Plasmodium vivax is the most widely distributed malaria parasite. Naturally acquired binding inhibitory antibodies (BIAbs) to region II of the Duffy binding protein (DBPII), a P. vivax ligand that is critical for reticulocyte invasion, are associated with a reduced risk of clinical malaria. Owing to methodological issues in evaluating antibodies that inhibit the DBPII–DARC interaction, a limited number of studies have investigated DBPII BIAbs in P. vivax-exposed populations. Based on the assumption that individuals with a consistent BIAb response are characterized by strain-transcending immune responses, we hypothesized that detecting broadly reactive DBPII antibodies would indicate the presence of BIAb response. By taking advantage of an engineered DBPII immunogen targeting conserved DBPII neutralizing epitopes (DEKnull-2), we standardized a multiplex flow cytometry-based serological assay to detect broadly neutralizing IgG antibodies. For this study, a standard in vitro cytoadherence assay with COS-7 cells expressing DBPII was used to test for DBPII BIAb response in long-term P. vivax-exposed Amazonian individuals. Taken together, the results demonstrate that this DBPII-based multiplex assay facilitates identifying DBPII BIAb carriers. Of relevance, the ability of the multiplex assay to identify BIAb responders was highly accurate when the positivity for all antigens was considered. In conclusion, the standardized DBPII-based flow cytometric assay confirmed that DBPII-BIAb activity was associated with the breadth rather than the magnitude of anti-DBPII antibodies. Altogether, our results suggest that multiplex detection of broadly DBPII-reactive antibodies facilitates preliminary screening of BIAb responders.Malaria remains a major public health problem worldwide, and  Plasmodium vivax, the most widespread human malaria parasite , and its cognate reticulocyte receptor, the Duffy antigen receptor for chemokines (DARC)  –12. Whil malaria , the mal malaria –16. Howe malaria , 17–19,  malaria  and redu malaria , 22. The malaria .In vitro evaluation of DBPII antibodies able to block P. vivax reticulocyte invasion has proven challenging, as short-term P. vivax blood-stage culture is not available for routine use in most malaria research laboratories , whose reactivity was previously associated with broadly reactive BIAb response . Using tTwo specific DBPII monoclonal antibodies (mAbs) were used to evaluate the ability of the multiplex assay to assess DBPII target epitopes. The 2D10 inhibitory mAb targets disulfide-bonded epitopes, which are critical for the maintenance of the native conformation required for erythrocyte receptor recognition in DBL domains , 30. TheP. vivax who had high levels of anti-DBPII antibody responses as characterized by the presence of BIAb activity (>90% of inhibition in the COS-7 cells) and ELISA-detected antibodies (reactive index >5 for different DBPII constructions); each positive pool included four individual samples; (ii) DBPII-negative pools from malaria endemic area, which were selected from individuals (four samples per pool) living in an endemic area (Amazon Basin) but with no detectable DBPII antibody response (negatives for BIAbs and ELISA); and (iii) DBPII-negative pools from individuals living in a nonendemic area of malaria  and who have never been exposed to malaria transmission (negative pool nonendemic area).To standardize the multiplexed microsphere-based flow cytometric assay, three types of pooled plasma samples were prepared: (i) DBPII-positive plasma pools obtained from individuals naturally exposed to P. vivax-exposed Amazonian individuals. The clinical, immunological, and epidemiological characteristics of the study population have been previously described  of them (asymptomatic infections).To evaluate the DBPII-multiplexed microsphere-based flow cytometric assay, the study included a total of 245 samples from 85 long-term escribed . Brieflyhttp://www.fiocruz.br/biosseguranca/Bis/manuais/biosseg_manuais.html).The ethical and methodological aspects of this study were approved by the Ethical Committee of Research on Human Beings from the Research Institute René Rachou . The current study was conducted according to the laboratory biosafety and biosecurity policy guidelines of the Fundação Oswaldo Cruz (FIOCRUZ), Brazilian Ministry of Health  of each sample to the mean OD plus three standard deviations of samples from 20 to 30 unexposed volunteers. RI values >1.0 were considered positive.A conventional enzyme-linked immunosorbent assay (ELISA) for antigen-specific IgG antibody response was carried out as previously described , 37 withP. vivax recombinant proteins according to the manufacturer’s protocol. Briefly, functional beads were incubated with dithiothreitol (1 M) for 1 h at room temperature. The beads were then washed and resuspended in a coupling buffer (BD Biosciences). Next, recombinant DBPII Sal-1, DBPII Brz-1, or DEKnull-2 at 1 mg/ml were activated by incubation with sulfosuccinimidyl 4- cyclohexane-1-carboxylate for 1 h. Next, the activated protein was added to the beads and allowed to conjugate for 1 h at at room temperature. N-Ethylmaleimide (2 mg/ml) was added, and the mixture was incubated for 15 min. The beads were then washed and resuspended in storage buffer (BD Biosciences). For assessment of antibody responses, 1 µl of coupled beads were washed and incubated with plasma diluted in 100 µl of washing buffer for 2 h. The unbound antibodies were washed off and then incubated with a biotin-goat anti-human IgG antibody  and streptavidin-PE . Simplex titration assay was performed to determine the best plasma sample dilution using twofold serial dilutions (starting at 1:50) was used in the test. Samples were acquired using a FACSVerse cytometer (Becton Dickinson) equipped with three lasers  capable of distinguishing the flex set of beads through the CBA Red and CBA NIR bead channels. The detection reagent was a phycoerythrin (PE)-conjugated antibody , which provides a fluorescent signal in proportion to the amount of bound antibodies (reporter channel PE). Antibody response was determined by obtaining the median fluorescence intensity (MFI) values using the FlowJo program (version 10.4.1.), following the analysis strategy described in x-axis represents the intensity of secondary staining. The exact value of MFI was obtained by selecting the statistical features of MFI and Comp-PE.The protocol described here was based on a multiplexed microsphere-based flow cytometric assay using the BD™ CBA Human Soluble Protein Flex Set System . This assay provides a method of capturing a set of analytes with beads of known size and fluorescence, allowing the coupling of different antigens to beads with different positions to create a multiplex assay. Here, functional beads (BD Biosciences) were coupled to 5 cells/well) were transfected with the plasmid (0.5 µg/well) liposome complexes (5% Plus-reagent and 3% lipofectamine) in Dulbecco’s modified Eagle’s medium  without fetal bovine serum . After 6 h of exposure to the DNA-liposome complexes , the transfection medium was replaced with DMEM supplemented with 10% fetal bovine serum. Twenty-four hours after transfection, the culture medium was replaced again, and the efficiency of transfection was assessed using an inverted fluorescence microscope . Forty-eight hours post-transfection, erythrocyte-binding assays were performed as previously described (2. Human erythrocytes O+ DARC+ (genotype Fy*A/Fy*B) in a 10% suspension were added to each well (200 µl/well), and the plates were incubated for 2 h at room temperature. After incubation, unbound erythrocytes were removed by washing the wells three times with DMEM medium without fetal bovine serum. Binding was quantified by counting rosettes around GFP-positive cells within 10 fields of view (×200). Binding for each plasma dilution was compared with the binding of transfected COS-7 cells incubated with negative control plasma at a 1:40 dilution (100% binding). The percent inhibition was calculated as 100 × (Rc − Rt)/Rc, where Rc is the average number of rosettes in the control wells and Rt is the average number of rosettes in the test wells. Percent inhibition values were considered positive at >50%. Samples were classified as high responders (HR) when the percentage inhibition values were >90%.The functional properties of DBPII antibodies (BIAbs) were assessed using COS-7 cell erythrocyte binding assays, as previously described , 26, 28.http://www.epidata.dk). Graphics and analyses were performed using GraphPad Prism version 7  and R statistical software (version 3.3.2). Differences in means and medians were tested using either one-way ANOVA, with Tukey’s post-hoc test, the Mann-Whitney test, or Kruskal-Wallis test, with Dunn’s post-hoc test, as appropriate. The performance parameters were defined using a 2 × 2 contingency table with 95% confidence intervals (95% CI), calculated using the OpenEpi open-source statistical calculator   . The perTo confirm the coupling of antigens and the ideal conditions to differentiate positive and negative samples in the flow cytometry-based DBPII assay, we first carried out a pilot assay, in which each recombinant protein was evaluated with serial dilutions of well-characterized positive and negative pooled samples. Based on the titration curve of DEKnull-2  that recognizes DBPII conformational inhibitory epitopes. Titration of both mAbs (2D10 and 3D10) demonstrated that the process of covalent coupling of the DEKnull-2 (n = 21) who presented with high levels of DBPII antibodies as detected by ELISA  and BIAb activity (>90% of binding inhibition); (ii) nonresponders from the endemic area , whose DBPII-specific antibodies (detected by ELISA and BIAbs) were undetectable. In addition, a subgroup of individuals living outside the endemic area and those with undetectable DBPII-specific antibodies were included (nonexposed (NE), n = 13). Regardless of the DBPII antigens used in the multiplex assay, the results confirmed that the HR subgroup was clearly discriminated from the NE and NR subgroups   samples were positive for at least one DBPII construct. A Venn diagram showed that a significant number of samples were positive for all assayed antigens   Figure 3p < 0.001) , particularly because of an increase in specificity (80% to 97%).As the ability of the DBPII-multiplex assay to identify BIAb responses depended on the number of antigens recognized in the assay, we evaluated the performance of the multiplex assay when identifying BIAb carriers considering the positivity for any antigen Table S1Plasmodium vivax DBPII-based vaccines aim to induce broadly neutralizing antibody responses that are able to block merozoite invasion of human reticulocytes  and noninhibitory (3D10) DBPII mAbs were correctly recognized. This is particularly relevant as the crystal structure of the DBPII/2D10 complex showed that 2D10 targets disulfide bond-dependent DBPII epitopes, which are critical for the maintenance of the native conformation of proteins required for DARC recognition . AlthougP. vivax samples for the presence of antibodies targeting DBPII-DARC interaction. The results demonstrate that the ability of the DBPII-based multiplex assay to correctly identify BIAb activity was associated with the