Background and Objectives: Thoracic ossification of the ligamentum flavum (OLF) often causes myelopathy and/or radiculopathy. The disease is frequently observed in East Asian populations. Although thoracic OLF in young athletes who have underwent decompression surgery has been reported, the removal of posterior spinal bony elements and ligamentous complex may often cause postoperative thoracolumbar instability. We established a novel surgical technique that preserves the posterior spinal elements, including the spinous processes, facet joints, and supraspinous and interspinous ligaments for thoracic OLF. This is the first case report to describe a navigation-assisted micro-window excision of thoracic OLF. Case: A 32-year-old male right-handed professional baseball pitcher with significant weakness and numbness in the left leg was referred to our hospital. The patient was diagnosed with thoracic OLF at T10-11 based on radiographic and magnetic resonance images in August 2022. After exposure of the left T10-11 laminae via a small unilateral incision, the location of T10-11 OLF was detected over the lamina by O-arm navigation. Then, the micro-window was made directly above the OLF using a navigated air drill, and the OLF was removed on the ipsilateral side. The contralateral side of OLF was also resected through the same micro-window, achieving complete spinal cord decompression. Results: The next day of the surgery, his leg weakness and numbness were significantly improved. Six weeks after the surgery, he started pitching. Three months after surgery, his symptoms had gone completely, and he pitched from the mound. Approximately 6 months after surgery, he successfully pitched in a professional baseball game. Conclusions: A navigation-assisted micro-window excision of thoracic OLF effectively preserved the spinal posterior bony elements and ligamentous complex. However, long-term clinical outcomes should be evaluated in future studies. The ossification of the ligamentum flavum (OLF) is a relatively rare disease that often causes thoracic myelopathy and/or radiculopathy. Previous reports have indicated that either posterior decompression or posterior decompression and fusion has been widely performed ,2,3,4. RA 32-year-old male right-handed professional baseball pitcher began to feel weakness and numbness in his left leg in January 2022. His left leg “gave way” while pitching in May 2022. In August, he developed walking difficulties with progressive numbness and weakness in the left leg and was referred to our hospital. His neurological examination in the left lower extremity revealed an increased tone, 4/5 weakness throughout, and decreased sensation to light touch. His proprioception was slightly abnormal. The Chaddock and Babinski signs on the left side were positive, and no ankle clonus was observed. Although the patient was ambulatory, it was difficult for him to stand on his left leg with his eyes closed for a prolonged period of time. Preoperative physical examinations  revealed severe spinal cord compression with a low-intensity-signal mass  setting was changed to perform a CT scan within 15 sec in order to minimize the radiation dose. StealthStation S7 (Medtronic) was used as the navigation system. After confirming the location of the OLF from outside the spinal canal, the micro-window was created on the left side of T10 lamina just above the OLF using a Stealth-Midas high-speed drill (Medtronic) under a Leica M530 neurosurgical microscope  . The driTo drill the OLF of the contralateral side, the surgical bed was rotated to tilt the patient’s body to the right. After partially exposing the normal dura mater, the ossification was resected piece by piece with Kerrison rongeurs while carefully separating the shaved ossification and the normal dura mater. The left and right outer edges of the dura mater and the remaining shaved ossification of the craniocaudal area were resected using curved Kerrison rongeurs and small curettes. Epidural hemorrhage gradually increased as decompression was performed, and the pulsation of the dura mater was confirmed. The adequacy of the OLF extirpation was frequently checked using the navigation probe. Finally, a nerve hook was used to confirm that there was no pressure on the dura mater in the spinal canal .The surgery lasted for 185 min, and the estimated blood loss was 10 mL. We achieved OLF resection and complete decompression of the spinal cord through the micro-window and completely preserved the posterior spinal elements, including the spinous processes, facet joints, and supraspinous and interspinous ligaments.The patient was allowed to stand and walk the next day after the procedure. After reviewing the postoperative MRI and CT images, spinal cord decompression and the complete removal of OLF were confirmed . In the Previously, various surgical techniques including posterior decompression  with/without fusion have been reported for thoracic OLF ,2,3,4. EIn recent years, unilateral posterior decompression using micro-endoscopic or full-endoscopic surgeries have also been reported in the literature. Even in unilateral endoscopic surgeries, either spinous processes, supraspinous, or interspinous ligaments are often sacrificed during surgery, or they involve unintentional fractures ,7,8,9. IThe advantages of our novel surgical technique not only include the preservation of the spinal posterior elements but also extend to the use of CT-based navigation, which allows for accurate identification of the OLF from a high position directly above the lamina, leading to the ipsilateral creation of a micro-window of the lamina and enhancing one’s ability to resect the OLF alone by burring a “trumpet-shaped” window. CT-based navigation has been introduced to ensure accurate and effective implant placement  and provThoracic OLF is typically seen in males in their 60s to 70s ,24, whilThoracic OLF is often observed in high-level baseball pitchers in East Asian countries. Surgical procedures that reduce postoperative spinal instability should be chosen. In the current study, we have proposed a novel surgical technique for thoracic OLF, which is named “navigation-assisted micro-window excision”. This procedure successfully preserved the spinal posterior bony elements and ligamentous complex. However, long-term clinical outcomes should be evaluated in future studies.


Post-acute sequelae of SARS-CoV-2 (PASC) is a condition characterized by persistent and debilitating symptoms that continue more than 28 days beyond the acute phase of COVID-19. A significant proportion of individuals who have contracted COVID-19 have reported long-term symptoms consistent with PASC. Despite the growing number of reports on PASC, its exact pathophysiology remains unclear, and there are currently no effective treatments available. Although various therapeutic interventions have been developed and used to treat COVID-19 patients, the effectiveness of these interventions in preventing the development of PASC is not yet established. Therefore, there is a pressing need to identify potential long-term health impacts and the relationship between therapeutic interventions and the development of PASC in COVID-19 survivors.A retrospective chart review was conducted at the reCOVer Clinic at Cleveland Clinic Foundation from March 2020 to November 2022. The study population consisted of patients diagnosed with PASC, who were matched with controls without PASC at a 1:1 ratio.We identified 57 patients that were deemed to have PASC in the study period. Multiple interventions that were given at the time of COVID-19 diagnosis were reviewed. Table 1 shows the characteristics of both patient groups. Our analysis did not find any significant differences in the baseline characteristics of patients who did and did not develop PASC, including hospital admission status, need for ICU, and need for intubation. Among those that received nirmatrelvir-ritonavir, tocilizumab, remdesivir and dexamethasone there were no statistical differences found in those that eventually developed PASC .The findings of our study suggest that treatment alone at the time of COVID-19 diagnosis is not protective against the development of PASC. Further studies are needed to identify other potential risk factors for PASC. Clinicians should consider future research focusing on identifying effective treatments for this debilitating condition.All Authors: No reported disclosures


Objective: This study aims to examine the impact of the COVID-19 pandemic on breast cancer screening in an underserved population, identify patient barriers, and discuss strategies to promote the importance of screening.Methods/operations: The Rutgers New Jersey Medical School Screening Access of Value for Essex (SAVE) program delivers cancer prevention services to the most vulnerable population in Essex County, New Jersey. The SAVE program was shut down from March 2020 to June 2020 due to COVID-19. The number of mammograms performed 18 months before the pandemic (September 2018 to March 2020) and 18 months after the shutdown of the program (July 2020 to December 2021) were recorded. A calling project was created in response to the pandemic to educate patients about COVID-19 precautions and provide healthcare and social services resources.Results: There was a 15.4% reduction in screening mammograms during the post-shutdown period . The number of diagnostic mammograms increased from 264 to 272. The calling project spoke with 1,548 patients and identified the following concerns: exposure to COVID-19, language barriers, and lack of health insurance.Conclusion: Although COVID-19 had a profound impact on most patients, especially in the realm of breast cancer screening, the implementation of the SAVE program's strategies such as transitioning to an appointment-only system has helped minimize the negative impacts. Reaching out to the patients, partnering with community organizations, and promoting SAVE services have played a vital role in encouraging more patients to have screening done. During the peak of the COVID-19 pandemic, all patients had a substantially harder time accessing various modalities of healthcare including annual follow-ups, elective procedures, screening exams, and other routine health services . The feaAll the barriers that prevented patients from accessing healthcare during the COVID-19 pandemic were magnified in underserved urban populations where a majority of residents require additional services such as public transportation, language interpreter services, and assistance-based payment programs . Due to It is especially important to acknowledge that this detrimental phenomenon is exacerbated in places such as Essex County, which house large populations of historically underserved communities due to longstanding barriers to healthcare that have persisted for years on end. Essex County is one of New Jersey’s most vulnerable patient populations with a demographic composed of over 60% of residents who identify as either Black or Hispanic . This paThe SAVE program was shut down from March 2020 to June 2020, which ultimately meant that patients were unable to obtain many essential preventive health services. To combat the potential harms of the pandemic on breast screenings, the SAVE program developed a calling project to educate patients about COVID-19 precautions and provide healthcare and social services resources. Strategies to minimize COVID-19 exposure such as wearing masks, social distancing, and proper hand hygiene were provided to patients prior to screening visits via phone. Lists of social service resources such as locations of outdoor “Fresh Produce Pop Up Markets” were created and distributed to patients especially those who were concerned about COVID-19 exposure from grocery stores. The objective of this study is to determine how breast cancer screenings were impacted in the underserved population of Essex County due to the COVID-19 pandemic.This article was previously presented in the form of an electronic poster presentation at the 2022 American College of Radiology Annual Meeting on April 24, 2022.Pre-COVID-19 operationsThe SAVE program is the only NJCEED screening program that operates in Essex County, New Jersey. This is the only health screening program in Essex County, which provides free breast and cervical cancer screenings for residents and has a primary focus on targeting the underserved population in this county. Traditionally, SAVE operates as a community outreach model. This is accomplished through the use of a mobile mammography van Figure  and morePost-COVID-19 operationsTo minimize contact and stop the spread of COVID-19, the traditional first-come-first-serve basis was transitioned to an appointment-only system. This was done to allow for adequate time between each appointment to reduce potential exposure from one patient to the next and to maximize social distancing. In a similar light, patient information and cancer education were provided via phone prior to the appointment to reduce the amount of time each patient would physically take in person.Calling projectIn response to the COVID-19 pandemic, a calling project was developed to provide COVID-19 precautions and receive direct feedback from our active and newly scheduled patients. Patients were reached via phone call and were provided different strategies to protect themselves from contracting COVID-19 including social distancing, wearing masks, and resources for COVID-19 testing. Patients were able to voice on the phone about the obstacles they currently faced to acquiring their breast and cervical screening services, which were obtained prior to appointments, and these verbal responses from patients were recorded.MetricsThe 18 months prior to the shutdown of the SAVE program from September 2018 to March 2020 were defined as the pre-COVID-19 time period, and the 18 months following the restart of the SAVE program after its shutdown from July 2020 to December 2021 were defined as the post-COVID-19 time period. The total number of screening mammograms and diagnostic mammograms were recorded for both the pre-COVID-19 and the post-COVID-19 time periods. Pre- and post-COVID-19 shutdown era screening and diagnostic mammograms were compared via Chi-squared tests. All analyses and descriptive statistics were conducted using Microsoft Excel . To assess the progress of the calling project, the total number of patients that were contacted as well as the major concerns each patient had on accessing their screening services were recorded.A 15.4% reduction in screening mammograms occurred subsequent to the shutdowns demarcating the post-COVID-19 period . Tangential to this reduction in screening mammograms, the number of diagnostic mammograms performed increased by 3% (264 pre-COVID-19 shutdown versus 272 post-COVID-19 shutdown).Subsequent Chi-squared tests sought to evaluate the differences between screening and diagnostic mammograms with respect to the pre- and post-COVID-19 shutdown time frame. Chi-squared analysis for the number of screening mammograms conducted pre-COVID-19 shutdown when compared to post-COVID-19 shutdown yielded a p-value of less than 0.001, while analysis for the number of diagnostic mammograms conducted in the same study time frame yielded a p-value of 0.0622 . However, the decrease in screening mammograms represents a similar trend that is observed in previous literature regarding COVID-19 and breast cancer screening nationwide. Expansion of SAVE services with additional community sites and more available appointments will be necessary to accommodate additional patients, especially with future emerging infectious diseases.


