The COVID-19 pandemic has presented many challenges that have spurredbiotechnological research to address specific problems. Diagnostics is one area wherebiotechnology has been critical. Diagnostic tests play a vital role in managing a viralthreat by facilitating the detection of infected and/or recovered individuals. From theperspective of what information is provided, these tests fall into two major categories,molecular and serological. Molecular diagnostic techniques assay whether a virus ispresent in a biological sample, thus making it possible to identify individuals who arecurrently infected. Additionally, when the immune system is exposed to a virus, itresponds by producing antibodies specific to the virus. Serological tests make it possibleto identify individuals who have mounted an immune response to a virus of interest andtherefore facilitate the identification of individuals who have previously encountered thevirus. These two categories of tests provide different perspectives valuable tounderstanding the spread of SARS-CoV-2. Within these categories, differentbiotechnological approaches offer specific advantages and disadvantages. Here we reviewthe categories of tests developed for the detection of the SARS-CoV-2 virus or antibodiesagainst SARS-CoV-2 and discuss the role of diagnostics in the COVID-19 pandemic. Identifying individuals who have contracted coronavirusdisease 2019 (COVID-19) and may be contagious is crucial to reducing the spread of thevirus. Given the high transmissibility of SARS-CoV-2 and the potential for asymptomatic orpresymptomatic individuals to be contagious [Since the emergence of ntagious ,4. Such The genetic sequence of the SARS-CoV-2 virus was first released by Chineseofficials on January 10, 2020 , and theMolecular tests detect either viral RNA or protein in a patient sample. They areessential to identifying infected individuals, which can be important for determiningcourses of action related to treatment, quarantine, and contact tracing. Tests for viral RNAare done by reverse transcription (RT) of viral RNA to DNA followed by DNA amplification,usually with polymerase chain reaction (PCR) .Tests fSerological tests, on the other hand, detect the presence of antibodies in bloodplasma samples or other biological samples, providing insight into whether an individual hasacquired immunity against SARS-CoV-2. Assays that can detect the presence of antibodies inblood plasma samples include ELISA, lateral flow immunoassay, and chemiluminescenceimmunoassay (CLIA) . To distMolecular and serological tests therefore offer distinct, complementaryperspectives on COVID-19 infections. Some of the same technologies are useful to bothstrategies, and different technologies have been employed to varying extents throughout theworld since the start of the COVID-19 pandemic. Two of the primary metrics used to evaluatethese tests are sensitivity and specificity. Sensitivity refers to a test’s abilityto correctly identify a true positive; for example, a test with 50% sensitivity wouldidentify SARS-CoV-2 in only one of every two positive samples. On the other hand,specificity refers to how well a test is able to identify a negative sample as negative.This metric can be relevant both in terms of understanding the risk of false positives andin discussing whether a test is susceptible to identifying other coronaviruses. Here, wereview the different types of tests within each category that have been developed andprovide perspective on their applications.4Molecular tests are used to identify distinct genomic subsequences of a viralmolecule in a sample or the presence of viral protein, and they thus can be used to diagnosean active viral infection. An important first step is identifying which biospecimens arelikely to contain the virus in infected individuals and then acquiring these samples fromthe patient(s) to be tested.Common sampling sources for molecular tests include nasopharyngeal cavity samples,such as throat washes, throat swabs, and saliva ,and sto4.1When testing for RNA from viruses like SARS-CoV-2, the first step involvespre-processing in order to create complementary DNA (cDNA) from the RNA sample using RT.The second step involves the amplification of a region of interest in the cDNA usingsuccessive cycles of heating and cooling. Depending on the application, this amplificationis achieved using variations of PCR. Reverse transcription polymerase chain reaction(RT-PCR) tests determine whether a target is present by amplifying a region of interest ofcDNA . Some te4.1.1t) determined byqPCR.In contrast to RT-PCR, quantitative, real-time PCR uses fluorescent dyes thatbind to the amplified DNA, thereby allowing a real time assessment of the amplificationprocedure  (in thisORF1b of theRNA-dependent RNA polymerase (RdRP), the envelope protein gene (E), andthe nucleocapsid protein gene (N) [The first test developed and validated for the detection of SARS-CoV-2 usedRT-qPCR to detect several regions of the viral genome: the gene (N) . The pubgene (N) . In collgene (N) . ImportaORF1b andN [N gene to screen patients, while the ORF1b generegion is used to confirm the infection [Severe acute respiratorysyndrome-related coronavirus 1 (SARS-CoV-1) and SARS-CoV-2 are the onlysarbecoviruses currently known to infect humans, a positive test can be assumed toindicate that the patient is infected with SARS-CoV-2, although this test is not able todiscriminate the genetics of viruses within the sarbecovirus clade. The fact that thetargets are so conserved offers the advantage of reduced concern about sensitivity inlight of the evolution of SARS-CoV-2.In January 2020, Chinese researchers developed a test that used RT-qPCR toidentify two gene regions of the viral genome, 1b andN . This asnfection . Thetest is below 33 or sometimes 35 [t corresponds to fewer qPCR cycles needed to reach adetectable level, indicating that higher amounts of virus were present in the initialreaction. Interpretations of the Ct values obtained from these tests haveraised some interesting questions related to viral load and contagiousness. LowerCt values correspond to a higher probability of a positive viral culture,but no threshold could discriminate all positive from all negative cultures [t is nota proxy for viral load [qPCR tests have played an important role in diagnostics during the COVID-19pandemic. For SARS-CoV-2, studies have typically considered a patient to be infectiousif the Ctimes 35 ,18. A loral load . Positivral load , meaningIn addition to the nuance required to interpret PCR results, there are alsofactors that influence their accuracy. The specificity of these tests is very high, meaning4.1.2Digital PCR (dPCR) is a new generation of PCR technologies offering analternative to traditional qPCR . In dPCRHigh sensitivity is particularly relevant for SARS-CoV-2 detection, since lowviral load in clinical samples can lead to false negatives. In one study, Suo et al. performet value was higher than the positive threshold of35 but lower than the negative threshold of 40. When the 61 negative and borderlinesamples were reanalyzed with ddPCR, 19 (31%) of the negative and 42 (69%) of theborderline samples were identified as positive. All of the suspected cases were laterconfirmed to be COVID-19 through a combination of symptom development and RT-qPCRresampling, indicating that ddPCR improved the overall detection rate compared toRT-qPCR from 28.2% to 87.4%.A second study  confirmeThey repeated this analysis in patient samples from contacts and closecontacts. Patients who tested negative with both methods (n = 48) were observed toremain healthy over a period of 14 days. Among the remaining 29 samples from contacts,RT-qPCR identified 12 as positive, 1 as negative, and 16 as borderline. All of thesamples that tested positive using RT-qPCR also tested positive using ddPCR. Incontrast, the negative result and all but one of the borderline results were identifiedas positive by RT-ddPCR, and these patients were later determined to be SARS-CoV-2positive based on clinical evaluation and repeated molecular sampling. Similarly, in thefinal group, 16 convalescent patients, RT-qPCR identified 12 as positive, three assuspect, and one as negative, but RT-dPCR identified all as positive. The evidence fromthis study therefore supports a lower limit of detection with ddPCR. Overall, thesestudies suggest that ddPCR is a promising tool for overcoming the problem of falsenegatives in SARS-CoV-2 RNA testing, but this method is unlikely to affect the currentpandemic due to its lack of availability.4.1.3In some cases, the DNA amplified with PCR is sequenced. Sequencing requires anadditional sample pre-processing step called library preparation. Library preparation isthe process of preparing the sample for sequencing, typically by fragmenting thesequences and adding adapters . In someSequencing has been an important strategy for discovery of SARS-CoV-2 variants. Sequen4.1.4Due to limited supplies and the need for more tests, several labs have foundways to pool or otherwise strategically design tests to increase throughput. The firstsuch result came from Yelin et al. , whorep0.4.2RT-PCR remains the gold standard for detection of SARS-CoV-2 RNA from infectedpatients, but the traditional method requires special equipment and reagents, including athermocycler. Loop-mediated isothermal amplification (LAMP) is an alternative to PCR thatdoes not require specialized equipment . Inthist value from RT-PCR of the same samples; RT-LAMP performs at 100%sensitivity for samples with a Ct from RT-PCR of 32 or less. The performance isworse when considering all RT-PCR positive samples . However, there is some evidence suggesting that samplesobtained from individuals that achieve Ct values >30 measured usingRT-PCR tend to be less infective that those that record a C t value <30 [in vitro transcribed N-gene RNAstandard were tested using quantities from 105 copies down to 10 copies. Theassay readout is the color of the dye changing from pink to yellow due to binding to theDNA product over 30 minutes. The RT-LAMP assay was then applied to clinical nasopharyngealsamples. For viral loads above 100 copies of genomic RNA, the RT-LAMP assay had asensitivity of 100% and a specificity of 96.1% from purified RNA. The sensitivity of thedirect assay of saliva by RT-LAMP was 85%. Sensitivity and specificity metrics wereobtained by comparison with results from RT-PCR. RT-LAMP pilot studies for detection ofSARS-CoV-2 were reviewed in a meta-analysis [One study showed that RT-LAMP is effective for detection of SARS-CoV-2 withexcellent specificity and sensitivity and that this method can be applied to unprocessedsaliva samples . This mealue <30 ,47,48, salue <30 . To deteanalysis .In the This test aims to bring the sensitivity of nucleic acid detection to the pointof care or home testing