detection of antibodies against both conserved (DEKnull-2) and polymorphic DBPII epitopes . Indeed, the odds ratio (OR) statistic showed that positivity for all three recombinant proteins in the multiplex assay increased the chances of identifying BIAb carriers by about 12 times. The accuracy of the multiplex assay to identify BIAb carriers was 90% when multiplex positivity took into account all three antigens. In our multiplex assay protocol, the results demonstrated that the “breadth,”  rather than the levels of DBPII-based antibodies, was related to the presence of BIAbs. Considering that BIAbs activity is associated with strain-transcending inhibitory immune responses, we can assume that the detection of anti-DBPII antibodies against a range of diverse allelic DBPII variants seems to be more relevant than levels of the antibody per se in our multiplex assay. In this scenario, it would be interesting to explore whether the inclusion of additional antigens involved in the reticulocyte invasion would increase the chance of detecting BIAbs; for example, the P. vivax reticulocyte-binding protein (PvRBPs) family that are involved in novel reticulocyte invasion pathways . Nevertheless, evaluating the protective nature of the antibody response was outside the scope of the current study. To properly address the clinical protection mediated by BIAbs, a long-term follow-up study is required with an estimated sample size that allows statistical power. Despite this, different studies have already confirmed the association between clinical protection and the presence of BIAb responses  that confirmed the specificity of the assay using different contrations of highly specific antihuman IgG conjugate. Of relevance, we previously used confocal microscopy to confirm that BIAb-positive plasma samples are characterized by high levels of IgG antibodies targeting DBPII-DARC interaction . Furtherulocytes . Neverthulocytes  demonstrulocytes . While ttibodies , 21, 22.In conclusion, the successful protocol described here indicates that a multiplex detection of broadly DBPII-reactive antibodies is a potential approach that could be useful to improve the detection of BIAb carriers in large-population cohort studies. For that, it is essential to adapt the multiplex platform to include allelic DBPII variants prevalent in the study area.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by The Ethical Committee of Research on Human Beings from the Rene Rachou Institute – Fiocruz Minas , according to the Brazilian National Council of Health. The study participants were informed about the aims and procedures and agreed with voluntary participation through written informed consent. The patients/participants provided their written informed consent to participate in this study.Conception and study design: JA, AT-C, FK, and LC. Fieldwork and recruitment of participants: JA, BL, LT, CP, and FK. Performance of experiments and data analysis: JA, FA, and CP. The drafting of the manuscript: LC, FK, CP, and JA. Provide critical advice and reagents: FN and JA. All authors contributed to the article and approved the submitted version.This work was supported by the National Research Council for Scientific and Technological Development-CNPq (422257/2016-8 by LC), the Research Foundation of Minas Gerais-FAPEMIG (APQ-02625-15 by LC), Programa Fiocruz de Fomento à Inovação: Inova Fiocruz (VPPCB-007-FIO-18-2-33 by FK), and the NIH Research Project Grant Program (R01AI064478 by JA and LC). LC and AT-C are research fellows from CNPq and the Coordination for the Improvement of Higher Education Personnel . Scholarships were sponsored by CAPES  and CNPq . The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Here we introduce a systems biology tool, the PHENotype SIMulator, which – by leveraging available transcriptomic and proteomic databases – allows modeling of SARS-CoV-2 infection in host cells in silico toi) determine with high sensitivity and specificity (both>96%) the viral effects on cellular host-immune response, resulting in a specific cellular SARS-CoV-2 signature and ii) utilize this specific signature to narrow down promising repurposable therapeutic strategies. Powered by this tool, coupled with domain expertise, we have identified several potential COVID-19 drugs including methylprednisolone and metformin, and further discern key cellular SARS-CoV-2-affected pathways as potential new drugable targets in COVID-19 pathogenesis.The current, rapidly diversifying pandemic has accelerated the need for efficient and effective identification of potential drug candidates for COVID-19. Knowledge on host-immune response to SARS-CoV-2 infection, however, remains limited with very few drugs approved to date. Viable strategies and tools are rapidly arising to address this, especially with repurposing of existing drugs offering significant promise.  Soon after its emergence in Wuhan (Eastern China) in late 2019, coronavirus disease 2019 (COVID-19) was declared a pandemic by the World Health Organization2, as it continuously spreads and holds the world hostage. As of February 2021, over 100 million COVID-19 cases and 2 million deaths have been reported worldwide. It is apparent that our civilized, well-organized and hitherto functioning societies were not adequately prepared nor equipped to deal with the high infectivity, transmissibility, mortality and global impact of the COVID-19 pandemic. While vaccine development and deployment is well underway, widespread distribution remains challenging, and at present only an antiviral (Remdesivir) and glucocorticoids (Dexamethasone/ Methylprednisolone) have been approved for treatment of severe COVID-19. Recently, the virus-neutralizing antibody cocktail  also received emergency use authorization for treatment of mild to moderate COVID-19 in high-risk patients3. Otherwise, no established drug is available to prevent or adequately treat COVID-19 and in the absence of a clear etiological understanding, treatment has remained largely supportive and symptomatic5. Making matters worse, the mutating virus is now posing additional challenges.The rapid emergence and spread of the virulent novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has hijacked and largely disrupted human civilization as we know it, bringing about countless global challenges but also the urgent need for innovative vaccine and drug discoveryin vitro studies, in silico studies are of great value for rapid and effective drug discovery. Indeed, computational structure-based drug design and immunoinformatics have recently resulted in identification of potential SARS-CoV-2 target proteins and drugs that are being selected for further testing7. Another promising avenue for obtaining effective and readily available therapeutic strategies is the repurposing of drugs already approved for other indications. Drug repurposing strategies provide an attractive and effective approach based on available drug characteristics – drug-related pharmacology and toxicology – for rapid therapeutic selection 8. If we could, with higher probability, identify and pre-select the most promising hypothesis-based candidates using in silico systems biology tools, prior to costly and laborious in vitro and in vivo experiments and ensuing clinical trials, we could significantly improve disease-specific drug development9.Therefore, next to in silico techniques have been developed, mainly making use of molecular modeling of key viral proteins for virtual screening of drug candidates simulating receptor-drug molecular dynamics6. In order to increase the effectivity of identifying candidate drugs for combating COVID-19, it is crucial to build on a more in-depth knowledge of the molecular basis of the immune signaling pathways regarding host-virus interaction and SARS-CoV-2-induced immunopathology. Only if we better understand how this particular virus affects host cells in detail, on a transcriptomic, proteomic level and beyond10, will we be able to effectively treat COVID-19 patients. It is becoming evident that treatment should not only focus on direct antiviral effects in mild cases but should also encompass potential (cytokine storm induced) aberrant host-response in severe cases12. Taken together, this points towards the importance of a more detailed and targeted approach for COVID-19, where antivirals or steroids alone might not suffice and specifically targeting the (aberrant) host-response is imperative8.Several 14. Here we aim to utilize our systems biology tool, the PHENotype SIMulator (PHENSIM), to leverage the power of pathway analysis by simulating tissue-specific infection of host cells of SARS-CoV-2 and subsequently perform in silico drug selection for potential repurposing. PHENSIM is a web-based user-friendly computational tool that allows phenotype prediction on selected cells, cell-lines and tissues, using a probabilistic algorithm15 via “message passing”16 across a network of meta-pathways17. These meta-pathways are obtained by joining all validated biological pathways, enriched with gene regulatory elements18. The algorithm thus computes, under-specified biological contexts, by iteratively propagating the effects and alterations of one or more biomolecules , proteins, microRNAs, or metabolites), thus making use of published virus-human interaction data7. Here we compare our results with available data from recently published in vitro studies based on transcriptomics and proteomics in different model systems10. Relevant and significantly affected pathways are further detailed on a protein interaction level. Finally, we show the potential of the PHENSIM in selecting promising hypothesis-driven COVID-19 drug candidates, which has applicability to other diseases and broader aspects of clinical practice, thereby outlining the potential power of PHENSIM in drug repurposing in COVID-19 and beyond.Recently in literature, tools and algorithms devised to perform simulation on biological networks have been describedin silico simulation of novel emerging pathogens, such as SARS-CoV-2, and it aims at elucidating distinct host-responses and molecular mechanisms triggered by that particular pathogen, all while defining possible candidate drugs for indication repositioning.Innovative approaches to rapidly elucidate a pathogens’ mechanism of action have proven crucial for containing the global burden of communicable diseases. The PHENSIM approach, described here, is based on the definition of a newly introduced protocol for in vitro) data that can be exploited to predict such interactions. To acquire this knowledge, we therefore employ transcriptomic and proteomic experiments of in vitro infected vs. normal, pathogen-free cell lines. When available, we leverage Differentially Expressed Genes (DEGs) as a means to simulate the direct and indirect effect of the virus on a host without a priori knowledge regarding the mechanism of infection. Using DEGs as input for our cell PHENotype SIMulator PHENSIM , targeting the JAK/STAT pathway using Baracitinib – approved for moderate to severe arthritis 46 – was recently shown to reduce time-to-recovery for hospitalized COVID-19 patients in combination with Remdesivir 47, however, caution is warranted 48.Drug repurposing towards COVID-19 is challenging, but also poses many new