Infection by coronavirus SARS-CoV2 is a severe and often deadly disease that has implications for the respiratory system and multiple organs across the human body. While the effects in the lung have been extensively studied, less is known about the impact COVID-19 has across other organs.Here, we contribute a single-nuclei RNA-sequencing atlas comprising six human organs across 20 autopsies where we analyzed the transcriptional changes due to COVID-19 in multiple cell types. The integration of data from multiple organs enabled the identification of systemic transcriptional changes.Computational cross-organ analysis for endothelial cells and macrophages identified systemic transcriptional changes in these cell types in COVID-19 samples. In addition, analysis of gene modules showed enrichment of specific signaling pathways across multiple organs in COVID-19 autopsies.Altogether, the COVID Tissue Atlas enables the investigation of both cell type-specific and cross-organ transcriptional responses to COVID-19, providing insights into the molecular networks affected by the disease and highlighting novel potential targets for therapies and drug development.The Chan-Zuckerberg Initiative, The Chan-Zuckerberg Biohub. COVID-19 (coronavirus disease 2019) is the most devastating infectious disease in recent history. The pandemic has impacted all parts of the globe and resulted in nearly 500 million infections and over 6,000,000 deaths . ApproxiThe systemic effects of severe COVID-19 are largely mediated through the immune response to SARS-CoV-2 infection and subsequent inflammatory response. Viral infection stimulates macrophages to overproduce pro-inflammatory cytokines, including IL-6, leading to the ‘cytokine storm’ that results in systemic inflammatory response syndrome . This heWe have previously shown that multi-organ, single-cell transcriptome-based approaches can yield significant insights into organ biology and cross-organ signaling . In addiHere, we report a COVID-19 single-nuclei RNA-sequencing (snRNA-seq) atlas comprising six organs and approximately 86,000 cells. We showed that transcriptional changes in severe COVID-19 infections were not restricted to the lung, the most severely affected organ upon SARS-CoV-2 infection, but to multiple organs, such as the liver and heart. In addition, we found significant changes in the transcriptional profiles of multiple cell types and identified a subset of recurrent molecular pathways commonly up-regulated in multiple cell types across organs. The COVID Tissue Atlas (CTA) represents a comprehensive resource to investigate the transcriptional changes resulting from COVID-19 in different tissues. Moreover, the scope of the CTA dataset enabled us to identify systemic transcriptional signatures that we would have missed by focusing on an individual organ. We anticipate our analysis and the CTA to be of significant value for future research, including identifying molecular targets for drug development and therapeutic applications.Organs from post-mortem control individuals and patients with COVID-19 were obtained at the time of autopsy from the University of California, San Francisco Medical Center, and the Saarland University Hospital Institute for Neuropathology. In both institutions, autopsies are exempt from local Institutional Review Board and Committee on Human Research oversight. All autopsies performed have informed consent from legal next of kin for use of tissues for research purposes. 3 cubes, flash-frozen in dry ice, and then stored at –80°C for single-nuclei extraction and total RNA extraction.During the autopsy, tissue samples were stored in ice-cold Wisconsin solution for transportation, then immediately processed as follows: tissues were rinsed twice with ice-cold PBS, then wiped off. Next, tissues were pre-cut into 1–2 mmCOVID-19 testing was performed on patients according to the testing procedure of host hospitals. For sample testing, total RNA was extracted using a hybrid TRIzol (Life Technologies #15596026) and RNeasy Mini kit (QIAGEN #74104) protocol . RT-qPCRg for 5 min and resuspended in 2 ml of cold Storage Buffer (S2Genomics), then centrifuged again at 500 × g for 5 min, resuspended in 2 ml of Storage Buffer, and filtered through a 40 µm Flowmi Tip Strainer filter. After centrifugation, nuclei were resuspended in 50–500 µl of Storage Buffer supplemented with 1 U/µl of RNAse inhibitor  and counted using a LUNA-FL Dual Fluorescence Cell Counter .The protocol for nuclei isolation was performed in a BSL2+ biosafety cabinet for the lung and in a BSL2 biosafety cabinet for all other organs wearing personal protective equipment. We carried out all procedures on ice or at 4°C. Single nuclei were generated from around 50 mg of flash-frozen tissues using the Singulator machine , following the manufacturer’s recommendations. The extended protocol was used for the ileum and colon, and the regular protocol was used for all other organs. After isolation, nuclei preparations were cleaned as follows: nuclei were centrifuged at 500 × For droplet-based snRNA-seq, libraries were prepared using the Chromium Next GEM Single Cell 3ʹ v.3.1 according to the manufacturer’s protocol (10x Genomics), targeting 10,000 nuclei per sample after counting with a TC20 Automated Cell Counter (Bio-Rad). We performed 12 cycles for cDNA amplification for all of the samples. To generate the final dual or single indexed 10X libraries, 13 cycles were performed.After library preparation, individual libraries were quality checked on an Agilent 4200 Tapestation using D5000 screen tape. These libraries were pooled equal molar into a total of seven pools ranging from 4 to 15 nM final concentration and quality checked again on an Agilent 4200 Tapestation using a D5000 screen tape, followed by qPCR on a Bio-Rad CFX96 RT PCR thermal cycler using the KAPA library quantification kit (# KK4923).Individual pools of 10x 3’ gene expression libraries were sequenced on Illumina’s Nextseq 2000 P3, Novaseq S2 and/or NovaSeq S4 flow cells with a targeted sequencing read depth of 20,000 reads per cell. Sequencing parameters were as follows: (1) for dual indexed libraries: Read 1=28 cycles, Index 1=10 cycles, Index 2=10 cycles, Read 2=90 cycles; (2) for single indexed libraries: Read 1=28 cycles, Index 1=8 cycles, Index 2=0 cycles, Read 2=91 cycles.Sequences were de-multiplexed using bcl2fastq version 2.20.0.4.22. Reads were aligned to an extended Gencode Reference 30 (GRCh38) genome containing SARS-CoV2 genes (kindly provided by Aviv Regev and Carly Ziegler) using CellRanger version 5.0.1, available from 10x Genomics, with default parameters.The count matrices generated by CellRanger were pre-processed by removing contamination of ambient RNA. We noticed high levels of contamination in single-nuclei data, which has been reported before , and appn_latent = 64 and n_layers = 2. We allowed each gene to have its own variance parameter by setting dispersion="gene". We then used the UMAP algorithm to visualize the resulting embedding in two dimensions. All UMAPs for each organ shown in the manuscript were generated in the same way. The UMAPs generated using scVI’s latent space showed minimal batch effect and allowed for the identification of cell populations based on known markers for each organ. For each organ, we first verified that individual clusters expressed known gene markers for the expected cell types. Some clusters, however, co-expressed multiple mutually exclusive markers, an indication of ambient RNA contamination, so we labeled these cells as doublets. Clusters that either expressed gene markers for multiple cell types (doublets) or did not express any markers for the cell types expected in the organ (unidentifiable cells) were systematically removed from the dataset. Finally, for each organ, we generated h5ad files with the cell-type annotations and the harmonized UMAP.For each organ, we first integrated the samples from different donors into a harmonized UMAP embedding using scVI  release We used the batch-corrected UMAPs for cell-type annotation. In brief, tissue experts at either UCSF or Stanford (from research labs focused on specific human tissues) analyzed the expression of cell type-specific markers and assigned identities to the clusters. Confident annotations for some clusters, however, were not possible due to high levels of RNA contamination or low expression of marker genes. We therefore only considered clusters for which a cell-type identity was clearly defined. The second round of quality control was applied based on feedback from tissue experts and their annotations. We increased the cut-off values for mitochondrial genes and filtered out putative doublets . After the second round of review with the tissue experts, we finalized the cell-type annotations for all organs and used them for all downstream analyses. We use the cell-type label annotations as ground truth for differential expression (DE) analysis, pathway enrichment, and ligand-receptor enrichment analysis .To annotate cell types in the kidney, we integrated our COVID-19 samples with a single-nuclei atlas of the kidney . We applpheatmap v1.0.12 with the clustering algorithm set to ward.D2.For the sub-clustering and analysis of lung epithelial cells, we independently integrated the CTA lung samples with the COVID-19 lung atlas published by the Broad Institute  and withzlm method as implemented by the MAST R package v1.20  and adjusted p-value <0.001.To identify gene sets enriched in COVID-19 donors, we selected the top DE genes for each cell type  and used them as input for pathfindR , a gene We used four different pathway databases as references for our analysis to be comprehensive, KEGG, Reactome, GO, and BioCarta. We then manually curated the enriched pathways, discussed them with tissue experts, and cross-validated them with existing literature to identify the signatures enriched in COVID-19 donors for each cell type and organ. We only considered enriched pathways with a p-value <0.001.abs(1)) in at least two-thirds of the organs. Some cell types appear in all organs, whereas some only appear in two or three. We, therefore, applied the coordination analysis only for cell types that appear in at least three organs .To identify transcriptional coordination in COVID-19 samples, we developed a custom analysis method to quantify shared responses across organs. First, we examined the set of genes that appear DE . For genes with the same sign in both organs, we assigned a value of 1; genes with opposing signs were assigned a value of 0. The coordination score for the pair of organs was then defined as