Host epigenome manipulation post coronavirus infection4 suggests that DNA methylation signatures may differentiate patients with SARS-CoV-2 infection from uninfected individuals, and help predict COVID-19 disease severity, even at initial presentation.Since the onset of the SARS-CoV-2 pandemic, most clinical testing has focused on RT-PCRWe customized Illumina’s Infinium MethylationEPIC array to enhance immune response detection and profiled peripheral blood samples from 164 COVID-19 patients with longitudinal measurements of disease severity and 296 patient controls.e.g.IRF7, OAS1). Using machine learning techniques, models built using sparse regression yielded highly predictive findings: cross-validated best fit AUC was 93.6% for case-vs-control status, and 79.1%, 80.8%, and 84.4% for hospitalization, ICU admission, and progression to death, respectively.Epigenome-wide association analysis revealed 13,033 genome-wide significant methylation sites for case-vs-control status. Genes and pathways involved in interferon signaling and viral response were significantly enriched among differentially methylated sites. We observe highly significant associations at genes previously reported in genetic association studies (In summary, the strong COVID-19-specific epigenetic signature in peripheral blood driven by key immune-related pathways related to infection status, disease severity, and clinical deterioration provides insights useful for diagnosis and prognosis of patients with viral infections. Viral infections affect the body in many ways, including via changes to the epigenome, the sum of chemical modifications to an individual’s collection of genes that affect gene activity. Here, we analyzed the epigenome in blood samples from people with and without COVID-19 to determine whether we could find changes consistent with SARS-CoV-2 infection. Using a combination of statistical and machine learning techniques, we identify markers of SARS-CoV-2 infection as well as of severity and progression of COVID-19 disease. These signals of disease progression were present from the initial blood draw when first walking into the hospital. Together, these approaches demonstrate the potential of measuring the epigenome for monitoring SARS-CoV-2 status and severity. Konigsberg et al. profile DNA methylation in blood samples from SARS-CoV-2 cases and controls. The authors use machine learning to classify infected vs. non-infected individuals and predict clinical outcomes related to disease severity. This pathogen is associated with coronavirus disease 2019 (COVID-19) first identified in Wuhan, China in 20196 and declared a pandemic on March 11, 20207. Since the onset of the pandemic, multiple tests for diagnosing COVID-19 have been launched, including real-time reverse transcriptase–polymerase chain reaction (RT-PCR), specific antibody detection, and next-generation sequencing assays that query for current or past infections1. With the exception of next-generation sequencing, which can discern viral subtypes, most diagnostic tests are viral strain dependent, can carry a high false negative rate, do not discern if the virus is viable and replicating, and do not predict clinical outcomes of infection9. For example, pre-symptomatic patients may test negative11 while patients who have recovered may continue to test positive though they are no longer infectious12. Accurate diagnostics are urgently required to control continued communal spread, to better understand host response, and for the development of vaccines and antivirals13.Coronaviruses (CoV) comprise a large group of human and animal pathogens, including the novel enveloped RNA betacoronavirus referred to as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)14, the U.S. ranks as one of the countries with the highest COVID-19 mortality rates15. Identification of which SARS-CoV-2-infected patients are most likely to develop severe disease would enable clinicians to triage patients via augmented clinical decision support. Having more information on disease severity has recently become critical due to widespread lack of hospital and intensive care unit (ICU) capacity, necessitating difficult decisions about resource triage. To our knowledge, no test can predict COVID-19 clinical course or severity, although work on cytokine abundance ratios after hospitalization has been proposed as a prognostic indicator of severe outcomes16.Individuals infected with SARS-CoV-2 have a variable course of infection, ranging from asymptomatic to death. Although the fatality rate varies tremendously according to demographic characteristics and co-morbidities3, specifically via DNA methylation. Viral-mediated antagonism of antigen-presentation gene expression in the case of Middle East respiratory syndrome coronavirus (MERS-CoV) was shown to occur via DNA methylation4. DNA methylation changes at cytosine-phosphate-guanine (CpG) sites have been increasingly leveraged in the emerging field of clinical epigenetics to characterize unique epigenetic signatures that diagnose disease. To date, considerable success has been demonstrated in developing highly accurate and robust machine learning (ML)-based disease classifiers using DNA methylation patterns to differentiate Mendelian disorders17, behavior disorders18, coronary artery disease19, and some cancers22. Consequently integration of a methylation-based disease classification can result in relevant improvement in clinical practice24.There is considerable evidence that enveloped RNA viruses such as CoV can manipulate the host’s epigenome via evolved functions that antagonize and regulate the host innate immune antiviral defense processesWith a goal to leverage Illumina’s Infinium MethylationEPIC Array to classify differential methylation signatures of SARS-CoV-2-positive  and control peripheral blood DNA samples (either confirmed SARS-CoV-2 negative or samples collected prior to the SARS-CoV-2 pandemic), we conducted this study to determine whether DNA methylation patterns could differentiate SARS-CoV-2-infected patients from non-infected patients from whole blood obtained from patients. Our secondary objective was to determine whether DNA methylation patterns could differentiate patients with SARS-CoV-2 infection who go on to develop severe disease. In this study, we identified a strong COVID-19-specific epigenetic signature in peripheral blood driven by key immune-related pathways related to SARS-CoV-2 infection status, disease severity, and clinical deterioration.https://research.cuanschutz.edu/university-research/covid-19-clinical-research/covid-19-biobank-specimen-repository) or the University of Colorado Emergency Medicine Specimen Bank (EMSB)25. Control subjects included patients from each study who tested negative for SARS-CoV-2 infection during the index visit. Through the University of Colorado COVID-19 Biorepository and the EMSB, patients tested were consented for blood collection and data abstraction from their electronic health record (EHR). Data obtained from EHR abstraction included demographics, past medical history, laboratory testing (including SARS-CoV-2), treatments, vital signs, hospital disposition, and clinical outcomes. In addition, previously collected samples from patients with acute upper respiratory viral infections  between February 5, 2018 and January 1, 2020 were obtained through the EMSB as SARS-CoV-2-negative controls. Additional biospecimens included discarded clinical samples from patients not approached for biorepository enrollment through the UCHealth University of Colorado Hospital Clinical Laboratory. Discarded samples were linked to a limited EHR dataset through the Colorado Center for Personalized Medicine’s health data warehouse, Health Data Compass, and then deidentified. The limited dataset included age, gender, race, ethnicity, viral test status (SARS-CoV-2 and other upper respiratory viruses), and clinical outcomes. The use of discarded samples and accompanying limited datasets was determined to be exempt from Institutional Review Board approval and the need for informed consent by COMIRB. All samples were frozen at −20 °C after collection prior to processing for methylation analyses.This protocol was reviewed and approved by the Colorado Multiple Institutional Review Board (COMIRB) and the research adheres to the ethical principles of research outlined in the U.S. Federal Policy for the Protection of Human Subjects. SARS-CoV-2+ were defined as those patients who tested positive for SARS-CoV-2 infection via a routine diagnostic RT-PCR assay in the Biobank at the Colorado Center for Personalized Medicine  or in the UCHealth University of Colorado Hospital Clinical Laboratory  of a nasopharyngeal swab collected in viral transport media; controls were defined as those who tested negative. Peripheral blood DNA samples were collected in EDTA tubes from patients seen at the UCHealth University of Colorado Hospital and tested for SARS-CoV-2 epigenetic signatures starting on March 1, 2020. Blood specimens were collected from patients consented to the University of Colorado COVID-19 Biorepository , to enhance the sensitivity of immune response detection. The custom panel targeted 262 genes with 7831 additional probes. While the majority of the additional probes targeted unique sequences within the genome, a number of probes were intentionally designed to target genomic sequences with a limited degree of repetitiveness. The list of genes and the Illumina IDs for the probes that target these genes are given in Supplementary Data Following a literature review of known epigenetic associations with respiratory viral infections from recent CoV outbreaks, we selected additional content to enrich Illumina’s Infinium MethylationEPIC ArrayBiospecimens were accessioned and tracked via the Colorado Anschutz Research Genetics Organization (CARGO) laboratory information management system (LIMS). Genomic DNA was extracted from SARS-CoV-2+ peripheral blood on the bead-based, automated extraction Maxwell(R) RSC System (Promega) in a biological safety cabinet in compliance with CDC safety guidelines and procedures for handling SARS-CoV-2 biospecimens (biospecimens from SARS-CoV-2+ cases) and from controls on the Autogen FlexSTAR+ using the Autogen’s FlexiGene Blood Extraction Kit . All DNA samples were quantified using both absorbance  and fluorescence-based methods  using standard dyes selective for double-stranded DNA, minimizing the effects of contaminants that affect the quantitation. DNA quality was assessed using an Agilent TapeStation . Samples were then uploaded to CARGO’s LIMS, barcoded, and labeled.28. The product of this process contains cytosine converted to uracil if it was previously unmethylated. The bisulfite-treated DNA was subjected to whole-genome amplification via random hexamer priming and Phi29 DNA polymerase, and the amplification products were then enzymatically fragmented, purified from dNTPs, primers, and enzymes, and applied to the Illumina chip as described elsewhere29.Purified DNA samples were processed using the Zymo EZ-96 DNA Methylation bisulfite conversion kits  as described previously28.The bisulfite-converted amplified DNA products were denatured into single strands and hybridized to the customized Infinium 850K BeadChip  via allele-specific annealing to either the methylation-specific probe or the non-methylation probe. Hybridization to the chip was followed by single-base extension with labeled di-deoxynucleotides according to Illumina’s Infinium protocol at the CARGO laboratoryThe hybridized BeadChips were stained, washed, and scanned to show the intensities of the un-methylated and methylated bead types using Illumina’s iScan System.30. Type I probe channel was empirically determined from signal intensities. Probe detection P values  were calculated for each color channel using pOOBAH, which leverages the fluorescence of out-of-band (OOB) probes. Normalization was performed using noob, which uses OOB probes