opportunities. Several innovative approaches have been used varying from structure assisted computer designed mini inhibitors of receptor binding domain (RBD) binding 49. In our current approach, we model SARS-CoV-2 infection in silico with the addition of CD147 into KEGG, in order to investigate the role of this extracellular matrix metalloproteinase inducer (EMMPRIN) in COVID-19  to the original simulator, if absent in KEGG. In the case of SARS-CoV2 infection, the absence of some important genes involved in SARS-CoV-2 infection of host cells in the KEGG database, was considered a limitation. One gene in particular is Basigin (BSG), also known as CD147. The exact role of CD147 in SARS-CoV-2 infection remains elusive to date, however, its role in spike-protein (SP) binding and viral infiltration of host cells has been described D-19 see .in silico model presented here provides an interesting framework that could be further developed and expanded, achieving a more complete cell signature with input of available data on processes such as cell-cell communication through ligand-receptor complexes 50 or viral immune evasion e.g. 51. As most in vitro studies are performed on cell lines, tissue tropism characteristics of viral infection seem key to better understanding viral activity 52. The same model could be adapted to study specific cells involved in viral infection like tissue-specific epithelial cells and immune cells (e.g. T cells and NK cells) 54. Moreover, many interesting avenues can potentially be explored using PHENSIM, such as modeling immune-related effects of this pathogen and others, in distinct tissue-specific non-immune epithelial cells, stem cells, and beyond 55. The system can be further adapted to include new data gathered on the viral translational landscape related to newly discovered open reading frames (ORFs) and potential novel polypeptides/proteins and infectivity potentiating cell surface structures like neuropilin 57. Interestingly, integration of all the aforementioned schemes could potentially yield novel and effective drug targets 58.As discussed, the Here we show distinct candidate drugs having a variable effect depending on the multiplicity of infection (MOI) of virus infection in A459-ACE2 expressing cells in low MOI 0.2;  and high14 has shown excellent performance by PHENSIM 15. PHENSIM creates and builds on interpretable and intervenable mechanistic bio-chemical models, rather than combinatorial and statistical “black-box” models for joint stationary distribution of biological data, as in, say protein-protein interaction (PPI) networks, Graphical or Deep-net models.As demonstrated by our results, we believe that the PHENSIM system provides a multitude of powerful systems biology functions and implements them easily and efficiently. PHENSIM is a simulation algorithm which follows the biological processes modeled by pathways. Therefore, PHENSIM is able to make a prediction of such processes and not only of the final effect, going beyond methods based on pathway enrichment. Furthermore, since pharmacological treatments may depend on the state of biological processes, PHENSIM may be of more appropriate use in this context. Comparison with other simulation algorithms such as BIONSIin vitro and in vivo experimental data 5 gives insight into drug efficacy 44 tracks specific host signal transduction pathways 4, in silico testing of single drugs and drug combinations and further delineation of future targets (e.g. CD147) and identification of specific pathways of action of both pathogen and therapeutic compound in healthy and infected systems. For cost efficiency, validated predictive methods and assays for early elimination of potential drug candidates are of great value 59. The overall efficiency  prompts to suggest implementing PHENSIM not only in viral acute pandemic settings 60, but in additional curative and noncurative diseases, especially complex chronic disorders, where clinical trials are time-consuming or impossible to reduce to practice. Optimally leveraging the power of pathway analysis by simulating host cell and tissue-specific infection and performing in silico drug selection, has a tremendous potential beyond COVID-19, with applicability to high global burden communicable diseases, translatable to pathogens of viral, bacterial and fungal origin, and potentially chronic disease such as inflamm-aging and diabetes. In conclusion, our PHENSIM approach will enable more rapidly initiated clinical trials and accelerated regulatory review of already pre-selected drugs with a high repurposable potential.PHENSIM gives rise to feasible validation and comparison of 20 The meta-pathway concept, introduced by us previously 61, has been devised to account for pathway cross-talk in analysis. Essentially, all KEGG pathways are merged in a single graph through common nodes, where the meta-pathway is a graph in which the nodes represent molecular entities  and the edges are the known biological interactions present in the KEGG database. The meta-pathway is further completed by adding validated miRNA-targets downloaded from miRTarBase (release 8.0), miRecords and TF-miRNA-interactions obtained from TransmiR (release 2.0) 64.PHENSIM is a systems biology approach, simulating the effect of the alteration of one or more biomolecules  in a specific cellular context using KEGG (Kyoto Encyclopedia of Genes and Genomes) meta-pathways 15. Such values are then propagated within the meta-pathway, using the MITHrIL (Mirna enrIched paTHway Impact anaLysis) pathway perturbation analysis 20. MITHrIL determines how local change can affect the cellular environment by computing a “perturbation”. For each gene in the meta-pathway, the perturbation reflects its expected change of expression/activity . Finally, these results are collected and synthesized using two values: the “Average Perturbation” and the “Activity Score” (AS). Given a node, the average perturbation is the mean for its perturbation values computed at each simulation step. It reproduces the expected change of expression for the entire process. The function of AS is twofold: 1) the sign gives the type of predicted effect (activation(+); inhibition(−)), 2) the value is the log-likelihood that this effect will occur. Together with AS, PHENSIM also calculates a p-value which determines how biologically relevant the predicted alteration is for the phenomena being simulated.To start the simulation, PHENSIM requires a set of biomolecules as input, their direction of deregulation (activation/up-regulation or inhibition/down-regulation), and a set of inactive biomolecules in the cellular context . The algorithm uses these details to compute synthetic Log Fold Changes (LogFC). Synthetic LogFCs are computed by sampling the normal distribution fitted to the actual LogFCs of a particular gene, as described previously 65. To determine this probability, PHENSIM randomly selects genes in the meta-pathway and runs the simulation on this random set. By repeating this procedure (n=1000 for our simulations), it is possible to empirically estimate the probability that a node has a higher activity score than the observed one. For this reason, we can employ such a value to determine which alterations are most specific for a particular infection, gaining novel hypotheses on the molecular action of the pathogen.All p-values computed by PHENSIM are corrected for multiple hypotheses using the q-value algorithm in silico simulation of emerging pathogen infection, aimed at defining candidate drugs for repositioning. First, we find a representation of the pathogen in the KEGG meta-pathway, which allows us to perform simulations. For a novel pathogen, such as SARS-CoV-2, interactions with the host genes might be unknown. Therefore, we can approximate this by employing expression data of pre-/post-infection samples. The rationale is that differentially expressed genes (DEGs) represent the downstream effects of the viral infection on the host; i) we compute DEGs between pre- and post-infection samples, ii) we extend the meta-pathway by adding a new node representing the virus, iii) the viral node is connected to each DEG with an activating (/inhibiting) edge if its LogFC computed between post- and pre-infection is positive (/negative), iv) we run a simulation by giving the upregulation of the viral node as input.Our approach defines a protocol for the pathogen signature, we use pathway endpoints; An endpoint is a biological element in a pathway whose alteration, based on current knowledge, affects the phenotype in a specific way20. Given the output of this simulation, we collect all Endpoint Activity Scores in a single signature, the ‘pathogen alterations profile’. This profile can be exploited to search for possible repositioning candidates, by building a drug signature database queried by means of a similarity measure (To build the  measure . When buGiven a particular drug identified through databases (i.e. Drugbank or Pubchem) and literature (Pubmed) searches, we define all known targets and their alterations (up/down-regulations caused by the drug), and these alterations are provided as input to PHENSIM together with the same cellular context specified for the viral simulation. The results are used to define a drug signature using pathway endpoints as described above, which are collected in a database used for repositioning.20, we can assume a Pearson correlation is sufficient to determine similarity between the viral and drug signature. A key characteristic of this approach is the capability to simulate both single and drug combinations. Furthermore, PHENSIM also provides a framework for extending pathways by adding new nodes and edges coming from results in the literature as well as other reputable sources.Our drug repurposing methodology is based on a similarity search performed on the drug signature database. Given a pathogen profile computed with PHENSIM, we use a correlation function to scan through each record in a drug profile database. This procedure yields a ranking on each drug in the range , where negative values indicate that the virus alteration profile is opposite to the drug and positive values indicate the reverse; drugs with a negative correlation are considered possible candidates for repositioning. In our experiments, we employ a Pearson correlation function to run the similarity search. Since PHENSIM is based on MITHrIL pathway perturbation analysis, which computes results in a log-linear space ρ can be written as equation (1):σ(·) is the standard deviation. Therefore, given a pathway P, we can sum the Pearson correlation components belonging to its endpoints to estimate how much it contributes to the final correlation value. More in detail, the partial correlation equation (2):Finally, to assess whether each drug candidate targets relevant infection processes, we decomposed the Pearson correlation in terms of KEGG pathways and reviewed the results. More in detail, let D and V be drug and pathogen alteration profiles, respectively. That is, D[e] is the activity score of endpoint “e” computed by PHENSIM for a drug simulation, and V[e] is the activity score for the same endpoint in the pathogen simulation. Pearson correlation To further evaluate whether the results of the correlation could be confirmed