the average value across shared DE genes . Thus, a coordination score of 1 indicates that all shared DE genes are jointly up- or down-regulated . In contrast, a score of 0 indicates that they are oppositely up- or down-regulated . For each cell type and for each pair of organs, we thus computed the coordination score.As a negative control, we repeated this analysis with a computationally shuffled dataset. Here, for each pair of organs for a particular cell type, we held the log2-FC values per gene in one organ fixed and randomly shuffled the log2-FC values per gene in the second organ. We reasoned that this shuffled dataset should possess near-zero coordination , with some small random deviation due to the finite size of the shared gene list. We generated N=1000 computationally shuffled datasets for each pair of organs and calculated the resulting coordination scores for each instance, producing a distribution of coordination scores as a negative control. We then averaged the results and retained the mean and standard error, to be compared with the coordination scores from the actual data. We then used the shared responses as input for pathway enrichment (see above), considering only the cell types that showed significant coordination compared to the random control .We applied CellPhoneDB  and idenWe collected data from 20 different autopsies  with an age range between 40 and 89 years (median age = 68 years), of which 15 tested positive for COVID-19 . The aveWe applied dimensionality reduction (PCA) and Leiden clustering for each organ while correcting batch effects across donors using scVI . FinallyMeasurements of SARS-CoV-2 mRNA by RT-qPCR showed high to moderate expression in the lung samples from COVID-19 donors . While sThe CTA lung dataset comprised 21,474 cells, of which 11,790 were collected from COVID-19 autopsies. After quality control and clustering (Methods), we identified 10 distinct cell types, including primary epithelial and immune cells . SeveralWe next focused on the effects that COVID-19 has on the different lung cell populations. In particular, significant epithelial cell damage resulting from COVID-19 is manifested as loss of alveolar type 1 (AT1) and alveolar type 2 (AT2) cells . To inveCOVID-19 is known to cause severe alveolar damage and loss of epithelial cells. During alveolar injury, AT2 cells are known to serve as progenitors to generate new epithelial cells. More recently, an additional cellular pathway has been identified where AT2-derived basaloid cells generate AT1 cells, termed alveolar basal intermediates. These cells were first identified in single-cell studies as KRT17+/KRT5- basaloid cells in human idiopathic pulmonary fibrosis (IPF) lungs  and indeInterestingly, while most of the transitional cells from Alternatively, the Broad atlas identified a pre-AT1 transitional cell state (PATS) population in COVID-19 lungs  that beaAcross all six cell types identified in the liver , hepatocTo identify DE genes for each cell type in the liver, we applied a negative-binomial model implemented in MAST  that corCoronavirus disease - COVID-19’ in the KEGG database), including TMPRSS2, EGFR, PLCG2, MAPK14, FOS, JUN, IFNAR1, C5AR1, CFB, C8G, MASP1, FGA, FGB, FGG in addition to multiple ribosomal-related transcripts, p<1e-6  indirect injury due to increased cytokines and immune inflammatory response, (2) direct invasion of cardiomyocytes by SARS-CoV-2, and (3) respiratory damage from the virus causing hypoxia leading to oxidative stress and injury to cardiomyocytes . To undeHeart samples yielded 20,091 cells after quality control (n=11 donors) . Across Coronavirus disease - COVID19 pathway in fibroblasts and macrophages, p<1e-5; Reactome: Influenza infection enriched in fibroblasts, p<1e-5; We then found significant transcriptional changes in cardiomyocytes, endothelial cells, and macrophages based on the number of DE genes in COVID-19 samples . ConsideSimilar to the liver, we observed insulin pathway enrichment in cardiomyocytes from COVID-19 samples . Heart fInterestingly, Notch signaling has been proposed as a target to prevent SARS-CoV-2 infection and interfere with the progression of COVID-19 associated heart and lung disease . Hippo sThe CTA provides a unique opportunity to identify systemic transcriptional responses across organs. As an indication of a systemic response to COVID-19, we found enrichment of the same signaling pathways in multiple cell types and across organs, including HIF-1, insulin, and Notch signaling  and 4D. In macrophages, we found a significant overlap in DE genes across organs compared to random sampling expectations . SpecifiTo further analyze these data, we defined the shared transcriptional response (STR) for a cell type as the set of genes that show DE in at least three organs from COVID-19 donors (p<1e-4 and log2-FC >1). We restricted the analysis to macrophages, endothelial cells, and stromal cells, which appear in multiple organs, and calculated the correlation between the log-FC values of all genes in the STR across pairs of organs . GeneralTo identify the cell types with high coordination in their STR across organs, we defined a coordination score by considering pairs of organs and the fraction of genes in the STR that showed the same direction in DE  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Aleksandra Walczak as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Single nuclei characterization of pervasive transcriptional signatures across organs in response to COVID19" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:Please provide more evidence to support the presence of ABI cells in the data.Please clarify the presence and potential role of neuronal cells in the datasets studied, and their fate during infection.Please indicate whether the increase of endothelial cells is related to blood vessel formation, e.g. through a cell subtype analysis. A higher resolution study of these subtypes and their potential role will add value to this resource.Please describe other variables that might be associated with changes in cell proportions and gene expression, like age and comorbidities.Please clarify the potential biases and limitations of this study, and how it compares with other previous similar studies, emphasizing the novel insights and/or complementarity of this study.Please clarify the nature of the coordinated response put forward in the manuscript. In particular, whether this is consistent across cell types, or specific to some of them, or not. Any orthogonal validation that could be used to confirm these claims will add further support to the principles put forward in the manuscriptReviewer #1 (Recommendations for the authors):The authors performed scRNA-seq of six human organs across 20 autopsies. The data quality and batch effects were well controlled in the analysis. Based on this, the authors identified specific and common changes in each organ after infection with SARS-CoV2. Specifically, the authors observed dysregulation in insulin and HIF signal pathways in the heart and lung. In addition to lung, the authors found significant transcriptional changes in macrophages and endothelial cells in heart and liver. This study provides a valuable data resource to study the systemic effects of severe COVID-19. However, it needs to address the following issues:1. The anchor integration alone was not sufficient to indicate the presence of ABIs cells in the data, did the authors observe the expression of ABIs cells markers in the cells they predicted as ABIs in their data?2. Considering that the nervous system may be affected by COVID-19 autopsies, did the authors detect neuronal cells in different organs? What happens to them after infection with SARS-CoV2?3. The analysis showed a much higher proportion of endothelial cells in the lungs and heart in COVID-19 than in control, were increased blood vessels observed in these organs? Do these extra vessels function normally? If yes, it would be interesting to explore why these organs need so many endothelial cells.4. Whether alterations in cell proportions and gene expression in COVID-19 are associated with age or some underlying diseases？Reviewer #2 (Recommendations for the authors):Expansion of hepatocytes in the liver is very interesting. Hepatocytes play a significant role in both glucose and lipid metabolism. These 2 pathways are tightly regulated by insulin signalling. Since the authors are reporting dysregulation of insulin signalling with potential hepatocytes regeneration caused by COVID-19, this could be further discussed. For example, did changes of insulin signalling lead to dysregulation of lipid metabolism?Differential abundance analysis (e.g. Milo) is a better alternative than plotting % of cell types in health vs COVID-19.The term COVID-19 has been inconsistently labelled throughout the paper, COVID-19, COVID, Cov19. It'd be great to have the term used consistently throughout the paper to avoid confusion. Essential revisions:Please provide more evidence to support the presence of ABI cells in the data.We thank the reviewer for the advice and agree that it is essential to provide more evidence of ABI cells’ existence in our dataset. Therefore, we computed the top differentially expressed genes of the ABI  cells from the Habermann et al. dataset and derived an ABI signature gene set. Next, we investigated this signature in the CTA and integrated datasets . Even though no subcluster showed enrichment for the ABI signature, a number of the ABI markers, in particular, those genes expressed in the KRT17+/KrRT5- cells, appeared in our basal population , suggesting that the ABI population  was present in the COVID-19 lung. We speculate that trans-differentiation from endogenous AT2s could account for the gain of abnormal basal cells in the alveoli. We revised the manuscript accordingly to reflect this finding. We included a new heatmap to show the expression of the ABI gene signature within the lung epithelial cells from the CTA .ABIs/Transitional AT2s/aberrant basaloid cells from IPF lungs , we did not detect any specific cell cluster in the CTA dataset enriched for the PATS signature. This result suggests that the PATS population is likely patient-specific ( or specific to sequencing technology differences) and, therefore, was not detected in the CTA donors. Accordingly, in the revised manuscript, we modified our PATS analysis description.Similarly, we derived a signature gene set of the pre-alveolar Type 1 transitional cell state (PATS) population in COVID-19 lungs from the Broad atlas. However, even though these PATS cells bear similarities to the