to perform a normal-exponential deconvolution of fluorescent intensities31. Finally, a common dye bias that results in greater intensities in the red color channel was corrected to ensure that the distribution of intensities in the two color channels were equal. Probes with detection P values >0.05 were removed, as well as probes overlapping single-nucleotide polymorphisms with global minor allele frequency >1% in dbSNP, probes with poor mapping, and probes containing non-unique sequence according to Zhou et al.32. Beta values were logit-transformed into M values for modeling. Probes with >25% missingness were removed. Remaining missing values were then imputed with mean probe M value.IDAT files were processed, filtered, and normalized using the SeSAMe R packageCase–control analyses were performed using the entire genotyped dataset passing epigenetics QC, with SARS-CoV-2 infection status determined as described above  and positive/negative status was randomized across the chip.33 package for association testing and estimating components to adjust for population structure (EPISTRUCTURE34) and we used ReFACTor35 to account for cell-type proportions. We chose ReFACTor to account for cell proportion information in a data-driven fashion. The linear mixed-effects model in GLINT was fit to each probe, testing for differences based on COVID-19 disease status while correcting for age, sex, chip position, 6 ReFACTor components, 1 EPISTRUCTURE component, and a variance component representing individual covariance36. Enrichment of top hits in common databases was performed using enrichR37. Probes were sorted by adjusted P value and the top 800 genes to which differentially methylated probes map were used as input to perform overrepresentation enrichment analysis within Gene Ontology (GO) categories, Kyoto Encyclopedia of Genes and Genomes pathways (KEGG), BioPlanet, and WikiPathways41. Probes were annotated to CpG island and genic regions using annotatr42.Preprocessing was performed using the GLINTClinical data were abstracted via detailed chart review for all EMSB patients. COVID-19 disease severity was determined by an ordered severity score of (1) discharged from emergency department; (2) admitted to inpatient care; (3) progressed to ICU; and (4) death. We also determined a hospital duration variable, where individuals without a measured hospital stay  were assigned 0 and individuals who died were removed from the cohort for length of stay analysis to minimize bias associated with timing of decisions to withdraw care.43 and Elastic Net44 algorithms for sparse penalized regression modeling available in the glmnet software package45. For each prediction model, only autosomal methylation probes passing QC were included, to remove potential confounding from sex-linked chromosomes. No demographic, clinical, or cell count variables were included in the predictive models, requiring the algorithm to pick CpG sites with strong enough associations to surpass the level of penalization of the hyperparameters across the entire least angle regression path. For each trait of interest, a separate model was created and best-fitting parameters were chosen after tenfold cross-validation either by maximizing area under the receiver-operator characteristic curve (AUC for dichotomous traits) or minimizing mean-squared error (MSE for quantitative traits). Each was fit across a grid of parameters representing various strengths of penalization and combination of L1 and L2 penalties under the weighted elastic net model. Both the days of hospitalization and case severity were modeled as continuous outcomes. To assess performance for quantitative traits in a manner comparable to dichotomous traits, we swept across potential cutpoints to estimate AUCs for this newly derived dichotomous variable. While case–control status was the primary phenotype of interest, measures of severity were assessed in SARS-CoV-2+ cases only.Predictive modeling was performed using the Lassoλ penalty via cross-validation, and the number of probes chosen in the final model. For the quantitative outcomes, we assessed overall performance using out-of-sample R2, the slope of the model, and λ number of probes. Finally, these were stratified each across the elastic net weights (α) from 0.01 to 1, representing the proportion of ridge (L2) vs Lasso (L1) penalty to choose a final model. All models included nonzero λ to encourage sparsity (a L2-only model would include prediction from the entire array). Final models described in results were chosen based on best-performing (maximum R2 or AUC) vs median values for each chosen set of hyperparameters. The final, out-of-sample best-fit prediction for each outcome was considered the “methylation score” used in downstream modeling, characterization of association, and determination of potential confounding with demographic and blood cell proportion characteristics.To estimate stability of estimation in parameters, we performed 100 iterations of model training and testing. Within each iteration for case–control and severity outcomes, we employed tenfold cross-validation to derive the model and a held-out set of 30% removed from train/test to gauge out-of-sample performance of the best-fitting model. Our train/test and validation splits were created within each stratum to preserve representation across all outcomes and reflect the distribution across the total dataset. For hospitalization duration, the train/test/validation models had instability in convergence and so we reverted to a train/test model using the tenfold cross-validation within the default cv.glmnet function. We assessed overall performance for the dichotomous COVID+/COVID− case–control status using out-of-sample AUC, the F1 score , the distribution of best-fit Further information on research design is available in the We identified 675 patients tested for either SARS-CoV-2 or other acute upper respiratory infections. Of these, 164 were SARS-CoV-2+ by RT-PCR, 58 historical EMSB patients had positive (non-SARS-CoV-2) acute upper respiratory viral RT-PCR tests, 7 had positive (non-SARS-CoV-2) acute upper respiratory viral RT-PCR tests during the pandemic, and 296 were negative for all viral infections and thus served as controls. We excluded 32 samples from the dataset as these were derived from a run with failed hybridization and removed 8 duplicates, resulting in a final cohort of 525 . Custom probes on the EPIC+ chip are enriched in significant EWAS results : specifically, 1.72% of EPIC probes are significant as opposed to 2.51% of custom probes. Principal component analysis of top associations reveals clustering by COVID-19 disease status , cell proportions via ReFACTor and ancestry via EPISTRUCTURE components, EWAS of COVID-19 disease status in 164 SARS-CoV-2+ compared to 296 controls yielded 13,033 significant CpGs mapping to 6117 unique genes at false discovery rate (FDR)-adjusted P value = 7.40 × 10−10) and Negative Regulation of Viral Genome Replication . IRF7 encodes a transcription factor that regulates the expression of interferon a and b, as well as interferon-stimulated genes. Other top CpGs are in genes relevant to viral response: OAS1 (2’-5’-oligoadenylate synthetase 1) is interferon-induced and activates RNase L, which degrades viral (and cellular) RNA . MX1 encodes an interferon-induced GTPase that inhibits viral replication. DTX3L and PARP9 form a complex that is involved in interferon-mediated antiviral defenses. This complex has also been shown to promote M1 polarization in macrophages by preventing STAT1 phosphorylation48. IFIT3 encodes another interferon-induced antiviral protein. Overall, we observe strong hypomethylation of interferon- and viral response-related pathways, which is expected as these pathways are activated transcriptionally in SARS-CoV-2+ individuals49.Top hypomethylated CpG sites show strong enrichment for interferon and viral response-related pathways including Type I Interferon Signaling Pathway   and very low correlations of effect sizes in SARS-CoV-2+ compared to control and other upper respiratory infections compared to control analyses   from the testing cohort were supplied to the classification model, with prediction optimized after the approach defined in “Methods.” Only methylation probes were used in feature selection. All models showed relative stability across iterations . When age and race/ethnicity categories were included in a multivariable model along with our prediction score, no additional covariates significantly predicted COVID-19 disease status . Similarly, BMI was not associated (P ~ 0.4).To combine methylation data across the genome into a single predictor, we employed ML models of sparse regression trained via cross-validated P value < 2 × 10−16 with or without adjustment), and in the larger CBC model , only hematocrit (P ~ 0.05) approached nominal significance. The inclusion of hematocrit moderately improved Akaike information criterion in logistic regression but with limited performance increase in multivariable modeling AUC (93.6 vs 94.1%).To determine the direct association of methylation with clinical outcomes, an additional logistic regression was performed for the subset of individuals with complete blood cell count (CBC) data . The inclusion of additional blood cell count data did not impact the association between the methylation score and outcome , as well as across the spectrum of severity . The best-fitting model for hospital duration had a cutpoint at 20 days, yielding an AUC of 79.6% with 14 individuals with longer stays vs 135 with shorter stays  Fig. . DichotoHere we report DNA methylation profiling in conjunction with analysis using ML techniques to identify a SARS-CoV-2-specific epigenetic signature in peripheral blood from a large cohort of individuals tested using conventional RT-PCR technology. We also describe the development of a classification algorithm that has high sensitivity and specificity in predicting infection and in-hospital clinical deterioration and that confidently rejects the probability of healthy individuals to be affected by SARS-CoV-2 infection. While any predictive signal invites concern of potential confounding, the methylation signature  we observe is not driven by confounding either from demographics or typical laboratory measurements . Our findings suggest that measurement of methylation signals that arise during and after SARS-CoV-2 infection may provide clinicians the ability to detect viral infection as well as predict patient clinical course after viral challenge. Unlike sequencing, RT-PCR, and antibody tests, the methylation array is able to predict the severity of SARS-CoV-2 infection and ultimately could provide clinicians with information on how to manage patients infected with SARS-CoV-2.56. In this study, we observed many methylation changes that are, on average, >10% differentially methylated in the SARS-CoV-2+ group, including IRF7 and MX1 interferon-related genes. These are much larger effect sizes than typically observed in EWAS in peripheral blood57 and similar to the clinical utility of epigenetics observed in cancer22. We did not observe confounding by cell proportions, measured by CBC from the EHR, providing strong support for the epigenetic signature of SARS-CoV-2. Although cell-type heterogeneity can be a strong confounder in epigenetic studies60, we did not pursue adjustment for cell proportions beyond adjustment for cell-type proportions using ReFACTor35 because our primary objective is to develop a COVID-19 disease-specific diagnostic methylation platform, rather than interrogate the underlying pathology.Our results support the hypothesis that the host epigenome, as measured in peripheral blood, is modified by infection from SARS-CoV-2 and can be used to identify novel biology and it is useful for clinical diagnosis, prognosis, and triage. Despite being a heterogeneous tissue, we relied on peripheral blood as the target tissue because it has proven to be