setting. It could be applied for screening, diagnostics, or as adefinitive test for people who are positive based on LFTs (see below). The estimated costper test is about 2 euros when RNA extraction is included. The main strength of this testover RT-PCR is that it can be done isothermally, but the main drawback is that it is about10-fold less sensitive than RT-PCR. The low cost, excellent sensitivity/specificity, andquick readout of RT-LAMP makes this an attractive alternative to RT-PCR. Alternativestrategies like RT-LAMP are needed to bring widespread testing away from the lab and intounder-resourced areas.4.3Technology based on CRISPR   has alsoLFT strips are simple to use and read, but there are limitations in terms ofavailability and cost per test. Another group therefore proposed the CREST  protocol, which uses a P51 cardboard fluorescencevisualizer, powered by a 9-volt battery, for the detection of Cas13 activity instead ofimmunochromatography . CREST cE and N genes or presumptive positive if thereis detection of either of them. The assay had 95% positive predictive agreement and 100%negative predictive agreement with the US Centers for Disease Control and PreventionSARS-CoV-2 RT-qPCR assay. The estimated limit of detection was 10 copies per μlreaction, versus 1 copy per μl reaction for the CDC assay.The DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter) method fromMammoth Biosciences involves purification of RNA extracted from patient specimens,amplification of extracted RNAs by loop-mediated amplification, and application of theirCas12-based technology. In this assay, guide RNAs (gRNAs) were designed to recognizeportions of sequences corresponding to the SARS-CoV-2 genome, specifically the N2 and Eregions . In the These results have been confirmed by other DETECTR approaches. Using RT-RPA foramplification, another group detected 10 copies of synthetic SARS-CoV-2 RNA per μlof input within 60 minutes of RNA sample preparation in a proof-of-principle evaluation. ThroughIt was also reported that electric field gradients can be used to control andaccelerate CRISPR assays by co-focusing Cas12-gRNA, reporters, and target . The autThermus thermophilus [All these methods require upstream nucleic acid amplification prior toCRISPR-based detection. They rely on type V (Cas12-based) and type IV (Cas13-based) CRISPRsystems. In contrast, type III CRISPR systems have the unique property of initiating asignaling cascade, which could boost the sensitivity of direct RNA detection. In type IIICRISPR systems, guide CRISPR RNAs (crRNAs) are bound by several Cas proteins  and can mophilus . TheautThis goal of amplification-free detection was later achieved for a Cas13a-basedsystem . This apFrancisella novicida known as FnCas9 achieved 100% sensitivity and 97%specificity in clinical samples, and the diagnostic kit is reported to have completedregulatory validation in India [There is an increasing body of evidence that CRISPR-based assays offer apractical solution for rapid, low-barrier testing in areas that are at greater risk ofinfection, such as airports and local community hospitals. In the largest study to date,DETECTR was compared to RT-qPCR on 378 patient samples . The autin India .4.4Immunoassays can detect molecular indicators of SARS-CoV-2 infection, such asthe proteins that act as antigens from the SARS-CoV-2 virus. They offer the advantage ofgenerally being faster and requiring less specialized equipment than other moleculartests, especially those involving PCR. As a result, immunoassays hold particular interestfor implementation at home and in situations where resources for PCR testing are limited.The trade-off is that these tests typically have a lower sensitivity, and sometimes alower specificity, than other molecular tests. However, these tests tend to return apositive result five to 12 days after symptom onset, which may therefore correlate moreclosely with the timeframe during which viral replication occurs . Immunoa4.4.1LFTs provide distinct value relative to PCR tests. They can return resultswithin 30 minutes and can be performed without specialized equipment and at low cost.They also do not require training to operate and are cheap to produce. Thus, they can bedistributed widely to affected populations making them an important public healthmeasure to curb pandemic spread. LFTs rely on the detection of viral protein with anantibody. Often this is done with an antibody sandwich format, where one antibodyconjugated to a dye binds at one site on the antigen, and an immobilized antibody on thestrip binds at another site . This deA recent review surveyed the performance of LFTs for detection of currentSARS-CoV-2 infection . This reDespite having lower sensitivity than PCR tests, LFTs occupy an importantniche in the management of SARS-CoV-2. Current infection detection by LFTs enables thescale and speed of testing that is beneficial to managing viral spread. LFTs wereavailable freely to citizens in the United Kingdom until April 1, 2022  and to c4.4.2ELISA is a very sensitive immunoassay that can be considered a gold standardfor the detection of biological targets, including antibodies and antigenic proteins. It can t. This study also assessed the performance of theELISA test under different conditions in order to evaluate how robust it would be to thechallenges of testing in real-world settings globally. Higher sensitivity was achievedfor samples that were stored under ideal conditions (immediate placement in−80° C). Therefore, while immediate access to laboratory equipment is anadvantage, it is not strictly necessary for ELISA to detect the antigen.One of these assays uses two monoclonal antibodies specific to thenucleocapsid of SARS-CoV-2 to evaluate the relationship between the effect of(estimated) viral load on the ability of the assay to detect the SARS-CoV-2 antigen. This st4.5Tests that identify SARS-CoV-2 using molecular technologies will identify onlyindividuals with current infections and are not appropriate for identifying individualswho have recovered from a previous infection. Among molecular tests, differenttechnologies have different sensitivities and specificities. In general, specificity ishigh, and even then, the public health repercussions of a false positive can generally bemitigated with follow-up testing. On the other hand, a test’s sensitivity, whichindicates the risk of a false-negative response, can pose significant challenge tolarge-scale testing. False negatives are a significant concern for several reasons.Importantly, clinical reports indicate that it is imperative to exercise caution wheninterpreting the results of molecular tests for SARS-CoV-2 because negative results do notnecessarily mean a patient is virus-free . ToreduAdditionally, the emerging nature of the COVID-19 pandemic has introduced somechallenges related to uncertainty surrounding interactions between SARS-CoV-2 and itshuman hosts. For example, viral shedding kinetics are still not well understood but areexpected to introduce a significant effect of timing of sample collection on test results. SimilarThere are also significant practical and logistical concerns related to thewidespread deployment of molecular tests. Much of the technology used for molecular testsis expensive, and while it might be available in major hospitals and/or diagnosticcenters, it is often not available to smaller facilities . At time5Although several molecular diagnostic tests to detect viral genetic material havehigh specificity and sensitivity, they provide information only about active infection, andtherefore offer just a snapshot-in-time perspective on the spread of a disease. Mostimportantly, they would not work on a patient who has fully recovered from the virus at thetime of sample collection. In such contexts, serological tests are informative.Serological tests use many of the same technologies as the immunoassays used todetect the presence of an antigen but are instead used to evaluate the presence ofantibodies against SARS-CoV-2 in a serum sample. These tests are particularly useful forinsight into population-level dynamics and can also offer a glimpse into the development ofantibodies by individual patients during the course of a disease. Immunoassays can detectantibodies produced by the adaptive immune system in response to viral threat. Understandingthe acquisition and retention of antibodies is important both to the diagnosis of prior(inactive) infections and to the development of vaccines. The two immunoglobulin classesthat are most pertinent to these goals are immunoglobulin M (IgM), which are the firstantibodies produced in response to an infection, and immunoglobulin G (IgG), which are themost abundant antibodies ,90. SeroIn general, SARS-CoV-2 infection will induce the immune system to produceantibodies fairly quickly. Prior research is available about the development of antibodiesto SARS-CoV-1 during the course of the associated disease, severe acute respiratory syndrome(SARS). IgM and IgG antibodies were detected in the second week following SARS-CoV-1infection. IgM titers peaked by the first month post-infection, and then declined toundetectable levels after day 180. IgG titers peaked by day 60 and persisted in all donorsthrough the two-year duration of study . Suchte5.1The application of ELISA to serological testing is complementary to its use inmolecular diagnostics (see above). Instead of using an enzyme-labeled antibody as a probethat binds to the target antigen, the probe is an antigen and the target is an antibody.The enzyme used for detection and signal amplification is on a secondary antibody raisedgenerally against human IgG or IgM. In March 2020, the Krammer lab proposed an ELISA testthat detects IgG and IgM that react against the receptor-binding domain (RBD) of the spikeproteins (S) of the virus . A subse5.2Another early approach investigated for detection of antibodies againstSARS-CoV-2 was CLIA. Like ELISA, CLIA is a type of enzyme immunoassay (EIA) . While t5.3The first serological test approved for emergency use in the United States wasdeveloped by Cellex . The Cel5.4Neutralizing antibody assays play a functional role in understanding immunitythat distinguishes them from other serological tests. The tests described above are allbinding antibody tests. On the other hand, rather than simply binding an antibody tofacilitate detection, neutralizing antibody assays determine whether an antibody responseis present that would prevent infection ,107. TheThe gold standard for assessing the presence of neutralizing antibodies is theplaque reduction neutralization test (PRNT), but this approach does not scale well . An earl5.5While the adaptive immune system produces antibodies in response to SARS-CoV-2viral challenge, these indicators of seroconversion are unlikely to remain in circulationpermanently. Previously, a