previously described Please clarify the presence and potential role of neuronal cells in the datasets studied, and their fate during infection.We identified a small subset of cells in the hearts of COVID-19 donors expressing high neuronal markers levels. Among the most enriched gene markers in this cluster compared to other cell types in the heart, we found the neuron-specific genes NRX1, NRX3, and XKR4, along with cancer-related genes such as ANK3  and BCL2 . We also found CDH19 among this cluster's top markers, typically in the brain and heart muscle. Therefore, the cluster comprises a strong signature of neuron-like cells expressing heart-related genes. While interesting, it is challenging to confidently assign an identity to this cluster due to a concern regarding RNA contamination.During our decontamination pipeline, we noticed that some of the cells expressing neuronal markers also expressed markers exclusively expressed in other cell types. We were able to fix some of this contamination. Still, we suspected that the expression of neuronal markers correlated with RNA contamination. Therefore, we decided to err on caution and not include the cluster in our downstream analysis. Finally, the Broad atlas also identified a small neuronal population in the heart of COVID-19 autopsies , suggesting that the cluster might represent a real population. However, further investigation and deeper sequencing might be necessary to assess the existence and relevance of such a neuron-like population.Please indicate whether the increase of endothelial cells is related to blood vessel formation, e.g. through a cell subtype analysis. A higher resolution study of these subtypes and their potential role will add value to this resource.To address this comment, we separately applied a sub-clustering of the endothelial cell populations from the lung and the heart . After unbiased clustering, we used a list of gene markers from recently published human lung and heart endothelial cell single-cell atlases to compare with the expression profiles of different endothelial cell clusters in our data. We identified four sub-clusters of endothelial cells in the lung and 4 in the heart. We found that the overall increase in endothelial cells in both organs is driven primarily by an increased proportion of capillary endothelial cells in COVID-19 samples compared to the controls . Consistently, a gene ontology analysis from capillary endothelial cells in the lungs showed angiogenesis significantly upregulated. Therefore, our data indicate that capillary endothelial cells are in COVID-19 infection. We attempted a similar analysis on our liver endothelial cell data, but due to the much smaller cell numbers encountered significant patient-specific batch effects that precluded further analysis (data not shown).We included two supplemental figures to investigate the changes in endothelial populations. Our results are preliminary but suggest a pivotal role of capillary endothelial cells.Please describe other variables that might be associated with changes in cell proportions and gene expression, like age and comorbidities.We appreciate the comment and are interested in exploring the potential correlations between transcriptional changes and clinical variables . We attempted regression analysis using factors including age, race, disease severity (ICU status), and infection length but couldn’t find a strong signal for correlations with gene expression changes. Unfortunately, we don’t have enough donors for the statistical analysis to be reliable. Notably, previous studies also failed to identify correlations with clinical metadata  due to similar issues of low sample size. While we believe those correlations exist and are crucial to understanding the disease, the current limitation is the number of donors in the dataset. By compiling data across multiple datasets, we anticipate that future studies will be able to explore these questions in more detail.Please clarify the potential biases and limitations of this study, and how it compares with other previous similar studies, emphasizing the novel insights and/or complementarity of this study.The main novelty of this study is the collection of multiple organs from the same donors and the cross-organ analysis of transcriptional responses. This feature allowed us to identify a coordinated transcriptional response in macrophages and endothelial cells by analyzing the transcriptional changes in multiple organs, which is often overlooked in other studies. We further explored these changes by identifying potential molecular interactions between these two cell types.In line with the CZ Biohub commitment to open science, our data is available for exploration through a web portal and can be easily accessed by the community.The main limitations of our study include the number of high-quality cells in the analysis, which is lower than in other studies. We sequenced up to 1 million nuclei for this project, but less than 10% passed our quality control. In addition, we believe a significant cause is the longer time between patient death and organ harvest for our samples, with an average time of 63 hours after death, with a few specimens collected more than 96 hours after death. Since our atlas is composed of single-nuclei data, another limitation is the potential transcriptional effects that we cannot observe due to technological limitations. However, our dataset should be complementary to the current body of knowledge regarding COVID-19, and we expect that future analyses, including multiple datasets, will overcome this limitation.A general bias we are aware of is the lack of race and sex diversity in our donors: 17 males vs. three females, and ethnicities Hispanic (n=5), African American (n=2), Asian (n=1), and White (n=12). Therefore, our results and conclusions are limited to the context of our donor population.We extended the discussion in the main text to include some of the novelties and limitations of the study.Please clarify the nature of the coordinated response put forward in the manuscript. In particular, whether this is consistent across cell types, or specific to some of them, or not. Any orthogonal validation that could be used to confirm these claims will add further support to the principles put forward in the manuscriptThe coordinated response is strong with only two cell types -> say that we tried.Orthogonal validation of the coordinated response would strengthen the validity of our results. We, therefore, applied our analysis to the COVID-19 dataset from the Broad Institute . The Broad’s single-nuclei atlas comprises samples from hearts, livers, and lungs from COVID-19-infected autopsies.Our main finding in the CTA is the coordinated transcriptional response in macrophages and endothelial cells across multiple organs, particularly in the heart, liver, and lung. Therefore, the Broad atlas provides an ideal opportunity for independent validation of our results. Here is a summary of the main results:By applying the same analysis to the Broad atlas, we identified a coordinated response in macrophages and endothelial cells across the heart, liver, and lung of COVID-19 autopsies.The list of genes showing a coordinated response in the Broad atlas showed high overlap with the coordinated response found in the CTA .Further, we found a strong correlation between the log-fold changes in gene expression for the overlapping set of genes. And we included scatterplots showing these correlations in Figure 5.We explain in detail the analysis in the text and emphasize the importance of this external validation.The additional panels and text strongly support our main finding of a coordinated transcriptional response in macrophages and endothelial cells across multiple organs in response to COVID-19.Reviewer #1 (Recommendations for the authors):The authors performed scRNA-seq of six human organs across 20 autopsies. The data quality and batch effects were well controlled in the analysis. Based on this, the authors identified specific and common changes in each organ after infection with SARS-CoV2. Specifically, the authors observed dysregulation in insulin and HIF signal pathways in the heart and lung. In addition to lung, the authors found significant transcriptional changes in macrophages and endothelial cells in heart and liver. This study provides a valuable data resource to study the systemic effects of severe COVID-19. However, it needs to address the following issues:1. The anchor integration alone was not sufficient to indicate the presence of ABIs cells in the data, did the authors observe the expression of ABIs cells markers in the cells they predicted as ABIs in their data?See the reply to Essential revisions comment #1 above.2. Considering that the nervous system may be affected by COVID-19 autopsies, did the authors detect neuronal cells in different organs? What happens to them after infection with SARS-CoV2?See the reply to Essential revisions comment #2 above.3. The analysis showed a much higher proportion of endothelial cells in the lungs and heart in COVID-19 than in control, were increased blood vessels observed in these organs? Do these extra vessels function normally? If yes, it would be interesting to explore why these organs need so many endothelial cells.See the reply to Essential revisions comment #3 above.4. Whether alterations in cell proportions and gene expression in COVID-19 are associated with age or some underlying diseases？See the reply to Essential revisions comment #4 above.Reviewer #2 (Recommendations for the authors):Expansion of hepatocytes in the liver is very interesting. Hepatocytes play a significant role in both glucose and lipid metabolism. These 2 pathways are tightly regulated by insulin signalling. Since the authors are reporting dysregulation of insulin signalling with potential hepatocytes regeneration caused by COVID-19, this could be further discussed. For example, did changes of insulin signalling lead to dysregulation of lipid metabolism?Differential abundance analysis (e.g. Milo) is a better alternative than plotting % of cell types in health vs COVID-19.Thanks, we appreciate the comment, although our limited number of samples prevents us from understanding the changes quantitatively. Integration with other studies in the future will facilitate the investigation of changes in cell populations.The term COVID-19 has been inconsistently labelled throughout the paper, COVID-19, COVID, Cov19. It'd be great to have the term used consistently throughout the paper to avoid confusion.Thanks. We reviewed the manuscript and edited it where needed.