a reliable source for generating epigenetic signatures and disease classifiers in other settings61. Our findings of altered DNA methylation in interferon response genes are in concordance with published results of changes in the expression of interferon response genes by SARS-CoV and MERS-CoV viruses through changes in histone modifications3. One of the most significant probes  is located in the gene encoding IRF7; loss-of-function variants in 13 genes including IRF7 were recently found to be associated with life-threatening COVID-19-associated pneumonia62. Another interferon-induced gene, OAS1, was similarly significant . In a recent GWAS on critical illness due to SARS-CoV-2, significant associations and replication were observed for variants in the OAS gene cluster, which includes OAS163, for which variants had previously been associated in candidate gene studies of SARS-CoV infection64. Also, in a Mendelian randomization study it was recently shown that increased circulating OAS1 proteins were associated with reduced SARS-CoV-2 susceptibility and disease severity65. Collectively, published genomics studies support several of the strongest associations observed in our study.To validate the customized EPIC methylation platform as a reliable tool for the clinical diagnosis of COVID-19 disease, we performed an EWAS with SARS-CoV-2 infection status. We observed that the epigenetic signature of SARS-CoV-2 infection is enriched for pathways related to host viral response, and specifically for Type I Interferon signaling that is a hallmark of host response to this virus4. While we did not observe genome-wide significant signals at classical HLA alleles, we observed six FDR q < 0.05 probes in the region, in HLA-V, DOA, DQA1, DQA2, and DRA, albeit with attenuated significance compared to top CpGs (minimum q ~ 0.0109), suggesting that the mechanism of host manipulation by SARS-CoV-2 may be different. However, these results should be interpreted with caution as interrogation of the HLA region is complex; HLA-V for example is a pseudogene66.Previous work also demonstrated that viruses that cause severe disease  alter host response by changing methylation landscape of antigen-presenting genes in the HLA region9, and may have reduced the classification accuracy. However, we have follow-up EHR information for the patients in this cohort, which minimizes the risk of misclassification bias. We do not expect this potential confounder to affect the measures of severity used in this study as these were determined directly from chart review, but we acknowledge that, for the initial analysis, the numbers of cases may have limited the statistical power and prognostic ability of ML. With additional cases that account for inherent genetic variability within the population, methylation patterns will become more refined and the AUC of these ML models to predict disease severity is likely to increase. While “duration of hospital stay” may not be as immediately actionable as predicting ICU admittance or ventilator use, and it is confounded by pre-existing frailty, social support (or lack of), socio-economic status, and need for ongoing care once the acute illness has receded, the increased variability in the continuous outcome provides improved signal as observed both in our EWAS and our ML modeling. For this analysis, the 11 individuals who died were removed from duration analyses, as their length of stay would be difficult to compare to those who survived. Although the emerging field of epigenetics has demonstrated actionable classification with much smaller sample sizes in contrast to traditional GWAS in other common disease domains67, we recognize that additional cases, and in particular understanding the less-severe end of the spectrum , will improve our understanding of outcomes across the spectrum of disease severity. We note that, even in our limited sample sizes, the AUCs for ICU admittance still indicate there is signal that can be resolved through future collections. Another limitation of our work is the specificity of the epigenetic signature to SARS-CoV-2 over other respiratory infections. Initial targeted epigenetic analyses demonstrate a trend toward differential methylation, though these findings are limited by low numbers. Currently, we are targeting the collection of biospecimens from patients with respiratory infections other than SARS-CoV-2 for these follow-up studies.As the signatures identified in this study appear to be reactive to the disease, aspects of the disease process are expected to impact these results. Namely, we anticipate these changes to be time-sensitive, as the infection will need to have spread enough to induce methylation changes. Similarly, our case–control variables were defined by RT-PCR, which can carry a high false negative rate depending on the stage of infection and timing of sample collection70. One of the advantages of DNA methylation analysis compared to RNA analysis arises from the relative stability of deoxyribonucleic acid over ribonucleic acid71. The inherent instability of RNA, due to its 2’-OH group and the ubiquitous presence of ribonucleases, requires the use of plasticware, buffers, and processing reagents that are devoid of chemical and enzymatic species that stimulate RNA hydrolysis. Contamination even with a small amount of ribonuclease can degrade RNA samples to the degree where they cannot be analyzed.Researchers have previously compared the robustness of DNA methylation profiling vs RNA transcriptome profiling in developing classifiers for different disease states72. While asymptomatic testing following exposure has increased in recent months, the current testing strategy in the U.S. still predominantly targets symptomatic patients despite estimates that asymptomatic patients represent 40–45% of infected individuals72. Transmission during the incubation period has been reported, and the viral load of symptomatic and asymptomatic patients is similar76. The relationship between SARS-CoV-2 viral shedding and risk of transmission is unclear, and the percentage of transmission attributable to asymptomatic or pre-symptomatic infection of SARS-CoV-2 is unknown77. We believe that the epigenetics platform may efficiently identify asymptomatic and pre-symptomatic infections, which may, if applied broadly, aid in limiting the spread of SARS-CoV-2.The strong signature of viral-driven epigenetic changes may have the ability to detect SARS-CoV-2 infection in patients who never develop symptoms (asymptomatic) and in patients who are not yet symptomatic (pre-symptomatic)78, anticoagulation12, or new pharmacologic targets may prevent mortality, guided by these epigenetics patterns. Although our findings must be complemented with further clinical assessment, our model has shown its capacity to leverage methylation quantification as an innovative strategy to generate epigenetic signatures that assess host response to SARS-CoV-2, which is scalable and may have the ability to confirm positive tests in asymptomatic patients and entire communities, and may ultimately differentially diagnose other viruses causing similar symptoms all within in a comprehensive high-throughput manner.Due to the widespread occurrence of SARS-CoV-2 and progression to COVID-19 disease, there is the need for scalable testing technologies that can deployed on the national level for surveillance, screening, and prognosis for those infected. The purpose of this study was to identify high-confidence host methylation biomarkers that are able to indicate SARS-CoV-2 infection and predict clinical course of the viral disease in a given patient. This study is a first step toward selecting biomarkers for inclusion on a high-throughput methylation beadchip array specifically for the clinical diagnosis of COVID-19 disease that is also cost-effective given the added value of predicting subsequent clinical outcomes. To that end, we focused on sparse predictive models. Notably, these models are not significantly confounded by demographics or blood cell count information, denoting their specificity to the current infection of the patient, and reducing concern of overfitting to one patient sub-population. These biomarkers can also be used in risk stratification of SARS-CoV-2-infected patients, an unmet need given that none of the existing testing modalities  can achieve this level of specificity. By identifying DNA methylation patterns associated with critical illness, we contend that a methylation test will provide patient-specific treatment targets before critical illness ensues. Pre-emptive dexamethasoneSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary InformationDescription of Additional Supplementary FilesReporting Summary


The Africanized honey bee commonly referred to as the "killer bee," is a hybrid of the East African lowland honey bee with various European honey bees. These bees tend to be more hostile as compared to other bee species. Their stings may have devastating clinical sequelae, including cardiovascular, cerebrovascular events, and fatalities. We report the first-in-Caribbean case of a middle-aged woman who experienced stress-related, Takotsubo cardiomyopathy  after being afflicted with innumerable Africanized honey bee stings.Key clinical message: The clinician should be cognizant of Takotsubo's cardiomyopathy as a potential sequela of Hymenoptera envenomation and anaphylaxis. The Africanized honey bee is rapidly looming as an environmental health care issue in the Caribbean . These cThese bee swarm invasions can precipitate cardiovascular and cerebrovascular events, even culminating in death . The litA 48-year-old Caribbean-Black female with no significant medical history presented to the emergency department via family members en extremis, obtunded and somnolent. Her vital signs indicated systolic blood pressures of 98 mm Hg, heart rate of 105 beats/min, respiratory rate of 26 breaths/min, and oxygen saturation of 94% on supplemental oxygen. Her physical examination revealed hundreds of stings throughout her body, including her face, upper chest, and lower extremities  revealed sinus rhythm with subtle 1-2 mm ST-segment myocardial elevation in V1-V3 with diffuse T-wave inversions Figure , 3b. SARThe patient was subsequently admitted to the cardiac care unit and initiated on a norepinephrine infusion, where her mental state improved to being oriented and fully conversant. She was immediately administered intravenous antihistamines (diphenhydramine 50 mg every six hours) and glucocorticoids (hydrocortisone 200 mg every eight hours). Her blood pressures stabilized hemodynamically within the ensuing hour, and she then proceeded to urgent coronary angiography, which revealed angiographically normal coronary arteries Figures -4c. AddiAfricanized honey bees belong to the Apoidea winged Hymenoptera family . EnvenomThe patient's pathophysiology may have resulted from either a direct envenomation effect, anaphylaxis, excessive exogenous or endogenous epinephrine. SRC has been observed with hymenopterous-induced anaphylaxis, but central to these reports has been the administration of exogenous epinephrine 0.2-5 mg) during the emergent resuscitation -14. It i mg durinThe patient was treated with supportive measures, including norepinephrine, histamine receptor blockade, and parenteral glucocorticoids before being tapered and transitioned to an oral regimen. However, there are no specific evidence-based guidelines to define an optimal medical regimen for SRC . PreemptWe report the first-in-Caribbean case of a middle-aged woman who experienced stress-related, Takotsubo cardiomyopathy after being afflicted with a cluster of Africanized honey bee stings which was successfully managed with emergency resuscitative and supportive measures followed by the gradual introduction of neurohormonal therapies. The clinician should be cognizant of Takotsubo's cardiomyopathy as a potential sequela of Hymenoptera envenomation and anaphylaxis.