two-year longitudinal study following convalesced SARS patientswith a mean age of 29 found that IgG antibodies were detectable in all 56 patientssurveyed for at least 16 months and remained detectable in all but 4 patients (11.8%)through the full two-year study period .These rIn order to study the persistence of SARS-CoV-2 antibodies, one study assessedsustained immunity using 254 blood samples from 188 COVID-19 positive patients . The samTiters of IgM and IgG antibodies against the RBD were found to decrease from 1.3to 6.2 months post infection in a study of 87 individuals . HoweverDetermining the potency of anti-RBD antibodies early in the course of aninfection may be important moving forward, as their neutralizing potency may be prognosticfor disease severity and survival . Thedur+ T cells were slightly decreased(50%) 6 months post-symptom onset. In this same subset of COVID-19 patients, 93% ofsubjects had detectable levels of SARS-CoV-2 memory CD4+ T cells, of which 42%had more than 1% SARS-CoV-2-specific CD4+ T cells. At 6 months, 92% of patientswere positive for SARS-CoV-2 memory CD4+ T cells. Indeed, the abundance ofS-specific memory CD4+ T cells over time was similar to that ofSARS-CoV-2-specific CD4+ T cells overall [Other immune indicators of prior infection have also been evaluated to see howthey persist over time. SARS-CoV-2 memory CD8 overall . T cell  overall ,126,127. overall , but fur overall ,130 to i5.6In addition to the limitations posed by the fact that antibodies are notpermanent indicators of prior infection, serological immunoassays carry a number oflimitations that influence their utility in different situations. Importantly, falsepositives can occur due to cross-reactivity with other antibodies according to theclinical condition of the patient . Due toThese limitations make serological tests far less useful for diagnostics and fortest-and-trace strategies; however, serological testing is valuable for public healthmonitoring at the population level. Serosurveys provide a high-level perspective of theprevalence of a disease and can provide insight into the susceptibility of a population aswell as variation in severity, e.g., between geographic regions . From a Early in the course of the pandemic, it was also hoped that serological testswould provide information relevant to advancing economic recovery. Some infectious agentscan be controlled through “herd immunity”, which is when a critical masswithin the population acquires immunity through vaccination and/or infection, preventingan infectious agent from spreading widely. It was hoped that people who had recovered anddeveloped antibodies might be able to return to work ,140. Thi6One possible alternative or complement to molecular and serological testing wouldbe diagnosing COVID-19 cases based on symptomatology. COVID-19 can present with symptomssimilar to other types of pneumonia, and symptoms can vary widely among COVID-19 patients;therefore, clinical presentation is often insufficient as a sole diagnostic criterion. Inaddition, identifying and isolating mild or asymptomatic cases is critical to efforts tomanage outbreaks. Even among mildly symptomatic patients, a predictive model based onclinical symptoms had a sensitivity of only 56% and a specificity of 91% . More prIn some cases, clinical signs may also provide information that can informdiagnosis. Using computed tomography of the chest in addition to RT-qPCR testing was foundto provide a higher sensitivity than either measure alone . X-ray din vitro culturing [Finally, in addition to genomic and serological surveillance, other types ofmonitoring have proven useful in managing the pandemic . One thaulturing  and can 7Deciding whom to test, when to test, and which test to use have proven challengingas the COVID-19 pandemic has unfolded. Early in the COVID-19 pandemic, testing was typicallylimited to individuals considered high risk for developing serious illness . This apChanges in public attitudes and the lifting of COVID-19 restrictions due to themultifactorial desire to stimulate economic activities has required a shift of testingparadigms in 2022, despite warnings from public health officials against a hard exit frompublic health restrictions ,156. An In contrast, LFTs were thought to have poor sensitivity and their value foridentifying infections and managing the pandemic was questioned ,162. HowThese limitations are demonstrated by the estimated 10-fold under-reporting ofcases in the United States in 2020 due to shortages in testing and slow rollout of testingand slow reporting of results . However8Diagnostic tools have played an important role during the COVID-19 pandemic.Different tests offer different advantages .SpecifiChildren are one segment of the population where the importance of the twoaforementioned conditions can be exemplified. This group is particularly vulnerable as thereare ongoing challenges with testing in schools, increased COVID-19 mortality rates, andCOVID-19-associated orphanhood. In this regard, although there is evidence of the efficacyof routine diagnostic testing to reduce the probability of having infectious students ,177, as The SARS-CoV-2 diagnostic tools presented in this paper are far less useful ifthey are difficult to obtain, or if their limited use results in biased data that would leadto ill-informed public health strategies. Under conditions of limited supply, differentstrategies for testing are needed . Thepan


The COVID-19 pandemic influenced individuals’ anxiety and depression across the United States over a short period, and some Americans relied on drugs for coping. This study examines American adults’ drug use trajectories in response to changing anxiety and depression levels during the COVID-19 pandemic and the moderating role of substance use disorder (SUD) services provided by non-profit facilities in anxiety/depression-induced drug use. Heterogeneity in such relationships is further explored based on race/ethnicity. This study used a nationally representative sample of 1,176 American adults who reported drug use between May 1, 2020, and June 30, 2021. Using individual-fixed effects Poisson estimators, the current study empirically modeled drug use changes according to changing anxiety/depression levels. Interaction terms between anxiety/depression levels and per capita spending by non-profit SUD facilities were used to explore the moderating effect of SUD service expenditures. Racial/ethnic disparities were explored in subgroup analyses on non-Hispanic White, non-Hispanic Black, Hispanic, and non-Hispanic Asian samples. We found more frequent drug use in response to elevated anxiety and depression during the COVID-19 pandemic. Greater spending on SUD service by non-profit facilities at the county level was associated with reduced drug consumption associated with anxiety and depression, with greater benefits for racial/ethnic minorities. Findings provide important policy implications for distributing public funds for non-profit SUD facilities for mitigating SUD risks, especially among racial/ethnic minorities. Individuals often rely on substances to adapt to and cope with stress . While sAvailability of SUD services in communities can improve access to the service and timely and continuous delivery of treatment and care, which are essential to address emerging and ongoing SUD problems , 9. DespFirst identified in 2020, the novel coronavirus COVID-19) spread quickly worldwide and was ultimately labeled as a pandemic leading to significant social, economic, and personal costs. The pandemic has contributed to elevated stress levels and worsened depression and anxiety symptoms worldwide. The United States was hit especially hard by the pandemic with the highest number of COVID-19 related deaths in the world  spread q. As AmerIn this study, we aim to examine the extent to which individuals have changed their drug use in response to changing anxiety and depression levels during the COVID-19 era and to what extent such anxiety/depression-related drug use is moderated by non-profit facilities’ SUD service provisions in their residential counties. We also explore heterogeneity in the moderating effect of nonprofit SUD facilities’ expenditures according to race/ethnicity.To our best knowledge, this is one of the first empirical studies examining to what extent individuals with the risk of SUD problems benefit from SUD services in their community while also uncovering racial/ethnic disparities in experiencing such benefits. While some previous studies provided important evidence for racial/ethnic disparities in the geographical distribution of the SUD facilities, they focused on racial/ethnic characteristics at the community level , 7, 14. To explore the moderating effect of SUD service expenditures in communities on the relationship between individuals’ anxiety/depression and drug use, the present study used an individual-level panel dataset of the Understanding America Study (UAS) COVID-19 survey. On March 10, 2020, the University of Southern California (USC) began administering a bi-weekly online survey to approximately 7,500 American adults aged 18 or older that asked about their perceptions, behaviors, and health conditions related to COVID-19 . To ensuhttps://uasdata.usc.edu/index.php). The current study used the 4–29 waves of the UAS between May 1, 2020, and June 30, 2021. The first three waves were excluded in this study because they do not include some of the key covariates .To mitigate sampling attribution associated with an online survey, the UAS provided tablets and internet subscriptions to those without them . ResearcIndividual-level data were merged to various county-level data  based on the UAS respondents’ county of residence. Because geographic information below the state-level is restricted data, the data administrator linked external data to the UAS based on a respondent’s county of residence and then shared the merged data after eliminating any identifiable geographical information of respondents. To prevent researchers from identifying each respondent’s county of residence, the data administrator further masked some variable values  by aggregating them into fewer categories or rounding them off.To measure anxiety and depression, the UAS used the four-item Patient Health Questionnaire (PHQ-4). The PHQ-4 asks how many days a respondent has been bothered by the following problems over the past two weeks: feeling nervous, anxious, or on edge; not being able to stop or control worrying; feeling down, depressed, or hopeless; and little interest or pleasure in doing things. The responses were ’not at all (zero),’ ’several days (one),’ ’more than half the days (two), and ’nearly every day (three).’ The Cronbach alpha for the four items was 0.92, ensuring the internal reliability of the measure. A total PHQ-4 score was calculated by summing up the four-item (score 0–12). To account for potential nonlinearity of the relationship between anxiety/depression and outcomes, the total PHQ-4 score was categorized into four levels: normal (score 0–2); mild (score 3–5); moderate (score 6–8); and severe (score 9–12) .As a proxy for SUD service provision at the county level, the current study used the total expenditure of non-profit SUD facilities in each county, which was then standardized by county population . This stThe expenditure information of non-profit SUD facilities was retrieved from the 2017 National Center for Charitable Statistics (NCCS) core files, including the information listed on the Form 990 that every non-profit organization must file to the U.S. Internal Revenue Service (IRS) each fiscal year for tax exemption. The NCCS data include various organizational information, such as financial information, National Taxonomy of Exempt Entities (NTEE) codes corresponding to primary activities, and geographical information of each non-profit organization. SUD facilities were identified if a non-profit organization’s NTEE code was F20 , F21 (Substance Abuse Prevention), or F22 (Substance Abuse Treatment). We aggregated expenditures spent by all the SUD facilities located in each county and divided them by the county population with selected non-profit SUD facilities. The latest available NCCS data was from 2017 because many non-profits submit their required reporting form to the IRS with one- or two-year delays, and their more recent information has not yet been updated in the data repository.To mask the exact value of expenditures per capita that otherwise can be used to identify the geographical location of respondents, the value was rounded to the nearest one if it was less than 10; to the nearest 10 if it was greater than or equal to 10 but less than 100; to the nearest 100 if it was greater than or equal to 100 but less than 300. If the value of expenditure per capita was greater than or equal to 300, it was consolidated at 300.The outcome variable of the current study was drug use. Drug use behavior was assessed using the question asking, "out of the past seven days, what is your best estimate of the number of days that you used recreational drugs other than alcohol or cannabis product?" The responses ranged from zero to seven.To explore heterogeneity in the moderating effect of SUD service availability by race/ethnicity, the present study conducted subgroup analyses by splitting the sample by non-Hispanic White, non-Hispanic Black, Hispanic, and non-Hispanic Asian adults. In the subgroup analyses, other racial categories, such as American Indian or Alaska Native, Hawaiian/Pacific Islander, and mixed races, were excluded from our analyses. We hereafter denote ‘non-Hispanic’ White, Black, and Asian adults by White, Black, and Asian adults.The present study controlled for various time-variant socio-demographic factors, such as marital and employment status, household income, health insurance ownership, number of household members, and the presence of children under 18 in the household. It is noteworthy that age and educational attainment were treated as time-invariant because they were absorbed in individual-fixed effects due to little variations during our short study period.We further controlled for psychological factors potentially associated with anxiety/depression and substance use, including resilience and perceived stress. The Brief Resilience Scale (BRS) was used to assess resilience . The sixConsidering the unique context of COVID-19, we also controlled for the community- and individual-level COVID-19 related risk factors that might trigger anxiety and depression. As community-level risk factors, we used daily county-level new case and death rates per capita, both of which were retrieved from the Johns Hopkins University Center for Systems Science and Engineering (JHU CSSE) data depository . Such daWe further controlled for COVID-19 related government benefits and state-level health guidelines that potentially mitigate or exacerbate anxiety and depression. The two indicators of receiving governmental benefits were whether a respondent or anyone in his/her household received economic stimulus funds in the past month and whether he/she received unemployment insurance benefits in the past two weeks. Three statewide health mandate indicators were also controlled: whether a respondent lived in a state with stay-at-home; non-essential business closure; or overnight business curfew orders on survey dates. The information about statewide orders was retrieved from OpenICPSR data depository .To model whether spending on SUD services played a moderating role in the relationship between anxiety/depression and drug use, the following individual-fixed effects Poisson model was estimated:Drugit is the number of days using recreational drugs by individual i in t. Poisson models keep only those individuals whose responses to the outcome variable  vary over time in the sample. Therefore, those who did not consume any drugs during the study period were excluded from the sample. The individual-fixed effects model enabled us to model changes in drug use in response to changes in anxiety/depression levels over time by controlling for time-invariant individual characteristics given SUD service expenditures in the community. For anxiety/depression levels (Depit), we included three indicators denoting ‘mild,’ ‘moderate,’ and ‘severe’ conditions by setting a ‘normal’ level as the reference group. SUDc indicates pre-existing level of per capita expenditures by non-profit SUD facilities in county c prior to the pandemic. SUDc and its coefficient because the effect of the variable is likely to be absorbed in individual-fixed effects of ii, with a county of residence not changing among a majority of the UAS sample. Xit denotes a vector of time-variant individual- and household characteristics. ii and tt are individual- and time fixed effects. εit denotes an idiosyncratic error term. The coefficient of interest is β2. It should be noted that time-invariant individual-characteristics, such as race/ethnicity, were absorbed into individual-fixed effects (ii). We further estimated This method is appropriate because the dependent variable of Related to our key dependent and independent variables, the average number of days using drugs was similar across racial/ethnic groups and the pooled sample (avg. = 0.54–0.55), except for Asians (avg. = 0.40), when including the days without using drugs. Conditional on any drug use, the average days of drug consumption was highest among Whites (avg. = 3.94) and lowest among Asians (avg. = 3.05). The community’s average non-profit SUD facilities expenditure per capita was highest among Asians (avg. = 24.17) and lowest among Blacks (avg. = 14.38). The percentage of observations that scored moderate or severe anxiety/depression was highest among Whites (17.87%) and lowest among Blacks (12.74%).In reporting some important covariates, the unemployment rate during the study period was highest among Hispanics (26.18%), followed by Blacks (22.83%) and Asians (21.18%). The percentage of observations earning household incomes of at least $60,000 was highest among Asians (51.02%), followed by Hispanics (39.06%) and Whites (37.39%). Health insurance ownership was also highest among Asians (90.79%), followed by Whites (84.21%).The average BRS score was similar across racial/ethnic groups and the pooled sample, except for Asians. Asian observations scored the lowest in the BRS (avg. = 18.18). Further, Asian observations scored the highest on the PSS-4 scale, with an average score of 10.28. Conversely, the average PSS-4 score was lowest among White observations (avg. = 9.51).COVID-19 new case rates per capita were similar across racial/ethnic groups and the pooled sample, while death rates per capita were higher among Blacks and Hispanics. Consistent with the objective data on higher new case and death rates in these communities, the perceived risk of infection and death due to COVID-19 was highest among Blacks. As the data on higher unemployment rates, Black and Hispanic observations reported a higher perceived risk of running out of money due to COVID-19 than other groups. Due to their economic insecurity, a greater proportion of Hispanic observations received stimulus checks (19.28%) and unemployment insurance benefits (13.85%).The current study found heterogeneity in the moderating effects of non-profit SUD facilities’ expenditures on the relationship between anxiety/depression and drug use across different racial/ethnic groups. White adults showed similar patterns to the pooled sample (Column II). Relative to the normal level times, White adults with anxiety/depression elevated to mild, moderate, and severe levels used drugs 37.39, 56.13, and 77.21 percent more days. For an additional $1 per capita spent by non-profit SUD facilities in their county of residence, White adults whose anxiety/depression increased to a moderate level used drugs 0.58 percent more days.Both Black and Hispanic adults showed more frequent recreational drug use when they experienced elevated anxiety/depression to mild (54.18% & 49.37%), moderate (90.65% & 155.52%), and severe (151.93% & 149.18%) levels (Columns III & IV). However, with higher SUD service expenditure per capita in the community, both groups exhibited lower drug consumption even with elevated anxiety/depression. Specifically, Black adults’ drug consumption was 2.30 and 1.13 percent lower for those whose anxiety/depression levels increased to moderate and severe levels if their county had higher levels of spending on SUD services. Similarly, with higher SUD service expenditure per capita in the community, Hispanic adults’ drug consumption was lowered by 0.70, 1.28, and 1.29 percent among those who experienced elevated anxiety/depression to mild, moderate, and severe levels.Asian adults’ drug consumption did not change even when they experienced elevated anxiety/depression to mild and moderate levels. However, those whose anxiety/depression elevated to a severe level showed a 365.46 percent increased drug use compared to when they showed a normal level (Column IV). With greater levels of spending on SUD services by non-profit facilities in the community, Asian adults with elevated anxiety/depression to mild reported 0.92 percent higher drug use relative to the times with a normal level, while those with increased anxiety/depression to a severe level related to a 7.28 percent lower drug consumption with higher SUD service spending.As a robustness check, we ran additional analyses by excluding the health insurance ownership variable from our original models . FinanciThe results from the models without health insurance ownership adjustment Click here for additional data file.