Wastewater-based epidemiology (WBE) has exhibited great utility in the early and rapid identification of SARS-CoV-2. However, the efficacy of wastewater surveillance under China's previous strict epidemic prevention policy remains to be described. We collected the WBE data of wastewater treatment plants (WWTPs) in the Third People's Hospital of Shenzhen and several communities to determine the significant effectiveness of routine wastewater surveillance in monitoring the local spread of SARS-CoV-2 under tight containment of the epidemic. The results of 1 month of continuous wastewater surveillance showed that positive signals for SARS-CoV-2 RNA were detected in the wastewater samples, and a significant positive correlation was observed between the virus concentration and the number of daily cases. In addition, the community's domestic wastewater surveillance results were confirmed even 3 days before, or simultaneously with, the infected patient being confirmed as having the virus. Meanwhile, an automated sewage virus detection robot, ShenNong No.1 robot, was developed, showing a high degree of agreement with experimental data, offering the possibility of large-scale multi-point surveillance. Overall, our results illustrated the clear indicative role of wastewater surveillance in combating COVID-19 and provided a practical basis for rapidly expanding the feasibility and value of routine wastewater surveillance for future emerging infectious diseases. In the post-epidemic era, an automated wastewater surveillance robot exhibits clear effectiveness and value for pathogenic biomonitoring, paving the way for routine wastewater monitoring to prevent future emerging infectious diseases. The coronavirus disease 2019 (COVID-19) pandemic, caused by a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still spreading globally and causing high levels of patient mortality, especially with the emergence of multiple SARS-CoV-2 variants [et al. performed viral tests on environmental fomites from patients with COVID-19, and they found both symptomatic and asymptomatic patients with COVID-19 can potentially spread SARS-CoV-2 environmentally [et al. found that SARS-CoV-2 can still be shed in excrement for a period of time, even after the COVID-19 patients have recovered from the respiratory symptoms [Accumulating evidence suggests that SARS-CoV-2 is mainly transmitted through respiratory droplets and contacted fomites, and it can enter sewage through excrement, leading to the potential risk of fecal-oral transmission . It has mentally . Furthersymptoms . Meanwhisymptoms . The sursymptoms . A recensymptoms . In addisymptoms . Actuallsymptoms  and norosymptoms ,17, so aIn this contribution, we investigated the WBE data collected from the wastewater treatment plant (WWTP) in The Third People's Hospital of Shenzhen and the corresponding emergency hospital area (where COVID-19 patients were quarantined)  and B , 3 times a day . It is worth noting that the wastewater samples were absent from September 3 to September 5 due to the closure and administration stages of the COVID-19 epidemic at that time. The numbers of daily clinical confirmed cases and new cases in the hospital were 117 and 8, respectively, at the beginning of the present study , and all confirmed and new cases were quarantined at the emergency quarantine area.For 1-month detection, the higher concentrations of SARS-CoV-2 RNA were obviously more observable in wastewater samples in area A than in area B Fig. , though The hospital accommodates approximately 5000 flow population  and 1000 inpatients per day. We assumed that one-tenth of the flow population defecated in the hospital toilet, and then assessed the daily flow number of defecations in the whole hospital. The distribution changes of SARS-CoV-2 RNA concentration in wastewater with daily confirmed cases and flow numbers in areas A and B from August 10, 2022 to September 9, 2022 are shown in Fig. To further investigate the association between wastewater SARS-CoV-2 RNA concentration and clinically reported cases, in addition to considering the normal distribution of the data, Pearson's correlation analysis was employed to assess the correlation. The observed concentrations of SARS-CoV-2 RNA in wastewater samples from area A were positively correlated with the presence of clinically reported cases Fig. . And theThrough investigating the association between sewage virus concentrations and clinically confirmed cases in area B, we found that the wastewater SARS-CoV-2 RNA concentrations were also positively correlated with the number of clinically confirmed cases  . Taking The actual SARS-Cov-2 loaded in domestic sewage is much lower than that in the Third People's Hospital of Shenzhen, especially under the previous strict epidemic prevention control in the city. Residents in Shenzhen are required to conduct throat swab testing at least every 2 days, and transfer immediately once being confirmed infected, while residents who are in close contact with them but do not test positive remain in the residence for isolation and observation. In this work, continuous sewage virus monitoring was carried out in the medium- and high-risk areas where an infection has occurred, and it was found that the positive status of sewage virus was almost synchronous with or up to 3 days delay to that of throat swabs Fig. . For theAn automated solution for wastewater SARS-CoV-2 detection is highly demanded due to the complexity and long time consumption of manual laboratory work. A detection robot named ShenNong No.1  was then used for fast detection  and, dueThe ongoing COVID-19 pandemic caused by SARS-CoV-2 was considered to be transmitted from person to person via droplet infections and fecal-oral transmission ,20,21. DHerein, 1-month wastewater surveillance for SARS-CoV-2 demonstrated a strong correlation between virus concentration in wastewater and the presence of confirmed cases. Furthermore, the strong correlation between virus concentrations upstream and downstream of the wastewater station also indicated that the virus spread over time. An additional valuable point is that since there is no significant regularity in the patient's detoxification time, community sewage monitoring should, as much as possible, have a certain sampling frequency to cover the whole day, otherwise the feces of some patients may be missed.The pilot project in Longhua District, Shenzhen, shows that the sensitivity of sewage monitoring is similar to that of throat swab detection. This provides a new perspective that through reasonable complementation and adjustment, such as the way adopted by the Hong Kong government, to conduct mandatory throat swab detection in the upstream area after sewage detection is positive, can minimize government costs while ensuring that the sources of infection can be found. Other virtues such as reduction of medical waste disposal caused by numerous throat swab tests, alleviation of residents queuing for the nucleic acid test, or even early warning by its nature of passive detection in some cases, makes it much more advanced, reliable, and recommendable.The automatic solution opens a new avenue for fast and online detection of SARS-COV-2 in wastewater, along with its high consistency and convenience. Virus signals can be easily missed due to inadvertent error in manual experimental operation, because the virus content in domestic sewage is extremely low. Fine and sophisticated automation operations can help to avoid deviation and improve the accuracy of the overall system.In summary, our 1-month wastewater surveillance study showed a significant linear correlation between wastewater virus concentrations and the presence of clinically reported cases, suggesting that even under strict government epidemic prevention and management, wastewater data continue to exert significant preventive effectiveness and affordability in combating COVID-19 or future infectious disease outbreaks.The whole procedure follows the national health industry standard WS/T 799–2022, method for the enrichment and nucleic acid detection of SARS-CoV-2 in sewage, including procedures of sample collection, sample enrichment, nucleic acid extraction, and viral detection. All the experiments were carried out in the P2 biosafety laboratory. Detailed methodological data can be found in the online supplementary files.The instantaneous sampling method was adopted in this work. In the Third People's Hospital of Shenzhen, wastewater from two spots, one is the influent of  WWTP in the emergency quarantine area and the other is the main WWTP of the whole hospital Fig. , were coThree methods for wastewater enrichment are recommended by the standard WS/T 799–2022 and the aluminum hydroxide adsorption–precipitation method was used in this work due to its simplicity and lower cost. Briefly, 30 min centrifugation of 50 mL water sample was first applied to remove suspended solids  and then 0.5 mL of AlCl3 solution (0.3 mol/L) was added to the supernatant fluid with pH adjusted to 6.0 to allow aluminum hydroxide colloid emerging and adsorbing the virus. After that the pellet containing virus particles was separated by 5 min centrifugation  and 0.2 g EDTA-2Na was then mixed with the pellet to get higher releasing efficiency and to ensure the stability of the RNA .The Nucleic Acid Extraction Kit  was used for viral extraction following the manufacturer's protocol.The 2019-nCoV Nucleic Acid Detection Kit  was used for viral detection and quantification following the manufacturers instructions, with N and Orf1ab target primers. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was used for the detection of viruses. The quantitative pseudovirus used for standard curve plotting and the positive control sample used for reference of enrichment, extraction, and PCR detection were all purchased from BDS company in this work, and the negative control samples were nuclease-free water.r) is defined as the ratio of the RNA concentration recovered in the concentrated solution after enrichment (Cenri) to the RNA concentration in the initial sewage sample before enrichment (Cinti), as shown in equation RNA) is determined by the Ct value of RT-PCR standard curve of quantitative pseudovirus (equation The recovery rate (Requation .The ShenNong No.1 robot contains several units, including automatic sewage sampling, automatic enrichment and concentration, automatic nucleic acid extraction, and automatic PCR detection. Samples were manually placed on the pallet then the whole process was accomplished automatically by the robot.p < 0.05 was considered for all tests.Statistical analyses were performed in Excel, SPSS 25.0 and R v 4.0.2. A statistical significance level of nwad089_Supplemental_FilesClick here for additional data file.


