination of CRISPR-Cas9 and piggyBAC technology to generate iPSCs homozygous for the 2 allele . Under tbination . Subsequ changes . Critica changes .CCR5Δ32 allele has since been linked to weakened immunity to other pathogens, alternate protective alleles with fewer detrimental effects may serve as the basis for future strategies [CCR5 editing efficiencies of 12.5% with single gRNA and 27% using two gRNA in human iPSCs [CCR5 mutations. These cells remained susceptible to HIV-1 infection by CXCR4-tropic strains but were resistant to challenge with CCR5-tropic strains, suggesting that their editing was CCR5-specific and did not overtly alter cell viability. A number of repair outcomes were sufficient for protection, many of which have not been observed in the human population. Determining the least invasive mutation that provides the strongest and long-lasting protection to infection remains a critical challenge.Note that while the rategies ,78. Relyan iPSCs . After cCCR5 knock-out itself. While sulfonation is a widely used post-translational modification for regulating proteins at the cell surface, other regulatory pathways that may be more specific to CCR5 might be more amenable for therapeutic targeting.Rather than directly targeting the co-receptor itself, host factors that regulate co-receptor expression have been explored as alternate therapeutic targets for preventing HIV-1 infection. For example, Park et al. identified SLC35B2 and TPST2 as HIV host factors in a genome-wide CRISPR screen . Both thCCR5, CRISPR-Cas9 has been used extensively to target CXCR4 and generate cells that are resistant to CXCR4-tropic HIV-1 infection in vitro. This has included editing in multiple T cell line models as well as in primary CD4+ T cells from both humans and rhesus macaques [CXCR4 editing as a therapeutic strategy is limited. In the course of natural infection, transmitted founder viruses are exclusively CCR5-tropic, with tropism switching only occurring in some patients after long-term infection [In addition to macaques ,81. Whilnfection . While iBesides the receptor and co-receptors, HIV is dependent on a large number of host proteins at each stage of its lifecycle to achieve successful replication. Modulation of these dependency factors has been shown to hamper HIV-1 infection and replication in vitro, several of which may serve as novel targets for therapeutic intervention. As discussed previously, the advent of CRISPR-Cas9 screening technologies has enabled discovery of several new factors, while targeted application of CRISPR-Cas9 in primary cell types and animal models has improved our understanding of host factor function . This haOne of the newest drug targets for therapeutic intervention has been the HIV viral core, which is deposited into the host cell cytoplasm following membrane fusion. The viral core is a protein shell made up of thousands of HIV capsid proteins that functions to protect the viral RNA genome during reverse transcription and transport to the nucleus . As suchLEDGF, which disrupted its interaction with HIV-1 integrase but otherwise allowed for normal interaction with host cellular binding partners [One of the major benefits of the CRISPR-Cas9 editing for gene knock-out is the ability to completely ablate protein expression and assign roles for host factors required at only a small fraction of their steady-state levels. For example, the dependency factor LEDGF (p75) is bound by the HIV-1 pre-integration complex (PIC) to tether it to the chromatin and target integration towards areas of active transcription. LEDGF has a long half-life and only small amounts are sufficient to direct proviral integration, so initial attempts to characterize its functional roles using RNAi required optimization of a stable, highly efficient knock-down strategy. CRISPR-Cas9 knock-out of LEDGF, on the other hand, enabled ready detection of an HIV replication defect in primary CD4+ T cells. Nevertheless, the cellular role of LEDGF in tethering host complexes to chromatin excludes LEDGF knock-out as a viable therapeutic strategy. To overcome this, Lampi et al. optimized a CRISPR-Cas9 strategy to induce a specific site-directed mutation, D366N, into partners . This chAs illustrated by the examples above, translating dependency factors into targets for host-directed therapies remains a substantial challenge. First, unlike the receptor/co-receptor interactions, few other dependency factors appear to be absolutely required for viral replication in vitro, but rather promote optimal replication. While the virus may rely on one dominant route to infection, host factor redundancies and flexibility in the route to infection may limit the effectiveness of inhibiting any one given dependency factor. It is unclear how these partial blocks to replication would manifest in vivo. Second, many of the known dependency factors are central players in host cell function and so are unlikely to be viable targets in a therapeutic strategy. For example, transcriptional elongation at proviral integration sites is promoted by recruitment of the positive transcription elongation factor b (P-TEFb) complex to sites of nascent transcription by the viral Tat protein . While PWhile dependency factors promote viral replication, host restriction factors act to inhibit viral replication through a wide variety of mechanisms . RestricTRIM5⍺ locus in the Jurkat T cell line. One isolated clone containing the R332G mutation in both alleles significantly enhanced protection against HIV-1 infection and exhibited a ~40-fold increase in antiviral activity.As mentioned above, one of the first restriction factors encountered by the HIV-1 following cell entry is TRIM5⍺. TRIM5⍺ assembles onto the capsid lattice of incoming cores and induces premature uncoating . This revif-deficient as well as wild-type HIV-1 in cell models of infection [As opposed to specific mutations that boost restriction factor activity, strategies to boost restriction factor expression have also been explored using CRISPRa. For example, Bogerd et al.  used a Cnfection . Similarnfection . BST-2/Tnfection .Several host restriction factors have been interrogated in T cells, but there are a number of restriction factors that play unique roles in macrophages and other infected myeloid cells. For example, the restriction factor SAMHD1 acts to inhibit HIV infection of resting T cells and macrophages by the depletion of dNTPs and the subsequent inhibition of reverse transcription ,98. To oMost described restriction factors are interferon-stimulated genes (ISGs) that act as part of the innate immune system to inhibit viral infection. Upon infection, host proteins known as pattern recognition receptors (PRRs) act to specifically recognize conserved pathogen-associated molecular patterns (PAMPs), and subsequently initiate a signaling cascade that triggers the interferon response and ISG expression. Depending on the cell type and the PAMP that is sensed, different ISGs are expressed under different circumstances. The collective activity of the expressed ISGs creates a less permissive environment for a broad array of pathogens. Indeed, recombinant interferon has sometimes been administered as a therapeutic agent in circumstances where specific antivirals may not be available, i.e., in the treatment of hepatitis B virus  before tWhile the innate immune response to HIV-1 is still being characterized, several groups have explored CRISPR-Cas9 as a tool to map the PRRs that sense infection and to identify the ISGs that directly impact HIV-1 replication. While RIG-I/MDA5 ,104 and One of the largest obstacles to an HIV cure is the persistence of the virus in long-lived cellular reservoirs. These reservoirs of latently infected cells evade immune clearance and can reseed infection upon ART cessation. While a large number of groups have investigated the use of CRISPR-Cas9 systems to directly target and excise integrated HIV-1 proviruses in latently infected cells ,111,112,HIV latency is mediated by several mechanisms that inhibit successful viral transcription, including epigenetic blocks, blocks to transcriptional initiation, blocks to transcriptional elongation, and blocks to transcriptional termination and RNA processing. These overlapping mechanisms can vary by cell type, cell state, as well as by proviral integration site, complicating their study. As highlighted above, several genome-wide knock-out studies have been performed in cell line models of HIV-1 latency to discover host factors that may contribute to latency reversal and maintenance. These have identified a number of host factors involved in the regulation of viral transcription. For example, P-TEFb is recruited to RNA Pol II at sites of nascent proviral transcription by the HIV-1 Tat protein to license transcriptional elongation. Ma et al. showed that P-TEFb’s catalytic subunit, CDK9, is SUMOylated by TRIM28 , inhibitWhile these cell line-based models of latency have been effective tools for understanding proviral transcription, it is unclear how translatable these findings are to latent reservoirs in PLWH. A number of primary cell models have been developed to reflect the latent state, but even these yield highly discordant results  and ofteCCR5Δ32 mutation ex vivo to enable autologous transplant of HIV-1-resistant cells.CRISPR-Cas9 gene editing has proven to be a powerful and versatile tool with applications throughout the therapeutic pipeline from target discovery to validation and application. The ease and accessibility of whole genome screening across a variety of model systems has revolutionized the small molecule drug discovery pipeline and has uncovered a variety of novel drug targets for a myriad of diseases . It has The use of CRISPR-Cas9 for the in vivo correction of in-born genetic defects or to attack drug-resistant pathogens or cancers has likewise been widely touted, though there are still a wide array of limitations and ethical considerations that must still be addressed as this technology advances. One of the overarching limitations to conceptualizing the use of CRISPR-Cas9 as a therapeutic in vivo is the difficulty of delivering the complexes to the right place in the body at the right time. Viral-based vectors such as adenoviral, adenoviral-associated, and lentiviral vectors can be used to deliver gRNA to target cells, but their application has been limited, and there has been mixed clinical success . FurtherWhile there are still a large number of limitations, it has only been a decade since it was first demonstrated that CRISPR-Cas9 could be used for precision editing of the human genome. Since then, this technology has revolutionized biomedical research, uncovering biological pathways that underlie diverse human diseases and unveiling new high-priority drug targets. With continued advances in the technology, we now stand at the cusp of a new era of genetic medicine with broad applications for fighting a diverse array of human disease. The use of CRISPR-Cas9 gene editing in HIV host factor discovery and validation has advanced the field and will undoubtedly inform the development of next-generation, host-directed therapeutic strategies in the decades to come.