Critically ill COVID-19 patients have pathophysiological lung features characterized by perfusion abnormalities. However, to date no study has evaluated whether the changes in the distribution of pulmonary gas and blood volume are associated with the severity of gas-exchange impairment and the type of respiratory support (non-invasive versus invasive) in patients with severe COVID-19 pneumonia.This was a single-center, retrospective cohort study conducted in a tertiary care hospital in Northern Italy during the first pandemic wave. Pulmonary gas and blood distribution was assessed using a technique for quantitative analysis of dual-energy computed tomography. Lung aeration loss  and the extent of gas:blood volume mismatch  were evaluated in critically ill COVID-19 patients with different clinical severity as reflected by the need for non-invasive or invasive respiratory support.p < 0.001); and a larger extent of gas:blood volume mismatch , due to higher shunt  and non-aerated/non perfused regions . The PaO2/FiO2 ratio correlated positively with normally aerated tissue  and negatively with the extent of gas-blood volume mismatch .Thirty-five patients admitted to the intensive care unit between February 29th and May 30th, 2020 were included. Patients requiring invasive versus non-invasive mechanical ventilation had both a lower percentage of normally aerated lung tissue (median [interquartile range] 33% [24–49%] vs. 63% [44–68%], In critically ill patients with severe COVID-19 pneumonia, the need for invasive mechanical ventilation and oxygenation impairment were associated with loss of aeration and the extent of gas:blood volume mismatch.The online version contains supplementary material available at 10.1186/s13054-021-03610-9. In December 2019, a disease (COVID-19) caused by a novel coronavirus (SARS-CoV-2) emerged in China; it has since spread globally, causing a pandemic. COVID-19 patients present with a variety of clinical manifestations, including severe hypoxemic respiratory failure requiring mechanical ventilation . AlthougThe radiological hallmarks of COVID-19 are ground-glass opacities, often overlapping with areas of lung consolidation . It has versus invasive respiratory support) and the degree of gas-exchange impairment, was associated with the extent of gas:blood volume mismatch.The association of changes in lung aeration and perfusion with gas-exchange impairment and disease severity in COVID-19 requires investigation. Within this context, we conducted a retrospective cohort study with the aim of quantifying lung aeration and perfusion changes in critically ill patients with severe COVID-19 pneumonia. We hypothesized that the severity of COVID-19 pneumonia, as reflected by the requested respiratory assistance  and REporting of studies Conducted using Observational Routinely-collected health Data (RECORD)  and 14% [8–18%], respectively (p = 0.272).Non-perfused regions were distributed in normally-, poorly- and non-aerated regions, as illustrated in Fig. p = 0.001). Pulmonary blood volume was distributed along a ventral-dorsal and an apical-caudal gradient and its distribution was similar in patients receiving non-invasive and invasive respiratory support  nor with the driving pressure . In a sensitivity analysis based on a linear regression model using type of respiratory support and aeration-perfusion compartments as covariates, shunt was the only variable independently associated with the PaO2/FiO2 ratio  the percentage of normally aerated tissue was lower, while the extent of gas:blood volume mismatch was higher in invasively compared to non-invasively ventilated patients; (2) the poorly aerated tissue was characterized by either high or low gas:blood volume ratio; (3) the non-aerated lung tissue mass was mostly perfused, with only a small proportion being non-perfused; and (4) the PaOThis is the first study to evaluate DECT findings quantitatively in severe COVID-19 pneumonia, with different degrees of clinical severity, as reflected by the type of respiratory assistance (non-invasive and invasive respiratory support). In our center, DECT was performed routinely in a high proportion of patients with COVID-19 pneumonia for clinical purposes. Radiological findings were analyzed according to requested level of respiratory support, as well as correlated with key physiological, biological, and clinical parameters. All patients included in the present study were severely hypoxemic and admitted to an ICU, where they received either non-invasive or invasive respiratory support. In other pulmonary diseases, contrast-enhanced DECT has been used to quantify blood volume, percentage of lack of perfusion, and different components of lung aeration and tissue mass , 24, 25.COVID-19 patients receiving invasive mechanical ventilation based on clinical indication, compared to those receiving non-invasive respiratory support, showed reductions in pulmonary and gas volume and increased areas of poorly aerated and non-aerated lung tissue. Furthermore, patients receiving invasive mechanical ventilation had a greater extent of shunt lung tissue and larger non-aerated/non-perfused lung regions but less tissue with a low gas:blood volume ratio compared to those receiving non-invasive respiratory support. Non-aerated regions were mostly perfused, with a small proportion being non-perfused. This is compatible with impairment of hypoxic vasoconstriction . On the COVID-19 pneumonia is characterized by a progressive deterioration of lung morphology, affecting both aerated and poorly/non-aerated compartments. Increased lung weight is potentially explained by increased edema, cellular infiltration, alveolar consolidation, or a combination thereof. According to a recent study , edema m2/FiO2 correlated positively with the extent of normally aerated tissue and inversely with the extent of gas:blood volume mismatch, and was independently associated with shunt. We hypothesize that hypoxemia in COVID-19 pneumonia can be explained by shunt due to higher blood volume in non-aerated lung tissue and higher blood volume distribution in normally and poorly aerated tissue, causing an overall reduction in gas:blood volume ratio. On the other hand, a higher percentage of hypoperfusion in non-aerated regions  coul vessels , 33. Thipandemic –9.Some limitations of our study should be addressed. Only stable patients with a clinical indication for DECT were included in this study, which may have introduced selection bias and resulted in a small sample size. DECT does not directly detect perfusion, but rather the relative amount of iodine concentration and, consequently, regional blood volume. However, it has been validated as an acceptable surrogate for regional blood flow in ARDS , 24. TheIn critically ill patients with severe COVID-19 pneumonia, loss of aeration and gas:blood volume mismatch were higher in patients requiring invasive mechanical ventilation compared to those receiving non-invasive respiratory support. The severity of hypoxemia was explained by the extent of loss of aeration and gas:blood volume mismatch.Additional file 1. Additional analyses.


Coronavirus disease 19 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is associated with various clinical manifestations, including skin lesions. In particular, during the COVID-19 pandemic lock-down period numerous chilblain-like lesions, mainly located on the feet, were observed in adolescents. The latter were often asymptomatic or associated with very mild respiratory symptoms. Here, we report three cases of acral nodular lesions in SARS-CoV-2 swab-negative adolescents with histological findings of chronic immune-mediated inflammation and immunohistochemical evidence of SARS-CoV-2 spike glycoproteins in endothelial cells and eccrine sweat glands. In one of these cases, the virus presence was confirmed by electron microscopy. During the COVID-19 pandemic, increasing numbers of skin lesions have been described in positive individuals ,3,4. VarIn this brief article, we present three cases of SARS-CoV-2 swab-negative adolescents, with no flu-like symptoms in the previous months, who developed nodular lesions of the feet reminiscent of deep granuloma annulare. A thorough histopathological analysis was carried out in these cases.Case 1: An 11-year-old boy was observed for multiple swelling of the feet associated with mild burning. His family and personal history was negative for connective tissue, autoimmune or other noteworthy diseases. A physical examination showed 7–8 lesions , sometimes completely occluding the vessel lumens. In the middle and deep dermis an ample area of edema, with extensive and marked fragmentation or even dissolution of the collagen and elastic fibers, was evident, as well as thrombotic foci c of the Case 3: An 11-year-old girl, with no notable medical history, developed acro-localized nodular lesions similar to those of cases 1 and 2 during the pandemic period. We again performed a punch biopsy. A histological examination showed acanthosis and mucinosis as in case 2. The vessels showed a narrow lumen with protruding endothelial cells and there was also sclero-hyalinosis of the wall and hemorrhagic suffusion.Methods. The samples were fixed in 20% formalin for light microscopy and after processing, paraffin-embedding and microtome cutting, sections were prepared for routine staining with Hematoxylin-Eosin.Then, 5 µm thick sections of all cases were prepared for immunostaining with anti-SARS-CoV-2 Spike glycoprotein S1 monoclonal antibody (ThermoFisher) for Coronavirus (MA5-36247), with 100 µg at 1 mg/mL concentration, isotype: IgG. Appropriate negative and positive controls were included in the procedure.In addition, a small portion of the sample of only one case was post-fixed with 1% osmium tetroxide in PBS for 2 h at 4 °C for electronic microscopy. The fixed specimens were processed for embedding in Epoxy-Resin-Araldite (M) CY212 . Semi-thin (two micron thick) sections were stained with toluidine blue. Ultrathin sections were mounted on formvar-coated nickel grids and routinely stained with uranyl acetate and lead citrate. Images of the semi-thin sections were captured using a Nikon photomicroscope equipped with a Nikon Digital sight DS-U1 camera . The ultra-thin sections were observed using a transmission electron microscope .Immunohistochemical analysis was done in all three cases using the SARS-CoV-2 anti-spike proteins antibody, yielding positive results in the eccrine sweat glands (An electron microscopy study was performed in only one case, showing marked hypertrophy of the endothelial cells, the nuclei being characterized by finely distributed chromatin and retiform nucleoli. The cytoplasm contained very few organelles, with few small globular mitochondria. The fibroblasts were remarkably elongated and surrounded by very short collagen fibers. Spherical structures ranging from 65 to 136 nm were visible in the cytoplasm of endothelial cells and fibroblasts , charactThe nodular lesions described in the present report differ from the chilblain-like lesions related to COVID-19 due to their frankly nodular appearance and chronic course. However, they recall the chilblain-like lesions that were reported with increasing frequency during the pandemic period ,19, in rOverall, we describe a hybrid pattern of dermatologic lesions likely related to SARS-CoV-2, confirmed only by EM and immunohistochemistry assay. In our opinion, the acral nodular lesions described herein may be related more to immune responses secondary to the viral infection than to the direct cytopathic effect of the virus. Many varied skin manifestations have been described in these months of the pandemic, sometimes not correlated with histopathological and immunohistochemical data. A careful evaluation of the time lapse since the presumed contact with SARS-CoV-2 is of fundamental importance to reconstruct the clinical history of patients with COVID-related skin manifestations. Further studies are needed to fully explore this issue and also to gain a more in-depth understanding of the spectrum of skin manifestations that can be caused by SARS-CoV-2.