The analytical sensitivity of the NAAT-RDT and other laboratory NAATs was comparable. During clinical evaluation, the overall sensitivity and specificity were 92.9% and 98.3%, respectively, with little variation between settings. The Lucira NAAT-RDT is a portable and self-contained device that provides an easily interpreted result within 30 minutes following a bilateral nasal swab collection. Its performance was shown to be acceptable for use in three settings in this quality improvement project, facilitating patient flow and management.In hospitals during the COVID-19 pandemic, laboratory testing was important to reduce SARS-CoV-2 transmissions, particularly for high-risk settings like the emergency department and pre-operative settings and for the safe return to work of exposed healthcare workers (HCWs). For these applications, delayed test results from laboratory nucleic acid amplification tests (NAATs) posed a barrier to maximizing efficient patient flow and minimizing staffing shortages. This quality improvement project sought to evaluate the performance of the Lucira Check-It COVID-19 Test, a rapid diagnostic test that used NAAT technology (NAAT-RDT). Using 10-fold serial dilutions of SARS-CoV-2, the analytical sensitivity of the NAAT-RDT was assessed against standard NAATs used for routine diagnostic testing. Clinical performance was assessed at two Nova Scotia hospitals in 405 cases with paired swabs tested by NAAT-RDT and laboratory-based NAATs. These represented three distinct populations: patients presenting to the emergency department  posed a barrier to maximizing efficient patient flow and minimizing staffing shortages. This quality improvement project sought to evaluate the analytical and clinical performance of the Lucira Check-It COVID-19 Test, a point-of-care test that used NAAT technology, in the perioperative setting, emergency department, and community testing sites. We found the Lucira Check-It to have comparable performance to laboratory NAATs. It can be employed with little training for specimen collection, processing, and interpretation, and at a cost justifiable from the resources saved from avoiding sample transport and laboratory testing. The COVID-19 pandemic disrupted the organization of patient care delivery in healthcare settings . In hospThis quality improvement project sought to determine the analytical and clinical performance of the Lucira NAAT-RDT in the pre-operative setting, ED, and community testing sites.n = 5) 10-fold serial dilutions in universal transport media  of a SARS-CoV-2-positive oropharyngeal/bilateral nares swab  with 95% confidence intervals (CIs).The clinical performance of the NAAT-RDT assay was determined among three distinct populations. First, asymptomatic patients in the pre-operative setting with no known exposures to SARS-CoV-2 were tested on the day of the planned surgery. Second, ED patients whether asymptomatic or symptomatic were tested at the discretion of the attending team when an immediate result contributed to patient disposition and patient flow. Finally, testing was made at community testing sites for asymptomatic close contacts of COVID-19 cases at least 72 h prior and for return to work in a healthcare setting. Healthcare workers with close contact with positive SARS-CoV-2 cases were instructed to stay home for 7 days following exposure. Asymptomatic testing using the NAAT-RDT at 72 h allowed these individuals to return to work if test results were negative, reducing concerns of subsequent transmission and helping maintain hospital staffing. As a quality project with retrospective data review, the collection of paired specimens was not consistent between the NAAT-RDT and clinical laboratory-based NAATs. Comparator NAATs included one of three standard kits  –15. OnThe analytical sensitivity of the Lucira NAAT-RDT was nearly identical to another isothermal NAAT-RDT  and a LDT, and these three were approximately one 10-fold dilution less sensitive than standard NAATs  , which wDuring COVID-19 outbreaks, preserving efficient patient flow and adequate hospital staffing is reliant on streamlined SARS-CoV-2 testing. The time to results for a SARS-CoV-2-positive specimen with a high viral load can be as little as 10 minutes, with negative or positive results from specimens with low viral loads taking up to 30 minutes. Given the transport time required for specimens to clinical laboratories for standard NAAT testing, rapid testing even at 30 minutes is a significant improvement. Quick, reliable, POC testing using the Lucira NAAT-RDT is one way to support patient flow and hospital staffing while facilitating effective infection prevention and control measures and reducing testing demand in clinical laboratories. This NAAT-RDT offered all these advantages, with sensitivity comparable to LDTs used in clinical laboratories . While tA single other study by Zahavi et al.  looked aOn the other hand, the other two false negative NAAT-RDT results had paired specimens with low Ct values of 18.4 and 18.9 when tested on a standard NAAT, suggesting the specimens had high viral loads seen during the infectious period of SARS-CoV-2. There are multiple possibilities that could explain these discrepant results. To ensure the NAAT-RDT was able to detect specimens with high viral loads, five specimens with Xpert Ct values spanning 16.5–17.5 were tested and were all detected within 10 minutes (data not shown). This excludes the possibility of template inhibition for the NAAT-RDT at high viral loads. Second, contamination could be possible in the clinical laboratory with the NAAT used as the comparator  leading to a false positive laboratory NAAT reference value. Given that the individuals with discrepant results between the NAAT-RDT and the standard laboratory NAAT were from different days a month apart, along with the robust quality control practices in clinical laboratories, contamination at high SARS-CoV-2 viral loads during the NAAT comparator testing is extremely unlikely. Another consideration is the timing between NAAT-RDT and standard NAAT testing. In this quality initiative, NAAT-RDT and standard NAAT results were no more than 24 h apart, but the test order was not documented. It is possible that both patients with discrepant results were in an early acute stage of illness detectable in the laboratory by a standard NAAT, but SARS-CoV-2 was not detected with the NAAT-RDT 24 h prior as the viral load was below the level of detection during this period . It is aThis quality initiative is not without limitations. Specimen numbers were low in the community setting, and no positives were obtained in the pre-operative setting. Additional comparative studies could be useful to strengthen the data for these settings. Next, the status of individuals as symptomatic or asymptomatic in the emergency department was also not available. Another main limitation of this quality initiative is the absence of data available to assess the cost–benefits and impact of NAAT-RDT implementation in the testing settings. Cost analyses were not possible given that NAAT-RDT kits and NAAT reagents and consumables for clinical testing were provided by provincial and federal initiatives to support SARS-CoV-2 testing. As for the assessment of impact, data from other studies using another NAAT-RDT show that rapid results facilitate clinical decision-making, improve the use of recommended treatments for COVID-19, and streamline patient flow . In the Overall, this quality initiative demonstrated that the Lucira NAAT-RDT offers a feasible option for rapid and portable SARS-CoV-2 testing, with comparable performance to laboratory NAATs. While this NAAT-RDT should be validated for each local application, it can be employed with little training for specimen collection, processing, and interpretation, and likely at a cost justifiable from the resources saved from avoiding sample transport and laboratory testing.


Background/aim: This study aimed to distinguish different phenotypes of long COVID through the post-COVID syndrome (PCS) score based on long-term persistent symptoms following COVID-19 and evaluate whether these symptoms affect general health and work ability. In addition, the study identified predictors for severe long COVID. Method: This cluster analysis included cross-sectional data from three cohorts of patients after COVID-19: non-hospitalized (n = 401), hospitalized (n = 98) and those enrolled at the post-COVID outpatient’s clinic (n = 85). All the subjects responded to the survey on persistent long-term symptoms and sociodemographic and clinical factors. K-Means cluster analysis and ordinal logistic regression were used to create PCS scores that were used to distinguish patients’ phenotypes. Results: 506 patients with complete data on persistent symptoms were divided into three distinct phenotypes: none/mild (59%), moderate (22%) and severe (19%). The patients with severe phenotype, with the predominating symptoms were fatigue, cognitive impairment and depression, had the most reduced general health status and work ability. Smoking, snuff, body mass index (BMI), diabetes, chronic pain and symptom severity at COVID-19 onset were factors predicting severe phenotype. Conclusion: This study suggested three phenotypes of long COVID, where the most severe was associated with the highest impact on general health status and working ability. This knowledge on long COVID phenotypes could be used by clinicians to support their medical decisions regarding prioritizing and more detailed follow-up of some patient groups. A proportion of patients infected with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) experience long-term persistent symptoms following coronavirus disease 2019 (COVID-19). These symptoms are referred to as long COVID . Long COSo far, there are no effective treatments for long COVID. Furthermore, the diagnostic criteria and clinical spectrum of this condition are not fully understood. Early identification of long COVID patients with poorer recovery and higher symptom load could lead to more individual symptomatic rehabilitation for these with poor outcomes . ExistinThis study is part of the longitudinal project called “COMBAT post COVID” that aimed to investigate long-term health after COVID-19 and its consequences ,18. The The studies were approved by the Swedish Ethical Review Authority  and conducted in accordance with the Helsinki Declaration. All the included study subjects gave informal consent to use their survey answers. The founding sources had no role in study design, data collection and data analysis, data interpretation or writing of the report.The cohort called “Non-hospitalized COVID” included subjects who underwent non-hospitalized/mild COVID-19. The enrolled subjects were symptomatic at onset and had a positive polymerase chain reaction (PCR) for SARS-CoV-2 in nasopharyngeal swabs between March and December 2020. The study population included mainly employees of Region Uppsala in particularly health care and resident care workers, since those groups were prioritized for early testing in the pandemic. Twelve months after infection, the patients answered a survey on their symptoms at onset and comorbidities. The survey was sent to 725 subjects 45–51 weeks after the COVID-19 diagnosis, by e-mail ,18.The cohort called “Hospitalized COVID” included subjects hospitalized for COVID-19 at the Department of Infectious Diseases at Uppsala University Hospital. All the subjects had a positive PCR for SARS-CoV-2 and were registered in the local register of patients hospitalized due to the COVID-19 infection. The survey was done 12 months after infection. The cohort was collected at two different time points. Between April and June 2020, 99 individuals received a survey to their home address and 46 (46%) responded. Between April and May 2021, of 140 subjects hospitalized, 52 subjects (37%) participated in a COVID-19 follow-up visit at the Department of Respiratory, Allergy and Sleep Research, Uppsala University Hospital. These patients responded to the survey in addition to a physical evaluation.The cohort called “post-COVID” included subjects enrolled to the post-COVID outpatient’s clinic in Uppsala. These patients were referred from primary care centers in Region Uppsala with remaining symptoms after at least 12 weeks of a microbiologically verified or probable COVID-19 infection  . All theThe study subjects answered question  on wheth2. The survey included questions regarding sociodemographic factors including education level, divided into university education (at least 3 years) or vocational collage  or upper secondary school; marital status ; country of birth included Sweden and countries other than Sweden; work status including current work, studies, parental leave, sick leave, unemployment or retirement; smoking status; use of snuff; and weight and height that were used to calculate the body mass index (BMI) kg/mThe participants were asked about the self-reported severity of symptoms at onset including very mild/mild, moderate, severe or very severe; as well as other disease diagnosed by a doctor including hypertension, heart disease , hypo-/hyperthyroidism, diabetes