ATP) channel high- vs low-affinity sulfonylureas and cardiovascular outcomes in patients with type 2 diabetes treated with metformin.This cohort study evaluates the association between mitochondrial adenosine triphosphate–sensitive potassium (K ATP) channel high-affinity sulfonylureas (glyburide and glipizide) vs low-affinity sulfonylureas (gliclazide and glimepiride) combined with metformin in type 2 diabetes (T2D)?Is there an increased risk of major adverse cardiovascular events (MACEs) associated with use of cardiac mitochondrial adenosine triphosphate–sensitive potassium  channels have been speculated to account for the intraclass difference in cardiovascular risk from in vitro and ex vivo studies; however, this hypothesis has not been assessed in a general population with diabetes receiving sulfonylureas added to metformin.Sulfonylureas are frequently used as add-on to metformin in type 2 diabetes (T2D), and individual sulfonylurea agents carry different risks of cardiovascular disease. Sulfonylureas’ different affinities to cardiac mitochondrial adenosine triphosphate–sensitive potassium , ischemic stroke, or cardiovascular death in patients with T2D treated with mitoKThis is a new-user, active-comparator, and propensity score–matched cohort study with analysis of the Taiwanese Diabetes Mellitus Health Database from 2006, to 2017. Data analysis was performed from August 2020 to July 2021.ATP channel high-affinity (glyburide and glipizide) and low-affinity (gliclazide and glimepiride) sulfonylureas combined with metformin.Cardiac mitoKPrimary outcome was major adverse cardiovascular events (MACEs), a composite of cardiovascular death or hospitalization for either MI or ischemic stroke. Secondary outcomes included individual MACE components, heart failure, arrhythmia, all-cause mortality, and severe hypoglycemia. Cox proportional hazards models were used to estimate adjusted hazard ratios (aHRs).ATP channel high-affinity sulfonylureas vs low-affinity sulfonylureas when combined with metformin were associated with an increased risk of MACE , MI , all-cause mortality , and severe hypoglycemia , but not with increased risks of ischemic stroke, cardiovascular death, arrhythmia, and heart failure. The duration analyses revealed the highest MACE risk during 1 to 90 days after initiation of mitoKATP channel high-affinity sulfonylureas .Each sulfonylurea group comprised 53 714 patients . MitoKATP channel high-affinity sulfonylureas vs low-affinity sulfonylureas was associated with an increased MACE risk in patients with T2D concomitantly receiving metformin, suggesting that high-affinity blockage of the mitoKATP channels could account for sulfonylurea-associated MACEs.Use of mitoK Although it has been investigated whether the specificity of sulfonylureas for pancreatic β-cells could account for these drugs’ associated cardiovascular risks,12 no studies have confirmed the proposed mechanism.Despite the availability of newer types of antidiabetic medications,ATP) by certain sulfonylureas may be an alternative mechanism underlying the intraclass difference in MACEs. Ischemic preconditioning is the most pivotal mechanism for cardiac protection and is involved in the opening of mitoKATP channels, which can be blocked by certain sulfonylureas, such as glyburide and glipizide.14 In vitro and ex vivo data have indicated that there were differential affinities to the mitoKATP channels among individual sulfonylureas,19 although this has not been translated to account for sulfonylurea-related adverse cardiovascular events in clinical settings, except for 1 observational study focusing on sulfonylurea monotherapy.20 As sulfonylureas are most used as add-on antidiabetic agents to metformin in management of type 2 diabetes (T2D), we aimed to assess whether use of cardiac mitoKATP channel high-affinity sulfonylureas (glyburide and glipizide) vs low-affinity sulfonylureas (gliclazide and glimepiride) when combined with metformin is associated with an increased risk of MACEs in a nationwide T2D population.Blockage of cardiac mitochondrial adenosine triphosphate–sensitive potassium (mitoK1c (HbA1c) levels, respectively. This study was approved by the institutional review board of TSGH, National Defense Medical Center and completed before the lead author became affiliated with the National Yang Ming Chiao Tung University. Written informed consent was waived because the study analyzed a deidentified database. The report of this study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.This was a population-based, propensity score (PS)–matched cohort study using the Taiwan Diabetes Mellitus Health Database (DMHD) from January 1, 2006 to December 31, 2017. The DMHD comprises patient diagnoses, medical procedures, and prescription refill records of all patients with new diagnoses of diabetes under the Taiwanese universal national health insurance (NHI) program, with a coverage rate of greater than 99% of Taiwanese inhabitants. In the database, patients with new diabetes diagnoses were defined as those with at least 3 outpatient visits or 1 inpatient visit for diabetes in a given year, and they did not have previous visits with any diagnoses of diabetes preceding the first visit related to diabetes. The nationwide death registry and the Tri-Service General Hospital (TSGH) electronic medical records were linked to the DMHD to obtain the causes of death and glycated hemoglobin AATP channel high-affinity sulfonylurea (glyburide and glipizide) or an add-on low-affinity sulfonylurea (gliclazide and glimepiride) to their metformin monotherapy, and marked the first prescription date of add-on sulfonylureas as cohort entry date. Patients who initiated sulfonylurea and metformin on the same day were also included. The study cohort was required to be aged 20 or more years at cohort entry. The 4 examined individual sulfonylureas comprised more than 99% of the sulfonylureas combined with metformin in the study period. Patients were excluded if they received both types of sulfonylureas at cohort entry; had lack of 1-year continuous NHI enrollment; or had a hospitalization with a primary diagnosis of myocardial infarction (MI), ischemic stroke, or pregnancy in the year preceding cohort entry. eTable 1 in We identified a base cohort of patients with newly diagnosed diabetes who initiated metformin monotherapy, defined as those without any prescription refill records of any antidiabetic medications in the year before the monotherapy commencement, from January 1, 2007, to December 31, 2016. The base cohort was required to have no type 1 diagnosis codes and to continuously receive metformin monotherapy, allowing a 30-day grace period between refills. From the base cohort, we identified the study cohort of patients with an add-on of a cardiac mitoKATP channel high-affinity or low-affinity sulfonylurea group), pregnancy, or December 31, 2017. Discontinuous use of sulfonylureas with metformin was determined according to the prescription refill records with more than a 30-day grace period. We extended the follow-up to 1 month after the dual therapy was discontinued to include the MACEs events that may have caused discontinuity in the dual therapy.The 2 sulfonylurea groups were followed from the cohort entry date until the earliest of the following events: occurrence of a primary cardiovascular outcome (defined later in this study), NHI enrollment discontinuation, metformin-sulfonylurea treatment discontinuation, switch or add-on of other antidiabetic medications , comorbidities , and health care utilizations. Comedications  were evaluated in the previous 6 months before cohort entry.P < .05. Data cleaning and statistical analyses were performed using SAS statistical software version 9.4 (SAS Institute). Data were analyzed from August 2020 to July 2021.Baseline characteristics were compared using the standardized difference, where a magnitude greater than 0.1 indicated an imbalance between groups. The Kaplan-Meier method with a log-rank test was used to compare the cumulative incidence of primary and secondary outcomes. Cox proportional hazards models with 95% CIs were used to estimate the hazard ratios (HRs) for each outcome, and the proportionality assumption was assessed through Schoenfeld residuals, with all assumptions being met. All analyses were also adjusted for PS deciles after the matching process to mitigate any residual confounding. Statistical significance was defined as a 2-sided value of 23 which combined PS and regression calibration, to address the unavailability of HbA1c in the DMHD by using HbA1c data obtained from a tertiary medical center, detailed in eMethods in 24 and high-dimensional PS-matched analyses , with re-estimated PS.Multiple predetermined sensitivity analyses were conducted. First, we performed the PS calibration analysis,ATP channel low-affinity sulfonylureas and high-affinity sulfonylureas in combination with metformin were identified. Overall mean (SD) patient age was 54.7 (12.1) years, and 31 962 (59.5%) were men  or treatment switching , with the differences between groups across all types of censoring no greater than 5%  and 0.78   . The cumATP channel high-affinity sulfonylureas were associated