Long non-coding RNA (lncRNA) is an important regulator of gene expression and serves a fundamental role in immune regulation. The present study aimed to develop a novel immune-related lncRNA signature to assess the prognosis of patients with colorectal cancer (CRC). Transcriptome data and clinical information of patients with CRC were downloaded from The Cancer Genome Atlas (TCGA) and UCSC Xena platforms. Immune-related mRNAs were extracted from the Molecular Signatures Database (MSigDB), and the immune-related lncRNAs were identified based on correlation analysis. Then, univariate, Lasso and multivariate Cox regression were applied to construct an immune-related lncRNA signature, and CRC patients were divided into high- and low-risk groups according to the median risk score. Finally, we evaluated the signature from the perspectives of clinical outcome, clinicopathological parameters, tumor-infiltrating immune cells (TIICs), immune status, tumor mutation burden (TMB) and immunotherapy responsiveness. In total, 272 immune-related lncRNAs were identified, five of which were applied to construct an immune-related lncRNA signature. The signature divided patients with CRC into low- and high-risk groups, the prognosis of patients in the high-risk group were significantly poorer than those in low-risk group, and the results were further confirmed in external validation cohort. Furthermore, the high-risk group showed aggressive clinicopathological characteristics, specific TIIC and immune function status, and low sensitivity to immunotherapy. The immune-related lncRNA signature could be exploited as a promising biomarker for predicting the prognosis and immune status of patients with CRC. Every year, more than 1.8 and 0.9 million estimated new CRC cases and deaths worldwide2. Recently, with the development of research, the pathophysiological and molecular mechanisms underlying the onset, invasion and metastasis of CRC have been extensively explored, and significant progress has been made in the diagnosis and treatment of CRC. However, CRC remains a severe health threat to humans because of the deficiency of effective biomarkers for diagnosis, especially in the early stage, and the prognosis of CRC patients is still poor. The 5-year survival rate for patients diagnosed with early stage is about 90%, while the 5-year survival rate sharply declines to 12% for patients diagnosed with advanced stage3. Considering the current situation of the diagnosis and treatment of CRC, it is imperative to seek more effective assessment models to evaluate the prognosis of CRC patients and provide optimized strategies for clinical CRC treatment.Colorectal cancer (CRC) is among the most diagnosed malignancies in digestive system and ranks second in terms of cancer-related death4. Immune cells are able to suppress the proliferation, invasion, and metastasis of cancer cells in the TME by perturbing the molecular signal pathway5. Nevertheless, some cancer cells also can inhibit the immune responses and create environments suitable for tumor growth, while these effects could be reversed by immunotherapy6. Therefore, immunotherapy provides an unprecedented opportunity for the effective treatment of cancers because of the crucial regulatory role of the immune cells in the TME7. Long non-coding RNAs (lncRNAs) are defined as transcripts with nucleotides more than 200 in length and have no ability to encode protein8. Previous studies have indicated that lncRNA can regulate gene expression at multiple levels, such as epigenetic, transcriptional or post-transcriptional regulation, and dysfunction of lncRNA is closely associated with the progression of multiple diseases, including malignancies11. Moreover, recent studies also indicated that lncRNA plays a significant regulatory role in different phases of tumor immunity, and several lncRNAs have been identified as potential biomarkers for many cancers13. For example, lncRNA SNHG1 inhibits the immune escape of breast cancer cells by suppressing the differentiation of Treg cells14. Similarly, NKILA regulates cytotoxic T lymphocyte sensitivity to activation-induced cell death by inhibiting NF-κB activity, thereby promoting tumor immune evasion of breast cancer15. These findings indicated that immune-related lncRNAs serve as potential tumor biomarkers and deserve more attention.In the past few years, accumulating studies have indicated that disorder of the immune response in the tumor microenvironment (TME) was significantly correlated with cancer development16. Recently, several studies have focused on developing a prognostic signature based on immune-related lncRNA and found that the signature could serve as a prognostic biomarker in human CRC18. However, most of these signatures are comprised of lncRNA pairs, which contain too much component. Besides, external validation was needed to improve the universal applicability of these developed signatures. Furthermore, the correlations between the developed signatures and immune cell phenotypes should be illustrated to identify which group of CRC patients is more likely to benefit from immunotherapy. Thus, we analyzed the entire RNA-sequencing profile data obtained from The Cancer Genome Atlas (TCGA) and developed a five-lncRNA immune-related signature for the prediction of prognosis in patients with CRC. In our study, we further investigated the predictive abilities of this signature in human CRC from the perspectives of clinicopathological parameters, tumor-infiltrating immune cells (TIICs), immune status, tumor mutation burden (TMB), and immunotherapy responsiveness. Importantly, the robustness of the immune-related lncRNA signature was validated in different populations from Gene Expression Omnibus (GEO) database.Previous research has indicated that multiple genes combinations is better than single gene in terms of the efficiency of a diagnostic model for tumorshttps://gdc.cancer.gov/). Then, the protein-coding genes and lncRNAs were classified by using the gene annotation function of the GENCODE project (https://www.gencodegenes.org/)19. In addition, the patient’s clinical parameters, including survival time, survival status, age, gender, American Joint Committee on Cancer (AJCC) stage, microsatellite instability, histological type and venous invasion status, were downloaded from the UCSC Xena (http://xena.ucsc.edu/) platform20. The gene expression profile matrix file and corresponding clinical information of GSE17536 were downloaded from GEO database (https://www.ncbi.nlm.nih.gov/geo/) and used as external validation cohort21. There is no need for approval by the ethics committee because all the data involved in the present study were obtained from the public database.The entire RNA-sequencing profile data of colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ) in the format of Fragments Per Kilobase Million (FPKM) were downloaded from TCGA (https://www.gsea-msigdb.org/gsea/msigdb/)22. Then, the correlation between lncRNAs and immune-related mRNAs was calculated using the Pearson correlation analysis. lncRNAs with Pearson correlation coefficient > 0.5 and P < 0.001, and meanwhile interacted with more than two immune-related mRNAs were identified as immune-related lncRNAs and selected for further analysis. To enhance the reliability of our study, the entire expression cohort of immune-related lncRNAs was randomly divided into the training cohort and the test cohort by the “caret” package in R.The immune-related mRNAs were extracted from immunomodulatory gene datasets IMMUNE RESPONSE (M19817) and IMMUNE SYSTEM PROCESS (M13664) based on the Molecular Signatures Database 23. Where the βi and Expi represented the coefficient index and the gene expression level, respectively. Patients were divided into high- and low-risk groups according to the median value of the risk score in the training cohort.To identify the survival-related lncRNAs, the prognostic significance of immune-related lncRNAs was analyzed by univariate Cox regression. Those immune-related lncRNAs with To evaluate the prognostic value of the immune-related lncRNA signature in human CRC, we depicted the survival curve between high- and low-risk groups by using the Kaplan–Meier method with a log-rank test. Additionally, we applied the time-dependent receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) to estimate the diagnostic efficacies. Similarly, we evaluate the prognostic value of this signature in the test cohort and the entire TCGA cohort to verify the reliability of the signature. To further confirm the clinical value of the immune-related lncRNA signature, we assessed the associations between the risk signature and clinical parameters. Finally, we compared the prognostic value of clinical parameters and risk score to determine whether the signature can be used as an independent prognostic factor.To further determine whether the immune-related lncRNA signature had a similar prognostic value in different populations, its prognostic capability was validated using external cohort.P < 0.001. After that, we used the “clusterProfiler” package in R to carry out Gene Ontology (GO) enrichment analysis, including biological process (BP), cell components (CC) and molecular function (MF) for the co-expressed network. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis is also carried out by the same tool24.To further investigate the biological function of the immune-related lncRNA signature, we constructed a co-expressed lncRNA-mRNA network by using Pearson correlation based on the correlation coefficient threshold > 0.5 and the corresponding 25.By using “rms”, “foreign”, and “survival” packages in R, we constructed a nomogram consisting of relevant clinical parameters and risk score based on the multivariate Cox regression analysis to predict survival for the patients with CRC. The calibration curve and ROC curves were applied to investigate the calibration and the discrimination of the nomogram26. Besides, we also analyzed the differences in TIIC abundances and immune-related biological functions between low- and high-risk groups by applying single sample GSEA (ssGSEA) algorithm27.To determine the correlation between the risk score and TIIC characteristics, we applied the QUANTISEQ algorithm to calculate the abundances of distinct TIIC among these samples from the TCGA project28.To identify the TMB of low-risk group and high-risk group, we obtained the information of somatic mutation profiles of CRC from TCGA sample. The differences in TMB between low- and high-risk groups were analyzed by performing Wilcoxon signed-rank test. Then, we analyzed and demonstrated the gene mutation patterns and frequencies in different subgroups by using the R package “maftool”http://tide.dfci.harvard.edu/) is a computational method to predict the clinical responses to immunotherapy and the function of T-cells in TME29. In this study, we applied the TIDE algorithm to predict the individual response to immunotherapy and compare the difference in TIDE scores between low- and high-risk groups.Tumor immune dysfunction and exclusion . The correlations of gene expression were evaluated by Pearson’s correlation coefficient. Cox regression and LASSO regression were used to evaluate the prognostic significance of the immune-related lncRNA signature and clinicopathological data. Chi-square test was used to evaluate the differences in clinicopathological features between two groups of patients. Fisher’s exact test was used while the sample size was less than five. Kaplan–Meier curves and log-rank tests were utilized to evaluate the survival data. The predictive accuracy of the risk model was determined by ROC curves. Wilcoxon test was mainly utilized for comparing two groups, and Kruskal–Wallis test was used for more than two groups. Fisher’s exact test was applied to evaluate the significant GO terms and KEGG enrichment pathways. P-value < 0.05 was considered as statistically significant.The statistical analyses were performed via the R software  + (0.52785 × ExpMIR181A2HG) + (0.46034 × ExpNIFK-AS1) + (0.05573 × ExpSNHG7) + (0.65167 × ExpZEB1-AS1).We carried out univariate Cox regression model to assess the prognostic significance of the 272 immune-related lncRNAs in the training cohort and found that 13 lncRNAs were significantly associated with the overall survival (OS) of CRC patients  and a high-risk group (n = 253) according to the median risk score. We assessed the predictive performance of the five-lncRNA immune signature by time-dependent ROC curves and found that the AUC for 1-, 3-, and 5-year OS were 0.790, 0.799, and 0.760, respectively . Besides, cox regression analysis also found that the risk score serves as a risk factor for the prognosis of CRC patients  , T classification , M classification , N classification , and risk score  were considered statistically significant  and risk score  were revealed as independent prognostic predictors , T CI 2.031–.201), T P < 0.001, we found that these five immune-related lncRNAs had an obvious correlation with 24 immune-related mRNAs, and the lncRNA-mRNA co-expression relationship network was constructed and visualized by using Cytoscape software  have developed rapidly in tumor treatment and achieved a certain therapeutic effect35. It is now well-established that patients with mismatch-repair-deficient (dMMR): microsatellite instability-high (MSI-H) tumors tend to be more sensitive to anti-tumor immunotherapy methods such as ICIs36. However, for the vast majority (~ 85%) of CRC patients whose tumors are MMR-proficient (pMMR): microsatellite stable (MSS), immunotherapy currently provides little benefit37. Therefore, identifying novel biomarkers for immunotherapy is needed.Colorectal cancer, which causes severe health burdens worldwide, is the most frequently diagnosed malignant tumor of the digestive system. Although surgical resection has been proven to be the only curable therapy for CRC patients diagnosed at the localized stage, the prognosis of patients diagnosed at the advanced stage remains largely unsatisfactorySATB2-AS1 has been reported to regulate TH1-type chemokines expression and immune cell density in CRC, thereby inhibiting tumor metastasis38. A recent study reported that lncRNA MALAT1 promoted tumorigenesis and immune escape of diffuse large B cell lymphoma by sponging miR-195 to induce apoptosis of CD8+ T cells39. GATA3-AS1 prevented the PD-L1 protein against ubiquitination through the miR-676-3p/COPS5 axis, thereby facilitating immune escape in triple-negative breast cancer40. These findings suggested that immune-related lncRNAs may be better prognostic biomarkers and serve as potential targets for anti-cancer therapy.Previous studies have revealed that lncRNA serves as an important regulator of the immune response in multiple cancer types. For example, lncRNA HCG11, MIR181A2HG, NIFK-AS1, SNHG7, and ZEB1-AS1) to predict the prognosis of CRC patients. Among these five candidate lncRNAs, SNHG7 has been reported to be expressed at high levels in CRC samples compared to normal control41. SNHG7 promoted proliferation and metastasis, and inhibited apoptosis of CRC cells via sponging miR-34a to regulate the PI3K/Akt/mTOR pathway42. It was reported that ZEB1-AS1 expression was significantly higher in CRC tissues than in adjacent normal tissues, and patients with high ZEB1-AS1 expression showed poorer prognosis than those with low expression43. In addition, ZEB1-AS1 activated the Wnt/β-catenin signaling pathway by sponging miR-181-5p, thereby promoting the proliferation of CRC cells in vitro44. LncRNA HCG11 has been experimentally demonstrated to facilitate the proliferation and migration of gastric cancer cells through the miR-1276/CTNNB1/Wnt pathway45. NIFK-AS1 was reported to function as a sponge for miR-146a to suppress the M2-like polarization of macrophages, thereby inhibiting the estrogen-induced proliferation, migration, and invasion of endometrial cancer cells46. A recent study has identified MIR181A2HG as a reliable component of an immune-related signature to predict the OS of patients with bladder cancer47. Given that these five lncRNAs serve vital roles in tumor progression, our study comprehensively considered their expression patterns and combined them to develop a novel prognostic signature in human CRC. Subsequently, survival analysis revealed that this immune-related lncRNA signature could distinguish patients with favorable prognosis from those with poor prognosis. Thus, our signature can be used to predict the prognosis of CRC patients.In this study, we constructed a survival-related signature comprised of five immune-related lncRNAs (48, TNF signaling49, RIG-I-like receptor signaling50, IL-17 signaling51 and JAK/STAT signaling52. Therefore, these results above suggested that the novel signature may participate in the progression of CRC through multiple signaling pathways.Functional enrichment analysis revealed that the immune-related lncRNA signature was mainly enriched in immunomodulatory pathways. Furthermore, KEGG enrichment analysis also revealed that the novel signature involved in various classical tumor-related pathways, which participate in the initial and progression of CRC, including NF-κB signaling53. The composition of TIICs within CRC tumors contained immunostimulatory and immunosuppressive components. The former included CD8+ T cells, dendritic cells, and natural killer cells, which are involved in the control of tumor growth, whereas the latter included CAFs, TAMs, and MDSCs, which promote tumor growth and metastasis55. During the progression of tumor, immunosuppressive TIICs hold the dominant position in TME and exert their oncogenic effects by inhibiting T cell functions56. CAFs modulate cancer growth and metastasis through secreting growth factors and re-modeling of the extracellular matrix57. In CRC, the expression intensity of CAFs was found to be increased at the invasive borders of the tumor, and high intensity of CAFs was significantly correlated with advanced disease stage58. Moreover, previous studies have indicated that CAFs were able to affect sensitivity to oxaliplatin and 5-fluorouracil in CRC cells59. In addition to CAFs, TAMs also play a significant role in regulating immune function in TME. According to the environmental stimuli, TAMs are often thought to polarize towards a pro-inflammatory “M1” or anti-inflammatory “M2” state60. In CRC, M1 TAMs exert anti-cancer effects by secreting pro-inflammatory cytokines such as TNF-α and IL-1, and increased densities of M1 TAMs are linked with a favorable clinical outcome61. Conversely, M2 TAMs exert tumor-supporting effects by secreting immunosuppressive cytokines such as IL-10, and high abundances of M2 TAMs are associated with a poor prognosis62. MDSCs are a heterogeneous population of immature myeloid cells which can be found in many types of tumors. MDSCs have been reported to promote the metastasis of CRC by suppressing the activity of anti-tumor T cells and producing pro-tumorigenic factors63. In our study, high densities of CAFs, M2 TAMs, and MDSCs were detected in the high-risk group, while the infiltration fractions of M1 TAMs were higher in the low-risk group. These results indicated that the lncRNA signature could predict the immune characteristics of CRC, and patients in the high-risk group infiltrated with more immunosuppressive TIICs compared to those in the low-risk group. We further analyzed the correlation between the lncRNA signature and TMB, an emerging biomarker for sensitivity to immunotherapies in multiple types of cancer independent of microsatellite status or immune checkpoint expression64. Tumors with high TMB are thought to harbor enhanced immunogenicity because of increased neoantigen burden, and therefore more likely to benefit from immunotherapy65. Interestingly, our study also found that the levels of TMB in the low-risk group were significantly higher than that in the high-risk group. In sum, the results above suggested that the immune-related lncRNA signature might be a promising reference for CRC immunotherapy.Studies have indicated that the initial and progression of CRC involves many aspects of immunodeficiency, and dysfunction of TIICs may affect the efficiency of immunotherapyInevitably, several limitations that existed in the present study should be pointed out. First and foremost, due to the lack of useful datasets containing lncRNA expression profiles and follow-up information of CRC in other public databases, the robustness of the immune-related lncRNA signature was validated in only one external cohort, multicenter perspective study will be performed to enhance the reliability of the signature in the future. Second, during the screening of candidate immure-related lncRNA, we took its prognostic value into consideration, which might omit some potentially valuable information and ultimately reduced the performance of the signature. Third, functional experiments should be conducted to further explore the functional roles of these immune-related lncRNAs in human CRC.In summary, the present study constructed and validated an immune-related five-lncRNA signature to predict the prognosis and immune characteristics of CRC patients based TCGA and GEO projects. There are significant differences in the clinical outcome and immune status of patients between low- and high-risk groups. This signature may provide new insights into the prognostic evaluation and individualized treatment for CRC patients.Supplementary Information.Supplementary Figure 1.