mellitus type 2, lung disease (including asthma and chronic obstructive pulmonary disease), conditions requiring immunosuppressive treatment, cancer, depression/anxiety and chronic pain. These diagnoses were selected as they were considered risk factors for severe COVID-19 infection and the symptoms were the most common symptoms at infection onset according to the WHO/International Soil Reference and Information Centre (WHO/ISARIC) platform . Age andGeneral health status after the infection (at the time of answering the survey) was assessed by a modified version of the EQ visual analog scale (VAS) . The nump-values for multiple comparisons were adjusted by the Benjamini and Hochberg (1995) method to control for the false discovery rate (FDR) [All statistical analysis was performed by using R  . For theThe study subjects were divided into different groups by the K-Means clustering method. The ordinal logistic regression models were used for developing the PCS scores and selecting the final predicting model. The area under the curve (AUC) of the multiple classes was used to calculate the overlap with the K-Means clustering and the PCS clustering. A backward selection procedure was used for the optimal variable selection. The Pearson correlation coefficients were calculated for assessing the association between the clinical functions and the predicted PCS scores. The confidence intervals were calculated by the bootstrap methods for the correspondingly statistics, for these correlation coefficients and also for the AUC of the multiple class receiver operating characteristic (ROC) curve . The floSensitivity analysis was carried out in two ways. One kind of sensitivity analysis was done by randomly splitting the total cohort into the training set with 80% of the individuals and the test set with 20% of the individuals.In total, 584 subjects (response frequency 52%) from three sub-cohorts completed the survey and were included in this study. The non-hospitalized cohort had the lowest mean age, highest proportion of women and more subjects with higher education, as seen in K, PCS_(K+1)), was r = 0.980 (p < 0.001) for K = 2, r = 0.992 (p < 0.001) for K = 3 and r = 0.989 (p < 0.001) for K = 4  of subjects in the non-hospitalized cohort and 13 (13%) in the hospitalized cohort that did not report any persistent long-term symptoms, while all the subjects in the post-COVID cohort reported at least one persistent long-term symptom. The most common persistent symptom among subject of the three cohorts was fatigue, as shown in The cohorts were randomly split into the training set  and the test set . Using the similar procedures as for the full dataset, three clusters were determined according to the PCS score correlations . Then PCThe association between PCS score and the difference between the reported work ability and general health in the survey and the retrospectively assessed work ability and general health before the COVID-19 infection was assessed. There was a statistically significant association between the PCS score and the decrease in both work ability, Twenty-two different factors were considered as possible predictors of the PCS score group severity . Through backward selection procedure to minimize the Akaike Information Criterion, 10 factors were ultimately selected: sex, BMI, smoking, snuff use, heart disease, lung disease, depression/anxiety, type 2 diabetes, chronic pain and self-reported symptom severity at onset. Of these, six factors  were found to be significant . The NagThe main result of our study was that we identified three distinct groups (phenotypes) of long COVID through the PCS score. These three phenotypes had a clear gradient of post-COVID symptoms’ frequency and severity and differed in sociodemographic, clinical factors and functional limitations. We further showed that higher PCS score was associated with higher reduction of self-reported general health status and working ability following COVID-19. Finally, we demonstrated that sex, BMI, smoking, snuff, coexisting heart disease, lung disease, depression/anxiety, diabetes mellitus type 2, chronic pain and self-reported severity COVID-19 onset were selected predictors of the PCS score groups.Two previous studies used similar clustering methods and PCS score ,16. BahmWe also found that individuals with highest PCS score (severe phenotype) had more persistent symptoms such as fatigue and memory and concentration impairment. This is in accordance with previous studies; however, it is still not known which symptoms have the most clinical relevance . FurtherIn the present study, we found that the subjects with the severe phenotype had the highest self-reported reduction of general health status and work ability following COVID-19 and were more frequently on sick leave. Our results are in general agreement with previous studies. More severely impaired quality of life and limitations with daily living was associated with a higher persistent symptom load ,27. An AIn our study, hospitalization due to COVID-19 was not associated with severity of persistent symptoms. This in accordance with previous cluster analysis using PCS score ,15. On tOur study identified six significant predictors of a higher PCS score: BMI, smoking, snuff, diabetes mellitus type 2, chronic pain and self-reported symptom severity at COVID-19 onset. Our finding was in general consistent with previous reports ,12,15. IThis study differs from other cluster analysis ,15,16 siThe strength of our study it was the use of three cohorts of patients that underwent COVID-19 of various severity and over different periods of the pandemic in Sweden. The same questionnaire was used and, therefore, sociodemographic and clinical data collected in each of the populations could be combined and compared. The PCS scores for each symptom calculated in our study might be used in clinical practice in patients with long-term remaining symptoms to estimate the risk zone for severe long COVID. For example, the patients with several remaining symptoms including fatigue, memory and concentration problems, dyspnea, heart palpitation, depression, anxiety and sleep problems have PCS score sum 40.6 and may have the most severe phenotype of long COVID. These patients may need more focused individual care and rehabilitate as expected to suffer functional impairments.A weakness of our study is that the response frequency was rather low and varied between populations. Another potential limitation was that several study subjects in the post-COVID cohort did not have a laboratory-confirmed COVID-19 infection. This was due to the limitation of laboratory testing in the early pandemic phase in Sweden. In addition, symptoms’ severity from the infection onset, self-reported general health status and work ability before the infection was assessed and rated retrospectively. Retrospective reporting on health-related quality of life may potentially introduce recall bias. Another limitation might be that the study subjects came from distinctive time periods with different availability to vaccine and various variants of virus that may change the severity and long-term outcomes of the infection.The results of this study suggested three phenotypes of long COVID, where the most severe symptom load was associated with the highest impact on general health status and working ability. Knowledge on long COVID phenotypes could be used by clinicians to support their medical decisions regarding prioritizing and more detailed follow-up of some patient’s groups. Further studies are needed to determine the underlying mechanism behind these phenotypes.


Little is currently known about HIV-related parameters that may increase the risk for oral ulcers during the COVID-19 pandemic. This study aimed to overcome this gap in research by assessing the associations between HIV viral load, antiretroviral adherence profile, co-morbidity status, SARS-CoV-2 infection and oral ulcers among people living with HIV (PLHIV).This was a secondary analysis of data generated from 21,206 to 18 years and above, recruited from 152 countries through an online survey between July and December 2020. Data were extracted for 874 people who reported living with HIV. The dependent variable was reporting having oral ulcer. The independent variables were the viral load, adherence to antiretroviral treatment and a history of SARS-CoV-2 infection. The confounding variables were age at last birthday and sex at birth. A multivariable logistic regression analysis was conducted to determine the associations between the dependent and independent variables after adjusting for the confounding variables.Of the 874 participants, 99 (11.3%) reported having oral ulcers during the first wave of the COVID-19 pandemic. The odds of PLHIV having oral ulcers during the first wave of the COVID-19 pandemic was significantly higher for people who did not know their viral load than those who had undetectable viral load ; and people who did not adhere to the use of antiretroviral treatment than those who adhered . Also, PLHIV who had SARS-CoV-2 infection had significantly higher odds of having oral ulcers than those who did not have the infection . PLHIV who had co-morbidities had non-significantly higher odds of having oral ulcers than those without co-morbidities .Oral ulcers may be an indicator of poor adherence to antiretroviral therapy and unsuppressed viral load among PLHIV. It may also be an indicator of SARS-CoV-2 infection and a signal to take prompt and critical care of affected individuals because of the risk for severe COVID-19 for these individuals.The online version contains supplementary material available at 10.1186/s12903-023-03330-2. Oral ulcers are a pathognomonic feature of COVID-19 . IndirecThe risk for people living with HIV (PLHIV) to COVID-19 induced pathologies is of major concern. This is because PLHIV, especially those with advanced HIV and those who are not on antiretroviral treatment, have higher rates of co-morbidities and behaviours that are associated with severe COVID-19. These include higher rates of inhalational drug use , greaterOral ulcers may be an early risk indicator for COVID-19 infection in PLHIV. They appear between 0 and 10 days after the start of COVID-19 symptoms with the most common sites being the tongue . Oral ulEthical approval for the study was obtained from the Human Research Ethics Committee at the Institute of Public Health of the Obafemi Awolowo University Ile-Ife, Nigeria (HREC No: IPHOAU/12/1557). Study participants provided consent before participating in the online survey.This was a secondary analysis of data generated from 21,206 participants recruited from 152 countries in a cross-sectional study through an online survey between July and December 2020. The details of the parent study, including participants’ recruitment process , 30 and Respondents had to be 18 years and above, understand the survey language  and could access the survey using an electronic device and an internet connection. There were no exclusion criteria. The study population were respondents who reported living with HIV by checking a box to indicate that they had HIV on a checklist of 27 medical ailments .The data of 874 participants who indicated they were living with HIV were extracted for analysis. The sample was considered statistically adequate as there was a minimum of 10 participants with complete responses for each of the independent variables in the study. This enabled us to perform regression analysis with a minimum probability level of 0.05 .www.mehewe.org) were asked to share the survey link with their contacts around the world. The link to the survey questionnaire was posted on social media groups , network email lists, and WhatsApp groups. Respondents were encouraged to share the link further with their networks. Details on study participants’ recruitment process had been published [This was a non-probability sample of respondents recruited using an online survey tool (Survey Monkey®). Initial participants reached by 45 members of the MEHEWE Study Group ; adherence - Some people find that they sometimes forget to take their medications to manage their HIV. Did you miss any doses of your HIV medications during COVID-19 ; and SARS-CoV-2 infection- whether they had tested positive for COVID-19 .Also, participants living with co-morbidities were identified. All participants who indicated they had any of the 23 listed health conditions presented on a checklist in addition to other health conditions not listed were identified as living with co-morbidities. These included medical conditions which put individuals at higher risk for severe COVID-19 disease  and those that might put people at moderate risk of COVID-19 disease . The list of health conditions was adopted from Marg et al. .The confounding variables were age at last birthday and sex at birth. Age is associated with viral load suppression and adherence to antiretroviral treatment , risk foA complete case approach was used, such that observations with missing data were removed from analysis . A multiTable The odds of PLHIV having oral ulcers during the COVID-19 pandemic was significantly higher for people