by PHENSIM, we devised a strategy to simultaneously simulate drug action and pathogen infection on a host cell line. First, we collected DEGs between pre- and post-infection samples as described in the previous section. Then, given a drug, we gather its known targets and their alterations (up/down-regulations caused by the drug) through databases (i.e. Drugbank or Pubchem) and literature (Pubmed) searches. Therefore, we extend the meta-pathway by adding two nodes, representing the virus and the drug, respectively. The virus node is connected to each DEG with an edge as described in the “To determine the efficacy of our model we used several datasets obtained in the context of SARS-CoV-2 infection. For each dataset, we computed the genome-wide Log Fold Changes (FC). As PHENSIM does not require any quantitative information, DEGs were termed upregulated if LogFC>0.6, and downregulated if LogFC<-0.6.49. In KEGG the latter gene is missing and to extend our simulation model, a new node, representing CD147 was added and connected with its known interactions and downstream nodes according to literature  66, and it has now been posed to recognize and internalize/endocytose SARS-CoV-2 in certain cell types 49.Prior to running all simulations, we verified if viral entry points were present in KEGG to better represent viral activity. SARS-CoV-2 is said to invade host cells via these two receptors: angiotensin-converting enzyme 2 (ACE2) and CD147  terature . The incii) the ability of PHENSIM to predict genes altered in the expression data, and ii) the ability to predict the correct direction of the alteration. In detail, we define as altered all genes having an absolute LogFC>0.6. The type of the alteration is given by the sign of the LogFC . Predictive power of PHENSIM was assessed by means of Positive Predictive Value (PPV), Sensitivity, and Specificity. The PPV is the proportion of true positive results with respect to all positive predictions, the sensitivity is the percentage of true positives with respect to the entire population, and the specificity is the percentage of true negatives with respect to all negative cases.To assess the reliability of the results, we focused on two fronts: et al. 2020 [GSE147507] 10 and proteomics data coming from Bojkova et al. 2020 5.For the evaluation of our strategy, we exploited transcriptomics data published in Blanco-Melo et al. dataset comprises RNA-seq data of infected vs. mock-treated cell-lines from human and ferret. The data were obtained by using the Illumina NextSeq 500 platform 10. In our analysis, we focused solely on human cell data. In detail, 4 cell-lines were evaluated: primary human lung epithelium (NHBE), transformed lung alveolar (A549) cells, transformed lung alveolar (A549) transduced with a vector expressing human ACE2 and transformed lung-derived Calu-3 cells. For all cell-lines, sequencing data of biological triplicates was obtained from mock treated or SARS-CoV-2 infected experiments. Furthermore, for both A549 cell lines different MOIs (multiplicity of infection) were used at low 0.2 and high MOI 2.0. Following the same procedure used by Blanco-Melo et al., raw counts were normalized and analyzed for differential expression using the DESeq2 pipeline. All genes with an FDR-adjusted p-value<0.05 and absolute LogFC>0.6 were considered differentially expressed. Non-expressed genes for a specific cell-line were defined as genes that showed an average read count lower than 10.The Blanco-Melo et al. 2020, namely proteome measurement by LC-MS/MS of control vs. SARS-CoV-2-infected human Caco-2 cell lines 5. All cell lines were analyzed in triplicates (n=3) at 2, 4, 10 and 24h. Log2-ratios between infected and normal differentially expressed proteins (DEPs)  were used as input for the simulation algorithm. Non-expressed proteins for the Caco-2 cells were taken from the Human Protein Atlas . Since PHENSIM uses Entrez Gene Identifiers, we mapped all proteins to their gene, yielding 5809 mapped proteins. Of these 5809 mapped proteins, we could find only 1914 in the KEGG meta-pathway. We therefore combined our PHENSIM analysis with an enrichment analysis to determine if a prediction made by our methodology was based on adequate data . Comparisons were performed using the Average Pathway Perturbations as reported by PHENSIM  and A549 transduced with ACE-2 (MOI 0.2 and 2) cells. Transcriptomics data for all cell lines were collected from GEO dataset GSE147507 and Log2-LogFCs were computed. Next, LogFCs were compared with our predicted Activity Scores (AS) by accounting for their direction of perturbation. We compared our predictions with the genes that Blanco-Melo et al. reports as important in the antiviral host-response to SARS-CoV-2. Furthermore, using an unbiased approach and to verify the accuracy of PHENSIM, we assessed the top-10 upregulated and top-10 downregulated genes for each cell-line. Finally, we assessed our viral simulation with results from Draghici et al. 2020 and Catanzaro et al. 2020 30.We compared our results with published in vitro experiments from Blanco-Melo 1) MITHrIL, 2) SPIA, 3) Reactome Pathways and 4) Gene Ontology Enrichment analysis. MITHrIL pathway analysis was performed as described in Alaimo et al., 2016 68. We used the LogFC of DEGs for all cell lines to perform MITHrIL perturbation analysis on the KEGG meta-pathway. Therefore, all values were aggregated on a pathway basis to compute an Accumulator and a p-value. Finally, p-values were adjusted for multiple hypotheses using the Benjamini-Hochberg FDR correction. Results were filtered by an FDR-adjusted p-value of 0.05 and ranked using the Accumulator. The top-25 significant pathways were reported in et al., 2009 69; the LogFC of DEGs and ranked pathways were calculated using FDR-adjusted p-values as computed by SPIA. Pathways with a p<0.05 were considered significant.Pathway analysis was applied to the transcriptomics data to determine which biological processes were altered by the viral infection. We used 4 pathway analyses approaches to assess the most impacted pathways: 70, and Gene Ontology, using the GOfuncR package 71. All results produced by the 4 pathway methodologies were collected and considered significant with an FDR-adjusted p-value<0.05 (Finally, to further expand our understanding of the biological processes affected by the infection, we performed enrichment analysis on both Reactome Pathways, using the ReactomePA package lue<0.05 .https://github.com/alaimos/phensim-covid19 . Website: https://phensim.atlas.dmi.unict.it/15.All input data, raw images, and source codes for PHENSIM are available at et al. respectively 10. For transcriptomic data, raw counts were normalized and analyzed for differential expression using the DESeq2 pipeline as previously described 72. All genes with an FDR-adjusted p-value<0.05 and absolute LogFC>0.6 were considered differentially expressed. In addition, we considered all genes showing an average read count<10 as non-expressed. All p-values computed by PHENSIM are corrected for multiple hypotheses testing, using the q-value algorithm 65. For proteomic data, Normalized LC-MS/MS data were downloaded and significance was tested using unpaired two-sided Student’s t-tests with equal variance assumed. All values were aggregated on a pathway basis to compute an Accumulator and a p-value, and p-values were adjusted for multiple hypotheses using the Benjamini-Hochberg FDR correction. Results were filtered by an FDR-adjusted p-value of 0.05 and ranked using the Accumulator.Statistical methods for transcriptomics and proteomics were applied as described by Blanco-Mello and Bojkova SupplementSupplementSupplementSupplement


Evidence-based guidelines have been developed outlining the concomitant use of anti-tumor necrosis factor alpha (anti-TNF) agents and immunomodulators including azathioprine (AZA) and methotrexate (MTX) in both adult and pediatric populations. However, there exists a paucity of data guiding evidence-based strategies for their withdrawal in pediatric patients in sustained remission. This narrative review focuses on the available pediatric evidence on this question in the context of what is known from the larger body of evidence available from adult studies. The objective is to provide clarity and practical guidance around who, what, when, and how to step down pediatric patients with inflammatory bowel disease (IBD) from combination immunotherapy. Outcomes following withdrawal of either of the two most commonly used anti-TNF therapies [infliximab (IFX) or adalimumab (ADA)], or immunomodulator therapies, from a combination regimen are examined. Essentially, a judicious approach must be taken to identify a significant minority of patients who would benefit from treatment rationalization. We conclude that step-down to anti-TNF (rather than immunomodulator) monotherapy after at least 6 months of sustained clinical remission is a viable option for a select group of pediatric patients. This group includes those with good indicators of mucosal healing, low or undetectable anti-TNF trough levels, lack of predictors for severe disease, and no prior escalation of anti-TNF therapy. Transmural healing and specific human leukocyte antigen (HLA) typing are some of the emerging targets and tools that may help facilitate improved outcomes in this process. We also propose a simplified evidence-based schema that may assist in this decision-making process. Further pediatric clinical studies are required to develop the evidence base for decision-making in this area. Given recent advances in the management of inflammatory bowel disease (IBD), a higher proportion of patients are being exposed to both biological and immunomodulator therapies earlier in their treatment course and for longer periods of time. First-line anti-tumor necrosis factor alpha (anti-TNF) treatment with infliximab (IFX)  for children with moderate-to-severe Crohn's disease (CD), for instance, has recently been shown to convey advantages over conventional therapy in a 2020 randomized control trial (RCT) . While eClinical observation and primarily adult-based data suggest that a significant proportion, up to 25–40%, of patients with quiescent disease on combination therapy may be stepped down to monotherapy with an immunomodulator and maintain remission for many years. Relapse rates approximate 50% between 1 and 2 years post step-down to monotherapy with either agent , 25–32. The current clinical challenge lies in the judicious selection of patients for whom drug withdrawal will prove beneficial. This involves a multifactorial assessment of where the threshold lies such that the benefits of cessation outweigh ongoing treatment. Holistically, any philosophy around “treat to target” and individualized medicine in IBD must assimilate this question around who can have therapy withdrawn, as well as the timing and manner of how this should best be done. The complex interplay between the perceptions and prejudices of both the physician and of patient (the child and their guardians) will also influence the decision –37.via avoidance of anti-drug antibodies and higher sustained drug levels, with additional synergistic effects likely  that demonstrated superiority of combination with AZA over IFX monotherapy based on proposed mechanisms