Radiotherapy omission is increasingly considered for selected patients with early-stage breast cancer. However, with emerging data on the safety and efficacy of radiotherapy de-escalation with partial breast irradiation and accelerated treatment regimens for low-risk breast cancer, it is necessary to move beyond an all-or-nothing approach. Here, we review existing data for radiotherapy omission, including the use of age, tumor subtype, and multigene profiling assays for selecting low-risk patients for whom omission is a reasonable strategy. We review data for de-escalated radiotherapy, including partial breast irradiation and acceleration of treatment time, emphasizing these regimens’ decreasing biological and financial toxicities. Lastly, we review evidence of omission of endocrine therapy. We emphasize ongoing research to define patient selection, treatment delivery, and toxicity outcomes for de-escalated adjuvant therapies better and highlight future directions.  The multimodal treatment of early-stage breast cancer includes breast-conserving surgery (BCS), radiation therapy (RT), and endocrine therapy (ET). As patients with breast cancer are living longer, it becomes increasingly important to minimize treatment-related morbidity. As such, there has been growing interest in the de-escalation of radiotherapy for very favorable-risk breast cancer. While there are ongoing trials evaluating the omission of radiotherapy, there are evolving data demonstrating decreased biological and financial toxicity of accelerated partial breast irradiation. The purpose of this article is to describe the strategies for de-escalated adjuvant therapies in early-stage breast cancer, including radiation omission, accelerated partial breast irradiation, and the role of endocrine therapy for very low-risk hormone receptor-positive (HR+) breast cancer. The concept of omission of RT for low-risk breast cancer dates to the early 1980s. At that time, the seminal NSABP B-06 trial had established the importance of RT in reducing the risk of an ipsilateral in-breast tumor recurrence (IBTR) after lumpectomy in women with node-positive or node-negative breast cancer , as wellThe NSABP B-21 study opened in 1989 and randomized 1009 women with tumors ≤1 cm to TAM alone, RT, or RT + TAM . There wp = 0.0008). However, there were no differences in regional recurrences, contralateral breast cancer, or distant metastases. These data suggest that the absolute risk of recurrence is lower when selecting patients of older age, with most dying of non-breast cancer-related causes. As a result, the National Comprehensive Cancer Networks Guidelines included adjuvant ET alone as a category one recommendation for women >70 years of age with early-stage HR+ breast cancer.In two subsequent modern randomized control trials, age was used as a criterion for omission after breast-conserving surgery (BCS) in patients with HR+ disease undergoing ET. In the CALGB 9343, 636 women ≥70 with clinical stage I, ER+ breast cancer, were randomized to TAM or RT + TAM . At 10 yOf note, despite modest absolute reductions in IBTR in PRIME II and NSABP B-21, the hazard ratio for the benefit of RT was similar (0.12 in PRIME II versus 0.19 in NSABP B21) and validated a persistent benefit of RT even in these early-stage breast cancer populations. The omission of RT based solely on age and HR status alone may lack appropriate risk-stratification power. It remains critical to identify a patient population with “low enough” IBTR risk following lumpectomy, for whom adjuvant RT may not provide a convincingly meaningful benefit. Does age remain an important criterion for RT omission once we account for tumor subtype? In a retrospective study of 1434 patients who underwent breast-conserving therapy from 1997–2006, the crude risk of locoregional recurrence (LRR) varied drastically by tumor subtype . PatientIn the Canadian study discussed above, investigators developed a low-risk clinical model of patients with T1, grade 1 or 2 breast cancer and ≥60 years, and reported a 10-year IBTR rate of 4.6% compared to 13.7% for the high-risk clinical group . Combinin = 895, placebo-treated: n = 355) and NSABP B-20 (n = 424), RS was found to be a significant predictor of LRR in TAM treated, placebo treated and chemotherapy + TAM treated patients. Of note, only 2% of patients enrolled in the NSABP 20 were over the age of 70 years, and no patients over the age of 70 years were enrolled in NSABP B14. On multivariable analysis, RS, age, and type of treatment were independent predictors of LRR. DEBRA is a phase III trial that randomizes patients between 50 to 70 years of age, with HR+ early-stage breast cancer, and with an Oncotype DX RS ≤18 to adjuvant RT versus observation. Moving beyond IHC, gene signatures based on multigene profiling assays are increasingly being used as predictive and prognostic markers for breast cancer recurrence and have been incorporated into ongoing clinical trials for the omission of RT . The PAMWe await results from these prospective clinical trials, which combine age and biology to optimize the selection of patients who may not benefit from adjuvant radiation.In the EBCTCG meta-analysis summarizing studies beginning before 2000, the rate of any first recurrence with BCS alone was 35% compared to 19.3% with BCS and RT, translating to a 10-year gain of 15.7% and a relative risk of 0.52 from RT . TherefoPartial breast irradiation (PBI) involves focused radiotherapy delivery to the lumpectomy cavity rather than the whole breast. The rationale is that residual microscopic disease tends to lie within 2 cm of the initial tumor ,14, and Accelerated RT involves higher doses delivered over a shorter interval of time. The rationale is that a shorter RT course with a higher dose per fraction can achieve the same therapeutic effect as a longer treatment course with a lower dose per fraction. A one-week schedule for whole breast or chest wall RT was evaluated in the UK FAST FORWARD study and found not to be inferior to standard fractionation without compromising normal tissue effects ,17. Accelerated partial breast irradiation (APBI) combines field reduction and acceleration of treatment time. APBI has been evaluated in phase III trials using percutaneous approaches, including multicatheter interstitial brachytherapy (MIB), balloon catheter intracavitary brachytherapy, electronic radiotherapy, and non-percutaneous approaches, including three-dimensional conformal external beam radiotherapy (3D-CRT), or intensity modulated radiation therapy (IMRT), proton beams or stereotactic PBI . The first randomized controlled trial of APBI came from the Hungarian National Institute of Oncology for patients with T1, N0-1 mi, >40 years of age with negative surgical margins. Investigators reported 10-year IBTR rates of 5.9% and 5.1% in the APBI and WBI arms, respectively, using HDR MIB or an electron beam . SubsequRegarding toxicity and cosmesis, results have been mixed with a potential confounding effect from the radiation components of the trials. For one, the interfraction recovery kinetics impact tissue repair for late fibrosis is estimated to have a half-life of repair of 4.4 hours. The RAPID trial from Canada demonstrated non-inferiority using 3D-CRT (38.5 Gy in 10 fractions BID) with an interfraction interval of 6 hours . This stn = 199, median follow-up 2.8 years) was 92.5% and 89.4%, respectively. There were low rates of telangiectasia (4.5% grade 1 and 1.5% grade 2), fibrosis (17.6% grade 1 and 3.0% grade 2), and retraction/atrophy . Overall, a once-per-day EBRT-APBI regimen with a sufficient interval between fractions appears to have a significantly less adverse effect on cosmesis. A notable finding of the NYU retrospective review was that when comparing patients with IBTR to those without IBTR, a higher proportion of those who did not receive endocrine therapy experienced IBTR . The NYU S14-0136 trial of prone APBI is a randomized controlled non-inferiority trial to compare radiation fibrosis with five (6 Gy × 5) versus three fractions (8 Gy × 3). The primary endpoint of the study is the rate of fibrosis at 2 years. The study closed accrual in June 2020, and we await the maturation of the data for the primary endpoint. A retrospective study of 345 patients treated at New York University (NYU) confirmed the low rates of toxicity and poor cosmesis with a five-fraction regimen . In thisThe side effect profile of APBI over WBI in the modern era will likely continue to improve as we modify delivery schedules, volumes treated, and treatment techniques. The data indicate that APBI not only reduces the biological toxicity of radiotherapy but also reduces the financial toxicity, based on Medicare fee-for-service global reimbursement patterns. Future directions to continue improving the therapeutic ratio include increasing the use of conformal treatments with IMRT, the use of even smaller volumes with preoperative treatments, and additional research regarding dose-volume parameters that allow even safer delivery of treatment with regard to long-term toxicity. Adjuvant ET, which includes TAM, a selective estrogen receptor modulator, or AIs, is another variable in the multimodal treatment of early-stage HR+ breast cancer with the opportunity to explore de-escalation in select patients. Compliance with ET can be challenging, particularly in patients with comorbid conditions . Rates op = 0.18). Overall, while local control with ET alone is excellent for patients who are adherent, the same may not be true for non-adherent patients. It is plausible that patients who opt for RT omission may also be more likely to have poor ET compliance for other medical or psychosocial reasons and personal preferences in their healthcare choices. All the radiation omission trials required ET. In the CALGB 9343 and PRIME II trials, the omission of radiotherapy discontinuation of ET rates were not reported, clouding our interpretation of results, and limiting the generalizability of the study findings. In a meeting for a multi-hospital retrospective review of 115 patients, the criteria for the LUMINA trial from the US, the ET non-adherence rate, defined as discontinuation before 5 years due to reasons unrelated to disease recurrence or death, was compared between women treated with RT versus RT omission . ET non-adherence was found to be 32% in the RT omission group versus 17% in the RT group. The rate of locoregional control was reduced to 83% from 100% in the RT omission groups among ET adherent versus non-adherent patients , which has an NCCN Category 1 rating in breast cancer for pN0 and 1–3 positive nodes, can identify a subgroup of patients (MammaPrint Index (MPI) > +0.355) as ultralow risk who have an excellent 20-year survival risk without any systemic therapy. The Stockholm tamoxifen (STO-3) study was a randomized clinical trial of TAM versus no therapy from 1976 until 1990 in postmenopausal women with node-negative, ER-positive breast cancer and tumors <3 cm who had received either adjuvant TAM for two years or no systemic therapy and were randomized to receive three additional years of TAM or no further treatment . Sixty-fIn a subset analysis of the prospective MINDACT trial , 1000 paThe Breast Cancer Index (BCI) is a gene expression-based biomarker developed using a cohort of ER+ lymph node-negative patients from the randomized prospective Stockholm trial using two biomarkers, the HOXB13:IL17BR ratio (H/I) and the molecular grade index (MGI) . InvestiAPBI alone and ET alone are potentially reasonable monotherapy strategies for patients with very low-risk breast cancer. In the Florence APBI trial of five fractions, it is notable that 35.8% of patients in the APBI arm and 28.8% in the WBI arm did not receive any adjuvant ET. Cost-effective analyses have revealed that both APBI alone and ET alone are appropriate options for patients older than 70 years of age with small cost differences . While AIn HR+ stage I breast cancer, older age combined with tumor subtype identifies patients at lower risk of local recurrence in whom the absolute benefit of both adjuvant radiotherapy and endocrine therapy remains small. Randomized trial evidence exists to support the omission of radiation based on age and tumor subtype, all of which required endocrine therapy. We are awaiting trials combining age and tumor biology, including utilizing multigene assays, to identify patients for whom omission is reasonable. De-escalation of radiotherapy in the form of reduced treatment fields and accelerated treatment time maintains a favorable therapeutic ratio, even with a decrease in the absolute benefit of radiation on local control in the modern era. There is emerging data on the omission of endocrine therapy when radiation is given in a low-risk patient population, and we await prospective trials to identify appropriate patients for endocrine therapy omission or de-escalation. Much progress has been made in reducing the overall treatment burden for early-stage breast cancer patients while maintaining excellent outcomes, and we look forward to continuing to refine the optimal treatment strategy for these patients.