with a 1.18-fold  increased risk of MACE compared with mitoKATP channel low-affinity sulfonylureas in combination with metformin , all-cause mortality , and severe hypoglycemia  but was not associated with higher risks of ischemic stroke, cardiovascular death, arrhythmia, and heart failure in patients with T2D concurrently receiving metformin.MitoKetformin . AdditioATP channel high-affinity sulfonylurea therapy . Among the 3 dose strata, only the use of mitoKATP channel high-affinity sulfonylureas greater than 1 defined daily dose was associated with an increased risk of MACEs . eTable 3 in The MACE risk varied by duration and dose of mitoK therapy . The dur1c in estimating the PS and using intention-to-treat analyses  increased risk of 3-point MACEs associated with mitoKATP channel high-affinity sulfonylureas vs low-affinity sulfonylureas although the prior study focused on sulfonylurea monotherapy, and the generalizability of the reported data might be limited, given that sulfonylureas are most prescribed as an add-on medication to metformin in clinical settings. Conversely, the 2 studies observed discrepant secondary outcome findings, such as a substantial excess in cardiovascular death in the prior study as opposed to no statistically increased cardiovascular mortality for use of mitoKATP channel high-affinity sulfonylureas compared with low-affinity sulfonylureas in this current study. Although there may be differences in study cohort characteristics and diabetes severity between the 2 studies, use of metformin may at least partly account for the contradictory data. Despite inconsistent findings,28 metformin could be associated with reduced risk of cardiovascular-related events and cardiovascular mortality in patients with T2D,30 and this benefit may offset or modify the risk of the cardiovascular events associated with use of mitoKATP channel high-affinity sulfonylureas. On the other hand, the Action in Diabetes and Vascular Disease: Preterax and Diamicron MR-Controlled Evaluation (ADVANCE) and The Cardiovascular Outcome Study of Linagliptin vs Glimepiride in Type 2 Diabetes (CAROLINA) trials found that the mitoKATP channel low-affinity sulfonylurea gliclazide modified release vs other antidiabetic medication and linagliptin vs the mitoKATP channel low-affinity sulfonylureas glimepiride, respectively, had comparable outcomes on the risk of MACEs.32 Although not directly comparable to this current study, the data from the 2 large prospective studies32 indirectly support our reported data.In accordance with this current finding, a recently published observational study,ATP channels, which could subsequently cause larger infarct sizes or lead to death during an acute ischemic event.34 Our findings are in line with these preclinical data, offering insights into the cardiovascular safety of sulfonylureas combined with metformin in patients with T2D.Overall, our findings are supported by biological plausibility. In vitro and animal data revealed that certain sulfonylureas could jeopardize or abolish ischemic preconditioning, the self-cardiac protection mechanism for minimizing a potentially lethal ischemic result, through antagonism of cardiac mitoK36 In our study, glyburide and glimepiride comprised 76.8% and 74.9% in the cardiac mitoKATP channel high-affinity sulfonylurea and in low-affinity sulfonylurea groups, respectively. This could explain why there was a higher risk of hypoglycemia in the high-affinity sulfonylurea users compared with the low-affinity sulfonylurea users. Additionally, the main findings persisted after adjusting for occurrence of hypoglycemia during follow-up, which may rule out the possibility that hypoglycemia acts as an intermediator for our observed associations.The findings on the hypoglycemia outcome are supported by the prior literature revealing that glyburide had the highest risk of hypoglycemia, followed by glimepiride and glipizide.ATP channel low-affinity sulfonylureas were found to have lower risks of both 3-point MACEs and hypoglycemia, as compared with cardiac mitoKATP channel high-affinity sulfonylureas, we recommend low-affinity sulfonylureas over high-affinity sulfonylureas in cases where sulfonylurea treatment is considered for patients with T2D. This recommendation about the choice of sulfonylureas is of great clinical importance, given that sulfonylureas are one of the most used antidiabetic drugs after metformin in current clinical settings, despite the presence of newer antidiabetic medications. Additionally, some newer antidiabetic agents have been compared to sulfonylureas as a class regarding the risk of cardiovascular diseases,38 but the reported comparative data may have been associated with the compositions of varying mitoKATP channel affinity sulfonylurea. Future comparative studies involving sulfonylureas are suggested to take the affinity of sulfonylureas to cardiac mitoKATP channels into account.Our data have implications in prescribing practice and comparative safety research of antidiabetic agents in T2D. As cardiac mitoKATP channels, which could account for the sulfonylurea intraclass difference in the risk of MACEs among sulfonylurea agents. The inclusion of a nationwide diabetic population with the most used sulfonylurea regimen as add-on therapy to metformin substantially increases the generalizability of our findings. The validity of the composite outcome of 3-point MACEs is expected to be high, as the coding algorithms for identifying hospitalization for MI or ischemic stroke were validated with high accuracy,22 and cardiovascular deaths were determined through a nationwide death registry.This study had several strengths. To our knowledge, this is the first study that showed pharmacological differences in sulfonylureas combined with metformin regarding their differential affinity to cardiac mitoKATP channel low-affinity sulfonylureas and high-affinity sulfonylureas on cardiovascular safety could not be assessed due to the fairly short follow-up period. The observed short-term use of sulfonylureas combined with metformin, however, reflects clinical medication use among patients with T2D. Fifth, the adoption of a PS-matching approach led to reductions in sample sizes, and we alternatively adopted the inverse probability of treatment weighting approach which maintained the original sample sizes and led to the consistent results. Sixth, we may not have sufficient statistical power in secondary and subgroup analyses including duration analyses, which needs to be taken into consideration when interpreting the findings.Several limitations of this study merit discussion. First, our study is potentially subjected to confounding by indication or disease severity. We attempted to mitigate this potential confounding by maintaining a balanced average daily dose of metformin and all proxies of diabetes severity between the 2 groups. Second, unmeasured confounders, such as smoking, could possibly confound the reported findings; however, we used an active-comparator design, and reached consistent findings when using the high-dimensional–PS approach. Third, the adopted as-treated exposure analyses were prone to potential informative censoring; nevertheless, the alternative intention-to-treat analysis yielded similar results as the main findings. Fourth, the long-term effect of cardiac mitoKATP channel high-affinity sulfonylureas vs low-affinity sulfonylureas, when combined with metformin, were associated with an increased risk of MACE, suggesting that the high-affinity blockage of cardiac mitoKATP channels may act as an important determinant of sulfonylurea-related adverse cardiovascular events in patients with T2D.MitoK


ICI therapy has greatly improved patient outcomes in melanoma, but at the cost of immune-related adverse events (irAEs). Data on the chronicity of irAEs, especially in real-world settings, are currently limited. We performed a retrospective chart review of 161 adult patients with melanoma treated with at least one cycle of ICI regimen in the adjuvant or metastatic setting: 129 patients received PD-1 inhibitor monotherapy and 32 received dual immunotherapy. Patients were grouped by duration of irAE: permanent (no complete resolution), long-term (resolution over a period ≥ 6 months), transient (resolution over a period < 6 months), or no irAEs. A total of 283 irAEs were reported in the whole patient population. Sixty-six (41.0%) patients developed permanent irAEs, fifteen (9.3%) experienced long-term irAEs as their longest-lasting toxicity, thirty-four (21.1%) developed transient irAEs only, and forty-six (28.6%) experienced no irAEs. Permanent irAEs occurred in 21 (65.6%) patients treated with dual immunotherapy and in 45 (34.9%) patients treated with monotherapy. The majority of permanent irAEs were endocrine-related (36.0%) or skin-related (32.4%). Grade 3–4 permanent irAEs occurred in 20 (12.4%) patients and included toxicities such as adrenal insufficiency, myocarditis, and myelitis. Fifty-three (32.9%) patients were still requiring treatment for long-term or permanent irAEs 6 months or more following the completion of ICI therapy, including twenty-four patients on thyroid hormone replacement and twenty-two on