described above. In this group of steroid refractory patients, who were naïve to both anti-TNF and thiopurines, induction therapy with the combination showed almost two-fold higher rates of mucosal healing at 6 months  with lower rates of serious adverse events (Both pediatric and adult guidelines (for CD) recommend combination therapy particularly where IFX is the anti-TNF agent used , 3. Thise events .p = 0.017 for IFX, 23.7% (18/76), p = 0.032 for AZA] , p = 0.001]. There was less anti-drug antibody formation in the combination arm [(3% (1/31) vs. 19% (7/37) in the IFX monotherapy group]. Short study duration and incompleteness of IFX-antibody analysis were important limitations of the study. However, it provides rare RCT level evidence in the adult UC context and, overall, favors combination IFX/AZA over monotherapy with these limitations in mind.The UC-SUCCESS trial—the solitary RCT in ulcerative colitis (UC) comparing combination therapy (IFX/AZA) vs. monotherapy with either IFX or AZA in 239 anti-TNF naïve adults—reported superior remission rates at 16 weeks  . Week 16n = 126] and the 2007 prospective study of Vermeire et al. of 174 CD patients with on-demand IFX dosing provide evidence for MTX in reducing anti-drug antibodies and enhancing anti-TNF levels, with the latter showing equivalent efficacy for MTX and AZA in this regard (p = 0.01) over the 12-month study period, it must be noted that there were no significant differences in clinical outcomes seen within that time frame. A caveat to the COMMIT results is that a large proportion of participants were given corticosteroids during the induction phase, followed by a prescribed taper and discontinuation by week 14. There is no published RCT comparing the two immunomodulators head to head in CD although a pediatric one is currently in progress . A caveat to extrapolating the outcomes with ADA treatment here into the pediatric population is that the vast majority of the 219 children included in the PANTS cohort were managed with IFX.Specific pediatric data are limited but some evidence for children can be extracted from the prospective PANTS cohort study with 12 months follow-up of 1,610 anti-TNF naïve patients with active luminal CD−14% of whom were pediatric (6–18 years) . Treatmep < 0.001)  . MTX co-European pediatric UC guidelines also recommend an immunomodulator where IFX is the biological agent used but, in contrast to their less prescriptive CD guidance, favor thiopurines over methothexate , 4. The p = 0.019). While mucosal healing was more likely attained sooner in the combination arm, this benefit was not maintained at 12 months . Meta-analyses of the adult literature on this question have not found significant benefit to combination therapy with ADA  showed no difference in clinical outcomes at 1 year follow-up when ADA was used with or without an immunomodulator. The longer-term outcomes of this patient cohort are pending publication and the effect of the significant reduction in immunogenicity when ADA is used in combination may become more apparent with time.A 2020 retrospective study from Targownik et al. examining long-term outcomes in more than 11,000 Canadian patients (6% of whom were pediatric) treated with an anti-TNF showed significantly improved clinical efficacy when adalimumab was combined with either immunomodulator in both UC and CD  (p = 0.035) , indicative of the now well-appreciated discrepancy between clinical and endoscopic outcome measures.A 2011 multicenter, United Kingdom retrospective study in 72 children with CD showed two-fold higher remission rates when ADA was used in combination rather than as monotherapy at 12 months follow-up  . A prosp= 0.035) . InteresImmunogenicity appears to be far less of an issue with newer biological agents such as vedolizumab and ustekinemab. Combination therapy with these agents generally has not shown improved outcomes, at least in part due to the low immunogenicity (<6% rates reported) of both drugs –52. HoweThe overall body of evidence favors commencement of combination therapy as the default regimen for achieving sustained disease remission, particularly for younger patients and in those with more severe disease. Enhanced anti-TNF efficacy, earlier achievement of mucosal healing, and long-term durability are especially important in these patients. While the data are certainly more compelling for combination with IFX, immunogenicity data and real-world clinical outcomes mean combination therapy with adalimumab may emerge as more standard care, rather than the exception in recalcitrant cases only. Patient selection for, and timing of, withdrawal follows as the next key decision for this ever-growing patient population on anti-TNF therapy managed with co-immunosuppression.Pariente et al. synthesized potential key risk factors for relapse post step-down from a combination regimen from various adult studies . Deep reWhile pediatric data on specific calprotectin cutoff levels and relapse risk post step-down are sparse, a level below the 100 mcg/g associated with “deep healing” in this population would be in keeping with the evidence around improved outcomes post withdrawal in the context of presumed mucosal healing . Pragmatp < 0.001)  with small bowel involvement –62. Up t< 0.001) . A recen< 0.001) .None of the studies analyzing predictive factors for relapse post step-down from combination to monotherapy evaluated transmural healing specifically, but this finding  may reasonably be assumed to help predict those patients who will tolerate therapeutic rationalization longer term.*05 and a variant in the gene C-X-C motif chemokine 12 (CXCL12) as two key markers predicting immunogenicity . Stratifying further, 90% of patients treated with IFX monotherapy carrying this “risk” allele developed drug antibodies by week 52. Conversely, those without this allele who were treated with ADA in combination with an immunomodulator had a 10% rate of anti-drug antibody development over the same period.There is emerging evidence around the potential utility of specific human leukocyte antigen (HLA) typing and other specific genetic markers in predicting risk of immunogenicity to anti-TNF therapies. These markers may factor in assessing risk of relapse, with or without drug withdrawal, at an individual level. Subsequent analysis from the PANTS cohort (adult and pediatric) and work from the adult European consortium ABIRISK  identified HLA-DQA1genicity , 66. Carn = 147, rheumatoid arthritis n = 229, IBD n = 184) on anti-TNF and other biologic therapeutics showed a 1.5- and 4-fold risk for immunogenicity in heterozygotes and homozygotes for HLA-DQA1*05, respectively. They found that patients homozygous for a minor allelic variant (rs10508884) in the CXCL12 gene also had four-fold higher rates of anti-drug antibody development and that CXCL12 protein levels above the median correlated with significantly higher rates of immunogenicity . This genetic profiling of immunogenicity risk could be used in concert with TDM to enhance the durability of anti-TNF therapies, which is crucial in pediatric IBD. It would prove an excellent example of the widespread and practical use of pharmacogenomics in the clinic. It would add further information to the decision matrix in determining who will benefit most from withdrawal and which therapy should be first withdrawn.The ABIRISK cohort of 560 patients with autoimmunity . Meta-analysis by Chalhoub et al. specifically addressed response and relapse with ADA monotherapy vs. combination in adult CD  and withdrawal groups . Overall safety data were equivalent between groups, and this study did not identify factors predicting either the need for intensification or successful withdrawal.Work by Kierkuś et al. provides one of the few pediatric randomized investigations into this question, albeit limited significantly by short follow-up intervals . Step-doArguments in support for earlier withdrawal of anti-TNF, including significant cost savings, reduced risk of opportunistic infections, and other serious side effects, are in part negated by the high relapse rates from clinical studies quoted here. Introduction of biosimilars into the therapeutic armamentarium has substantially reduced the cumulative financial costs. A 2016 United Kingdom IBD audit reported savings of 10–30%, exceeding £5,000/patient/year by switching to biosimilars, with larger savings likely to follow longer term , 72. Fur. showed re-induction required in 45% (54/121) at a median of 6 months post withdrawal and 9% (5/54) of relapsers requiring surgery by 1 year follow-up  at a median of 4 months, with 6% (1/18) of relapsers requiring colectomy.. A similollow-up . HoweverPediatric data on anti-TNF withdrawal are limited to small observational studies. The retrospective study of Wynands et al. in children (10.7 ± 2 years) was an early warning against premature de-escalation to sole immunomodulator treatment (AZA) in those with more severe CD . Of 36 pp = 0.009) and incomplete mucosal healing  predicted relapse. Retreatment with IFX in relapsers showed efficacy rates of 91% in keeping with larger studies in adults.Kang et al. found relapse rates at 1, 4, and 6 years of 19, 62, and 75%, respectively, in children with moderate-severe CD who had IFX withdrawn after 1 year of sustained clinical remission on combination IFX and AZA —reported a five-fold increased risk of infection [odds ratio (OR) 14.5 (95% CI 4.9–43) vs. 2.9 (95% CI 1.5–5.3)] for two or more immunosuppressives  vs. monotherapy with either of the three . Of noteConcerns around lymphoma risk associated with AZA use for >2 years in young males and the role of primary EBV in contributing to this risk of lymphoproliferative disease (particularly hemophagocytic lymphohistiocytosis) that have arisen from extensive population studies such as CESAME and DEVELOP have prompted both North American pediatric and European adult guidance for gastroenterologists to “consider” EBV status and gender in the combination therapy decision , 78–80. With conflicting results on the efficacy of combining methotrexate with biologicals mentioned previously, step-down to anti-TNF monotherapy represents a reasonable step for selected children in sustained remission as long-term thiopurines carry a small but additional, long-term risk. This is reflected in current, real-world PIBD practice as indicated in a 2021 survey from 62 pediatric gastroenterology centers where withdrawal of immunomodulators as the initial step-down from combination therapy was the preferred option for 88% (59/67) of physicians .In the realization that relapse is a possible, if not probable, outcome for many patients who have undergone treatment rationalization (even in those stratified as the lowest risk) beyond a 5-year follow-up period, it is worth considering the re-treatment outcome data. Most of the data here focus on re-treatment outcomes following anti-TNF withdrawal given concerns around immunogenicity, loss of response, and potential adverse events on re-introduction. In terms of success of retreatment with the withdrawn anti-TNF, the body of evidence from the aforementioned studies in adults and pediatrics alike consistently indicates efficacy rates exceeding 85%, with negligible rates of serious adverse events with re-induction –56, 