Cefiderocol is a recently FDA approved, novel siderophore beta-lactam antibiotic. Conventional antimicrobial susceptibility testing (AST) suggests that a variety of Gram-negative pathogens, including carbapenem-resistant (CR) isolates, are overwhelmingly susceptible to cefiderocol.. This antibiotic performed well in the APEKS-NP trial for the treatment of nosocomial pneumonia caused largely by carbapenem susceptible isolates. However, in the CREDIBLE-CR trial involving exclusively CR Gram-negative bacteria, cefiderocol was associated with a higher rate of all-cause mortality. We hypothesized one explanation for these discrepant data might be undetected cefiderocol heteroresistance (HR).in vivo models.HR is a form of antibiotic resistance in which an isolate harbors a minority resistant subpopulation of cells co-existing with a majority susceptible population, and is often undetected by standard AST. Isolates exhibiting undetected HR, with as low as 1 in 1 million resistant cells, can cause treatment failure in Acinetobacter, 180 Klebsiella, and 108 Pseudomonas isolates collected in Georgia, USA.We quantified HR to cefiderocol by population analysis profile (PAP) of 161 We observed CR isolates exhibited a high frequency of HR, which was largely undetected by standard AST, and correlated with all-cause mortality in the CREDIBLE-CR study (Table). Carbapenem-susceptible isolates exhibited no or low rates of cefiderocol HR (Table). Cephalosporin-resistant bacteria mostly exhibited increased rates of cefiderocol HR, but below those of CR strains. These differences in rates of cefiderocol HR correlated with the mortality data from the APEKS-NP and CREDIBLE-CR trials, across the bacterial species tested (Table).Table: Surveillance rates of cefiderocol heteroresistance correlate with all-cause mortality in the APEKS-NP and CREDIBLE-CR trialsin vitro susceptibility profile and increased patient mortality in the CREDIBLE-CR trial.These data suggest that the lower rates of cefiderocol HR in carbapenem-susceptible isolates that predominated the APEKS-NP trial may explain the enhanced efficacy of the drug in that study as compared to the CREDIBLE-CR trial. Importantly, the widespread, undetected cefiderocol HR observed among CR pathogens may explain the discordance between this drug’s excellent All Authors: No reported disclosures


Osteoarthritis (OA) is a prevalent chronic whole-joint disease characterized by low-grade systemic inflammation, degeneration of joint-related tissues such as articular cartilage, and alteration of bone structures that can eventually lead to disability. Emerging evidence has indicated that synovium or articular cartilage-secreted extracellular vesicles (EVs) contribute to OA pathogenesis and physiology, including transporting and enhancing the production of inflammatory mediators and cartilage degrading proteinases. Bioactive components of EVs are known to play a role in OA include microRNA, long non-coding RNA, and proteins. Thus, OA tissues-derived EVs can be used in combination with advanced nanomaterial-based biosensors for the diagnostic assessment of OA progression. Alternatively, mesenchymal stem cell- or platelet-rich plasma-derived EVs (MSC-EVs or PRP-EVs) have high therapeutic value for treating OA, such as suppressing the inflammatory immune microenvironment, which is often enriched by pro-inflammatory immune cells and cytokines that reduce chondrocytes apoptosis. Moreover, those EVs can be modified or incorporated into biomaterials for enhanced targeting and prolonged retention to treat OA effectively. In this review, we explore recently reported OA-related pathological biomarkers from OA joint tissue-derived EVs and discuss the possibility of current biosensors for detecting EVs and EV-related OA biomarkers. We summarize the applications of MSC-EVs and PRP-EVs and discuss their limitations for cartilage regeneration and alleviating OA symptoms. Additionally, we identify advanced therapeutic strategies, including engineered EVs and applying biomaterials to increase the efficacy of EV-based OA therapies. Finally, we provide our perspective on the future of EV-related diagnosis and therapeutic potential for OA treatment. Osteoarthritis (OA) is a highly aging-related disease that involves entire joint disorder and is typically associated with irregular chronic pain, which seriously affects the life quality of patients Figure ) The degeneration of articular cartilage is referred to as one of the hallmarks of OA As an alternative, extracellular vesicles (EV) or exosomes that are produced by stem cells and contain potent cytokines, growth factors, and miRs may be powerful in mediating inflammation and enhancing progenitor cell proliferation Apart from building up effective treatment strategies, early diagnosis of OA is of pivotal importance to limit further progression of cartilage damage via paracrine signallings Extracellular vesicles (EVs) are membrane vesicles with diameters of 30-5000 nm secreted from various cells that communicate with each other EV-related materials in the pericellular cartilage matrix and growth plate cartilage have long been described in the previous literature Figure ). For instance, evidence showed that OA chondrocytes  actively released EVs with enriched miR-372 lncRNA and a low level of a lncRNA, HULC (highly upregulated in liver cancer), while EVs from normal articular chondrocytes showed a reverse trend via releasing EVs in the synovial space. Ni et al. and Withrow et al. have summarized these interactions that can be referenced for probing OA pathogenesis Recent research has illustrated the involvement of EVs in the pathological and physiological processes of OA Several types of EV readouts, including the size, amount, and biological contents of EVs from the diseased sites, can be the representative biomarkers. Mustonen and colleagues recently showed that synovial fluid (SF) from the human knee joint with rheumatoid arthritis (RA) has a significantly higher proportion of hyaluronan (HA)-positive EVs at size range 101-200 nm but a much lower proportion of HA-positive EVs at size range >501 nm than those in OA and control groups via EVs. For instance, Liu and colleagues have demonstrated that synovial fibroblast secreted EVs containing miR-126-3p are responsible for suppressing apoptotic cell death, inflammation, and osteophyte formation in chondrocytes, and this miR expression was significantly reduced in OA patients Table 1). Therefore, it is highly desirable to develop biosensors, especially with current advances in nanotechnology and biomaterials, for detecting OA-related EVs and the EV contents to probe the progression of OA Non-coding RNA is an important biologically active molecule in EVs, including miR, lncRNA, and circular non-coding RNA (circRNA) Despite the biochemical contents of EVs, biophysical properties of OA-related EVs have been rarely explored. A recent report revealed the mechanical difference between non-malignant and malignant cell lines EVs by employing quantitative nanomechanical mapping atomic force microscopy (QNM AFM) Plain radiography is traditionally the gold standard for morphological assessment of OA knee with K-L score analysis of the images Figure ) A number of analytical assays have been employed to detect EV sizes by nanoparticle tracking analysis (NTA) and EV contents, including the mentioned biomarkers by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and RNA-sequencing (RNA-seq) for nucleic acids Figure ). For instance, Zhan et al. developed a self-standard ratiometric fluorescence resonance energy transfer (FRET) nanoprobe, consisting of Cy3-CD63 aptamer adsorbed onto 2D MXene nanosheets (fluorescent quencher) via hydrogen bonds and metal chelate interactions for quantifying the EVs in the solution  3 particles mL-1, which was 1000x lower than that of ELISA. This method offers a rapid and ultrasensitive approach to detect EVs. On the other hand, Shin and colleagues reported a SERS-based platform comprised of aggregated and positive charge gold nanoparticles (AuNPs) coated on a glass substrate for capturing the negative charge surface of EVs  Figure ) Numerous studies applied nanomaterials typically conjugated with anti-CD63 antibody, CD63 aptamer, or EV-related surface marker capturing molecules to bind EVs and switch on particular physical or chemical signals, including fluorescence Figure ) -1 (0.48 pM) and 34.3 pg mL-1 (1.56 pM), respectively. This platform was more sensitive and more rapid (< 10 min) than that of the conventional ELISA (cut-off LOD at ~100 pg mL-1 with handling time ~6 h). These findings demonstrate the possibility of probing OA biomarkers by rapid and sensitive biosensors. Point-of-care, non-invasive, and real-time biosensors can be highly attractive for clinical applications towards screening OA. However, very limited literature reports the usage of biosensors to detect the content of EVs from OA joints. Moreover, biosensors that can simultaneously probe both EV membranes and EV-associated contents in fluid samples in OA patients have been rare. We believe that biosensors with this detection ability are novel, attractive, and cost-effective without the need for EV isolation to understand the stage of OA, as OA-related markers can be concentrated in EVs.Similarly, recent reviews have discussed various biosensors for detecting OA and RA biomarkers based on the mechanisms above for signal amplification Table 2). However, the isolation of bone marrow MSCs from other tissues requires invasive procedures that increase pain and cost for the patients. Therefore, alternative sources for isolating EVs from other tissues, including synovial MSCs, platelet-rich plasma, infrapatellar fat pad, and umbilical cord-derived MSCs, have been emerging and showing promising tissue engineering results and treating/inhibiting OA symptoms. This section highlights and explores the recent findings of EVs derived from non-bone marrow MSC sources as potential options for treating knee OA and cartilage injuries.MSC-EVs exhibit great therapeutic potential for treating OA, as previous literature shows that the paracrine factors of MSCs provoke chondrocyte proliferation in vivo than that of P-PRP Platelet-rich plasma (PRP) is an autologous derivative of whole blood via suppressing the Wnt/β-catenin signal pathway in interleukin-1β (IL-1β)-stimulated chondrocytes, which were harvested from the terminal of tibia and femur of 4-week-old New Zealand white rabbits A recent study demonstrated that activated PRP upregulated the expression of platelet-derived growth factor-AB (PDGF-AB), transforming growth factor-β (TGF-β), and vascular endothelial growth factor (VEGF). These factors were secreted in PRP-EVs to promote cell proliferation (with reduced apoptosis) and cartilaginous matrix secretion via MHC class II molecules that potentially trigger an alloresponse, rejecting foreign transplanted cells Human infrapatellar fat pad (IPFP)-derived MSCs can generally be obtained by a single arthroscopy during knee arthroplasties in vitro and in vivo. Moreover, their mechanistic study showed that miR-100-5p from IPFP-EVs was able to bind to the 3'-untranslated region (3'UTR) of mTOR that inhibited the autophagy activity signalling pathway, such as the expression of ADAMTS5 and MMP13 that are responsible for OA progression. Therefore, IPFP-EVs may provide an alternative therapeutic source for treating OA.Recently, Wu et al. reported that EVs derived from IPFP (IPFP-EVs) contained abundant miR-100-5p, as evident by RNA-sequencing (RNA-seq) Synovial MSCs (SMSCs) have a remarkable proliferative and chondrogenic potential for cartilage repair via SMSC-EV delivery, Wang et al. overexpressed SMSCs with miR-155-5p and harvested their EVs (SMSCs-155-5p-EVs) as the miR-155-5p enriched cargoes To further enhance chondrocyte ECM secretion Human bone marrow-derived MSCs (hBMMSCs) are the most commonly used MSCs for research and clinical purposes. However, there are some limitations of hBMMSCs. For example, the relative number of hBMMSCs in the marrow and their differentiation potential decreases significantly with the age of donors in vitro with IL-1β stimulation (osteoarthritic chondrocyte model). Also, they employed a rat cartilage defect model with a drill bit to assess the efficacy of UCMSC-EVs treating damaged cartilage tissues in vivo. Their results further showed that UCMSC-EVs treated model exhibited the highest score by International Cartilage Repair Society (ICRS) macroscopic assessment and the highest pain persistent level, compared to those in the PBS control group and H19 silencing UCMSC-EVs group. A similar study was reported by the same group employing a hollow-fiber bioreactor  to simulate 3D culture for enhancing the production of UCMSC-EVs Yan et al. have shown that a rotary cell culture system creates microgravity for the 3D culture of UCMSCs by cell aggregation in vivo.Although non-MSC sources can be considered as alternative options for harvesting regenerative EVs, several limitations arising from the cell source and methodology of EV isolation may restrict the application of these sources. For instance, the volume of autologous concentrates of PRP is limited, and the protocol of harvesting PRP lacks reproducibility as the separation methods may not be standardized in vivoInjection of specific EVs alone into the articular space may not be optimal towards potential therapeutic outcomes due to low half-lives (<6 hours) Figure ). Subsequently, the engineered EVs can carry an elevated level of specific contents or cell-targeting ligands to enhance the therapeutic effects and also insert imaging molecules for long-term tracking purposes. Several reviews have comprehensively described the details of these engineering strategies for targeted drug delivery Recent research has attempted to incorporate therapeutic agents into EVs or EVs mimetics through passive or active loading. Passive encapsulation only requires a simple incubation of EVs with desired drugs or cells that can secrete desired drugs spontaneously through hydrophobic interactions Figure ). Recent approaches realize the conversion of dominant phenotype from M1 to M2 to foster a restorative environment in RA As the infiltration of inflammatory cells plays a vital role in cartilage destruction and bone erosion, the modulation of the immuno-environment may help reverse OA progression Activated macrophages also express folic acid (FA) receptors (FRs), especially FRβ, on the membrane surface. Thus, FRβ can be a target to mediate cellular uptake Figure ) in vitro. Moreover, the intra-articularly injected EVs remained around the cartilage tissues within the DMM model ~1-fold more than the non-tagged EVs within 24 or 48 h. These findings demonstrate the possibility of genetically modified EV surfaces for prolonged retention in wound sites Similarly, EVs can be engineered to target chondrocytes. Xia and colleagues recently demonstrated a chondrocyte-targeted miR-140 delivery platform based on EVs  into EVs/exosomes is challenging. Lin et al. have developed exosome-liposome hybrid nanoparticles through merging CRISPR/Cas9 expression vectors-bearing liposomes with exosomes at 37 oC overnight to achieve membrane fusion in vivo.CRISPR/Cas9 system has been a promising and powerful gene therapy tool by genome editing via suppressing JAK2/STAT3 pathway, which was shown to play a pathological role in OA Besides the mentioned EV modification methods, MSCs can also be primed/pre-conditioned to produce EVs with desirable miR profile/contents useful for OA treatment via subcutaneous injection are rapidly cleared in situ by circulating innate immune cells and subsequently require repeated administration to obtain their desired effect in vivoThe therapeutic efficacy of EVs is directly related to the exposure time of EVs to targeted cells and surface receptors. Similar to the challenges with cell or soluble factor delivery, EV delivered intravenously, intraperitoneally, or via binding or affinity-based mechanisms locally via integrins and to hyaluronic acid via CD44 interacting with hyaluronan Extracellular matrix-derived natural biopolymers and proteins found in native tissues such as hyaluronic acid, sulfated glycosaminoglycans, collagen, and fibronectin contain motifs have been exploited to retain EVs Figure 4) Biomaterials of various forms, including hydrogels, sponges, membranes/matrices, scaffolds, and decellularized tissues, have been used to incorporate and deliver EVs. Most commonly used biomaterials for EV release are hydrogels and ECM-based scaffolds/matrices; hydrogels are  polymer networks swollen in water with very well-established and diverse fabrication techniques, chemical modifications, drug delivery release/diffusion kinetics, while ECM-based materials  retain native architectures and chemical compositions and also have a variety of well-established fabrication methods to produce complex 3D structures and drug-loading. Natural or synthetic polymers can be combined to generate hybrid or composite biomaterials with more tunable functionality with respect to fabrication, physical or mechanical properties, or the addition of therapeutics. Chemical modifications of biopolymers for hydrogel formulation have been extensively reviewed via biomaterials for bone regeneration via intra-articular injection. Additionally, a full-thickness osteochondral defect in New Zealand rabbits is the most heavily used animal model to assess treatment efficacy for cartilage regeneration, followed by an osteoarthritis model transecting the anterior cruciate in combination with a medial meniscectomy in Sprague-Dawley rats. Lastly, all studies EVs derived from human cells (primary or cell line) plated on tissue culture plastic used ultracentrifugation for isolation, collection, and a combination of nanoparticle tracking analysis or dynamic light scattering, transmission electron microscopy (TEM), and western blotting or flow cytometry for EV characterization prior to use in vivo. Experimental details, including EV source, isolation technique, biomaterials, in vivo models, and outcomes, are summarized in Table 3.While there are numerous studies (and reviews) of EV delivery in vivo. While there are challenges in working with decellularized materials as described above, combining decellularized tissues with synthetic or chemically modified natural polymers has enabled a high level of tunability, bioactivity, and manufacturability. For example, decellularized porcine cartilage combined with GelMA was used to generate a unique 3D printable bioink to support the controlled release of MSC-EVs in vitro, EVs were found to provide unique mitochondrial proteins to rescue the damage in vivo analysis in a rabbit osteochondral defect model showed that the EV-containing hydrogel significantly increased ICRS macroscopic scores  and immunohistochemistry of COL2A1 and decreased MMP13 expression after the 6 and 12 week time points compared to all other groups, demonstrating a clear impact of local EV retention for cartilage tissue regeneration.Decellularized extracellular matrix continues to be a widely used and attractive raw material for biomaterial scaffold fabrication due to the retention of native proteins and matrix architecture while effectively eliminating cells or debris known to cause a detrimental immunological response Surface modification of biomaterial scaffolds with EVs or employing a biomaterial scaffold as a reloadable depot for EV retention and release are other methods recently investigated for cartilage tissue engineering. EVs isolated from human umbilical Wharton's jelly MSCs  intra-articularly injected weekly into rabbit osteochondral defects pre-implanted with freeze-dried, and EDC-crosslinked decellularized porcine cartilage scaffolds enhanced osteochondral regeneration compared to the scaffold alone HA hydrogels have recently been investigated in conjunction with EVs for enhanced cartilage repair and regeneration, including soluble high molecular weight HA viscosupplements alone. Treatment of critical-sized osteochondral defects in five-month-old New Zealand White rabbits immediately following wound closure with either 1mL intra-articular injections of high molecular weight HA  or 1mL of high molecular weight HA and 200 µg of EVs isolated from clonal immortalized E1-MYC 16.3 human embryonic stem cell-derived MSC line, with repeated injections performed 7 and 14 days post wound closure resulted in significantly increased ICRS score at 6 and 12 weeks Freeze-dried EDC/NHS-crosslinked chitosan and hyaluronic acid hydrogels (CS-HA) combined with EVs and adipose-derived MSCs injected into full-thickness osteochondral defects immediately following surgery promoted significantly increased ICRS scores of MRI imaging compared to the hydrogel alone, MSCs alone, MSC-EVs, CS-HA/MSC, or the CS-HA/MSC-EVs group after 4 weeks and 24 weeks in situ covalent crosslinking; this design was recently exploited by Liu et al. to entrap and deliver EVs derived from human induced pluripotent (cell line iPS-S-01)-derived MSCs to cartilage defects in rabbits 12 DiI-labeled EVs in fresh PBS daily; supernatants were analyzed with a particle analyzer and subtracted from the loaded EV total to determine percent retention in vitro compared to crosslinked GelMA alone or GelMA/gelatin hydrogels in vitro mechanistic analysis on their EVs; microarray analysis demonstrated that miR-23a-3p was highly enriched, and bioinformatic analysis suggested