who did not know their viral load than those who had undetectable viral load ; and people who did not adhere to the use of antiretroviral than those who adhered . Also, PLHIV who had SARS-CoV-2 infection had significantly higher odds of having oral ulcers than those who did not have the infection . The presence of co-morbidities had a non-significant association with having oral ulcers .The study findings indicate that PLHIV who do not know their viral load, do not adhere to the use of antiretrovirals and who had SARS-CoV-2 infection may have significantly higher likelihood of having oral ulcers during the pandemic. Having a co-morbidity did not seem to be associated with oral ulcers in PLHIV during the pandemic. Though oral ulcers are a common feature of COVID-19, we provide evidence to suggest that there may be differential risk for oral ulcers among PLHIV during the COVID-19 pandemic.One of the strengths of the study is the population level analysis of the risk indicators of oral ulcers. There are, however, limitations associated with the study. First, this is a cross sectional study and so causal inferences cannot be made. Second, the convenience sampling and use of an online survey, with the unintended exclusion of persons without smartphones and people whose languages were not covered by the study instrument, limit the generalisability of findings. Third, the self-reported parameters are susceptible to recall and social desirability bias , 44, morFirst, having an unknown viral load is suggestive of poor monitoring of one’s viral load. Reasons why an individual may not know their viral load range from poor access to viral load testing to lack of appropriate action on viral load results especially in low-resource settings . Oral leSecond, though very few PLHIV had SARS-CoV-2 infection, the odds of reporting oral ulcers by PLHIV who had SARS-CoV-2 infection was 14 times higher than those who did not report SARS-CoV-2 infection. Prior studies highlighted that PLHIV do not have higher risk of SARS-CoV-2 infection, but once exposed, have higher risk of severe COVID-19 outcomes . The risHowever, little is currently little understood about the pathophysiology of oral ulcers associated with COVID-19. Oral ulcers may result from the opportunistic infections due to prolonged use of steroids to treat SARS-CoV-2 infection. It also may result from immunosuppression and poor oral hygiene or due to direct infection of the oral tissues . StudiesIn conclusion, although the presence of oral ulcers are a pathognomonic feature of COVID-19, oral ulcer in PLHIV during the COVID-19 pandemic may be an indicator of poor adherence to antiretroviral therapy and unsuppressed viral load as suggested by individuals reporting they do not know their viral load status. It may also be an indicator of SARS-CoV-2 infection and a signal to take prompt and critical care of affected individuals because of the risk of severe COVID-19 for these individuals. Clearly there is scope for more work to be done to further understand these associations.Below is the link to the electronic supplementary material.Supplementary Material 1


To describe the updated coronary computed tomographic angiography (CCTA)-based coronary artery bypass graft (CABG) anatomic SYNTAX Score (aSS) and assess its utility and reproducibility for assessing the completeness of revascularization after CABG. The CCTA-CABG aSS is a visual assessment using CCTA post-CABG which quantifies the failure in effectively grafting stenotic coronary segments, and therefore assesses the completeness of surgical revascularization. It is calculated by subtracting the aSS of successfully anastomosed coronary segments from the aSS of the native coronary tree. The inter-observer reproducibility of the CCTA-CABG aSS was evaluated in 45 consecutive patients with three-vessel disease with or without left main disease who underwent a CCTA 30 days (± 7 days) after CABG. The CCTA-CABG aSS was evaluated in 45 consecutive patients with 117 bypass grafts and 152 anastomoses. The median native coronary aSS was 35.0 [interquartile range (IQR) 27.0–41.0], whilst the median CCTA-CABG aSS was 13.0 (IQR 9.0–20.5). The inter-observer level of agreement for the native coronary aSS and the CCTA-CABG aSS were both substantial with respective Kappas of 0.67 and 0.61. The CCTA-CABG aSS was feasible in all patients who underwent CABG for complex coronary artery disease with substantial inter-observer reproducibility, and therefore can be used to quantify the completeness of revascularization after CABG.The online version contains supplementary material available at 10.1007/s10554-023-02978-9. The angiographic coronary artery bypass graft (CABG) anatomic SYNTAX Score (aSS) was developed and used to evaluate the patency of bypass grafts at mid-term follow-up after CABG in the LE MANS cohort of the SYNTAX trial . Since iComputation of the CABG aSS consists of two steps: the first being to calculate the aSS of the native coronary arteries, and the second to evaluate the patency of the bypass grafts; wherein if a stenotic lesion in a coronary segment is successfully bypassed, then the segment’s weighting points are subtracted from the aSS of the native circulation. In cases of narrowed or occluded bypass grafts, the deduction of points is reduced or nullified.The diagnostic accuracy of coronary computed tomographic angiography (CCTA) has improved through technological advancements , and it CCTA is an established imaging modality to evaluate bypass grafts , with hiGiven the high diagnostic accuracy of CCTA for detecting graft and native coronary artery stenoses, it is a suitable modality to compute the CABG aSS. The purpose of this methodological report is, therefore, to describe the CCTA-CABG aSS, together with assessing its utility and reproducibility for evaluating the completeness of revascularization after CABG.The CCTA-aSS of the native coronary arteries was calculated as a summation of lesion-based aSS Table  5]. The. The5]. 2.Whenever the graft and anastomoses were patent, the segment-weighting points of the bypassed coronary segment, including any points for adverse lesion characteristics, were subtracted Fig. .3.When the vessel  proximal to the anastomosis had > 1 stenotic lesion then the aSS points of those coronary segments distal and closest upstream of the anastomosis : no segment-weighting points were subtracted since the lesions were considered flow limiting : the segment-weighting factor was reduced from × 5 to × 2 since the flow to the myocardial bed had improved ]. Typically, an additional acquisition was needed to image the origin of the IMA, whilst a three-dimensional volume rendering image was useful to understand the anatomy of the bypass grafts and their topological relationship with the native coronary arteries, whereas curved multiplanar reconstructions enabled luminal assessment of any stenoses. The score was calculated following the sequential steps:To evaluate inter-observer reproducibility, two blinded readers in the independent core laboratory  with experience in the interpretation of more than 200 mentored cases of CCTA in clinical research calculated the CCTA-CABG aSS using CCTAs performed 30 days (± 7 days) after CABG in 45 consecutive cases from the FASTTRACK CABG trial (NCT04142021) . Vessel t-test. Inter-observer variability (tercile partitioning) was determined with weighted kappa statistics  using linear weights on the native coronary aSS and the CABG-aSS [Continuous variables are presented as mean ± standard deviation (SD) or as median and interquartile range (IQR) depending on their distribution and compared using the Student’s The CCTA-CABG aSS was successfully evaluated in all 45 patients who had a total of 117 bypass grafts, made up of 65 arterial (55.6%) and 52 saphenous vein (45.4%) grafts, and 152 anastomoses. The median CABG aSS was 13.0 , with a score > 22 in 22.2% of cases Fig. . The medThe inter-observer level of agreement for the native coronary aSS by tercile partitioning  was moderate . When the native coronary aSS was stratified based on the tercile of the original SYNTAX trial , the agreement was substantial . The intraclass correlation (ICC) for inter-observer variability of the native coronary aSS was 0.91 (95% CI 0.83–0.95).The inter-observer level of agreement in the CCTA-CABG aSS by tercile partitioning  was substantial . The ICC for inter-observer variability of the CCTA-CABG aSS was 0.92 (95% CI 0.85–0.96). Similarly, when the CCTA-CABG aSS was divided into < 22 or ≥ 22, the agreement was substantial .The main findings from this study were that the CCTA-CABG aSS was feasible in all study patients, with substantial inter-observer reproducibility.Reproducibility analyses for the native aSS, stratified according to terciles of the original SYNTAX trial, and the CCTA-CABG aSS demonstrated substantial inter-observer agreement . In previous publications, Kappa values for the inter-observer agreement of the aSS based on ICA and assessed by a core lab ranged from 0.52 to 0.82 –12 , with no patient having a score of zero. In the SYNTAX-LE MANS sub-study, those patients with an ICA-based CABG-aSS ≥ 22 post-CABG had significantly higher rates of 5-year death  and the composite of all-cause death, cerebrovascular accident and myocardial infarction (log-rank p = 0.025) compared to those with the score < 22 [In the recent AHA/ACC/ACCP/ASPC/NLA/PCNA guidelines for the management of patients with chronic coronary disease, CCTA to evaluate bypass graft in patients with symptoms was recommended as 2a . In addiCCTA-CABG aSS could aid planning pre-procedure and assessment post-procedure as the universal index for the completeness of the revascularization after CABG. Furthermore, when we consider the revascularization for patients with chest pain who previously underwent CABG, this scoring to assess the severity of coronary artery disease could be a useful tool to share the assessment as objective metrics for the Heart team discussion for decision-making. In terms of evaluating the completeness of revascularization, previous ICA-based definitions were ambiguous in cases of sequential lesions, diffuse disease, and bifurcations. Given the high complexity of CAD in the FASTTRACK CABG study  , the resA comprehensive and universally accepted definition of complete revascularization after CABG and PCI is a major and somewhat utopic challenge since it depends very much on the modality used for the assessment: vascular and segmental, involving patency and narrowing; or myocardial and segmental, investigating viable myocardium at risk of ischemia. The ultimate scientific goal is to combine these two facets of mechanical revascularization. In our current methodological approach, the scientific target is more specifically vascular and segmental, without speculating on the amount of flow needed by the myocardium subtended by a native vessel or graft. The data from 1,162 patients who underwent off-pump CABG for three-vessel disease showed that functional complete revascularization, defined as constructing bypass grafts to coronary artery territories where ischemia was identified by pre-operative myocardial SPECT (single-photon emission computed tomography), had a significant impact on long-term survival , whereasIn the present assessment, theoretical weighting factors established by Leaman et al. and based on literature existing at the time of the score’s design were used, and these constitute the foundation of the aSS . In thisCT (FFR derived from CCTA), as illustrated in Fig.  Although the prognostic value of the aSS in PCI has been repeatedly demonstrated over more than one decade, data on residual aSS post-CABG and its prognostic value are scarce , 15. TheThe prognostic value of the aSS stemmed from prospective testing in the landmark Syntax trial , and wasCT becomes common practice, the challenge will be to prospectively re-evaluate and re-establish a new scale of risk assessment related to the decision-making process.If the method of quantifying the myocardium at risk and FFRCT and simulated blood flow measurements might allow better evaluation of flow-limiting lesions before and after CABG [Anatomical evaluation of residual disease after CABG is still currently based on historical and theoretical weighing scales related to the presence of a narrowing in a specific coronary segment, regardless of the caliber, length, and amount of myocardium actually supplied by that specific vessel . CCTA-bater CABG .The assessment of the area of ischemic myocardium using a purely anatomical evaluation remains challenging. After graft placement, antegrade coronary flow through and beyond the native coronary artery stenosis can be reversed , 32. ComThe CCTA-CABG aSS was feasible in all patients who underwent CABG for complex coronary artery disease with substantial inter-observer reproducibility, and therefore can be used to quantify the completeness of revascularization after CABG. The potential of a threshold residual value that identifies high-risk patients post-CABG should be investigated in prospective studies.Supplementary material 1 (DOCX 36 kb)Below is the link to the electronic supplementary material.