oral steroids. ICI treatment was temporarily interrupted for 64 (22.6%) irAEs and permanently discontinued due to irAEs in 38 patients ; additionally, 4 (2.5%) patients died of irAEs. Our findings show that ICI treatment in melanoma is associated with a wide range of toxicities that can be permanent and may have long-lasting impacts on patients, which should therefore be discussed when obtaining consent for treatment. The development of immune checkpoint inhibitor (ICI) therapy has transformed the treatment landscape for high-risk and advanced melanoma over the past decade. Novel ICI agents including ipilimumab , pembrolizumab, and nivolumab (both anti-PD-1 antibodies) are now routinely employed and have greatly improved patient outcomes . SeveritOne area of growing research in the field of irAEs is the duration of toxicity. As clinicians accumulate more experience with ICIs, it is becoming increasingly apparent that many irAEs can have long-lasting or even permanent effects . While sThe present study reports data from our local experience at the Cancer Centre of Southeastern Ontario (CCSEO) with the aim of characterizing the long-term and permanent irAEs associated with adjuvant ICI treatment in patients with high-risk resected melanoma or palliative ICI treatment in patients with unresectable advanced or metastatic melanoma. We also report on the management of irAEs in these settings at our centre as well as the association with survival outcomes in patients who received single-agent ICI in the first-line palliative setting.We conducted a retrospective chart review of 161 adult patients with melanoma treated with at least one cycle of an ICI regimen in the adjuvant or palliative setting. We obtained approval from our local research ethics board prior to study commencement. One hundred and twenty-nine patients received PD-1 inhibitor monotherapy, and thirty-two received dual immunotherapy . We collected baseline patient, tumour, and treatment characteristics including age, sex, Eastern Cooperative Oncology Group (ECOG) performance status, baseline autoimmune disease history, melanoma stage, BRAF mutation status, baseline neutrophil-to-lymphocyte ratio (NLR), baseline platelet-to-lymphocyte ratio (PLR), ICI regimen, and ICI treatment line.We grouped patients by duration of irAE: permanent (no complete resolution), long-term (resolution over a period ≥ 6 months), transient (resolution over a period < 6 months), or no irAEs. The start date for irAE duration was determined by the date of clinic or hospital visit at which signs/symptoms of irAE were first identified. The end date for irAE duration was determined by the date of the clinic or hospital visit at which the irAE was declared completely resolved based on no further signs/symptoms and no further treatment required. Patients who experienced more than one irAE were categorized according to their longest-duration irAE. The characteristics of individual irAEs were also collected, including type, severity as graded by CTCAE v4.0 , treatmep-values. Statistical analysis was performed using Microsoft Excel in Microsoft Office 365 and IBM SPSS Statistics Version 26.We conducted descriptive and univariate analyses to provide an overview of the baseline study population. Chi-square or Fisher’s Exact test, if appropriate, were utilized for the univariate analyses. A logistic regression model was utilized to identify predictors of long-term or permanent irAEs. Objective response rates were analyzed using one-way analysis of variance. Overall survival was analyzed as a time-to-event endpoint using the Kaplan–Meier method with log-rank testing to calculate Thirty-eight patients received ICI therapy in the adjuvant setting alone, and one hundred and twenty-three patients received ICI therapy in the palliative setting; 10 patients received ICI therapy in both adjuvant and palliative settings. Sixty-six (41.0%) patients developed permanent irAEs, fifteen (9.3%) experienced long-term irAEs as their longest-lasting toxicity, thirty-four (21.1%) developed transient irAEs only, and forty-six (28.6%) experienced no irAEs. Permanent irAEs occurred in 21/32 (65.6%) patients treated with dual immunotherapy and in 45/129 (34.9%) patients treated with monotherapy. The baseline characteristics of patients in each irAE duration group are reported in The characteristics of the irAEs in each duration group are summarized in Patterns of irAE development in patients who received anti-PD-1 monotherapy are shown in Patterns of irAE development in patients who received dual anti-CTLA-4/anti-PD-1 immunotherapy are shown in Among 26 patients with baseline autoimmune disease, 21 (81%) experienced an irAE, including 11 (42.3%) who experienced a baseline disease flare, 9 (34.6%) who developed a permanent irAE, and 4 (15.4%) who developed a long-term irAE. Four patients required a long-term dose increase to an existing replacement therapy, namely thyroid replacement (three patients) or chronic prednisone (one patient).p = 0.717), suggests that patients may be more likely to develop long-term or permanent irAEs if they are in the older age group, receive dual immunotherapy, or experienced grade ≥3 toxicity, whereas patients may be less likely to develop long-term or permanent irAEs if they have stage IV disease at the time of first ICI therapy or had baseline PLR ≥ 200 . This result was consistent in a 6-month landmark analysis performed to account for time bias . A similar trend in objective response rate was also observed and is shown in The subgroup analysis of patients who received single-agent ICI in the first-line palliative setting, shown in We present here a retrospective observational study describing the real-world experience of an academic cancer centre with chronic toxicities secondary to ICI therapy in the treatment of adjuvant and palliative melanoma patients. Long-term or permanent irAEs, of which the majority were endocrine- or skin-related, occurred in 50.3% of patients and may be more likely in patients experiencing higher-grade irAEs. In the subgroup of patients who received single-agent anti-PD-1 as frontline therapy for advanced unresectable or metastatic disease, long-term or permanent irAEs were associated with better survival outcomes.Treatment with an ICI regimen became the standard of care for advanced melanoma relatively recently ,8,9, witIn a previous study by Johnson et al. , which wA more recent study by Patrinely et al.  reportedThe prospect of long-lasting repercussions from irAEs naturally leads to discussions around the optimal duration of ICI therapy in advanced melanoma to balance efficacy and toxicity. It has been postulated that treatment may be safely discontinued in certain patients who have demonstrated robust responses, but the timing and identification of the best candidates for this strategy remain unclear . The resAdditional future areas of focus may include an analysis of the amount of healthcare resources spent on treating irAEs. As the usage of ICI therapies expands, so too will the demands for immunosuppressive therapies and irAE-related healthcare visits. Additionally, the impact of irAEs and irAE treatment on fertility and sexual potency, such as hypophysitis and long-term corticosteroids, remains yet unexplored and warrants investigation. Reproductive health is another important domain which can be affected in this population, especially as ICI therapies see increasing use in the adjuvant setting and younger patients and could be an area of focus for future studies.The results of the current study are limited by its retrospective study design, small sample size, and inability to accurately attribute irAEs to a specific line of immunotherapy in patients who received multiple lines of immunotherapy. Particular care should be taken when interpreting the survival data, which are still affected by time bias despite the 6-month landmark analysis; of note, our sample size is too small to conduct a meaningful 12-month landmark analysis. Therefore, our findings need to be interpreted cautiously and should be validated with larger, multi-centre studies.Nonetheless, our study provides merit in generating real-world long-term toxicity data in advanced and high-risk resected melanoma treated with ICI therapy. While ICI clinical trials typically do not include patients with known risk factors for toxicity, such as comorbid baseline autoimmune disorders, these patients do receive ICI therapy in the real world. Additionally, the risks of cosmetic or long-term toxicities, even if low grade, can have significant patient impacts that may not be captured in clinical trials.The significant benefits of treatment with ICIs in melanoma are tempered by the wide range of potential toxicities that can be permanent and may have long-lasting impacts on patients. It is essential that the risk of long-term toxicities be discussed when obtaining consent for ICI therapy, and both patients and clinicians must be prepared to respond to them appropriately. The management of irAEs is becoming increasingly important as ICIs are being used more commonly in the adjuvant setting and for longer periods in the palliative setting due to the impact of survivorship.