81. Looking beyond biochemical and histopathologic parameters, the developmental phase and patient factors around the acceptability of relapse at a specific time must also be carefully considered before embarking on a planned withdrawal. Optimization of growth and pubertal development, avoidance of interruption of educational/vocational requirements, and consideration of the psychosocial implications of potential relapse should factor in the decision-making conversations with the patient and their family.In order to optimize outcomes for pediatric patients with IBD and improve the evidence base for decision-making on this topic, some of the unmet research questions include, but are not limited toshort-, medium-, and longer-term outcomes after immunomodulator withdrawal from a combination regime;risk stratification and approach to clinical relapse in this context;head-to-head studies of co-immunosuppression strategies (thiopurines and methotrexate) with anti-TNF therapies in both pediatric CD and UC;efficacy of co-immunosuppression with newer biologics and small molecules;outcomes, risk stratification, and optimization of timing and approach to withdrawal of anti-TNF therapies for patients in long-term remission;clinical utility of genetic profiling including HLA typing in predicting individual response, durability, immunogenicity to anti-TNF treatment, and how to tailor co-immunosuppression based on these data; andclarification of treatment targets including “deep” healing and transmural healing and how these may be incorporated into routine clinical practice around the approach to treatment monitoring and withdrawal.Based on the available adult and pediatric data, it is reasonable to suggest step-down to anti-TNF monotherapy after at least 6 months of sustained clinical remission in a select group of patients . UnaccepSummarizing the available published evidence, the best predictors of a successful step-down to monotherapy include:low or undetectable anti-TNF trough levels in the context of quiescent disease;FCP < 100 mcg/g, or other combined indicators of mucosal and/or transmural healing;no significant predictors for severe disease ;no previous escalation of the anti-TNF regimen required; andminimum of 6 months of combination therapy prior to step-down.Further long-term, high-quality evidence is required to help guide the decision-making process around this important question for patients and physicians alike. HLA and genetic risk profiling for immunogenicity to biologics may help fine-tune the patient selection process. We are currently undertaking a large, multi-center clinical study, as part of the Paediatric IBD Porto Group, into immunosuppressive withdrawal and hope to provide further information including long-term follow-up outcomes in this area of interest, and at times controversy, in PIBD.JM and RR conceptualized, designed and produced the initial manuscript and revisions for the final submission. PH and DW provided critical revisions to the manuscript up to the final submission. All authors contributed to the article and approved the submitted version.RR and PH were supported by an NHS Research Scotland Career Researcher Clinician award. JM was supported by a joint Edinburgh Children's Hospital Charity/Catherine McEwan Foundation research fellowship (Grant No. 2019-66).DW has received speaker's fees, travel support, and participated in medical board meetings with AbbVie, Roche, and Nestle Heath Sciences. RR has received speaker's fees, travel support, and participated in medical board meetings with Abbvie, Janssen, Takeda, Celltrion, Pharmacosmos, and Nestle. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The appropriate characterization of burn depth and healing is paramount. Unfortunately, the accuracy of approximating thermal injury depth among all physicians is poor. While tools to improve detection accuracy, including laser doppler imaging and laser speckle imaging exist, these technologies are expensive and limited to specialized burn referral centres. They also do not provide an easy means for quantitative, interval tracking of burn healing. Considering these limitations, the application of artificial intelligence has garnered significant interest. We herein present the use of three novel machine learning and computer vision-based algorithms to track burn wound healing.Convolutional neural network (CNN) models, were trained on 1800 2D color burn images, to classify them into four burn severities. These CNNs were used to develop saliency algorithms that identify the highest “attention” pixels used to recognize burns. Image-based algorithms that count these attention pixels of the CNN, count pixels representing red granulation of burns, and measure burns, were also developed. As proof-of-concept, we tracked the healing of a localized burn on a 25-year-old female patient. The patient suffered a scald on the dorsum of the foot, resulting in a deep partial-thickness burn. Opting out of surgical intervention, the patient visited the hospital over a 6-week period for treatment with non-adhesive dressings and silver nitrate. High-resolution images of the burn, with and without a fiducial marker, were captured with a smartphone camera every 7-days. Images were taken under institutional lighting and used as algorithmic inputs.Data analyses indicate that the healing of the open-wound area was accurately measured in millimetres (+/- 1.7 mm error) using a fiducial marker (18.3 mm diameter). The open-wound area shrank consistently from week 1 to week 6 seen in . The normalized, 2D colour images, where the “red” pixel value was counted , confirms the reduction of the red granulation in the wound. The saliency algorithm also measured a percentage reduction in the machine learning model’s total attention pixels over the 6-week period . This suggests that the model was less discerning of the healing burn wound over time, suggesting burn healing, which was also clinically validated.


Background: Home treatment of patients affected by COVID-19 is still a matter of daily debate. During the clinical evolution of the disease, there are high risks of lung failure, which requires oxygen therapy. Here, we report our clinical experience with at-home treatment using high-flow nasal cannula in non-hospitalised patients with confirmed COVID-19. Patients and methods: In this study, 18 patients with moderate-to-severe respiratory failure secondary to COVID-19 were monitored at home daily for temperature and SpO2 measurements. Other parameters such as saturation of peripheral oxygen (SpO2), SpO2/FiO2 (fraction of inspired oxygen), temperature, and lung performance were monitored periodically. Depending on oxygen requirements, the patients also received either standard oxygen via a face mask or, if higher FiO2 required, high-flow nasal cannula (HFNC). Results: All 18 patients had favourable outcomes and recovered from COVID-19. No death was recorded in this group. Conclusion: Our clinical experience proves that high-flow nasal cannula oxygen therapy may be considered for at-home treatment of COVID-19 patients with moderate lung failure. This could be useful for further treatment during the pandemic and may also be considered in future epidemics. COVID-19 is a recent, ongoing pandemic which has spread quickly, overwhelming hospitals and increasing demand for critical care services. It has a high mortality rate which ranges from 62% to 97% in patients requiring advanced respiratory support [We describe the at-home monitoring and the clinical course of 18 patients affected by COVID-19—14 male and 4 female, with a mean age of 68.7 years. All patients had a nasopharyngeal swab positive for SARS-COV-2. Patients were enrolled consecutively, and the observation period was between October and November 2020. According to the guidelines for home care of SARS-COV-2 patients, all the necessary treatments—azithromycin, methylprednisolone, low-molecular-weight heparin—were administered by health workers and nurses specialised in the management of patients affected by COVID-19 . We evalFor at-home monitoring, patients used a thermometer, a blood pressure monitor, and a pulse oximeter. Temperature, saturation, heart rate, and blood pressure were measured 3 times per day. The parameters were measured daily even when symptoms were not present due to the well-known clinically significant hypoxemia in the absence of dyspnoea. The patients transmitted the data via text messages to their referring physician. A doctor was available to provide advice by phone, conduct an online consultation, modify treatment, order additional medication , change the dosage of treatment, organise laboratory tests, or determine a change in patient support with oxygen therapy. Patients received therapy with azithromycin 500 mg daily for 6 days, methylprednisolone 0.5 mg/kg/day, and low-molecular-weight heparin (enoxaparin) 4000 IU once a day until SpO2 normalised. In case of persistent irritating cough, patients were given paracodeine. When respiratory failure occurred in the presence of one or more risk factors for thromboembolism, an intermediate or high dose of enoxaparin 6000 or 8000 UI) was administered after the assessment of the risk of bleeding [000 UI waRespiratory support was provided with standard oxygen via nasal cannula, Venturi mask, and mask with reservoir to 9 patients, while the other 9 patients (SF < 144) were treated with HFNC  supplemeComorbidities with an associated increased risk of worse outcome for COVID-19 were also recorded.2, (34%), systemic arterial hypertension (82%), and pre-existing respiratory diseases such as COPD and pulmonary interstitial disease (34%). All patients were former smokers. At the onset of symptoms, the average temperature was 38.7 °C  devices. These patients presented with severe dyspnoea and persistent irritating cough at night. HFNC was chosen as an alternative therapy that enhances gas exchange and reduces work of breathing [The use of HFNC for patients with COVID-19 and lung failure has already been reported in other studies ,11 with We present our approach to home care for patients with moderate or severe respiratory failure secondary to COVID 19. In the coming months, we hope to learn whether our programme is effective in increasing the efficiency of health workers, to mitigate the health crisis and alleviate suffering. Of course, this study’s great limitation is the choice of patients to be treated at home, which is different to a possible selection of patients who could be treated at home or in hospital. Therefore, we look forward to learning from other groups of patients who implement similar therapeutic interventions, or alternatively, we may hypothesize a study in which a control group of selected patients treated in a different place or way may be included. We observed that an adequate respiratory support with close clinical and instrumental monitoring proved to be an effective clinical response in patients with moderate to severe acute respiratory failure, and this further encourages at-home treatment of COVID-19 pneumonia. Furthermore, if these results are confirmed, other types of infective/viral pneumonia could be evaluated for at-home treatment in the future, even with severe respiratory failure, thus reducing the need for hospitalisation.