that miR-23a-3p may bind to the 3'UTR coding sequence of the gene PTEN. The authors then cultured miR-23a-3p with 293 T-cells transfected with luciferase reporter constructs containing the predicted 3'UTR of PTEN and found that luciferase activity was decreased and subsequently abolished when cultured with mutated 3'UTR of PTEN, confirming EV function via miR-23a-3p targeting PTEN via western blotting. Collectively, these two studies highlight the clinical efficacy of using photocrosslinkable networks for EV retention and delivery from hydrogels.HA modified with o-nitrobenzyl alcohol (HA-NB) generates aldehyde groups under light irradiation that can interact with amines on other biopolymers, enables in vitro via a transwell assay  and nanoflow cytometric analysis. Furthermore, EV release kinetics were accelerated under increasing concentrations of hyaluronidase treatment, confirming a degradation-dependent control of EV release in vivoAs previously described, covalent/supramolecular interactions can be exploited to entrap and control the delivery of biomolecules such as EVs. Here, Diels-Alder crosslinked hyaluronic acid/PEG hydrogels (DAHP) were developed using a furyl functionalized HA and a maleimide -PEG-Mal crosslinker that enables orthogonal crosslinking with low reactivity with amine groups via Safranin O/Fast Green and Toluidine Blue, and Col II staining in vitro over 36 days performed on a rocker at 37 ºC and detected using a CD63 ELISA kit. Mechanistic in vitro studies of the circRNA3503-overexpressed EVs highlighted their efficacy in attenuating inflammation-induced apoptosis and provided a balance between ECM synthesis and degradation by acting as a sponge for miRs that regulate expression of target functional genes such as MMPs and SOX9 In addition to EVs being a potent and independent therapeutic for treating OA or promoting cartilage repair, EVs have also been shown to be unique bioactive carriers for nucleic acids in vitro and in vivo be tissue- or application-specific . Decoupling these effects will be critical to validate EV/biomaterial efficacy.Together, these studies showcase a wide variety of biomaterials for EV delivery to promote cartilage tissue repair and regeneration. Collectively and independently, each of these materials indicates potential for their biomaterial delivery vehicle to release EVs and subsequently enhance cartilage repair compared to the materials alone and in some cases compared to EV treatment alone. As discussed in many review articles, there are significant limitations to translating EV/biomaterial therapeutics to clinical application, first, in standardizing cell/tissue/species source for EV generation, scalability, isolation techniques, application techniques  and in designing appropriate pre-clinical studies to thoroughly identify and qualify that the effects of EV-loaded materials are primarily driven by EVs. Second, there also remains challenges in standardizing techniques to evaluate EV release and subsequent synergistic effects of bioactivity of EVs released from biomaterials. Important to note that many of the specific functional outputs via material degradation-mediated EV release or EV diffusion from biomaterials. It will be essential to elucidate the effects of EV/biomaterial degradation further and release with respect to timing and dosing to achieve a therapeutic effect for OA. One area of biomaterials that has shown promise in cartilage tissue engineering (and many other applications) that may be advantageous for EV delivery is granular hydrogels and microparticles Lastly, based on several reviews and publications showing the robust effects of intra-articular delivery of EVs to promote cartilage repair and regeneration, studies here suggest that a single application of EV-loaded biomaterials may provide a similar therapeutic effect controlled In summary, recent research has made significant progress in overcoming major barriers to using EVs as a delivery system and a marker for OA pathology diagnosis. EVs are ideal systems for delivering osteoarthritis therapeutics, owing to their size, surface expression profiles, low immunogenicity, low cytotoxicity, and long-term safety. EVs from modified cells or engineered EVs with drug loading technologies have been shown to improve the therapeutic effect. However, the side-effect of using EVs for OA therapy is unknown, and safety evaluation research is required before clinical translation. Recent advances in nanomaterials-based offer great sensitivity and rapid biosensors for detecting EVs. Tissue engineering techniques are also used in EV-based OA therapy/cartilage repair. Biological scaffolds, especially hydrogels, have been shown to have a good sustained EV-release effect in cartilage repair. We believe that theranostic platforms will be the main direction of EV-based OA therapy/cartilage repair in the near future.The mentioned exosome-liposome nanohybrid system also inspires us to consider its potential application to detect both EVs and the content of EVs


Fibromyalgia is a chronic pain condition that is frequently accompanied by comorbid conditions, including depression. Depression is associated with reduced physical functioning and health disproportionately affecting middle-aged and older adults with fibromyalgia. This study examined depressive symptoms as a mechanism through which FM status is associated with BMI and physical performance among adults in mid-to-late-life. Participants included 250 community-dwelling middle-aged and older adults  with (59%) or without (41%) fibromyalgia . Depressive symptoms were measured using the Beck Depression Inventory-II, BMI was objectively assessed, and physical performance was measured using the Fullerton Advanced Balance scale, 6-Minute Walk Test, 30-Second Chair Stand, and 8-Foot Up and Go Test. Physical performance measure analyses were adjusted for age. Asymptotic mediation analyses revealed that fibromyalgia status was indirectly associated with higher BMI , and poorer performance in the Fullerton Advanced Balance , 6-Minute Walk Test , 30-Second Chair Stand , and 8-Foot Up and Go test  via depressive symptoms. Participants with fibromyalgia reported greater depressive symptoms which was subsequently associated with greater BMI and reduced physical performance. Findings support depressive symptoms as one factor through which fibromyalgia status is associated with higher obesity risk and reduced physical function in middle-aged and older adults with fibromyalgia. This study supports fibromyalgia status as a critical consideration when evaluating the health and disability risk of aging adults.


Exosomes or other extracellular vesicles released from cells play an important role in cell-to-cell communication by transferring bio-information , which indicates parental cell status to recipient cells in the extracellular environment. Increasingly, evidence shows that tumor-derived exosomes mediate tumor pre-metastatic niche (PMN) remodeling to establish a supportive and receptive niche to promote tumor cell colonization and metastasis. Uptake of genetic information by target cells in the extracellular environment triggers epigenetic changes that contribute to PMN formation. Here, we provide a comprehensive overview of the current understanding of exosomes-mediated reprogramming of cells in PMN formation. After the (PMN) .An important question regarding the PMN is how a tumor induces PMN formation in a specific organ. Of the various factors secreted by tumors to initiate PMN formation, exosomes are key components that carry bio-information  from parental cells to communicate with cells in the primary tumor and the microenvironment of distant organs . As an eExosomes are extracellular vesicles with a diameter of 30–150 nm that can be secreted by most cells into the extracellular environment. Exosomes exist widely in blood, urine, saliva, and other bodily fluids. Unlike budding, the exosomes biogenesis is complex and involves endocytic pathways, including endosomal sorting complex required for transport (ESCRT) and non-ESCRT dependent pathways . The mosGenerally, exosomes are reported to be widely involved in physiological and pathological processes, such as antigen presentation and immune response, cell apoptosis and senescence, material transportation and signal transduction between cells, tumor cell proliferation and invasion, and epithelial mesenchymal transition (EMT)  Figure . Increaset al. found that in the pre-metastatic lung, pro-inflammatory mediators S100A8/A9 induced the expression of serum amyloid A (SAA) three and recruited Mac1+ myeloid cells via TLR-dependent manner to promote inflammatory PMN formation and facilitate metastasis  in colorectal cancer patients without lymph node metastases was 87%, while the 5-years OS rate in patients with lymph node metastases decreased to 60.6%  derived-exosomal macrophage migration inhibitory factor (MIF). This process induces PMN formationand promotes liver metastasis  can induce CCRCC specific lung metastasis . They found that the tissue distribution and accumulation of EXO-DOX were to their companion exosomes. Importantly, the EXO-DOX inhibited breast cancer lung metastasis. This study proposed a new approach for disease chemoprevention by targeting specific organs using exosomes’ organotropism mechanism  are crucial for vascular remodeling. Most studies on vascular remodeling use human umbilical vein endothelial cells (HUVECs) as a laboratory model system for the study of the function and pathology of ECs. Tumor-derived exosomes mainly interact with ECs and lead to reprogramming of ECs to regulate the level of zonula occludens-1 (ZO-1), occludin, and Claudin5. These three proteins are central components of tight junctions (TJs) and comprise a major group of cell-cell adhesion complexes in ECs and epithelial cells. Both exosomes-associated non-coding RNAs and proteins are associated with vascular remodeling, especially in lung PMN .et al. found that brain endothelial and microglial cells uptake of breast brain-tropic cell-derived CEMIP+ exosomes triggered the inflammation pathway  and promoted vascular remodeling and angiogenesis  derived exosomal miRNAs  downregulated ECs expression of ZO-1 and vascular endothelial-cadherin (VE-cadherin) and increased vascular permeability to promote intrahepatic tumorigenesis  activated lung fibroblasts to facilitate tumor extrahepatic metastasis via enhancing angiogenesis and pulmonary endothelial permeability (Hepatocellular carcinoma (HCC) with high recurrence and metastasis is related to poor prognosis. However, the mechanism of HCC intrahepatic and extrahepatic metastasis remains unclear. Yokota, Y. igenesis . Additioeability . In melaeability .et al. found that CRC-derived