BackgroundThe copy number of the oligonucleotide 5’-purine-uridine-uridine-purine-uridine-3’ (purUUpurU) motif in a viral genome was previously shown to correlate with the severity of acute illness. This study aimed to determine whether purUUpurU content correlates with virulence in other single-strand RNA (ssRNA) viruses that vary in clinical severity.MethodologyWe determined the copy number of purUUpurU in the genomes of two subtypes of human respiratory syncytial virus (RSV), respiratory syncytial virus A (RSV-A), and respiratory syncytial virus B (RSV-B), which vary in clinical severity. In addition, we determined the purUUpurU content of the four ebolaviruses that cause human disease, dengue virus, rabies virus, human rhinovirus-A, poliovirus type 1, astrovirus, rubella, yellow fever virus, and measles virus. Viral nucleotide sequence files were downloaded from the National Center for Biotechnology Information (NCBI)/National Institutes of Health website. In addition, we determined the cumulative case fatality rate of 20 epidemics of the Ebola virus and compared it with that of the other human ebolaviruses.ResultsThe genomic purUUpurU content correlated with the severity of acute illness caused by both subtypes of RSV and human ebolaviruses. The lowest purUUpurU content was in the genome of the rubella virus, which causes mild disease.ConclusionsThe quantity of genomic purUUpurU is a virulence factor in ssRNA viruses. Blood hyperviscosity is one mechanism by which purUUpurU causes pathology. Comparative quantitative genomic analysis for purUUpurU will be helpful in estimating the risk posed by emergent ssRNA viruses. We recently reported that the number of copies of the oligonucleotide 5’-purine-uridine-uridine-purine-uridine-3’ (purUUpurU) within the genome of several single-strand RNA (ssRNA) viruses correlates with the potential severity of acute illness caused by several of those viruses . ssRNA vPurUUpurU activates innate immunity by binding to Toll-like receptor 8 (TLR8). Acting through the adaptor protein myeloid differentiation primary response 88 (MYD88) and nuclear factor kappa B (NF-κB), this causes the production of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6) . Both TNThis paper will report the findings of quantitative comparative genomic analysis for purUUpurU in several other viruses. First, we examine two subtypes of respiratory syncytial virus (RSV) that differ in the severity of the illness they cause. In our previous report, we noted a high copy number of purUUpurU in RSV, which can cause a fatal acute illness in infants and the elderly. There are two major subtypes of RSV, respiratory syncytial virus A (RSV-A) and respiratory syncytial virus B (RSV-B). These differ in the severity of illness they cause, with RSV-A causing more severe disease -9. Thus,Next, we will examine the species of ebola virus that cause disease in humans. Epidemics of these viruses, namely, Ebola virus (EBOV), Sudan virus (SUDV), Bundibugyo virus (BDBV), and Taï Forest virus (TAFV), differ in their case fatality rate. Thus, they provide another opportunity to test the pathogenicity of purUUpurU. Finally, we will also report the results of quantitative comparative genomic analysis of other viruses that cause acute illnesses of varying severity, namely, yellow fever virus (YFV), rabies virus, measles virus, human rhinovirus A (HRV-A), astrovirus, rubella, dengue virus, and poliovirus 1.https://www.ncbi.nlm.nih.gov/labs/virus/) in fasta format. All complete  nucleotide sequences cumulatively available at the time of analysis were studied. Several curations were performed to remove incomplete sequences. All data analyses were conducted in Python programming language (Python3.7) and Bio.Seq, SeqIO, motifs modules of Biopython toolbox (https://biopython.org/).Viral nucleotide sequence files were downloaded from the National Center for Biotechnology Information (NCBI)/National Institutes of Health website (The purUUpurU motifs were counted in the following manner: because purine bases are either adenine (A) or guanine (G), the possible purUUpurU motifs are AUUAU, AUUGU, GUUGU, and GUUAU. The complementary DNA sequences are ATTAT, ATTGT, GTTGT, and GTTAT. The strand present in viral particles  was used for analysis.If two motifs overlapped, both motifs were tracked. The results report the non-overlapping motif counts with the incidence rate of the overlap. For instance, AUUGUUGU would be detected as two motifs, AUUGU and GUUGU, but there is one non-overlapping motif. The following viruses were examined: RSV-A, RSV-B, BDBV, SUDV, TAFV, EBOV, measles virus, HRV-A, dengue virus, rabies virus, astrovirus, rubella, poliovirus type 1, and YFV.Definitive outcome data from the West Africa 2013-2016 EBOV epidemic were used to calculate the fatality of that epidemic. Those data were taken from a published source and collected by the World Health Organization (WHO) during the earliest phase of that epidemic, from December 2013 to September 2014 . FatalitThe mean number of purUUpurU and genome size  were calculated. The coefficient of variation (CV) of both was also calculated. Pearson’s correlation coefficient was used to determine the correlation between genome size and purUUpurU count. This statistic was performed using Microsoft Excel . The Mann-Whitney U test was used to determine the significance of the difference between the purUUpurU counts of RSV-A and RSV-B and EBOV and BDVB. A chi-square test was used to determine whether there was a significant difference between the case fatality rates of EBOV and SUDV. These calculations were performed with the Python statistics package (SciPy.stats from SciPy 1.5.2).Results of the purUUpurU counting are presented in Figure The purUUpurU count was a strong discriminant between the RSV-A and RSV-B Figure . The genFigure The mean purUUpurU count decreased in the following order: TAFV > EBOV > BDBV > SUDV. Case fatality rates were as follows: EBOV, 69.2% , SUDV, 53.7% , BDBV, 32.0% , and TAFV, 0 deaths (one case). Using these data, the case fatality rate for the disease caused by EBOV was higher than for the disease caused by SUDV .The percentages of overlapping motifs ranged from 0.1% (rubella virus) up to 9.0% (poliovirus 1)  . Based oNevertheless, very similar figures are cited in the literature -18. ThisElucidation of genomic purUUpurU content as a virulence factor provides a biological basis for a higher case fatality rate in epidemics caused by EBOV. Further, a case fatality rate of 39.5% is lower than any other epidemic caused by EBOV. Thus, we regard data from the definitive outcomes of smaller outbreaks and the beginning of the West Africa 2013-2016 outbreak to be the most reliable and that EBOV is the most virulent ebolavirus Figure .Dengue virusThe moderate copy number of purUUpurU in the genome of the dengue virus may seem surprising given its association with severe diseases such as “break-bone fever” and viral hemorrhagic disease. However, of the estimated 390 million annual dengue virus infections, 293.9 million are estimated to be clinically inapparent . Severe Nonspecific viral syndromesAt both ends of the spectrum, in many cases, the genomic purUUpurU content accounts for the potential severity of acute illness caused by the virus. SARS-CoV-2 has 247 copies of purUUpurU in its genome  and can Significance of PurUUpurUPurUUpurU can be viewed as a viral equivalent of N-formylmethionine, a product of bacterial metabolism that activates innate immunity. It is a driver of the cytokines that cause the symptoms seen in the initial nonspecific phase of many viral illnesses. If the infection progresses, continued production of cytokines driven by purUUpurU can lead to elevated blood viscosity with subsequent organ dysfunction due to reduced tissue perfusion, as seen in the disease caused by RSV and ebolaviruses. This outcome is likelier if the host interferon response is deficient. In infections caused by viruses with the greatest purUUpurU content, inflammation driven by that oligonucleotide can lead to marked elevations of cytokines, hyperviscosity syndrome, and thromboinflammation. The high copy number of purUUpurU in SARS-CoV-2 suggests that the cytokine storm seen in COVID-19 is caused by the virus, not dysregulation of the host immune system. This may also be the case in the disease caused by EBOV. Thus, the purUUpurU content of a viral genome influences the inflammatory potential of that virus.Quantitative comparative genomic analysis has contributed to the understanding of how much of the variation in a clinical outcome is determined by the virus and how much is due to variation in the host immune response. PurUUpurU data suggest that viruses can cause cytokine storm syndrome in a normally functioning immune system. Quantitative comparative genomic analysis has revealed that the genomic purUUpurU content is an important characteristic of any ssRNA virus. A 2007 publication reported that 49 of the 87 new human pathogens identified since 1980 are RNA viruses . Pri. Pri1]. A meta-analysis of inflammatory markers in COVID-19 showed that in critically ill patients, acute phase reactants and related biomarkers are elevated to a greater degree than cytokines. The authors questioned the relevance of cytokine storm in organ dysfunction in COVID-19. Instead, the degree of elevation of D-dimer, the degradation product of cross-linked fibrin, was the strongest discriminant between critically ill COVID-19 patients and those with sepsis . IncreasAlong with impaired myocardial contractility and capillary leak, blood hyperviscosity should be recognized as a severe adverse effect of marked cytokine elevations. All of these factors decrease tissue perfusion. Cytokine elevations change from being beneficial to pathologic when they cause decreased tissue perfusion in the host.The quantity of genomic purUUpurU is a virulence factor in ssRNA viruses. Blood hyperviscosity is one mechanism by which purUUpurU causes pathology. Comparative quantitative genomic analysis for purUUpurU will be helpful in estimating the risk posed by emergent ssRNA viruses.