Access to recreational physical activities, particularly in outdoor spaces, has been a crucial outlet for physical and mental health during the COVID-19 pandemic. There is a need to understand how conducting these activities modulates the risk of SARS-CoV-2 infection. In this case–control study of unvaccinated individuals conducted in San Francisco, California, the odds of testing positive to SARS-CoV-2 were lower for those who conducted physical activity in outdoor locations  in the two weeks prior to testing than for those who conducted no activity or indoor physical activity only. Individuals who visited outdoor parks, beaches, or playgrounds also had lower odds of testing positive to SARS-CoV-2  as compared with those who did not visit outdoor parks, beaches, or playgrounds. These findings, albeit in an unvaccinated population, offer observational data to support pre-existing ecological studies that suggest that activity in outdoor spaces lowers COVID-19 risk. The COVID-19 pandemic has caused widespread decreases in individual physical and recreational activity, particularly in outdoor spaces  and chroSeveral ecological studies have observed an association between the availability of green space and reduced COVID-19 case rates ,6,7,8,9.We conducted a case–control study to investigate whether outdoor physical and recreational activities were associated with SARS-CoV-2 transmission in the city and county of San Francisco (CCSF) from April to June 2021, when average daily cases ranged from 43 to 10 and the proportion of residents who were fully vaccinated increased from ~32% to ~65% . In thisWe identified participants from the San Francisco Department of Public Health surveillance database of SARS-CoV-2 test records during the study period. Using a case–control design, we recruited unvaccinated individuals aged 13 years and older who had tested positive for SARS-CoV-2 (“cases”), as well as a random sample of those who tested negative (“controls”). Given the narrow time window, we did not match for the date of the test result. The study was approved by the University of California, San Francisco, Committee on Human Research. Informed consent was required of all study participants, including participants younger than 18 years and their parents/guardians.Exclusion criteria included having received ≥ 1 dose of a COVID-19 vaccine, residing outside of CCSF, living in a congregate setting , or being unable to confirm birth date or test date. Cases were also excluded if they were unaware of their SARS-CoV-2 status or had not yet been interviewed by the health department’s case investigation team. To minimize exposure recall bias, we contacted subjects within three weeks of their test dates.We attempted to reach all remaining eligible study participants by telephone and completed an interviewer-led, closed-ended questionnaire on activities during the two-week period prior to their test dates . InterviWe examined the sociodemographic characteristics of the cases and controls, and tested for differences using Pearson’s Chi-squared and Fisher’s exact tests. We then fitted logistic regression models to generate odds ratios for SARS-CoV-2 status by outdoor sport and outdoor park use, adjusting for potential confounding variables including age, race/ethnicity, ZIP code per capita income, and household occupancy. Household occupancy was defined by occupants per room: under-occupied was defined as fewer than one occupant per room, balanced was defined as one per room, and over-occupied was defined as more than one per room. In addition, we performed exploratory interaction analyses to test for the effect modification of SARS-CoV-2 status and outdoor sport and park use by household occupancy and ZIP code per capita income. ZIP code estimated per capita income was obtained as a computed average from the publicly available American Community Survey 2019 Census Dataset  and dichAll statistical analyses were conducted using R version 4.0.5  .Among the sampled participants from a pool of 267,464 records, 982 cases and 1433 controls were reached by phone. In total, 139 cases and 35 controls consented, met the eligibility criteria, and completed questionnaires. The median age for the sample was 31.5 years (interquartile range [IQR] 25–43), 51% were male, and 35% identified as Latinx.p = 0.002) and race/ethnicity (p = 0.04)  . Cases wIn the multivariable analysis, cases were less likely to engage in outdoor sports and less likely to use outdoor parks, beaches, or playgrounds, regardless of activity . There wp ≥ 0.25 for all interactions) .Interaction analyses found no statistically significant effect modification of SARS-CoV-2 status and outdoor sport and park use by household occupancy or ZIP code per capita income (actions) . AdditioIn this case–control study conducted among unvaccinated individuals in San Francisco, California, from 7 April to 8 June 2021, we found that the odds of testing positive for SARS-CoV-2 were significantly lower for those who conducted physical activity in outdoor locations in the two weeks prior to testing than those who conducted no activity or indoor physical activity only. The protective effect of outdoor locations was further highlighted by the association between low odds of SARS-CoV-2 infection and visiting outdoor parks, beaches, or playgrounds.These findings, based on individual-level observational data, support ecological studies that found an association between the availability of green space and reduced COVID-19 case rates ,6,7,8,9.First, recreational activities in outdoor spaces may prevent SARS-CoV-2 transmission insofar that they offer safe, healthy outlets that may replace other, riskier activities indoors. This relationship might partly explain the disproportionate burden of COVID-19 on marginalized populations in lower-income, higher-density urban settings, who historically have little or no access to safe and clean green spaces . Even thSecond, individuals who reported any physical activity in outdoor spaces in the two weeks prior to testing might be healthier overall. Green space use, which has been shown to have decreased individual stress levels during the pandemic , and conLastly, high vaccine uptake in San Francisco  and B.1.1.529 (Omicron). Additionally, challenges in the recruitment of controls due to increased vaccination rates during the time of the study and the limited success of COVID-19-safe recruitment methods  limited the size of the control group, regardless of oversampling controls in the study participant selection phase. Selection bias might have affected the results, as people who did not participate in the study might have been different from individuals included in this study, for example, due to different lifestyle factors. Moreover, we acknowledge that the regression models may not have adjusted for all possible confounders. Interviewees might have been prone to recall bias, as awareness of their COVID-19 test result might have influenced their responses. Finally, our questionnaire did not include the duration and frequency of outdoor activities.In this case–control study of unvaccinated individuals, the adjusted odds of testing positive for SARS-CoV-2 were 84% lower among individuals who conducted outdoor recreational physical activity in the two weeks prior to testing than those who conducted no activity or indoor activity only. The adjusted odds of testing positive for SARS-CoV-2 were 72% lower among individuals who visited outdoor parks in the two weeks prior to testing than who did not. These findings support pre-existing ecological studies which suggest that proximity to green spaces and activity in outdoor spaces might lower the overall COVID-19 risk. Efforts to increase green space and its use, particularly in areas with decreased access, are therefore urgent: equity in access to outdoor space may not only be crucial for individual physical and mental health, but also may be an important protective factor in decreasing the spread of respiratory infections, further contributing to health of communities overall.


Quality care is a key component of the health care system. The gap between actual care received and ideal care quality in the existing healthcare system is enormous. Although healthcare spending is remarkably rising than any other global economy, the healthcare system is still facing immense challenges in inaccurate diagnoses, medication errors, inappropriate or unnecessary treatments, and insufficient clinical practices. The World Health Organization (WHO) report 2020 shows that global spending on healthcare has reached US$ 8.3 trillion, ~10% of the global GDP  system to correctly classify long-term cardiovascular outcomes in patients with normal ejection fraction. Echocardiographic data from 61,525 patients were collected to develop an AI model, which was later internally and externally validated using data from 3,810 and 5,760 patients. This AI-based system was able to stratify patients with a left ventricular end-diastolic diameter (LV-D) and predicts ECG-EF accurately with high AUCs. Nowadays, stereotactic body radiotherapy (SBRT) is considered one of the key treatment options for patients with early-stage lung cancer. It has shown a beneficial effect in improving tumor control and overall survival rate. A recent study tested the performance of the Mask R-CNN-based algorithm for evaluating the dose accuracy of a lung SBRT treatment plan with the target of a newly predicted internal target volume (ITVpredict) and the feasibility of its clinical application . The cone-beam CT (CBCT) images were collected from early-stage 45 lung cancer patients who underwent SBRT at Huadong Hospital. This AI-enable tool was able to predict the ITV volume of large tumors more accurately, which ensures the feasibility of this automated model in making an appropriate treatment plan.A shift to value-based care from fee-for-service is not a dream because of the availability of patient data in the electronic health record (EHR) systems, standardization framework, and advanced algorithms. Clinicians can collect overwhelming amounts of patient data and utilize advanced analytical tools to make accurate predictions and actionable insights to improve overall provider performance, decrease medical errors, and reduce healthcare waste . Chen etJiang et al.). A total of 417 patients were included in the study, and MRI data of those patients were used to divide patients into pre-AD  and control groups  based on a standard uptake ratio >1.18. AI model achieved state-of-the-art performance in classifying pre-AD and normal control with an accuracy of 89.85% ± 1.12%. It is now established that advanced AI algorithms have surpassed traditional statistical methods in image recognition and being extensively used in medical image analysis. In the last decade, AI-based radiomic models have made meaningful contributions to detecting chronic diseases, including lung cancer. A systematic review and meta-analysis included a total of 19 published studies to evaluate the diagnostic accuracy of AI models for lung cancer staging . The findings of AI models have the potential to improve diagnostic accuracy for lung cancer staging in terms of sensitivity, specificity, and the area under the receiver operating curve (AUROC).Alzheimer's disease (AD) is a critical global health problem contributing to a substantial financial burden. A previous study reported that ~6.5 million aged 65 or older are living with AD in the USA. Early identification of AD patients significantly reduces healthcare costs and improves patients' quality of life. Since AI techniques based on MRI are being used in the early diagnosis of AD, a novel deep learning radiomics (DLR) model was developed to classify cognitively normal adults at risk of AD from normal control using T1-weighted structural MRI images (AI models have tremendous potential to reduce medical errors, effectively utilize limited resources, and ultimately improve value by making accurate and effective clinical decisions. Recently, several studies internationally validated AI tools and achieved classification accuracy performance that outperformed human performance , 4. TranAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.