As the COVID-19 pandemic unfolded, emergency departments (EDs) across the world braced for surges in volume and demand. However, many EDs experienced decreased demand even for higher acuity illnesses. In this study we sought to examine the change in utilization at a large Canadian community ED, including changes in patient demographics and presentations, as well as structural and administrative changes made in response to the pandemic.This retrospective observational study took place in Ontario, Canada, from March 17–June 30, 2020, during province-wide lockdowns in response to COVID-19. We used a control period of March 17–June 30 in 2018–2019. Differences between observed and expected values were calculated for total visits, Canadian Triage and Acuity Scale (CTAS) groups, and age groups using Fisher’s exact test. Length of stay (LOS), physician initial assessment time (PIA), and top primary and admission diagnoses were also examined.Patient visits fell to 66.3% of expected volume in the exposure period . CTAS-1 (highest acuity) patient volumes dropped to 86.8% of expected (P = 0.1964) while CTAS-5 (lowest acuity) patient volumes dropped to 32.4% of expected (P <0.0001). Youth (0–17), adult (18–64), and senior (65+) visits all decreased to 37.4%, 71.7%, and 72.9% of expected volumes, respectively (P <0.0001). Median PIA and median ED LOS both decreased . The most common primary diagnosis in both periods was “other chest pain.” Viral syndromes were more prevalent in the exposure period. The top admission diagnoses were congestive heart failure in the control period (4.8%) and COVID-19 in the study period (3.5%).ED utilization changed drastically during COVID-19. Our ED responded with wide stakeholder engagement, spatial reorganization, and human resources changes informed by real-time data. Our experiences can help prepare for potential subsequent “waves” of COVID-19 and future pandemics. The first case of COVID-19 in Canada was identified in Toronto, Ontario, on January 25, 2020. Over the next few months, every province and territory in Canada declared a state of emergency and instituted lockdowns in response to increasing infection spread, which reached a total of 1,155,834 cases as of April 23, 2021.3Based on challenging international experiences, many Canadian emergency departments (ED) sought to prepare for a high flux of patients with infectious symptoms.14Given the large role of EDs in the response to the COVID-19 pandemic, it is important to investigate the landscape of ED utilization during the initial period. The objective of this study was to characterize the utilization of a large community hospital in Ontario, the most populous province, after the declaration of province-wide lockdowns, as well as to explore observed local changes in response to these effects. This included changes in incoming patient demographics, common presentations, and internal indicators of operations such as length of stay (LOS) and physician initial assessment (PIA) times.These insights can help inform stakeholders in the provision of emergency care by examining how populations respond to pandemics in their decisions to visit EDs. The findings have implications for human resource planning, hospital resource management, supply chain manufacturing and procurement, and local continuing education initiatives.This was a retrospective, population-based cohort study conducted in the Mackenzie Health (MH) Hospital ED. The study period was from March 17–June 30 in the years 2018–2020. The exposure time period starts March 17, 2020, during which the provincial government of Ontario declared a state of emergency response to COVID-19 and instituted lockdowns of non-essential services. Data collection ended on June 30, 2020. We used a control period of March 17–June 30 in the years 2018–2019 to obtain baseline characteristics for examined variables. This study was approved by the Southlake Regional Health Centre Research Ethics Board.What do we already know about this issue?Emerging evidence shows that COVID-19 significantly disrupted demand for healthcare internationally.What was the research question?How has COVID-19 changed the demand for emergency care in large community hospitals?What was the major finding of the study?We found decreased volumes, increased acuity, older demographics, and higher proportions of infectious presentations.How does this improve population health?By understanding the impact of pandemics on population-level demand for emergency care, we can better prepare for future outbreaks.Mackenzie Health is a large 506-bed community hospital in Richmond Hill, Ontario. The ED received 111,384 visits in 2019. Richmond Hill is a city of 195,022 in Ontario, a province with a population of 13,448,494.We retrieved data from the hospital electronic database . We examined total daily visits and acuity level via the Canadian Emergency Department Triage and Acuity Scale (CTAS).International Classification of Diseases, 10th rev (ICD-10) format. We compared the volume and proportion of the top five primary diagnoses to the 2018–2019 control period. This was also done for the top five admission diagnoses. Finally, the volume and proportion of acute myocardial infarction (AMI) and acute stroke were examined for the purpose of comparison to emerging literature on the impact of COVID-19 on those conditions in other jurisdictions.18Primary diagnoses were defined as the most responsible diagnosis coded in the hospital electronic database according to the th percentiles. Categorical variables of primary diagnoses and admission diagnoses were summarized by percentages. We performed statistical analysis using SAS version 9.4 software .We compared observed numbers of visits to expected numbers as projected by the method in Johnston et al (2002) (Box A1).P<0.0001)  .P<0.0001 for all groups).During the exposure period the volume of all CTAS categories fell compared to expected values (32.4%–86.8%) . The volP-values <0.0001)  volumes during the exposure period were below expected  . Youth vThe median ED LOS decreased from 3.3 hours (Q1–Q3: 1.9–5.6) across 2018–2019 to 3.0 hours (Q1–Q3: 1.7–5.1) during the exposure period . The 90tR073 - Other chest pain   . In the The proportion of AMI and acute strokes both increased . The absolute number of AMIs increased from an average of 45 per year over 2018–2029 to 83 in 2020 (84% increase). The absolute number of acute strokes decreased from an average of 188 in 2018–2019 to 177 in 2020 (5.9% decrease).Top admission diagnoses are outlined in In this study we noted significant decreases in ED volumes, particularly for youth patients (aged 0–17 years) and lower acuity patients (CTAS 4–5). This was accompanied by decreases in PIA and LOS. COVID-19 entered the most common primary diagnoses overall as well as the most common primary diagnoses causing admission. Infectious primary diagnoses became more common as well. We also noted a large increase in AMIs and a slight decrease in acute strokes, although they both increased in terms of the proportion of total ED visits. Additionally, there was a slight decrease in the number of patients who were dead on arrival to the ED during the study periods in 2018 and 2019 to 2020 .The decreases in ED volumes are similar to international reports.There were significant drops in low-acuity patients, similar to what was observed in the SARS ourbreaks, 24The proportion of influenza-related conditions and respiratory tract infections increased during COVID-19, similar to the increase in influenza-like illness volumes seen in the US during the H1N1 outbreak.14As the makeup of emergency visits changed at MH, department leadership instituted a number of structural and human resource changes to meet the newly changing landscape. This undertaking necessitated ongoing wide stakeholder engagement both within the ED and with the hospital administration. In response to the significant decrease of patient volumes, more agile emergency-physician staffing adjustments were made. The changes resulted in almost monthly changes to the ED staffing template in terms of physician hours and zone coverage to meet demand. These adjustments required ongoing real-time data analysis and feedback, as well as constant engagement with the clinicians in the ED.Although there was a decrease in visits of all triage levels, lowest severity patients visits (CTAS 4–5) decreased the most in both volume and in proportion. At MH, this led to the conversion of the minor treatment zone into an admitted isolation patient outflow area to support the sub-acute zone. Additionally, the sub-acute zone was fitted with high-efficiency particulate air (HEPA) filters and added COVID-19 screening capabilities resulting in the zone in its entirety becoming a positive pressure area. Lower acuity patients were treated in the ambulatory zone, which was equipped with physically distanced chairs. Patient educational material was presented with signage and on media screens to remind patients of proper hand hygiene and physical distancing in the ED. Hallway beds were also removed to further allow for distancing. However, these changes decreased the total number of available acute care beds from 64 to 37 in the ED. Therefore, surge planning was frequently updated in the case of sharp increases in patient visits.Additionally, MH experienced an increased proportion of patients presenting with respiratory symptoms. To meet this demand, the resuscitation area was converted into an ambulatory isolation area with eight chairs, two beds, and a new HEPA filtration system. Moreover, a dedicated and physically separate COVID-19 assessment centre was opened at MH, which experienced drastic increases in volumes during the study period . This asAn ongoing focus on infection control was maintained throughout this time period and a multitude of infection control changes were undertaken in response to the pandemic. These included updating triage screening questions; installation of HEPA ventilation in COVID-19 areas; increases in cleaning frequency and use of full-spectrum UV disinfection machines; reserving negative pressure rooms for aerosol-generating medical procedures; and the use of a dedicated multidisciplinary intubating team for high-risk intubations.Delayed presentations of critical illnesses mean that clinicians and administrators will need to prepare with downstream exacerbations of time-sensitive conditions that would have otherwise been managed earlier. Therefore, continued proactive advocacy and messaging informing the public of the safety of EDs ahead of subsequent “waves” is crucial.Additionally, emergency care resources can be distributed to meet new demands. Changes in patient demographics, i.e., a shift towards older patients, can be met with increased geriatric support, such as extended geriatric nurse practitioner hours. Likewise, emergency care resources can be shifted to accommodate for increases in influenza-like or infectious presentations, such as increasing patient isolation capacity.The data collection method ensured a complete sample of all patient visits during the exposure period. Additionally, the method for calculating expected values accounted for both seasonal variation and year-to-year variation.Z038 – Encounter for observation for other suspected diseases and conditions ruled out, which limits inferences about most common presentations. Finally, the observed trends may have been influenced by changes in local care provision. Anecdotally, some local family physician offices closed, which may have redirected some low-acuity complaints to the ED. However, no local hospitals closed or were designated as “COVID-19 hospitals”.However, the exposure period was subjectively chosen by expert opinion to be based on Provincial Government actions and may not precisely capture the period in which shifts in public sentiment and emergency care demand occurred. Additionally, the determination of presenting diagnoses was abstracted from a hospital database using ICD codes and were not confirmed via chart review. Some of these common primary diagnoses are subject to interpretation, such as Emergency department utilization changed significantly during COVID-19. This large Canadian ED experienced lower volumes, decreased proportions of lower-acuity and younger patients, and an increase in viral illness presentations. The experiences of this local ED can help equip ED administrators with structural and process-based changes for potential subsequent “waves” of COVID-19 and future pandemics.