P < 0.001, FDR = 0.01) was found. The significant reduction of Actinobacteria was identified in all taxonomic levels down to the genus (P < 0.01) using the ANOVA test. Indeed, a significantly reduced relative abundance of Corynebacterium was found in the patients compared to healthy controls (P = 0.001). Reduced abundance of Corynebacterium has been widely associated with anosmia, a common symptom of COVID-19 as suffered from our patients. Contrastingly, the Corynebacterium genus was highly represented in the nasal mucosa of healthy subjects. Further investigations on larger cohorts are necessary to establish functional relationships between nasal microbiota content and clinical features of COVID-19.The evolution and the development of the symptoms of Coronavirus disease 19 (COVID-19) are due to different factors, where the microbiome plays a relevant role. The possible relationships between the gut, lung, nasopharyngeal, and oral microbiome with COVID-19 have been investigated. We analyzed the nasal microbiome of both positive and negative SARS-CoV-2 individuals, showing differences in terms of bacterial composition in this niche of respiratory tract. The microbiota solution A (Arrow Diagnostics) was used to cover the hypervariable V1–V3 regions of the bacterial 16S rRNA gene. MicrobAT Suite and MicrobiomeAnalyst program were used to identify the operational taxonomic units (OTUs) and to perform the statistical analysis, respectively. The main taxa identified in nasal microbiome of COVID-19 patients and in Healthy Control subjects belonged to three distinct phyla: Proteobacteria , Firmicutes , and Actinobacteria  with a relative abundance > 1% in all groups. A significant reduction of Actinobacteria in Cov19 group compared to controls (The online version contains supplementary material available at 10.1007/s00284-022-03106-x. The WHO reported that pandemic Coronavirus Disease 2019 (COVID-19) has caused to date about six millions of deaths worldwide . Recentln = 4 controls (CO)  which resulted as negative to the SARS-CoV-2 molecular assay and n = 18 symptomatic COVID-19-positive patients . All the enrolled subjects underwent a nasal swab, performed by a trained otolaryngologist, at admission within the Department of Infectious Diseases at Teaching Hospital of Naples Federico II of Naples, Italy. The inclusion criteria provided that each patient was not being treated with antibiotics, pro- and pre-biotics, antiviral, or corticosteroid medications, for at least one month prior to sample collection. The clinicians evaluated the severity of the symptoms according to the Clinical Status Ordinal Scale as reported by Beigel et al. (2020) .The present pilot study described the nasal microbiome changing in Cov19 patients compared to not-affected subjects. Interestingly, the nasal microbial composition resulted different between the study groups, as shown by the beta-diversity. Furthermore, our data showed a significantly increased microbial diversity in affected individuals compared to healthy subjects, as already reported in similar settings –31. NoteCorynebacterium genus, we hypothesized a possible relationship between the reduced abundance of this taxon and anosmia in Cov19 patients. The loss of smell was a common symptom associated with SARS-CoV-2 infection caused by several factors determining both acute and chronic olfactory dysfunctions [Corynebacterium was known to be reduced in individuals with loss of smell, even in the absence of SARS-CoV-2 infection [By evaluating the clinical features of our study group, we observed in all Cov19 patients the presence of a common symptom, the loss of smell. Consequently, focusing on the unctions . In factunctions , the infunctions –31. In tnfection . More renfection .Corynebacterium as highly represented in nasal swabs of healthy individuals. Besides, to reinforce our data, we compared the HC group’s microbial profile with one described in healthy subjects by Bassis et al., showing a similar abundance of Actinobacteria [Even though our control group was very small, we confirmed data published by De Boeck et al.  which shbacteria .In keeping with our data, we underlined that also the findings by Ngo et al.  indicateIn addition to that, Koskinen et al. found increased diversity in subjects with partial or complete smell impediments compared to normosmic participants . This waOur evidence was also supported by the literature and thus we can speculate that SARS-Cov-2 could play a role in changing the protective microbiome film of nasal mucosa, increasing the quantity of species able to produce butyrate and giving olfactory dysfunctions, like loss of smell.Corynebacterium could act as a potential biomarker linked to the loss of olfactory function in COVID-19 patients. Reasonably, we are conscious that further studies are required to understand the mechanisms underlying the loss of olfactory function that we herein associate with the reduction of Corynebacterium in COVID-19 patients. We are aware of the small cohort enrolled in the study and so we were unable to draw general conclusions in differentiating the composition among HC and Cov19, as well as female and male. Nevertheless, we would underline the difficulty in enrolling “naïve” patients without evident confounding factors influencing the nasal microbiome content, particularly during the COVID-19 pandemic spread. Here we present the data gained from our pilot study aiming to give a first insight into the nasal microbiota of COVID-19 patients. As a matter of course, further studies are required.In our study, we are not able to establish who drives who, but this incidental finding could open a new field of investigation in the setting of potential interactions between SARS-Cov-2 and microbiome milieu. We highlight how the Supplementary file1 (TIF 6579 kb)S1 Fig. Beta-diversity of bacteria identified in the COVID-19 patients divided by gender  and Healthy Control (HC) groups. Principal Component Analysis (PCoA) plots using the Bray–Curtis distance measures. Results of PERMANOVA (ADONIS), ANOSIM, and PERMADISP tests showed significant separation between all the centroids and differences between groups (p<0.001). Green dots indicate HC subjects; pink and blue dots indicate Female and Male patients, respectively.Supplementary file2 (XLSX 15 kb)Supplementary file3 (DOCX 21 kb)Supplementary file4 (DOCX 18 kb)Below is the link to the electronic supplementary material.