exosomal miR-25-3p could be transferred to ECs, upregulating the expression of VEGFR2 and downregulating the level of ZO-1, occludin, and Claudin5 by silencing KLF2/KLF4. Thus, exosomal-miR-25-3p promotes CRC liver/lung metastasis by inducing angiogenesis and vascular leakiness/permeability in liver/lung PMN  was significantly downregulated in CRC tumor. After being taken up by Kupffer cells, CRC-derived exosomal ANGPTL1 regulated the secretion pattern of Kupffer cells and decreased the MMP9 expression by inhibiting the JAK2-STAT3 signaling pathway. Thus, in the liver PMN, vascular leakiness was prevented, and CRC liver metastasis was attenuated , the role of exosomes in vascular remodeling in PMN remains controversial. Zeng, Z. lung PMN . Howevertenuated .et al. discovered a new circular RNA-Cholangiocarcinoma-associated circular RNA 1 (circ-CCAC1), a key player in cholangiocarcinoma progression. Circ-CCAC1 was increased in cholangiocarcinoma-derived exosomes. Exosomal circ-CCAC1 entered human umbilical vein endothelial cells (HUVECs) and sequestered EZH2 to modulate SH3GL2/ZO-1/Occludin signaling and reduce TJs protein levels. Consequently, vascular endothelial barriers were destroyed and angiogenesis was induced  and eukaryotic translation initiation factor 2 alpha (eIF2α). This increase resulted in the downregulation of TJ protein in HUVECs . Consequently, angiogenesis was enhanced. Additionally, the tight junction protein ZO-1 was inhibited, thus increasing vascular permeability and cancer transendothelial migration  cells, Treg cells, neutrophils, macrophages, and MDSCs. In this process, tumor-derived exosomes have a close relationship with those cells and serve as a double-edged sword . IncreasBack in 2005, David Lyden’s group first revealed that tumor-specific chemokines and/or cytokines induce fibronectin production from lung resident fibroblasts to recruit bone marrow-derived cells (BMDCs) . These specific BMDCs express vascular endothelial growth factor receptor 1 (VEGFR1) and VLA-4 (integrin α4β1) to form cellular clusters and a pre-metastatic permissive niche for incoming tumor cells. Their findings demonstrated the critical role of BMDCs in PMN formation; however, the role of cancer-derived exosomes was not revealed . In 2015et al. found that primary tumor (Lewis lung carcinoma or B16/F10 melanoma)-derived exosomal RNAs induce chemokine secretion and neutrophil recruitment in the lung by activating TLR3 in lung epithelial cells. Thus, lung PMN was formed and lung metastasis was promoted  monocytes Ly6Clow cells subpopulation, producing the anti-metastatic function. They observed that non-metastatic exosomal pigment epithelium-derived factor (PEDF) increased the Nr4a1 transcription factor expression in PMo and promoted PMo differentiation into macrophages. Additionally, non-metastatic exosomes induce macrophage M1 polarization and recruit NK cells to prevent melanoma cell metastasis  glucose uptake through downregulation of the glycolytic enzyme pyruvate kinase (PKM2) and GLUT1. Thus, the nutrient competition bias leaned toward cancer cells. Glucose availability was increased in cancer cells in the PMN, and cancer proliferation and colonization were also promoted . The auts in PMN . Additiotuations .et al. found that nasopharyngeal carcinoma-derived exosomal membrane protein 1 (LMP1) can induce normal fibroblast transformation into cancer-associated fibroblasts (CAFs) via the NF-κB p65 pathway. Accordingly, the metabolic status of CAFs and tumor cells were also changed. In activated CAFs, aerobic glycolysis and autophagy increased. Energy-rich nutrients, including lactate and β-hydroxybutyrate (β-HB), were exported to “feed” cancer cells for the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Moreover, EV packaged LMP1-activated CAFs promoted the proliferation, migration, and radiation resistance of tumor cells  are a novel class of endogenous non-coding RNAs first reported to exist in exosomes by Long non-coding RNAs (lncRNAs) were also found to be enriched in exosomes. Exosomal LncRNAs have numerous functions and also showed the potential to be diagnostic biomarkers . ExosomeSome studies reported the existence of DNA in exosomes. One study indicated that double-stranded DNA (dsDNA) in exosomes could represent the whole genomic DNA. The study’s experiments on mouse models proved that exosomal dsDNA were able to detect the BRAF (V600E) mutation in the serum isolated from melanoma-bearing mice. This result suggested the possibility of exosomal dsDNA to act as biomarkers for cancer genetic mutations, which may provide individualized protocols for targeted therapy .et al. revealed that circulating exosomal glypican-1 (GPC1) was detected in the serum of patients with pancreatic cancer. Exosomal GPC1 was significantly related to tumor burden and the survival of patients because it distinguishes healthy people from pancreatic cancer patients. Interestingly, mutant KRAS mRNA was only detected in GPC1 positive circulating exosomes . Collectively, non-coding RNAs , double-stranded DNAs, and proteins were all discovered to be present in exosomes, revealing their potential to be diagnostic biomarkers.Proteins enriched in exosomes also had clinical diagnostic value. In 2015, Melo SA exosomes . TherefoBesides diagnostic values, exosomes are also reportedly predictors of cancer patient prognoses. A study indicated that the amount of exosomes derived from serum of CRC patients was significantly higher than in healthy controls. Moreover, high-level serum exosomes correlated with high levels of CEA and more poorly differentiated tumors. Although there was no significant difference, patients with high levels of serum exosomes tended to have shorter OS than patients with low levels of serum exosomes (3-years OS 55% vs 89%) . AnotherIn recent years, researchers have searched for novel exosomal applications to treat disease, especially tumors. Inhibiting exosome release has been proved to suppress tumor proliferation, metastasis, and chemoresistance mediated by increased exosomes in cancer. For example, inhibition of CAFs-derived exosomes secretion decreased the percentage of CSCs and suppressed tumor proliferation. It was also associated with enhancing the chemotherapy sensitivity . Intriguvia a caveola-dependent endocytosis pathway, meaning that knockdown of caveolin-1 can significantly suppress the internalization of exosomes. This protein may be a promising therapeutic target in cancer therapy (Inhibiting the process of exosome uptake is another way to limit upregulated proliferation and exosome-mediated metastasis. Recent work indicated that the anti-hypertensive drug reserpine could inhibit the uptake of tumor-derived exosomes and influence the formation of PMN, thus suppressing metastasis . Another therapy . Likewis therapy .via suppression of the PTEN/PI3K/AKT signaling pathway. Moreover, miRNA-126 loaded exosomes could inhibit the formulation of lung metastatic foci in vivo (Recently, researchers explored the feasibility of using exosomes as a vehicle to deliver anti-tumor drugs. Exosomes have potential as drug carriers for three main reasons. Firstly, their double membrane structure can protect drugs from degradation. Secondly, exosomes can be selectively transported to specific organs and tissues, thus may provide a novel idea for targeted therapy. Thirdly, exosomes with low toxicity, are widespread in extracellular fluid, blood, saliva, semen, breast milk, and even penetrate the blood-brain barrier .,Exosome in vivo . CollectOverall, exosomes have revealed their potential as diagnostic or prognostic markers. They may even also play vital roles in targeted cancer therapy. However, their complex composition, uncertain biological functions, and even safety concerns limit their translation into clinical applications. Further research is needed to transform basic research results of exosomes into clinical applications.In summary, studies have increasingly confirmed that tumor-derived exosomes have multiple functions in PMN remodeling to promote distant metastasis. However, the cargo, function, and mechanism of exosomes still need to be further explored. Some important issues need to be solved urgently. For example, what is the interaction effect between tumor-derived exosomes and stromal/immune cell-derived exosomes in the PMN? Do the different components enriched in exosomes interact with each other? How long-lasting is the effect of tumor-derived exosomes on the PMN? How reliable and valid are tumor-derived exosomes as biomarkers? How feasible is the prospect of clinical application of tumor-derived exosomes? We believe there is strong evidence that these challenges will be answered.


We developed a testing program for severe acute respiratory syndrome coronavirus 2 in an urban Latinx neighborhood in Providence, Rhode Island, USA. Approximately 11% of Latinx participants (n = 180) tested positive. Culturally tailored, community-based programs that reduce barriers to testing help identify persons at highest risk for coronavirus disease. As of May 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had infected >154 million globally and caused >3.2 million deaths . Sexual minorities accounted for ≈40% of the sample, and gender minorities accounted for 12% of the sample. However, sexual and gender minorities had far lower rates of COVID-19 infection; 90% of persons who tested positive for SARS-CoV-2 were cisgender and heterosexual.Our experience suggests that SARS-CoV-2 testing models that reduce barriers to care can successfully reach medically underserved communities that have high rates of COVID-19 infection. Culturally tailored approaches might be critical for identifying Latinx populations unaware of their SARS-CoV-2 infection (Our program provides a useful framework for reducing barriers to SARS-CoV-2 testing services in underserved communities, including sexual and gender minorities and Latinx populations who otherwise might not be tested for SARS-CoV-2. Perhaps the greatest challenge to replicating and sustaining this model is developing a viable funding model. Despite our program’s success in enabling testing for persons at elevated risk for COVID-19, the human and financial resources needed to maintain this testing site design might limit its ability to be implemented in resource-limited environments. The need for culturally tailored testing programs will continue even as vaccination programs are enacted across the country. Currently, reimbursement-based and traditional medical service delivery models often operate at a financial loss; greater public funding support is needed to sustain culturally tailored, low-barrier testing models that address ethnic and racial disparities in SARS-CoV-2 infection.