The global coronavirus disease 2019 (COVID-19) pandemic has drastically changed the current practice of medicine worldwide. As more clinical data is collected and processed, we are beginning to have an understanding of which patients are more at risk for severe complications of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Preliminary data has shown that patients with lung cancer are disproportionally affected by the current COVID-19 pandemic. Furthermore, studies have shown that lung cancer patients are also significantly more likely to be admitted to the ICU and need mechanical ventilation. A specific subset of patients that are even more at risk for severe COVID-19 are those that require lung cancer surgery. To minimize the risk of SARS-CoV-2 infections in patients undergoing surgery, new treatment guidelines and preventive measures are necessary. In this review, we summarize the latest evidence regarding recommendations for patients undergoing lung cancer surgery in the COVID-19 era.The current coronavirus disease 2019 (COVID-19) pandemic has forced healthcare providers worldwide to adapt their practices. Our understanding of the effects of COVID-19 has increased exponentially since the beginning of the pandemic. Data from large-scale, international registries has provided more insight regarding risk factors for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and has allowed us to delineate specific subgroups of patients that have higher risks for severe complications. One particular subset of patients that have significantly higher risks of SARS-CoV-2 infection with higher morbidity and mortality rates are those that require surgical treatment for lung cancer. Earlier studies have shown that COVID-19 infections in patients that underwent lung cancer surgery is associated with higher rates of respiratory failure and mortality. However, deferral of cancer treatments is associated with increased mortality as well. This creates difficult situations in which healthcare providers are forced to weigh the benefits of surgical treatment against the possibility of SARS-CoV-2 infections. A number of oncological and surgical organizations have proposed treatment guidelines and recommendations for patients planned for lung cancer surgery. In this review, we summarize the latest data and recommendations for patients undergoing lung cancer surgery in the COVID-19 circumstance. The global coronavirus disease 2019 (COVID-19) pandemic and its resultant strain on healthcare systems have drastically changed the current practice of medicine worldwide . As moreOne particular subset of cancer patients that may be at higher risk for severe COVID-19 are those that require lung cancer surgery for non-small cell lung cancer (NSCLC). Earlier data has shown that COVID-19 in patients with NSCLC that underwent lung resection is associated with higher likelihood of respiratory failure and mortality ,22. CurrAn increasing number of studies have shown that cancer patients are disproportionally affected by the current COVID-19 pandemic ,15,28. IFor lung cancer patients and patients with hematological malignancies, the risk of mortality due to SARS-CoV-2 infections is the highest among all cancer patients ,32. In tAside from tobacco-related damage, lung cancer has been shown to be an independent risk factors for COVID-19 morbidity and mortality as well . SeveralOne important hypothesis as to how lung cancer increases the risk for severe COVID-19 disease is that the SARS-CoV-2 is able to modulate the lung tumor microenvironment (TME). The TME is the immediate surrounding environment of a primary tumor or metastasis and is mainly composed of stromal cells such as T-cells, B-cells, natural killer (NK) cells, fibroblasts, adipocytes, vascular endothelial cells, and pericytes. In general, these cells secrete signals that allow tumors to survive, grow, invade, and migrate, a process often referred to as oncomodulation . EarlierThere are several other factors that could contribute to higher risks for SARS-CoV-2 infections in lung cancer patients. Advanced age is a well-known factor that is associated with an increased risk of mortality in COVID-19 patients, with mortality rates steadily rising from 65 years onwards. This age group also encompasses the majority of patients with lung cancer ,49. A laThe main goal of the treatment of patients with lung cancer during the COVID-19 pandemic is to provide high-quality oncological care while simultaneously minimizing the risks of contracting SARS-CoV-2 or the risk of severe complications should they develop COVID-19 . In the The ACS COVID-19 guidelines are divided into three triage levels: a semi-urgent setting (phase I) in which there are few COVID-19 patients and sufficient hospital resources, an urgent setting (phase II) in which there are many COVID-19 patients with limited ICU capacity or hospital resources, and a highly urgent setting (phase III) in which (nearly) all hospital resources are routed to COVID-19 patients. In phase I, ACS recommends deferring NSCLC cases with predominantly ground glass nodules (<50% solid), solid nodules <2 cm, or cases with an indolent histology. Patients with (predominantly) solid lung cancers >2 cm and negative clinical nodes, patients with node positive NSCLC, and patients postinduction treatment need to be treated as soon as feasible. In phase II, the guidelines recommend deferring all elective thoracic procedures. Tumor-associated infections and surgical complications in hemodynamically stable patients should be treated at this time. Alternatively, healthcare providers can also choose to transfer patients to hospitals in phase I or to offer alternative treatments if possible, e.g., neoadjuvant therapy, SBRT, or definitive chemoradiation therapy (CRT). Finally, the ACS recommends for phase III that healthcare providers defer all cases except for patients with threatened airways, tumor-associated sepsis, or hemodynamically unstable patients with surgical complications . The ASC3, pleural solid nodules > 1 cm, a volume doubling time <400 days, or a new solid component in a previously nonsolid nodule. Low priorities include discordant biopsies that are likely to be benign, operable pure ground glass opacifications (T1A), and the diagnostic workup or resection of nodules >500 mm3 with a known volume doubling time >600 days. For medium and low priorities, the guidelines state that SBRT is an acceptable alternative treatment if surgical treatment is indicated when no surgical capacity is available [The ESMO guidelines for lung cancer surgery divide cases into three priority levels: high, medium, and low. High-priority procedures include drainage with or without pleurodesis of pleural effusion, treatment of pericardial effusions, and evacuation of pleural empyema. In addition, T2N0 disease, resectable T3/T4 tumors, and resectable N1/N2 disease naïve from treatment after neoadjuvant chemotherapy are all considered high priority as well. Finally, procedures including mediastinoscopy, thoracoscopy, biopsy, and endoscopy for diagnostic or staging purposes are high priority as well. Medium priority includes discordant biopsies that have high chances of being malignant and resectable T1aN0 disease. Furthermore, the diagnostic workup or surgical treatment of nodules with the following characteristics are considered medium priority as well: solid nodules or solid components in partially solid nodules >500 mmvailable . HoweverA few other noteworthy organizations that have published guidelines for lung cancer surgery are the British Thoracic Society (BTS), the International Association for the Study of Lung Cancer (IASLC), and the Thoracic Surgery Outcomes Research Network. The BTS and IASLC guidelines are less specific and offer more general recommendations for patients undergoing lung cancer surgery. In the guidelines of the BTS, the authors have given a set of general recommendations for the diagnosis and treatment of lung cancers. Regarding surgery, the guidelines recommend minimizing the hospital length of stay for patients planned for surgery by using minimally invasive approaches or day of surgery admission. Furthermore, patients with higher risks—more specifically, those not fit for a lobectomy—should be considered for referral for radiation therapy or radiofrequency ablation. In addition, surgeons may opt for surgical treatment without preoperative biopsy, using Herder score and frozen sections intraoperatively . The IASIn addition to these specific recommendations for patients undergoing surgery for NSCLC, the majority of guidelines include general guidance protocols as well. Patients planned for surgery should undergo reverse transcription polymerase chain reaction (RT-PCR) testing prior to treatment. If the COVID-19 test is positive, surgical treatment should be delayed for 2 to 3 weeks or until the patient is asymptomatic. All patients should be tested after their isolation period. Physicians and all operating room personnel should be screened daily for fever or COVID-19 symptoms. Healthcare providers with symptoms should be tested for COVID-19 and be quarantined for 2 weeks if the results are positive. In urgent surgical cases, procedures can be performed in specialized negative-pressure operating rooms. Surgeons and operating personnel should use full PPE. Postoperatively, patients should be treated in negative-pressure isolation rooms. Furthermore, the number of visitors should be drastically reduced or visitors should be prohibited altogether for patients after surgical treatment during their hospital stay ,59,61,62Since the beginning of the COVID-19 pandemic, healthcare providers worldwide have strived to offer non-COVID patients optimal treatments, despite the many strains the current pandemic has brought on our healthcare systems. In the case lung cancer patients, many endeavors have been made to ensure that patients receive treatments according to the latest therapeutic guidelines. For patients with early-stage or locally advanced NSCLC, surgical treatment is often recommended as primary modality or as treatment after induction chemotherapy. However, these patients have significantly higher risks for severe respiratory complications and mortality if they contract SARS-CoV-2, especially in the postoperative period ,34,50,51p = 0.03) and with increased rates of readmission (p = 0.004). There were no significant differences in 30-day morbidity or mortality rates. In addition, no differences were observed regarding oncological outcomes such as R0 resections, rates of nodal upstaging, or need for adjuvant chemotherapy [Several studies have investigated the safety and outcomes patients that underwent surgical resections for lung cancer. In a small retrospective study by Chang et al., 21 patients underwent pulmonary resection for lung cancer at NYU Langone Health between 13 March and 4 May 2020. Surgical treatments included 13 lobectomies, 6 segmentectomies, and 2 pneumonectomies. In this period, a total of 2087 COVID-19 patients were admitted to the same hospital as well. The authors investigated the safety of the patients that received surgical treatment, as well as healthcare provider safety, by using COVID-19 screening and by evaluating symptoms and early treatment outcomes. No major and five minor complications were observed in this patient cohort. The median hospital length of stay was 2 days. Furthermore, no previously COVID-19 negative patients or healthcare providers developed any symptoms or tested positive for SARS-CoV-2 infections in this period. The authors concluded that lung cancer surgery is safe, even if the surgical procedures are performed by healthcare providers who care for COVID-19 patients in hospitals with high rates of hospitalized COVID-19 patients . In a siotherapy . Other cotherapy .Despite our growing understanding of COVID-19 on the increasing numbers of studies regarding the effects of COVID-19 on our healthcare systems, many questions are still unanswered. For lung cancer surgery, the long-term epidemiological effects of treatment delays are yet to be seen. Furthermore, many patients have not visited hospitals despite symptoms due to concerns for contracting COVID-19 or due to healthcare providers discouraging them due to limited resources, resulting in an increasing number of preventable deaths. The aim of the numerous international guidelines and recommendations is to obviate this problem and to allow patients to receive the appropriate treatments that they need without excessive delays and in a safe manner. Furthermore, the collection of data by international registries such as TERAVOLT and CCC-19 will provide us with more insight into how (lung) cancer treatments should be tailored during this pandemic. The main hope of healthcare providers worldwide is that the vaccines will help slow the spread of COVID-19, allowing healthcare systems and hospitals to resume all treatments for non-COVID pathologies without further delays. Further research is still necessary to understand the pathophysiological mechanisms that lead to increased susceptibility and mortality rates of lung cancer patients that contract COVID-19. In addition, the safety of our surgical and nonsurgical treatments during COVID-19 surges need to be evaluated in larger, multicenter studies as well.