COVID-19 is associated with increased risks of neurological and psychiatric sequelae in the weeks and months thereafter. How long these risks remain, whether they affect children and adults similarly, and whether SARS-CoV-2 variants differ in their risk profiles remains unclear.In this analysis of 2-year retrospective cohort studies, we extracted data from the TriNetX electronic health records network, an international network of de-identified data from health-care records of approximately 89 million patients collected from hospital, primary care, and specialist providers . A cohort of patients of any age with COVID-19 diagnosed between Jan 20, 2020, and April 13, 2022, was identified and propensity-score matched (1:1) to a contemporaneous cohort of patients with any other respiratory infection. Matching was done on the basis of demographic factors, risk factors for COVID-19 and severe COVID-19 illness, and vaccination status. Analyses were stratified by age group  and date of diagnosis. We assessed the risks of 14 neurological and psychiatric diagnoses after SARS-CoV-2 infection and compared these risks with the matched comparator cohort. The 2-year risk trajectories were represented by time-varying hazard ratios (HRs) and summarised using the 6-month constant HRs , the risk horizon for each outcome , and the time to equal incidence in the two cohorts. We also estimated how many people died after a neurological or psychiatric diagnosis during follow-up in each age group. Finally, we compared matched cohorts of patients diagnosed with COVID-19 directly before and after the emergence of the alpha (B.1.1.7), delta (B.1.617.2), and omicron (B.1.1.529) variants.vs just before) the emergence of the delta variant (n=44 835 in each cohort), increased risks of ischaemic stroke, epilepsy or seizures, cognitive deficit, insomnia, and anxiety disorders were observed, compounded by an increased death rate. With omicron (n=39 845 in each cohort), there was a lower death rate than just before emergence of the variant, but the risks of neurological and psychiatric outcomes remained similar.We identified 1 487 712 patients with a recorded diagnosis of COVID-19 during the study period, of whom 1 284 437  were adequately matched with an equal number of patients with another respiratory infection. The risk trajectories of outcomes after SARS-CoV-2 infection in the whole cohort differed substantially. While most outcomes had HRs significantly greater than 1 after 6 months , their risk horizons and time to equal incidence varied greatly. Risks of the common psychiatric disorders returned to baseline after 1–2 months  and subsequently reached an equal overall incidence to the matched comparison group . By contrast, risks of cognitive deficit (known as brain fog), dementia, psychotic disorders, and epilepsy or seizures were still increased at the end of the 2-year follow-up period. Post-COVID-19 risk trajectories differed in children compared with adults: in the 6 months after SARS-CoV-2 infection, children were not at an increased risk of mood (HR 1·02 [95% CI 0·94–1·10) or anxiety (1·00 [0·94–1·06]) disorders, but did have an increased risk of cognitive deficit, insomnia, intracranial haemorrhage, ischaemic stroke, nerve, nerve root, and plexus disorders, psychotic disorders, and epilepsy or seizures (HRs ranging from 1·20 [1·09–1·33] to 2·16 [1·46–3·19]). Unlike adults, cognitive deficit in children had a finite risk horizon (75 days) and a finite time to equal incidence (491 days). A sizeable proportion of older adults who received a neurological or psychiatric diagnosis, in either cohort, subsequently died, especially those diagnosed with dementia or epilepsy or seizures. Risk profiles were similar just before versus just after the emergence of the alpha variant (n=47 675 in each cohort). Just after (This analysis of 2-year retrospective cohort studies of individuals diagnosed with COVID-19 showed that the increased incidence of mood and anxiety disorders was transient, with no overall excess of these diagnoses compared with other respiratory infections. In contrast, the increased risk of psychotic disorder, cognitive deficit, dementia, and epilepsy or seizures persisted throughout. The differing trajectories suggest a different pathogenesis for these outcomes. Children have a more benign overall profile of psychiatric risk than do adults and older adults, but their sustained higher risk of some diagnoses is of concern. The fact that neurological and psychiatric outcomes were similar during the delta and omicron waves indicates that the burden on the health-care system might continue even with variants that are less severe in other respects. Our findings are relevant to understanding individual-level and population-level risks of neurological and psychiatric disorders after SARS-CoV-2 infection and can help inform our responses to them.National Institute for Health and Care Research Oxford Health Biomedical Research Centre, The Wolfson Foundation, and MQ Mental Health Research. Since the early stages of the pandemic, COVID-19 has been known to be associated with an increased risk of many neurological and psychiatric sequelae.First, we do not know if or when the risks of different post-COVID-19 outcomes return to baseline. This information is important to patients , clinicians , and health policy makers (who must plan appropriate service provision). Previous studies on the neurological and psychiatric sequelae of COVID-19 did not investigate this question.Evidence before this studyWe searched PubMed (Medline) on March 21, 2022, for publications since database inception in English using the terms “(neuropsychiatr*[Title/Abstract] OR neurologic*[Title/Abstract] OR psychiatric[Title/Abstract] OR depress*[Title/Abstract] OR anxiety*[Title/Abstract] OR cognit*[Title/Abstract] OR brain[Title/Abstract]) AND (variant*[Title/Abstract] OR omicron[Title/Abstract] OR delta[Title/Abstract] OR evolution[Title/Abstract]) AND (COVID[Title/Abstract] OR COVID-19[Title/Abstract] OR SARS*[Title/Abstract])”. We found cohort studies and systematic reviews reporting neuropsychiatric sequelae persisting up to 10 months after COVID-19. We found one large, 6-month electronic health records study of neuropsychiatric disorders after a COVID-19 diagnosis. This study reported increased incidence and relative risk of cognitive symptoms and anxiety or depression 6 months after COVID-19, compared with influenza. We are not aware of any large-scale data regarding long-term COVID-19 sequelae beyond 12 months, or the evolution of incidence or relative risk of neuropsychiatric diagnoses in patients recovered from COVID-19 throughout the pandemic, stratified by COVID-19 variant or vaccination status.Added value of this studyTo our knowledge, this is the first study with a comparator cohort that assesses the risks of a range of neurological and psychiatric outcomes of COVID-19 up to 2 years after the index SARS-CoV-2 infection. We found that the risks of post-COVID neurological and psychiatric outcomes follow different trajectories: the risk of cognitive deficit, dementia, psychotic disorder, and epilepsy or seizures remain elevated 2 years after SARS-CoV-2 infection, while the risks of other diagnoses  subside after 1–2 months and show no overall excess over the whole 2-year follow-up. We also found that risk trajectories differ somewhat in children: they are not at an increased risk of mood or anxiety disorders (even over the first 6 months) and their risk of cognitive deficit is transient, but they share adults’ risk of several other diagnoses and are notably at risk of epilepsy or seizures. Finally, we found that the risks of neurological and psychiatric outcomes remain similar after the emergence of the omicron (B.1.1.529) variant as with the delta (B.1.617.2) variant, but are offset by a significantly lower death rate.Implications of all the available evidenceThe persisting increased risk of post-COVID-19 cognitive deficit, dementia, psychotic disorders, and epilepsy or seizures 2 years after the index infection calls for enhanced service provision to diagnose and manage these sequelae, and research to understand the mechanisms. The differing profile of post-COVID-19 neurological and psychiatric diagnoses in children informs the risk–benefit association of policies aimed at preventing COVID-19 in paediatric populations and suggests that underlying mechanisms might in part be different from those in adults. The observation of comparable neurological and psychiatric risks just after (compared with just before) emergence of the omicron variant suggests an ongoing neuropsychiatric burden of COVID-19 even with variants that lead to otherwise less severe disease.Second, the risk profile in different age strata, especially in children, has not been well characterised.Third, whether or not risk profiles have changed with the emergence of different variants is unknown; in particular, whether or not the omicron (B.1.1.529) variant, which has a lower mortality rate and better acute outcomes than the alpha (B.1.1.7) and delta (B.1.617.2) variantsWe used electronic health records to investigate these three questions. We assessed the 2-year risk trajectories of 14 neurological and psychiatric diagnoses in three age groups , and if and when these risks returned to baseline. Then we compared these risks between patients diagnosed just after versus just before the emergence of the alpha, delta, and omicron variants.In this analysis of retrospective cohort studies, we used two electronic health records networks. The primary source was the TriNetX Analytics Network a federated network recording anonymised data from electronic health records of approximately 89 million patients in 62 health-care organisations located mainly in the USA but also in Australia, the UK, Spain, Bulgaria, India, Malaysia, and Taiwan. The participating health-care organisations in the networks are hospitals, primary care, and specialist providers who contribute data from uninsured and insured patients. From this data source we compiled our primary cohorts of patients with COVID-19 and any other respiratory infection. The secondary network was TriNetX's US Collaborative Network (comprising approximately 78 million patients), which is restricted to the USA. We used this network when comparing outcomes just after versus just before the emergence of new SARS-CoV-2 variants, because the time of emergence of variants varied across countries. Both networks contain the same range of data, including demographics, diagnoses represented by ICD-10 codes, medications, and procedures. Our study design was similar to that described in previous publications using the TriNetX platform,Using the TriNetX user interface, we created cohorts based on our inclusion and exclusion criteria. The primary cohort was defined as all patients who had a confirmed diagnosis of COVID-19 (ICD-10 code U07.1). We constructed a contemporaneous propensity-score matched comparator cohort of patients diagnosed with any other upper or lower respiratory infection (hereafter referred to as other respiratory infection) who did not have a COVID-19 diagnosis or a positive test for SARS-CoV-2 before their infection or during follow-up. The dates of diagnosis of COVID-19 and other respiratory infection are referred to as index events. The cohorts included patients of any age who had an index event on or after Jan 20, 2020 (the date of the first recorded COVID-19 case in the USA), up to the end of follow-up .Using TriNetX's US Collaborative Network, we created primary alpha, delta, and omicron cohorts comprising patients who had a first diagnosis of COVID-19 within the time windows corresponding to the dominance of a particular SARS-CoV-2 variant, and the comparator pre-alpha, pre-delta, and pre-omicron cohorts comprising those with a first diagnosis of COVID-19 in the period just before the emergence of that variant. These time windows were defined to be minimally separated in time  while retaining sufficient statistical power to detect group differences . As a reData de-identification is formally attested as per section §164.514(b)(1) of the Health Insurance Portability and Accountability Act Privacy Rule. Because we used anonymised and routinely collected data, no participant consent was required. Additional details on cohorts are provided in the The COVID-19 and other respiratory infection cohorts were stratified by age group  and by date of the index events in 2-monthly periods. Within each stratum, cohorts were propensity-score matched (1:1) for 82 covariates: sociodemographic factors and comorbidities representing risk for COVID-19, for more severe COVID-19 illness,We used a greedy nearest-neighbour matching approach with a caliper distance of 0·1 pooled SDs of the logit of the propensity score. Characteristics with a standardised mean difference (SMD) between cohorts lower than 0·1 were considered to be adequately matched.We investigated neurological and psychiatric sequelae of COVID-19 in terms of 14 outcomes occurring up to 2 years after the index event or the last day of follow-up , whichever came first: anxiety disorder; mood disorder; psychotic disorder; insomnia; cognitive deficit . We made comparisons between the matched COVID-19 and other respiratory infection cohorts using the log-rank test. The 2-year risk trajectories were represented by time-varying hazard ratios (HRs) calculated using natural cubic splines fitted to the log-cumulative hazard.For the analysis of the risks of outcomes by SARS-CoV-2 variants, we calculated constant HRs over the whole follow-up  using the Cox proportional hazard ratio. As in the other analyses, both the individual outcomes and their composite with death are reported.Additional details of the statistical analyses are in the The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the manuscript.We extracted data for 1 487 712 patients with a recorded diagnosis of COVID-19 between Jan 20, 2020, and April 13, 2022, from the TriNetX Analytics database, of whom 1 284 437 were adequately matched  to an equal number of patients with another respiratory infection. Both matched cohorts comprised 185 748 children (aged <18 years), 856 588 adults (aged 18–64 years), and 242 101 older adults (aged ≥65 years). In the matched COVID-19 cohort, mean follow-up was 213 days , and in the matched other respiratory infection cohort, the mean follow-up was 223 days  for outcomes with persistent or ongoing risk trajectories were reflected differently across age groups: the difference in incidence of cognitive deficit and dementia between cohorts was more noticeable for older adults than for adults or children; that of myoneural junction or muscle diseases was predominantly seen in children and adults but not older adults; that of epilepsy or seizures was significant only in children, and the risk of psychotic disorder was more evident for children and older adults than for adults.The endpoints of the 2-year risk trajectories can be summarised in terms of cumulative incidence at the end of the 2-year follow-up period and the proportion of patients with each diagnosis who subsequently died during the follow-up . Interprvs just before) the emergence of the omicron variant were at an increased risk (over 140 days of follow-up) of dementia, mood disorders, and nerve, nerve root, and plexus disorders, and at a broadly similar risk of most other outcomes. However, risks were more than offset by a substantially lower risk of death, such that the composite risks of each outcome and death were significantly lower than among those diagnosed just before the emergence of omicron.For our analysis of risk of outcomes with new variants, we compiled six additional US COVID-19 cohorts: the primary alpha (n=47 675), delta (n=44 835), and omicron (n=39 845) cohorts, and matched control cohorts of equal size diagnosed just before the emergence of alpha, delta, and omicron, respectively  to its occurrence, but beyond 2 months, other causes should be actively considered. Risk trajectories are also informative for public health. An increase in the number of new cases of COVID-19 is likely to lead to an increase in the number of cases of mood and anxiety disorders but this will be short lived. By contrast, the absence of risk horizons within the first 2 years of a COVID-19 diagnosis  for some diagnoses  indicate that patients and clinicians must remain vigilant about the possibility of these delayed sequelae. These findings also suggest that service provision needs to be reinforced and sustained, because new cases are likely to occur for a considerable time after the pandemic has subsided. The time to equal incidence informs us about what happens after the risk horizon has been reached. On the one hand, the risks might become approximately equal in the two cohorts so that a so-called COVID excess remains throughout follow-up and the time to equal incidence is never reached , which was the case for insomnia and myoneural junction or muscle disease. On the other hand, the risks might reverse, with more new diagnoses in the other respiratory infection cohort than in the COVID-19 cohort after the risk horizon, so that a time to equal incidence is eventually reached , as seen for anxiety and mood disorders.Another important aspect of outcome trajectories is the proportion of people who received a neurological or psychiatric diagnosis who subsequently died. All-cause mortality was substantial among older adults diagnosed with neurological or psychiatric sequelae both after COVID-19 diagnosis and after another respiratory infection—notably, those with epilepsy or seizures, dementia, cognitive deficit, and psychotic disorder. The fact that similar proportions of patients with these outcomes died in both cohorts suggests that this high mortality reflects general physical ill health rather than being related to SARS-CoV-2 infection itself.The mortality rate in older patients also raises the issue of death as a competing risk.Compared with adults and older adults, children were at a particularly increased risk of epilepsy or seizures, encephalitis, and nerve, nerve root, and plexus disorder, leading to significantly higher cumulative incidence after 2 years  in this age group. The persistence and severity of these outcomes cannot be determined from our study, but some will probably have deleterious consequences for children's health and physical and educational development. Therefore, these findings inform the risks and benefits of vaccination (and other preventive measures) against COVID-19 in paediatric populations. Reassuringly, unlike adults, children were not at an increased risk of mood and anxiety disorders after SARS-CoV-2 infection (even in the first 6 months) and cognitive deficit in children had a transient risk trajectory rather than ongoing risks as seen in older groups. The difference in profiles and trajectories of risks in children might indicate that the pathogenesis of COVID-19 sequelae is different in some respects from that of adults.The risk of neurological and psychiatric diagnoses of COVID-19 was greater with the emergence of the delta variant  than just before its emergence. These risks were compounded by an increased risk of death  the emergence of each variant might have decreased the observed HRs. Sixth, children and adolescents were grouped together, so further studies are needed to characterise the risks in different paediatric subgroups. Seventh, although in-hospital mortality data are well captured in TriNetX, out-of-hospital mortality reporting is more variable and linkage with mortality indices is only partial, so our incidence estimates will be underestimates and should be interpreted cautiously; but HRs for composite outcomes should not be affected to the same extent. Eighth, we do not know the severity or course of each disorder after diagnosis, or whether or not these are similar after COVID-19 and after other respiratory infections.Our study has specific limitations in addition to those inherent in electronic health records studies. First, our COVID-19 cohorts are probably enriched for symptomatic cases because self-diagnosed or asymptomatic COVID-19 is less likely to be coded in the health record. This is also true of the comparator cohort and their respiratory infections, and so HRs are less affected by this limitation than are incidences. Second, COVID-19 appeared to be more severe than other respiratory infections, but the mediation of our results by severity of the illness was not analysed. However, mediation by several markers of severity has been tested in our previous study,In summary, post-COVID neurological and psychiatric outcomes followed different risk trajectories: the risk of cognitive deficit, dementia, psychotic disorder, and epilepsy or seizures remained increased at 2 years after a COVID-19 diagnosis, while the risks of other diagnoses  subsided early and showed no overall excess over the 2-year follow-up. Children are not at increased risk of mood or anxiety disorders (even over the first 6 months) but share adults’ risk of several other diagnoses. The comparable risks seen after the emergence of omicron indicate that the neurological and psychiatric burden of COVID-19 might continue even with variants that lead to otherwise less severe disease. These findings are relevant for policy makers involved in anticipating and addressing the health burden of the pandemic, for researchers seeking to identify the mechanisms underpinning brain sequelae of COVID-19, and for patients and clinicians wishing to know the neurological and psychiatric risks following SARS-CoV-2 infections.https://osf.io/snfhw. The data used for this Article were acquired from TriNetX. This study had no special privileges. Inclusion criteria specified in the Methods and The TriNetX system returned the results of these analyses as csv files, which we downloaded and archived. Aggregate data, as presented in this Article, can be freely accessed at US Centers for Disease Control and Prevention COVID data tracker see https://covid.cdc.govFor more on TriNetX website see https://www.trinetx.com/platformFor more on aggregate data see https://osf.io/snfhwFor more on TriNetX website see https://www.trinetx.com/platformFor more on We declare no competing interests.